IBRUTINIB 依鲁替尼 A Btk protein inhibitor.

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Feb 272014

IBRUTINIB 依鲁替尼

A Btk protein inhibitor.

1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one

1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

CAS number	936563-96-1

Ibrutinib, PCI 32765, PCI32765, ibrutinibum, IMBRUVICA,

CRA-032765
Ibrutinib
Imbruvica
Pc-32765
PCI 32765
PCI32765
UNII-1X70OSD4VX

Molecular Formula: C₂₅H₂₄N₆O₂

Molecular Weight: 440.49706

Company: Pharmacyclics
Approval Status: Approved February 2014US FDA:link
Treatment Area: chronic lymphocytic leukemia

Bruton’s tyrosine kinase (Btk) inhibitor

U.S. Patent No: 7,514,444 , 7,718,662
patent validity: December 2026

An orally bioavailable small-molecule inhibitor of Bruton’s tyrosine kinase (BTK) with potential antineoplastic activity. Ibrutinib binds to and inhibits BTK activity, preventing B-cell activation and B-cell-mediated signaling and inhibiting the growth of malignant B cells that overexpress BTK. BTK, a member of the src-related BTK/Tec family of cytoplasmic tyrosine kinases, is required for B cell receptor (BCR) signaling, plays a key role in B-cell maturation, and is overexpressed in a number of B-cell malignancies.

Imbruvica (ibrutinib) is an orally available, selective inhibitor of Bruton’s tyrosine kinase (Btk), a gene that is disrupted in the human disease X-linked agammaglobulenemia (XLA). BTK is a signaling molecule of the B-cell antigen receptor (BCR) and cytokine receptor pathways.

Imbruvica is specifically approved for chronic lymphocytic leukemia in patients who have received at least one prior therapy.

Imbruvica (Ibrutinib, previously known as PCI-32765) was approved as a “breakthrough therapy” on November 13, 2013 by the US Food and Drug Administration (FDA) for the treatment of mantle cell lymphoma (MCL), a rare and deadly form of blood cancer.

IBRUTINIB

Ibrutinib, a first in class oral Bruton’s tyrosine kinase (Btk) inhibitor, was launched in the U.S. for the treatment of patients with mantle cell lymphoma in 2013, and for the treatment of chronic lymphocytic leukemia in 2014. In the E.U., the product candidate is awaiting registration for both indications. Additional phase III clinical trials are ongoing for the treatment of these indications in combination with bendamustine and rituximab and for the treatment of relapsed or refractory marginal zone lymphoma (MZL). Janssen and Pharmacyclics are conducting phase II clinical trials for the treatment of refractory follicular lymphoma. Early clinical development is also under way at Pharmacyclics for the treatment of recurrent B-cell lymphoma, relapsed/refractory MCL, and relapsed or relapsed and refractory multiple myeloma. The company filed an IND seeking approval to commence clinical evaluation of ibrutinib for the treatment of autoimmune disease. Preclinical studies had been under way for rheumatoid arthritis; however, no recent development has been reported. Ibrutinib is also active against Lyn and LCK tyrosine kinases.

In 2011, a codevelopment agreement was signed between the National Cancer Institute (NCI) and Pharmacyclics for the treatment of hematologic/blood cancer. Also in 2011, a worldwide codevelopment and comarketing agreement was signed by Janssen and Pharmacyclics for the treatment of cancer. In 2012, orphan drug designation was assigned in the U.S. and the E.U. for the treatment of CLL. This designation was also assigned by the FDA in 2012 for the treatment of mantle cell lymphoma. In 2013, several orphan drug designations were assigned in the U.S.; for the treatment of small lymphocytic lymphoma, for the treatment of Waldenstrom’s macroglobulinemia and for the treatment of diffuse large B-cell lymphoma. For this indication, orphan drug designation was assigned also in the E.U. the same year. In 2012, fast track designation was assigned by the FDA for the treatment of CLL. In 2013, breakthrough therapy designations were assigned to the compound in the U.S.: for the treatment (as monotherapy) of patients with chronic lymphocytic leukemia or small lymphocytic lymphoma, for the treatment of relapsed or refractory mantle cell lymphoma who have received prior therapy and for the treatment of Waldenstrom’s macroglobulinemia.

Imbruvica is supplied as a capsule for oral administration. The recommended dose is 420 mg taken orally once daily (three 140 mg capsules once daily). Capsules should be taken orally with a glass of water. Do not open, break, or chew the capsules.

The FDA approval of Imbruvica for chronic lymphocytic leukemia was based on an open-label, multi-center trial of 48 previously treated patients. Imbruvica was administered orally at 420 mg once daily until disease progression or unacceptable toxicity. The overall response rate (ORR) and duration of response (DOR) were assessed using a modified version of the International Workshop on CLL Criteria by an Independent Review Committee. The ORR was 58.3%, all partial responses. None of the patients achieved a complete response. The DOR ranged from 5.6 to 24.2+ months. The median DOR was not reached.

Imbruvica (ibrutinib) is an orally available, selective inhibitor of Bruton’s tyrosine kinase (Btk). Ibrutinib forms a covalent bond with a cysteine residue in the BTK active site, leading to inhibition of BTK enzymatic activity. BTK is a signaling molecule of the B-cell antigen receptor (BCR) and cytokine receptor pathways. BTK’s crole in signaling through the B-cell surface receptors results in activation of pathways necessary for B-cell trafficking, chemotaxis, and adhesion.

Ibrutinib (USAN,^[1] also known as PCI-32765 and marketed in the U.S. under the name Imbruvica) is an anticancer drug targeting B-cell malignancies. It was approved by the US FDA in November 2013 for the treatment of mantle cell lymphoma^[2] and in February 2014 for the treatment ofchronic lymphocytic leukemia.^[3] It is an orally-administered, selective and covalent inhibitor of the enzyme Bruton’s tyrosine kinase (BTK).^[4]^[5]^[6]Ibrutinib is currently under development by Pharmacyclics, Inc and Johnson & Johnson‘s Janssen Pharmaceutical division for additional B-cell malignancies including diffuse large B-cell lymphoma and multiple myeloma.^[7]^[8]^[9]

Mechanism

In preclinical studies on chronic lymphocytic leukemia (CLL) cells, ibrutinib has been reported to promote apoptosis, inhibit proliferation, and also prevent CLL cells from responding to survival stimuli provided by the microenvironment.^[12] In this study, treatment of activated CLL cells with ibrutinib resulted in inhibition of Btk tyrosine phosphorylation and also effectively abrogated downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. Additionally, ibrutinib inhibited proliferation of CLL cells in vitro, effectively blocking survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement and stromal cell contact.

In early clinical studies, the activity of ibrutinib has been described to include a rapid reduction in lymphadenopathy accompanied by a transient lymphocytosis, suggesting that the drug might have direct effects on cell homing or migration to factors in tissue microenvironments.^[13]

Ibrutinib has been reported to reduce CLL cell chemotaxis towards the chemokines CXCL12 and CXCL13, and inhibit cellular adhesion following stimulation at the B cell receptor.^[14]^[15] Together, these data are consistent with a mechanistic model whereby ibrutinib blocks BCR signaling, which drives cells into apoptosis and/or disrupts cell migration and adherence to protective tumour microenvironments.

History

Ibrutinib was first designed and synthesized at Celera Genomics which reported in 2007 a structure-based approach for creating a series of small molecules that inactivate BTK through covalent binding to cysteine-481 near the ATP binding domain of BTK.^[4] These small molecules irreversibly inhibited BTK by using a Michael acceptor for binding to the target cysteine. In April 2006, Pharmacyclics acquired Celera’s small molecule BTK inhibitor discovery program, which included a compound, PCI-32765 that was subsequently chosen for further preclinical development based on the discovery of anti-lymphoma properties in vivo.^[16] Since 2006, Pharmacyclics’ scientists have advanced the molecule into clinical trials and identified specific clinical indications for the drug. It also has potential effects against autoimmune arthritis.^[17] It was approved by the US FDA on November 13, 2013 for the treatment of mantle cell lymphoma.^[2] On Feb. 12, 2014, the U.S. Food and Drug Administration expanded the approved use of the drug ibrutinib to chronic lymphocytic leukemia (CLL). ^[18]

Ibrutinib is an inhibitor of Bruton’s tyrosine kinase (BTK). It is a white to off-white solid with the empirical formula C₂₅H₂₄N₆O₂ and a molecular weight 440.50. Ibrutinib is freely soluble in dimethyl sulfoxide, soluble in methanol and practically insoluble in water.

The chemical name for ibrutinib is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1Hpyrazolo[ 3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one and has the following structure:

IMBRUVICATM (ibrutinib) Structural Formula Illustration

IMBRUVICA (ibrutinib) capsules for oral administration are supplied as white opaque capsules that contain 140 mg ibrutinib as the active ingredient. Each capsule also contains the following inactive ingredients: croscarmellose sodium, magnesium stearate, microcrystalline cellulose, sodium lauryl sulfate. The capsule shell contains gelatin, titanium dioxide and black ink. Each white opaque capsule is marked with “ibr 140 mg” in black ink.

PCI-32765 (ibrutinib) is disclose d in U.S. Patent No. 7,514,444, issued on April 7, 2009, and has the following structur

Ibrutinib is an orally available drug that targets Bruton’s tyrosine kinase (BTK).

Ibrutinib is an irreversible small molecule BTK inhibitor that is in Ph Ib/II of clinical trials in a variety of B-cell malignancies including chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (cancer of plasma cells, a type of white blood cell present in bone marrow). At present ibrutinib is administered orally in clinical trials, via the gastrointestinal tract, at high clinical doses (420 mg/day or 840 mg/day) to patients with CLL and SLL to obtain the desired thereapeutic effect. The need for such high doses of ibrutinib may be due to low bioavailability (the oral bioavailability of ibrutinib is reported to be 22.8% in rats) and may be responsible for the adverse side effects associated with the use of ibrutinib such as nausea or emesis, dizziness and diarrhea. Moreover, low bioavailability results in more variable absorption and potential variability of the desired therapeutic response.

As stated above, at present ibrutinib is administered orally, via the gastrointestinal tract, at high clinical doses (420 mg/day or 840 mg/day) to patients to obtain the desired clinical benefit. It is presently disclosed that when ibrutinib is administered intraduodenally versus via the gastrointestinal tract in rats, the oral bioavailability of ibrutinib unexpectedly increased from 21 % to 100% as determined by AUC.

This unexpected increase in oral bioavailability of ibrutinib can translate into a number of desirable practical benefits. The increase in oral bioavailability should enable administration of ibrutinib at a significantly lower therapeutically effective dose than is currently being used. The lower variability associated with this greater bioavailability should lead to a more reliable therapeutic response as well as more predictable drug absorption.

And avoidance of exposure of Ibtrutinib to the stomach and/or use of lower therapeutically effective dose of ibrutinib can reduce or altogether eliminate potential adverse side effects of this drug such as diahrrea, nausea or emesis, and dizziness. U.S. Patent No. 7,514,444, mentioned above, discloses administration of 0.02-5000 mg/kg andl-1500 mg of ibrutinib/per day and in clinical trials 420 or 840 mg/day of ibrutinib is being administered to the patients with CLL and SLL.

There is no reasonable expectation in the art that ibrutinib can be adminstered orally at lower efficacious doses to the patients with CLL and SLL, particularly as evidenced by the 420 or 840 mg/day of ibrutinib being administered in clinical trials to those patients. Moreover, other than for active agents that are unstable in the stomach or at acidic pH delivery of any active agent with low bioavailability further along in the gastrointestinal tract reduces the path length for drug absorption and would be expected to reduce bioavailability. Therefore, it was unexpected to achieve delivery of ibruntinib directly to the small intestine with greater bioavailability.

PC1-32765 (Ibrutinib), chemical name: 1_ [(3R) _3-[4_-3 – (4 – phenoxy-phenyl)-1H-pyrazolo [3,4-d] pyrimidine – 1 – yl] – 1-piperidinyl]-2 – propen-1 – one, and its structural formula is as follows:

PC1-32765 is an oral medication that inhibits B cell as the main receptor tyrosine kinase signaling and promote cell death process, preventing cell migration and adhesion in malignant B cells.

US20080108636 basic patent has been disclosed a synthetic route:

This synthetic route with 4 – phenoxy-benzoic acid as raw material, after eight-step reaction the final product, the following reaction steps:

The above method has the following disadvantages:

1, eight single-step reaction, long route, the economy is bad; i1, to use synthetic intermediates 4:00 trimethylsilyl diazomethane (TMSCHN2), this material easy to blow up, the risk coefficient is large, so large-scale production greatly reduces the possibility;

ii1, synthetic intermediates 7:00, set out to use polymer-supported triphenylphosphine, non-industrial raw materials used, the price is expensive, the cost of smell;

iv, the final step of acylation, the selectivity is poor, a large amount of negative product, purification is difficult, amplification reaction is difficult.

In summary, the route material is not common, expensive step, high costs, the reaction dangerous side reactions, purification difficult, limiting the possibility of industrial production of the route.

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WO2013184572A1

Polymorphs

EXAMPLES

[00438] The following ingredients, formulations, processes and procedures for practicing the methods disclosed herein correspond to that described above. Example 1; Preparation of Crystalline Forms of l-((R)-3-(4-amino-3-(4-phenoxyphenyl)- lH-pyrazolo[3,4-dlpyrimidin-l-yl)piperidin-l-yl)prop-2-en-l-one (Compound 1)

Form A – Route 1:

[00439] Amorphous Compound 1 (ca. 15 mg) was measured into a vial. Ten volumes (150 μΐ) of solvent [methyl tert-butyl ether (MTBE), diisopropyl ether (DIPE), ethyl acetate, isopropyl acetate, isopropyl alcohol, methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), acetone, methanol, nitromethane, 10% aqueous acetone, or 10% aqueous isopropyl alcohol] were added to the vial. The vial was sealed and placed in a shaker at 50 °C for one hour. If a slurry was obtained, an additional thirty volumes (total of 600 μΐ) of solvent was added, then the slurry was returned to 50 °C for another hour. If the sample remained as a slurry at this point, no further solvent was added. The solution/slurry was stirred at 50 °C for one hour, then cooled to 0 °C at 0.1 °C/min, then held at 0 °C overnight. If a slurry was obtained, the solids were filtered under vacuum to provide Compound 1 , Form A; the solution was returned to ambient temperature for slow evaporation through a pin-hole to furnish Compound 1, Form A.

“Compound 1” or “l-((R)-3-(4-amino-3-(4-phenoxyphenyl)-lH-pyrazolo[3,4- d]pyrimidin- 1 -yl)piperidin- 1 -yl)prop-2-en- 1 -one” or “1 – {(3i?)-3-[4-amino-3-(4-phenoxyphenyl)- lH-pyrazolo[3,4-JJpyrimidin-l-yl]piperidin-l-yl}prop-2-en-l-one” or “2-Propen- 1 -one, 1- [(3R)-3-[4-amino-3-(4-phenoxyphenyl)- lH-pyrazolo[3,4-<f]pyrimidin- 1 -yl] – 1 -piperidinyl-” or ibrutinib or any other suitable name refers to the compound with the following structure:

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Synthesis

US20080214501

Synthesis of Compound 3—Btk Activity Probe

4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate 2) is prepared. Briefly, 4-phenoxybenzoic acid (48 g) is added to thionyl chloride (100 mL) and heated under gentle reflux for 1 hour. Thionyl chloride was removed by distillation, the residual oil was dissolved in toluene and volatile material removed at 80° C./20 mbar. The resulting acid chloride was dissolved in toluene (200 mL) and tetrahydrofuran (35 mL). Malononitrile (14.8 g) was added and the solution and stirred at −10° C. while adding diisopropylethylethylamine (57.9 g) in toluene (150 mL), while maintaining the temperature below 0° C. After 1 hour at 0° C., the mixture was stirred at 20° C. overnight. Amine hydrochloride is removed by filtration and the filtrate evaporated in vacuo. The residue was taken up in ethyl acetate and washed with 1.25 M sulphuric acid, then with brine and dried over sodium sulfate. Evaporation of the solvents gave a semisolid residue which was treated with a portion of ethyl acetate to give 4.1 g of 1,1-dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene as a white solid (m.p. 160-162° C.). The filtrate on evaporation gave 56.58 (96%) of 1,1-dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene as a grey-brown solid, which was sufficiently pure for further use.

1,1-Dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene (56.5 g) in acetonitrile (780 mL) and methanol (85 mL) is stirred under nitrogen at 0° C. while adding diisopropylethylamine (52.5 mL) followed by 2M trimethylsilyldiazomethane (150 mL) in THF. The reaction is stirred for 2 days at 20° C., and then 2 g of silica is added (for chromatography). The brown-red solution is evaporated in vacuo, the residue dissolved in ethyl acetate and washed well with water then brine, dried and evaporated. The residue is extracted with diethyl ether (3×250 mL), decanting from insoluble oil. Evaporation of the ether extracts gives 22.5 g of 1,1-dicyano-2-methoxy-2-(4-phenoxyphenyl)ethene as a pale orange solid. The insoluble oil is purified by flash chromatography to give 15.0 g of a red-orange oil.

1,1-Dicyano-2-methoxy-2-(4-phenoxyphenyl)ethene (22.5 g) and 1,1-dicyano-2-methoxy-2-(4-phenoxyphenyl)ethene oil (15 g) are treated with a solution of hydrazine hydrate (18 mL) in ethanol (25 mL) and heated on the steambath for 1 hour. Ethanol (15 mL) is added followed by water (10 mL). The precipitated solid is collected and washed with ethanol:water (4:1) and then dried in air to give 3-amino-4-cyano-5-(4-phenoxyphenyl)pyrazole as a pale orange solid.

3-Amino-4-cyano-5-(4-phenoxyphenyl)pyrazole (29.5 g) is suspended in formamide (300 mL) and heated under nitrogen at 180° C. for 4 hours. The reaction mixture is cooled to 30° C. and water (300 mL) is added. The solid is collected, washed well with water, then with methanol and dried in air to give of 4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate 2).

Synthesis of 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl (Intermediate 4); a) triphenylphosphine (TPP), diisopropyl diazodicarboxylate (DIAD), tetrahydrofuran (THF); b) TFA/CH₂Cl₂.

To a solution of 1-boc-3-(S)-hydroxypiperidine (3.98 g, 19.8 mmol) and triphenylphosphine (5.19 g, 19.8 mmol) in THF (150 ml) was added DIAD (3.9 ml, 19.8 mmol). The yellow solution was stirred 1 minute then Intermediate 2 (4.0 g, 13.2 mmol) was added and the reaction was heated with a heat gun (3-5 minutes) until the solid had dissolved. After stirring for 1 hour at room temperature, the solvent was removed and the resulting brown oil was subjected to flash chromatography (30% then 50% THF/hexanes) to provide 4.45 g (69%) of Intermediate 3 (trace of triphenylphosphine oxide is present) as a light brown foam.

To a solution of Intermediate 3 (4.4 g, 9.0 mmol) in CH₂Cl₂(20 ml) was added TFA (2.8 ml, 36.2 mmol). After stirring 2 hrs at room temperature, the solvent was removed and the residue was partitioned between ethyl acetate (250 ml) and dilute aq. K₂CO₃. The organic layer was dried (MgSO₄), filtered and concentrated to 70 ml. The resulting solution was stirred and 4.0M HCl in dioxane (4 ml) was added to provide a thick light orange precipitate. The precipitate was collected by filtration and washed with ethyl acetate (50 ml). The material was then partitioned between ethyl acetate (300 ml) and dilute aq. K₂CO₃. The organic layer was dried (MgSO₄), filtered and concentrated to provide 2.78 g (80%) of Intermediate 4 as a light yellow foam.

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SYNTHESIS

US7514444

Compounds described herein may be prepared using the synthetic methods described herein as a single isomer or a mixture of isomers.

A non-limiting example of a synthetic approach towards the preparation of compounds of any of Formula (A), (B), (C) or (D) is shown in Scheme I.

Halogenation of commercially available 1H-pyrazolo[3,4-d]pyrimidin-4-amine provides an entry into the synthesis of compounds of Formula (A), (B), (C) and/or (D). In one embodiment, 1H-pyrazolo[3,4-d]pyrimidin-4-amine is treated with N-iodosuccinamide to give 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine. Metal catalyzed cross coupling reactions are then carried out on 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine. In one embodiment, palladium mediated cross-coupling of a suitably substituted phenyl boronic acid under basic conditions constructs intermediate 2. Intermediate 2 is coupled with N-Boc-3-hydroxypiperidine (as non-limiting example) via Mitsunobu reaction to give the Boc (tert-butyloxycarbonyl) protected intermediate 3. After deprotection with acid, coupling with, but not limited to, an acid chloride, such as, but not limited to, acryloyl chloride, completes the synthesis to give compound 4.

Example 1 Synthesis of Compounds Preparation of 4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate 2)

4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate 2) is prepared as disclosed in International Patent Publication No. WO 01/019829. Briefly, 4-phenoxybenzoic acid (48 g) is added to thionyl chloride (100 mL) and heated under gentle reflux for 1 hour. Thionyl chloride is removed by distillation, the residual oil dissolved in toluene and volatile material removed at 80° C./20 mbar. The resulting acid chloride is dissolved in toluene (200 mL) and tetrahydrofuran (35 mL). Malononitrile (14.8 g) is added and the solution and stirred at −10° C. while adding diisopropylethylethylamine (57.9 g) in toluene (150 mL), while maintaining the temperature below 0° C. After 1 hour at 0° C., the mixture is stirred at 20° C. overnight. Amine hydrochloride is removed by filtration and the filtrate evaporated in vacuo. The residue is taken up in ethyl acetate and washed with 1.25 M sulphuric acid, then with brine and dried over sodium sulfate. Evaporation of the solvents gives a semisolid residue which is treated with a little ethyl acetate to give 4.1 g of 1,1-dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene as a white solid (m.p. 160-162° C.). The filtrate on evaporation gives 56.58 (96%) of 1,1-dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene as a grey-brown solid, which is sufficiently pure for further use.

Example 1a Synthesis of 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 4)

- Synthesis of compound 4; a) polymer-bound triphenylphosphine (TPP), diisopropyl diazodicarboxylate (DIAD), tetrahydrofuran (THF); b) HCl/dioxane; then acryloyl chloride, triethylamine (TEA).

Compounds described herein were synthesized by following the steps outlined in Scheme 1. A detailed illustrative example of the reaction conditions shown in Scheme 1 is described for the synthesis of 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 4).

101 mg of 4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine and 330 mg of polymer-bound triphenylphosphine(TPP) (polymerlab) were mixed together with 5 mL of tetrahydrofuran (THF). tert-Butyl 3-hydroxypiperidine-1-carboxylate (200 mg; 2.0 equivalents) was added to the mixture followed by the addition of diisopropyl diazodicarboxylate (0.099 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered to remove the resins and the reaction mixture was concentrated and purified by flash chromatography (pentane/ethyl acetate=1/1) to give intermediate 3 (55 mg).

Intermediate 3 (48.3 mg) was treated with 1 mL of 4N HCl in dioxane for 1 hour and then concentrated to dryness. The residue was dissolved in dichloromethane and triethylamine (0.042 mL) was added followed by acryl chloride (0.010 mL). The reaction was stopped after 2 hours. The reaction mixture washed with 5% by weight aqueous citric acid and then with brine. The organic layer was dried with MgSO₄, and concentrated. Flash chromatography (with CH₂Cl₂/MeOH=25/1) gave 22 mg of compound 4 as a white solid. MS (M+1): 441.2; ¹H-NMR (400 MHz): 8.26, s, 1H, 7.65, m, 2H, 7.42, m, 2H, 7.1-7.2, m, 5H, 6.7-6.9, m, 1H, 6.1, m, 1H, 5.5-5.7, m, 1H, 4.7, m, 1H, 4.54, m, 0.5H, 4.2, m, 1H, 4.1, m, 0.5H, 3.7, m, 0.5H, 3.2, 1,1H, 3.0, m, 0.5H, 2.3, m, 1H, 2.1, m, 1H, 1.9, m, 1H, 1.6, m, 1H.

Example 1b Synthesis of 1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 13)

The synthesis of compound 13 was accomplished using a procedure analogous to that described in Example 1a. EM (calc.): 440.2; MS (ESI) m/e (M+1H)⁺: 441.1, (M−1H)⁻: 439.2.

Example 1c Synthesis of 1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 14)

The synthesis of compound 14 was accomplished using a procedure analogous to that described for Example 1a. EM (calc.): 440.2; MS (ESI) m/e (M+1H)+: 441.5, (M−1H)−: 439.2.

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US7718662

Synthesis of Compounds Example 1 Preparation of 4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (2a)

Example 1a Synthesis of 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (4)

Synthesis of compound 4; a) polymer-bound triphenylphosphine (TPP), diisopropyl diazodicarboxylate (DIAD), tetrahydrofuran (THF); b) HCl/dioxane; then acryloyl chloride, triethylamine (TEA)

Compounds described herein were synthesized by following the steps outlined in Scheme III. A detailed illustrative example of the reaction conditions shown in Scheme II is described for the synthesis of 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 4).

101 mg of 4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine and 330 mg of polymer-bound triphenylphosphine (TPP) (polymerlab) were mixed together with 5 mL of tetrahydrofuran (THF). tert-Butyl 3-hydroxypiperidine-1-carboxylate (200 mg; 2.0 equivalents) was added to the mixture followed by the addition of diisopropyl diazodicarboxylate (0.099 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered to remove the resins and the reaction mixture was concentrated and purified by flash chromatography (pentane/ethyl acetate=1/1) to give intermediate 3a (55 mg).

Intermediate 3a (48.3 mg) was treated with 1 mL of 4N HCl in dioxane for 1 hour and then concentrated to dryness. The residue was dissolved in dichloromethane and triethylamine (0.042 mL) was added followed by acryl chloride (0.010 mL). The reaction was stopped after 2 hours. The reaction mixture was washed with 5% by weight aqueous citric acid and then with brine. The organic layer was dried with MgSO₄, and concentrated. Flash chromatography (with CH₂Cl₂/MeOH=25/1) gave 22 mg of compound 4 as a white solid. MS (M+1): 441.2; ¹H-NMR (400 MHz): 8.26, s, 1H, 7.65, m, 2H, 7.42, m, 2H, 7.1-7.2, m, 5H, 6.7-6.9, m, 1H, 6.1, m, 1H, 5.5-5.7, m, 1H, 4.7, m, 1H, 4.54, m, 0.5H, 4.2, m, 1H, 4.1, m, 0.5H, 3.7, m, 0.5H, 3.2, m, 1H, 3.0, m, 0.5H, 2.3, m, 1H, 2.1, m, 1H, 1.9, m, 1H, 1.6, m, 1H.

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SYNTHESIS

CN 103121999

To solve the above problems, the present invention adopts a technical solution is: to provide a tyrosine kinase inhibitor PC1-32765 synthesis method, the reaction steps are as follows:

The beneficial effect of the present invention: The invention relates to a tyrosine kinase inhibitor synthesis of PC1-32765, as the B cell to inhibit the tyrosine kinase receptor signaling key, not only can inhibit the formation of blood cells and less side effects and mild reaction conditions, simple operation, easy purification, low cost, environmentally friendly, suitable for large-scale production.

A tyrosine kinase inhibitor PC1-32765 synthesis method comprising the steps of:

1, the compound 10 and the coupling reaction of compound 15 to give compound 6;

2, the compound 6 obtained by reacting compound 16 with compound 11 in the process, we have chosen a more perfect catalyst;

3, compound 11 to give compound 12 by protecting;

4, selective deprotection of Compound 12 Compound 13; 5, Compound 13 for Compound 17 only attack only remaining position to obtain a very pure compound 14;

6, take off the protecting group to obtain PC1-32765

Wherein the compound can 10,15,16,17 agent or industrial grade reagent compound or the use of methods and techniques related to synthesis.

Example 1 Preparation of Compound 6

Under nitrogen and the 0.1moL 1.5 equivalents of compound 10 Compound 15 and 800mL of dioxane was added to 2L reaction flask, and then 1.5 equivalents of sodium acetate was added and the catalyst PdC12 (PPh3) 2 0.2 equivalents, 50_60 ° C for 5 hours , filtered hot and the filter residue was washed three times with ethanol, the combined filtrate was concentrated to give a solid, rinsed with ethanol to give the pure product 16.2 g, yield 60%

Example 2 Preparation of Compound 6

Under nitrogen and the 0.1moL 1.5 equivalents of compound 10 Compound 15 and 2L 800mL DMF was added to the reaction flask, and then 1.5 equivalents of sodium acetate was added and the catalyst PdCl2 (PhCN) 2 0.2 equivalents, 50_60 ° C for 5 hours, hot filtered, the filter residue was washed three times with ethanol, the combined filtrate was concentrated to give a solid, which was rinsed with ethanol to give pure product 21.5 g, yield 71%.

Example 3 Preparation of Compound 11

The compound 0.1moL 1.2 equivalent of compound 6 and 16, and 2L IOOOmL THF was added to the reaction flask, 1.5 equivalents of cesium carbonate was added, refluxed for 24 hours, after the reaction, most of the solvent was concentrated and the remaining water was poured into a large, precipitated solid was filtered, washed with water to afford compound 36.9 g compound 11, yield 76%, used without further purification.

Example 4 Preparation of Compound 12

The compound will be to 0.1moL 11 and 1.2 equivalent of compound IOOOmL THF trifluoroacetyl chloride and the reaction was added to 2L flask, then triethylamine was added 2.5 ,30-40 0C for 24 hours, after the reaction, the solvent was concentrated, diluted with water, extracted with ethyl acetate, washed with water, saturated sodium chloride each time, and concentrated to obtain the product 50.1 g of ethyl acrylate, 86% yield, used directly in the next reaction.

Example 5 Preparation of Compound 13

The compound 0.1moL 12 and 500mL of methanol and 50mL 6N hydrochloric acid was added to IL reaction flask, stirred at room temperature for 3 hours to complete the reaction quickly, and a solid precipitates, filtered and the solid was washed several times with ethyl acetate, obtain 38.5 g of pure compound 13 in 80% yield.

Example 6 Preparation of Compound 14 ‘

The 0.1moL compound 13 and 1.2 equivalents of acrylic acid chloride was added to 2L of methylene chloride IL reaction flask ,20-40 ° C was added dropwise 1.2 equivalents of triethylamine was added dropwise, at room temperature for 3 hours after the reaction with two chloride extraction and concentrated to give the product 47.7 g, yield 89%. Without further purification.

Example 7 PC1-32765 Preparation

Compound 14 with the 0.1moL 160mL 800mL of methanol and a saturated solution of sodium carbonate small, 50_60 ° C for 5 hours,

After completion of the reaction was diluted with water, concentrated and then extracted with methylene chloride, concentrated to obtain crude product was recrystallized from toluene to give the final product 28.6 g, yield 65%. HPLC purity 98.6%, ee%> 98%.

The present invention relates to a tyrosine kinase inhibitor of the synthesis of PC1-32765, as the B cell to inhibit the tyrosine kinase receptor signaling key, not only can inhibit the formation of blood cells and less side effects, and the reaction conditions gentle, simple operation, easy purification, low cost, environmentally friendly, suitable for large-scale production.

…

Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase

ChemMedChem

Volume 2, Issue 1, pages 58–61, January 15, 2007

http://onlinelibrary.wiley.com/doi/10.1002/cmdc.200600221/full

http://www.wiley-vch.de/contents/jc_2452/2007/z600221_s.pdf

SYN OF COMPD 4

To 101 mg of a known intermediate 2 [WO 2001019829] and 330 mg polymer-bound Triphenylphosphine (polymerlab) in 5 ml THF, 200 mg (2.0 eq.) of 3-OH N-Boc piperidine was added followed by 0.099 ml diisopropyl diazodicarboxylate. The reaction mixture stirred at room temperature overnight. After filtered off resins, the reaction mixture was concentrated and purified with flash chromatography (pentane/ethyl acetate = 1/1) to give 55 mg of intermediate 3. This compound (48.3 mg) was treated with 1 ml of 4N HCl in dioxane for 1 hour and concentrated to dryness, which was dissolved in dichloromethane and 0.042 ml of triethylamine, followed by 0.010 ml of acryl chloride. The reaction was stopped after 2 hours. The reaction mixture was washed with 5wt% citric acid (aq.) and brine, dried with MgSO4, and concentrated. Flash chromatography with (CH2Cl2/MeOH = 25/1) gave 22 mg of compound 4 as white solids. MS (M+1): 441.2; 1H-NMR (400MHz): 8.26, s, 1H; 7.65, m, 2H; 7.42, m, 2H; 7.1-7.2, m, 5H; 6.7-6.9, m, 1H; 6.1, m, 1H; 5.5-5.7, m, 1H; 4.7, m, 1H; 4.54, m, 0.5H; 4.2, m, 1H; 4.1, m, 0.5H; 3.7, m, 0.5H; 3.2, m, 1H; 3.0, m, 0.5H; 2.3, m, 1H; 2.1, m, 1H; 1.9, m, 1H; 1.6, m, 1H

……………..

References

Statement on a Nonproprietary Name Adopted by the USAN Council
FDA Press Release
Azvolinsky, PhD, Anna. “FDA Approves Ibrutinib for Chronic Lymphocytic Leukemia”. Cancer Network. Retrieved 14 February 2014.
Pan, Z; Scheerens, H; Li, SJ; Schultz, BE; Sprengeler, PA; Burrill, LC; Mendonca, RV; Sweeney, MD; Scott, KC; Grothaus, Paul G.; Jeffery, Douglas A.; Spoerke, Jill M.; Honigberg, Lee A.; Young, Peter R.; Dalrymple, Stacie A.; Palmer, James T. (2007). “Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase”. ChemMedChem 2 (1): 58–61.doi:10.1002/cmdc.200600221. PMID 17154430.
Celera Genomics Announces Sale of Therapeutic Programs to Pharmacyclics
United States patent 7514444
Janssen Biotech, Inc. Announces Collaborative Development and Worldwide License Agreement for Investigational Anti-Cancer Drug, PCI-32765
Clinical trials involve PCI-32765
Clinical trials involve ibrutinib
“Imbruvica (Ibrutinib)”. Medscape Reference. WebMD. Retrieved 13 January 2014.
“IMBRUVICA (ibrutinib) capsule [Pharmacyclics, Inc]”. DailyMed. Pharmacyclics, Inc. November 2013. Retrieved 13 January 2013.
Herman SE, Gordon AL, Hertlein E, Ramanunni A, Zhang X, Jaglowski S, Flynn J, Jones J, Blum KA, Buggy J.J., Hamdy A, Johnson AJ, Byrd JC, SE (2011). “Bruton’s tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765”. Blood 117 (23): 6287–6296. doi:10.1182/blood-2011-01-328484. PMC 3122947. PMID 21422473.
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Ponader S, Chen SS, Buggy JJ, Balakrishnan K, Gandhi V, Wierda WG, Keating MJ, O’Brien S, Chiorazzi N, Burger JA (2012). The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo 119. Blood. pp. 1182–1189.
de Rooij MF, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ, Pals ST, Spaargaren M (2012). “The clinically active BTK inhibitor PCI-32765 targets B-cell receptor (BCR)- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia”. Blood 2012: 2590–2594.
Honigberg, LA; Smith, AM; Sirisawad, M; Verner, E; Loury, D; Chang, B; Li, S; Pan, Z; Thamm, DH; Miller, RA; Buggy, JJ (2010). “The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy”.Proceedings of the National Academy of Sciences of the United States of America 107 (29): 13075–80. doi:10.1073/pnas.1004594107. PMC 2919935. PMID 20615965.
Chang, BY; Huang, MM; Francesco, M; Chen, J; Sokolove, J; Magadala, P; Robinson, WH; Buggy, JJ (2011). “The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells”. Arthritis Research & Therapy 13 (4): R115.doi:10.1186/ar3400. PMC 3239353. PMID 21752263.
http://cancer.osu.edu/mediaroom/releases/Pages/Ohio-State-Cancer-Research-Played-a-Significant-Role-in-FDA-Approval-of-Important-New-CLL-Drug.aspx#sthash.3o9uyt78.dpuf

BTK inhibitor PCI-32765, National Cancer Institute Drug Dictionary
Official Imbruvica patient website

MORE
1) E · Werner, L · Honeywell Berg, Z · Pan; Bruton tyrosine kinase inhibitor; drugs circulating Company; filing date: 2006.12.28, open) No. (Notice: CN101610676A CN101610676B, CN101805341A, CN101805341B, CN102746305A, CN102887900A

2) Pan, Z ; Scheerens, H; Li, SJ; Schultz, BE; Sprengeler, PA; Burrill, LC; Mendonca, RV; Sweeney, MD; Scott, KC; Grothaus, Paul G.; Jeffery, Douglas A.; Spoerke, Jill M. ..; Honigberg, Lee A.; Young, Peter R.; Dalrymple, Stacie A.; Palmer, James T. (2007) “Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase” ChemMedChem 2 (1): 58-61 ( article link )
3) Celera Genomics Announces Sale of Therapeutic Programs to Pharmacyclics , April 10, 2006
4) Honigberg; Lee, Verner; Erik, Pan; Zhengying; Inhibitors of Bruton’s tyrosine kinase, U.S. Patent 7,514,444 ; US 20080108636 A1; CA2663116A1, CN101610676B, CN101805341A, CN101805341B, CN102746305A, CN102887900A, EP2081435A2, EP2081435A4, EP2201840A1, EP2201840B1, EP2443929A1, EP2526771A1, EP2526933A2, EP2526933A3, EP2526934A2, EP2526934A3, EP2529621A1, EP2529622A1, EP2530083A1, EP2532234A1, EP2532235A1, US7514444, US7732454, US7825118, US7960396, US8008309, US8088781, US8158786, US8232280, US8236812, US8399470, US8476284, US8497277, US8501751, US8552010, US20080076921, US20080108636, US20080139582, US20090181987, US20100004270, US20100022561, US20100041677, US20100324050, US20100331350, US20110008257, US20110039868, US20110184001, US20110257203, US20110281322, US20120088912, US20120095026, US20120108612, US20120115889, US20120122894, US20120129821, US20120129873, US20120135944, US20120214826, US20120252821, US20120252822, US20120277254, US20120283276, US20120283277, US20130005745, WO2008039218A2, WO2008039218A3
5) Buggy, Joseph J. Chang, Betty Y.; Methods and Compositions . for inhibition of Bone resorption, PCT Int Appl, WO2013003629, 03 Jan 2013.
6) Wei Chen, David J. Loury, Tarak D. Mody; Preparation of pyrazolo-pyrimidine Compounds as Inhibitors of Bruton’s tyrosine kinase; U.S. Patent Number 7,718,662 , 18 May 2010; Also published as CA2776543A1, CN102656173A, EP2393816A2, EP2393816A4, EP2650294A1, US7741330, US20110086866, WO2011046964A2, WO2011046964A3
7) Wei Chen, David J. Loury, Tarak D. Mody; Inhibitors of Bruton’s tyrosine kinase; WO2013010136 A3
8) John C. Byrd, et al;. Targeting BTK with Relapsed Ibrutinib in Chronic Lymphocytic Leukemia; N Engl J Med 2013; 369:32-42
9) Michael L. Wang, MD, et al, Targeting with BTK. Ibrutinib in Refractory or Relapsed Mantle-Cell Lymphoma; N Engl J Med 2013; 369:507-516

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What Does 100% of Your Daily Value of Cholesterol Look Like?

Uncategorized Comments Off

Feb 262014

Healthline just published an interesting infograph that gives a visualization of what your daily value of cholesterol looks like. In the graphic, you can see what 300 mg of cholesterol looks like for 20 high cholesterol foods: http://www.healthline.com/health/high-cholesterol/daily-value

This is a very informative resource as it helps us visualize what their cholesterol intake look like

What Does 100% of Your Daily Value of Cholesterol Look Like?

It’s no secret that eating fatty foods raises your bad cholesterol level, also known as LDL. An elevated LDL clogs up your arteries and makes it difficult for your heart to do its job. Potentially, it could lead to heart disease.

The USDA recommends consuming no more than 300 mg of cholesterol a day. While a deep-fried Twinkie at the county fair is an obvious no-no, other high cholesterol culprits may be sneaking into your diet. Check out what that number looks like in terms of everyday food items.

Warning: you may need to revise your grocery list—and your eating habits!

Fried Chicken:

4 pieces=300mg cholesterol

Croissants:

6 2/3 rolls=300mg cholesterol

Cheddar Cheese:

12 3/4 slices=300mg cholesterol

Prosciutto:

28 slices=300mg cholesterol

Corned Beef:

14 thin slices=300mg cholesterol

Butter:

1 1/5 sticks=300mg cholesterol

read at

http://www.healthline.com/health/high-cholesterol/daily-value

CANGRELOR

Uncategorized Comments Off

Feb 242014

Cangrelor, AR-C69931MX

[dichloro-[[[(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-(2-methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl)purin-9-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]methyl]phosphonic acid

N-[2-(Methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5¢-adenylic acid monoanhydride with (dichloromethylene)bis[phosphonic acid]

163706-06-7 cas no

Also known as: AR-C69931XX, 163706-06-7, Cangrelor (USAN/INN), Cangrelor [USAN:INN:BAN], UNII-6AQ1Y404U7, cangrelor (AR-C69931MX),

Molecular Formula: C17H25Cl2F3N5O12P3S2

Molecular Weight: 776.359196

Cangrelor

MAR 09, 2013

The Medicines Company said yesterday it will pursue marketing approvals for its anti-clotting drug candidate Cangrelor after it met its primary efficacy endpoint in a Phase III clinical trial of improvement compared with Plavix (clopidogrel).

The intravenous small molecule antiplatelet agent reduced by 22% the likelihood of patients experiencing death, myocardial infarction, ischemia-driven revascularization, or stent thrombosis within 48 hours of taking it—to 4.7% from 5.9% of subjects randomized during CHAMPION PHOENIX. The Phase III trial compared Cangrelor to oral Plavix in 11,145 patients undergoing percutaneous coronary intervention.

Cangrelor also showed a 38% reduction (0.8% compared with 1.4%) over Plavix in the likelihood of patients experiencing the key secondary endpoint, incidence of stent thrombosis at 48 hours.

Cangrelor is designed to prevent platelet activation and aggregation that leads to thrombosis in acute care settings, including in patients undergoing percutaneous coronary intervention. During CHAMPION PHOENIX, Cangrelor made its best showing in patients with Q-wave myocardial infarction (QMI), lowering by 39% (to 0.2% compared with 0.3%) the incidence of QMI. Cangelor’s most disappoint showing was its inability to lower the odds of death compared with Clopidogrel; both drugs showed a likelihood of 0.3%.

“Our next step is to submit for market approvals in the U.S. and Europe. We anticipate submitting these data for a new drug application to the U.S. Food and Drug Administration in the second quarter with findings of prior trials, including the BRIDGE trial in patients awaiting open heart surgery,” Simona Skerjanec, PharmD, senior vp and innovation leader for antiplatelet therapies at The Medicines Company, said in a statement.

Cangrelor is a P2Y12 inhibitor under investigation as an antiplatelet drug[1] for intravenous application. Some P2Y12 inhibitors are used clinically as effective inhibitors of adenosine diphosphate-mediated platelet activation and aggregation.[1] Unlike clopidogrel (Plavix), which is a prodrug, cangrelor is an active drug not requiring metabolic conversion.

Poor interim results led to the abandonment of the two CHAMPION clinical trials in mid 2009.[2] The BRIDGE study, for short term use prior to surgery, continues.[3] The CHAMPION PHOENIX trial was a randomized study of over 11,000 patients published in 2013. It found usefulness of cangrelor in patients getting cardiac stents. Compared with clopidogrel given around the time of stenting, intravenous ADP-receptor blockade with cangrelor significantly reduced the rate of stent thrombosis and myocardial infarction.[4]Reviewers have questioned the methodology of the trial.[5]

One particularly preferred example of a reversible, short-acting P2Y12 inhibitor is cangrelor. Cangrelor is a potent, direct, and reversible antagonist of the platelet P2Y12 receptor. Cangrelor has a half-life of approximately less than 10 minutes, allowing for a return to normal platelet function in a very short period of time upon discontinuation of the drug. By reducing the need for a compound to be metabolized for activity, and by having a relatively short half-life, reversible, short-acting P2Y12 inhibitors are considered “reversible,” meaning that full platelet functionality may return rather quickly as compared to thienopyridines.

The binding of cangrelor to the P2Y12 receptor inhibits platelet activation as well as aggregation when mediated in whole or in part via this receptor. Cangrelor can be derived completely from synthetic materials, and is an analogue of adenosine triphosphate (ATP). ATP is a natural antagonist of the P2Y12 receptor sites and is found in humans.

The chemical structure for cangrelor is depicted below as Formula I.

Cangrelor is clinically well tolerated and safe and has no drug-drug interaction with aspirin, heparin or nitroglycerin. Unlike orally dosed thienopyridines, cangrelor can be administered intravenously and binds directly to P2Y12 receptor sites of platelets. In each of the embodiments of the present invention, the term “cangrelor” encompasses the compound of Formula I as well as tautomeric, enantiomeric and diastereomeric forms thereof, and racemic mixtures thereof, other chemically active forms thereof, and pharmaceutically acceptable salts of these compounds, including a tetrasodium salt. These alternative forms and salts, processes for their production, and pharmaceutical compositions comprising them, are well known in the art and set forth, for example, in U.S. Pat. No. 5,721,219. Additional disclosure relevant to the production and use of cangrelor may be found in U.S. Pat. Nos. 5,955,447, 6,130,208 and 6,114,313, as well as in U.S. Appln. Publication Nos. 2006/0270607 and 2011/0112030.

Invasive procedures means any technique where entry to a body cavity is required or where the normal function of the body is in some way interrupted by a medical procedure and/or treatment that invades (enters) the body, usually by cutting or puncturing the skin and/or by inserting instruments into the body. Invasive procedures can include coronary artery bypass grafting (CABG), orthopedic surgeries, urological surgeries, percutaneous coronary intervention (PCI), other general invasive procedures, such as endarterectomy, renal dialysis, cardio-pulmonary bypass, endoscopic procedures or any medical, surgical, or dental procedure that could result in excessive bleeding or hemorrhage to the patient.

Perioperative means the period of a patient’s invasive procedure which can occur in hospitals, surgical centers or health care providers’ offices. Perioperative includes admission, anesthesia, surgery, to recovery.

Thrombosis is the formation of a blood clot (thrombus) inside a blood vessel obstructing the flow of blood through the circulatory system. When a blood vessel is injured, the body uses platelets and fibrin to form a blood clot to prevent blood loss. Some examples of the types of thrombosis include venous thrombosis which includes deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, cerebral venous sinus thrombosis, cerebral venous sinus thrombosis and arterial thrombosis which includes stroke and myocardial infarction.

The compound cangrelor from the Medicines Company is represented by the structure

Figure imgf000013_0002

TETRASODIUM SALT

Cangrelor sodium, AR-C69931MX

Cangrelor Tetrasodium [USAN]
RN: 163706-36-3

Platelet P(2T) receptor antagonist.

5′-O-[[[Dichloro(phosphono)methyl](hydroxy)phosphoryloxy](hydroxy)phosphoryl]-N-[2-(methylsulfanyl)ethyl]-2-(3,3,3-trifluoropropylsulfanyl)adenosine tetrasodium salt

C17-H21-Cl2-F3-N5-O12-P3-S2.4-Na,

864.2899

The Medicines Co. (Proprietary), AstraZeneca Charnwood (Originator)

CARDIOVASCULAR DRUGS, Treatment of Disorders of the Coronary Arteries and Atherosclerosis, P2Y12 (P2T) Antagonists

2-Mercaptoadenosine (I) was S-alkylated with 1-chloro-3,3,3-trifluoropropane (II) in the presence of NaH to give trifluoropropyl sulfide (III). Subsequent acetylation of (III) with Ac2O at 80 C provided (IV), which was N-alkylated with methylthioethyl iodide (V) and NaH yielding (VI).

Further hydrolysis of the resulting (VI) with 0.1 M NaOH in refluxing MeOH furnished adenosine derivative (VII). The 5′-hydroxyl group of (VII) was then phosphorylated by reaction with phosphoryl chloride in cold triethyl phosphate followed by aqueous work-up.

The resulting 5′-monophosphate (VIII) was treated with carbonyl diimidazole and tri-n-butylamine to produce the phosphoryl imidazole intermediate (IX), which was finally condensed with dichloromethylenebis(phosphonic acid) (X).

The target compound was isolated as the tetrasodium salt upon treatment with NaI in methanol-acetone.

Alkylation of mercaptopurine (I) with 3-chloro-1,1,1-trifluoropropane (II) in the presence of NaH gave thioether (III).

After protection of the amino group of (III) as the acetamide (IV) by means of Ac2O and NaOAc, N-alkylation with 2-(methylthio)ethyl iodide (V) yielded (VI),

which was deacetylated by hydrolysis with NaOH in refluxing MeOH. Subsequent treatment with POCl3 produced the intermediate phosphoryl chloride (VIII).

Then, condensation of this acid chloride with dichloromethylene bisphosphonic acid (IX) in the presence of tributylamine in triethyl phosphate yielded the title compound, which was isolated as the tetrasodium salt.

Alternatively, hydrolysis of acid chloride (VIII) in the presence of ammonium bicarbonate gave phosphate salt (X), which was treated with carbonyldiimidazole, and the activated intermediate (XI) was then condensed with bisphosphonate (IX) to furnish the target compound.

…………

J. Med. Chem., 1999, 42 (2), pp 213–220

http://pubs.acs.org/doi/full/10.1021/jm981072s

10l (AR-C69931MX)

N6–(2-Methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5‘-adenylic Acid, Monoanhydride withDichloromethylenebis(phosphonic acid) (10l). Prepared as the triammonium salt in 4% yield from 3l: 1H NMR δ(D2O) 8.30 (1H, s, H8), 5.97 (1H, d, J = 5.5 Hz, H1‘), 4.65 (1H, m, H2‘), 4.47 (1H, m, H3‘), 4.28 (1H, m, H4‘), 4.17 (2H, m, H5‘a and H5‘b), 3.67 (br s, NHCH2), 3.21 (2H, t, J = 7.6 Hz, SCH2), 2.72 (2H, t, J = 6.6 Hz, SCH2CH2CF3), 2.58 (2H, m, NCH2CH2), 2.04 (3H, s, SCH3);31P NMR δ(D2O) 8.80 (d, 1P, J = 18.6 Hz, Pγ), 0.42 (dd, 1P, J1 = 18.9 Hz, J2 = 28.9 Hz, Pβ), −9.41 (d, 1P, J = 29.0 Hz, Pα). Anal. (C17H34Cl2F3N8O12P3S2·3H2O) H, N, S; C: calcd, 23.16; found, 23.66.

References

Cangrelor Attenuates Coated-Platelet Formation
CHAMPION Trials With Cangrelor Stopped for Lack of Efficacy
What Cangrelor Failure Means to Medicines
Effect of Platelet Inhibition with Cangrelor during PCI on Ischemic Events (2013) Bhatt, DL etal. New England Journal of Medicine March 10, 2013 DOI: 10.1056/NEJMoa1300815 (published initially online).
The Duel between Dual Antiplatelet Therapies (2013) Lange, RA and Hillis, LD. New England Journal of Medicine March 10, 2013 DOI: 10.1056/NEJMe1302504
15th European Federation for Medicinal Chemistry International Symposium on Medicinal Chemistry (Sept 6 1998, Edinburgh)1998,:Abst P.281
Specific P2Y12 purinoceptor antagonist; inhibits ADP-induced platelet aggregation. Prepn: A. H. Ingall et al., WO 9418216 (1994 to Fisons); eidem, US 5721219 (1998 to Astra); and in vivo antithrombotic activity: idem et al., J. Med. Chem. 42, 213 (1999).
In vivo antithrombotic effects in canine arterial thrombosis: J. Huang et al., J. Pharmacol. Exp. Ther. 295, 492 (2000).
Mechanism of action study: A. Ishii-Watabe et al., Biochem. Pharmacol. 59, 1345 (2000).
Clinical safety assessment and evaluation in acute coronary syndromes: R. F. Storey et al., Thromb. Haemostasis 85, 401 (2001); in angina pectoris and non-Q-wave myocardial infarction: F. Jacobsson et al., Clin. Ther. 24, 752 (2002).
Clinical pharmacodynamics compared with clopidogrel: R. F. Storey et al., Platelets 13, 407 (2002).
Review of clinical development: S. C. Chattaraj, Curr. Opin. Invest. Drugs2, 250-255 (2001).
WO2013/103567 A2,
Journal of Medicinal Chemistry, 1999 , vol. 42, 2 p. 213 – 220

RAMELTEON, TAK 375 ..Melatonin MT1/MT2 receptor agonist

Uncategorized 1 Response »

Feb 232014

RAMELTEON

ACN-S001714, ZINC00007031

HSDB 7787
Ramelteon
Rozerem
TAK-375
UNII-901AS54I69

Molecular Formula: C₁₆H₂₁NO₂ Molecular Weight: 259.34344

CAS number	196597-26-9

	(S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]propionamide

(5)-N-[2-(l,6,7,8-tetrahydro-2H-indeno-[5,4-ό]furan-8- yl)ethyl]propionamide

United States

US 6034239

1999-07-22

expiry 2019-07-22

EP885210B1 , EP1792899A1 and J. Med Chem. 2002, 45, 4222-4239

NMR

[PPTData Supplement – Drug Metabolism and Disposition

dmd.aspetjournals.org/content/suppl/…/Supplemental_Information.pptx‎

May 17, 2010 – Ramelteon NMR Assignments. COSY: Black Arrows. HMBC: Red Arrows. Figure S-1b. 1H NMR Spectrum of Ramelteon. Figure S-1c.

Ramelteon is the first in a new class of sleep agents that selectively binds to the melatonin receptors in the suprachiasmatic nucleus (SCN). It is used for insomnia, particularly delayed sleep onset. Ramelteon has not been shown to produce dependence and has shown no potential for abuse.

Ramelteon, marketed as Rozerem by Takeda Pharmaceuticals North America, is the first in a new class of sleep agents that selectively binds to the MT₁ and MT₂ receptors in the suprachiasmatic nucleus (SCN), instead of binding to GABA _A receptors, such as with drugs like zolpidem,eszopiclone, and zaleplon. Ramelteon is approved by the U.S. Food and Drug Administration (FDA) for long-term use.

Ramelteon does not show any appreciable binding to GABA_A receptors, which are associated with anxiolytic, myorelaxant, and amnesic effects.

Rozerem (ramelteon), FDA Approved 07.04.05, can be used for insomnia, particularly delayed sleep onset. Ramelteon has not been shown to produce dependence and has shown no potential for abuse, and the withdrawal and rebound insomnia that is typical with GABA modulators is not present in ramelteon. Some clinicians also use ramelteon for the treatment of Delayed sleep phase syndrome.

Ramelteon

Mechanism of action

Ramelteon is a melatonin receptor agonist with both high affinity for melatonin MT₁ and MT₂ receptors and selectivity over the MT₃ receptor. Ramelteon demonstrates full agonist activity in vitro in cells expressing human MT₁ or MT₂ receptors, and high selectivity for human MT₁ and MT₂receptors compared to the MT₃ receptor.^[1]

The activity of ramelteon at the MT₁ and MT₂ receptors is believed to contribute to its sleep-promoting properties, as these receptors, acted upon by endogenous melatonin, are thought to be involved in the maintenance of the circadian rhythm underlying the normal sleep-wake cycle. Ramelteon has no appreciable affinity for the GABA receptor complex or for receptors that bind neuropeptides, cytokines, serotonin, dopamine, noradrenaline,acetylcholine, and opiates. Ramelteon also does not interfere with the activity of a number of selected enzymes in a standard panel.

The significance of ramelteon’s lack of affinity for the MT₃ receptor is not clear, despite the manufacturer’s emphasis of this fact in commercial advertisements. The MT₃ receptor appears almost exclusively in the gut and might not have any relationship to sleep or wakefulness.

The major metabolite of ramelteon, M-II, is active and has approximately one tenth and one fifth the binding affinity of the parent molecule for the human MT₁ and MT₂ receptors, respectively, and is 17 – 25-fold less potent than ramelteon in in vitro functional assays. Although the potency of M-II at MT₁ and MT₂ receptors is lower than the parent drug, M-II circulates at higher concentrations than the parent producing 20 – 100 fold greater mean systemic exposure when compared to ramelteon. M-II has weak affinity for the serotonin 5-HT2B receptor, but no appreciable affinity for other receptors or enzymes. Similar to ramelteon, M-II does not interfere with the activity of a number of endogenous enzymes.

All other known metabolites of ramelteon are inactive.

No published studies have indicated whether ramelteon, in humans, is more or less safe or effective than the hormone melatonin which it mimics; melatonin is much less expensive and is widely available over-the-counter in the US and Canada. The biological action of melatonin is similar to that of ramelteon. Ramelteon has been directly compared to melatonin in cats, and Ramelteon had a significant (3x) longer effect and had a more profound effect on the EEG of the sleeping cats.^[2]

Introduction

ROZEREM (ramelteon) is an orally active hypnotic, chemically designated as (S)-N-[2- (l,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]propionamide, and contains one chiral center. The compound is produced as the (S)-enantiomer, with an empirical formula of C₁₆H₂₁N0₂, molecular weight of 259.34, and the following chemical structure (I):

(I) -Ramelteon

Ramelteon is used to help patients who have sleep-onset insomnia (difficulty falling asleep) to fall asleep more quickly. It is the first in a new class of sleep agents that selectively binds to the MT] and MT₂ receptors in the suprachiasmatic nucleus (SCN), in a class of medications called melatonin receptor agonists with both high affinity for melatonin MT_! and MT₂ receptors and selectivity over the MT₃ receptor. It works similarly to melatonin, a natural substance in the brain that is needed for sleep.

Ramelteon was first disclosed in European patent EP 885210, which also disclosed a process for synthesizing ramelteon, as shown in scheme 1 : Scheme 1

Ramelteon

The processes of the prior art suffer from many disadvantages, some of which result from the fact that they involve several steps.

For instance, in US patent US 6034239, which is related to EP 885210, there is disclosed a process for preparing an intermediate compound of Formula (IV), which involves conversion of diethylcyano methyl phosphonate in the presence of 60% sodium hydride. Disadvantages of this particular reaction include the need for the highly flammable and corrosive base sodium hydride, the use of toxic triethyl phosphate for the formation of diethylcyano methyl phosphonate (which also has a high boiling point), and low yield of 60%. Such disadvantages mean that the disclosed process is difficult to implement industrially or economically. A further problem associated with prior art preparation techniques is the formation of dimeric impurities at the nitrile reduction stage (i.e. where the intermediate of Formula (IV) is reduced). For instance, US 6034239 discloses reduction of (l,2,6,7-Tetrahydro-8H-indeno-[5,4- b]furan-8-ylidene)-acetonitrile of formula (IV) by means of H₂ over Raney nickel in in a solvent medium of ethanol NH₃ to provide compound of formula (IIA). The reaction is carried out by applying 5 kg of hydrogen pressure, which results in the formation of the byproduct and impurity Dimer A, which in turn affects the yield and purity of the product of formula (IIA).

Dimer A

Similarly, (l,2,6,7-Tetrahydro-8H-indeno-[5,4-b]furan-8-ylidene)-acetonitrile of formula (IV) may be reduced by means of H₂ over Raney cobalt in a solvent medium of ethanol/ NH₃ to afford compound of formula (IIB). The reaction, which is carried out by applying hydrogen pressure, is not selective, and results in the formation of the by-product and impurity Dimer B, which in turn affects the yield and purity of product of formula (IIB).

Dimer B

Repeated purifications are required to remove impurities such as Dimer A and B to obtain ramelteon having the desired purity, which results in the poor yield of ramelteon.

Several other approaches are also described in the literature to make ramelteon and related compounds in WO2006030739, WO208062468, WO2008106179, US 2010152468, WO2009106966 and WO2010/055481. However, all processes of the prior art for the preparation of ramelteon are cumbersome; the processes employ a plurality of reagents and involve multiple steps, which make the overall processes uneconomical. Therefore there is a need for a more economical, efficient and industrially suitable method of making ramelteon, whereby address the problems associated with prior art, some of which are discussed above.

Ramelteon is the active ingredient in trademarked ROZEREM^®, and is approved by the United States Food and Drug Administration for the treatment of insomnia characterized by difficulty with sleep onset.

Different processes for preparing (S)-N-[2-(l,6,7,8-tetrahydro-2H-indeno-[5,4- b]furan-8-yl)ethyl]propionamide, i.e. ramelteon, are disclosed in US 6034239, JP 11080106, JP 11140073 and WO 2006/030739. U.S. Patent No. 6034239 describes the following processes for the preparation of ramelteon:

Japan Patent Publication No. 11080106 reports the following process for the preparation of ramelteon:

Ru(OCOCH₃)[(R)-BI NAP] IOOatm H₂/50 temp

BF₃ DEE Complex

Japan Patent Publication no. 11140073 reports the following process for the preparation of an intermediate of ramelteon:

PCT Publication No. WO/2006/030739 reports the following process for the preparation of ramelteon:

POCI3/DMF (EtO)₂P(O)CH₂CO₂Et Toluene NaH/Toluene

Purification in Ethanol water 95 7%

United States Patent No. 6,034,239 discloses the formation of chiral intermediates (S)-(- )-N-[2-(l,6,7,8,-tetrahydro-2H-indeno[5,4-b]furan-8-yl)ethylamine (sometimes referred to as compound S-2 or intermediate compound S-2) by the catalytic asymmetric hydrogenation of 2- (l,2,6,7,-tetrahydro-8H-indeno[5,4-b]furan-8-ylidene)ethylamine (compound 3 in the reaction scheme shown below) in the presence of a catalytic amount of BINAP-ruthenium complex in approximately 89% e.e. (enantiomeric excess). Following the catalytic reaction, the product is purified by preparing acid salts and acylated with propionyl chloride (compound 4 in the reaction scheme shown below) to obtain ramelteon (compound 1 in the reaction scheme shown below) in its pure (S) isomer form.

|S)*2

An alternate process for preparing ramelteon is disclosed in the Journal of Medicinal Chemistry, Vol. 45, pp. 4222-4239 (2002), wherein the exo double bond of intermediates (A) shown below was asymmetrically reduced using (S)-2, 2′-bis-(diphenylphosphino)-l, 1 ‘- binaphthyl (binap)-Ru complex as the catalyst to obtain the enantiomerically pure compound (B). Compound (B) is subsequently converted to ramelteon (1) through the intermediate steps of Claisen condensation, ozonolysis and cyclization.

m Both of the above processes uses expensive catalyst and give poor enantioselectivity. Additionally, these processes are expensive due to the need to perform multiple purifications steps in order to achieve an enantioselectivity of at least about 99% or greater of the desired isomer.

PCT Patent Publication No. WO 2008/062468 A2 discloses the following process for the preparation of ramelteon:

RAMELTEON

WO 2008/062468 teaches that separation of the enantiomers of intermediate (2) may be accomplished by: i) optical resolution of the racemic amine intermediate (2) by preparing acid salts with chirally pure acids; or ii) chromatographic techniques using chiral and/or achiral stationary phases for batch process, super critical or sub critical chromatography and/or continuous process chromatography. Although WO 2008/062468 mentions the possible use of optical resolution with chirally pure acids, there is no further teaching, discussion or disclosure of this method. WO 2008/062468 does, however, provide detailed descriptions of chromatographic methods for separating the isomers of intermediate compound (2). The disclosed chromatographic process suffers the following disadvantages:

• Preparative chromatography is time consuming & expensive;

• Highly sophisticated instrumentation required; • Not commercially feasible.

PCT Patent Publication No. WO 2008/106179 discloses a process for the preparation of ramelteon that involves the following reaction steps:

wherein X= O-alkyl or NH₂ and chiral reduction of the compound of formula IV in the presence of Ru-BINAP complex under hydrogen atmosphere in an organic solvent.

IV V

The process disclosed in WO 2008/106179 is similar to the process disclosed in United States Patent No. 6,034,239 and the Journal of Medicinal Chemistry, Vol. 45 in that a Ru-BINAP complex is employed.

Resolution of racemic mixtures via reaction with optically active acids and the subsequent crystallization of the resulting salts is preferably employed when the chiral carbon of the racemic compound is an alpha carbon {i.e., one carbon removed) to the functional group forming the acid addition salt. As the distance between the chiral carbon of the racemic compound to the functional group of the racemic compound increases to beta (i.e., two carbon removed) & gamma (i.e., three carbon removed), the resolution of the diastereomeric salt becomes more difficult and not very useful.

Ramelteon has a chiral center at the gamma carbon, which makes the separation of the isomer with an optically active acid quite a daunting task. Similarly, N-[2-(l, 6, 7, 8,- tetrahydro-2H-indeno [5, 4-b]furan-8-yl)]ethylamine (compound T), an intermediate useful in the production of ramelteon has a chiral center at the gamma carbon which would lead a skilled artisan to believe that optical resolution with an optically active acid could prove difficult.

Synthesis

Chilman-Blair, K.; Castañer, J.; Silvestre, J.S.; Bayés, M. (2003). “TAK-375”. Drugs of the Future 28 (10): 950. doi:10.1358/dof.2003.028.10.763214.

………………..

SYNTHESIS

Scheme 1 :

XIV (S)-XII

……………………………………..

SYNTHESIS

WO2012035303A2

Scheme 2

0-30°C

Metal salt Propionyl halide/

Propionc anhydride

Ramelteon (I)

Synthesis of ramelteon

Preparation 1

N-[2-(l,6,7,8-Tetrahydro-2H-indeno[5,4-b]furan-8-yl)-ethyl]-propionamide (2.0 gm) was dissolved in 50.0 ml (n-Hexane:IPA:DEA) (as used herein, “IPA” stands for isopropyl alcohol, and “DEA” stands for diethylamine)

and optically resolved by high performance column chromatography on CHTRAL PACK IA-3 using Mobile phase : n-Hexane:IPA:DEA Flow rate: 1.0ml/min UV:285 nm; at a column temperature of 25°C;sample concentartion: lmg/ml and, eluted with mobile phase. Both the enantiomers were collected separately and after evaporation of solvent under vacuum, enantiomerically pure ramelteon (I) was obtained. Preparation 2- using Supercritical Fluid Chromatography (SFC)

N-[2-(l₅6,7,8-Tetrahydro-2H-indeno[5,4-b]furan-8-yl)-ethyl]-propionamide (2.0 gm) was dissolved in 50.0 ml (n-Hexane:Ethanol:DEA) and optically resolved by Supercritical Fluid Chromatography (SFC) on CHIRAL PACK AD-H using a mobile phase : C02/(Methanol/ Diethylamine[DEA]) and eluted with mobile phase. Both the enantiomers were collected separately and after evaporation of solvent under vacuum, enantiomerically pure S- ramelteon of Formula (I) and R-ramelteon were obtained with isomeric purity>99%.

………………………………

SYNTHESIS

WO2010055481A1

synthesis of ramelteon that comprises the step of separating N-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8- yl)]ethylamine (compound 2) into its isomers using an optically active acid to achieve high enantioselectivity of the desired isomer. This embodiment may further include the step of acylating the substantially pure enantiomer, (S)-N-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8-yl)]ethylamine (compound (S)-2) using a suitable acylating agent, such as propionyl chloride) to provide (S)-7V-[2-(l,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl]ethyl]propionamide (ramelteon or compound 1) substantially free of the (R)-isomer.

One embodiment of the present invention for the preparation of ramelteon is shown below in Scheme 1.

Example 1

Preparation of (S)-N-2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8-yl) ethylamine (Compound (S)-2)

A solution of N-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8-yl)ethylamine (45 g; 0.22 mol) in methanol (225 ml) is added to a solution of S-(+)-2-(4-isobutylphenyl)propionic acid (41 g; 0.20 mol) in methanol (205 ml) at 25-30⁰C. The reaction mixture is concentrated to dryness under reduced pressure. The crude salt precipitated is recrystallized in methanol to give a diastereomeric salt of (S)-N-2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8-yl) ethylamine with (S)-(+)-2-(4-isobutylphenyl) propionic acid having a chiral purity of greater than 90% enantioselectivity. The product obtained is recrystallized from methanol to give the pure salt having chiral purity of 99% or greater enantioselectivity.

The purified salt is suspended in water and the pH of the suspension is adjusted to 11-12 using aqueous sodium hydroxide. The reaction mixture is extracted with dichloromethane, washed with water and evaporated to give the pure (S)-N-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8-yl)]ethylamine (compound (S)-2), substantially free from its (R) isomer.

Example 2

Preparation of (S)-N-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan-8-yl)ethyl] propionamide (ramelteon)

Triethyl amine (15.15 g, 0.15 mol) and propionyl chloride (13.66 g, 0.15 mol) were added to a solution of S-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5,4-b]furan-8-yl)]ethylamine (25 g, 0.12 mol) (compound (S)-2) (prepared in Example 1) in dichloromethane and stirred at room temperature for 2 hours. 75 mL water was added to the reaction mixture, and the layers were separated. The dichloromethane layer was concentrated under reduced pressure and purified from a mixture of acetone and hexane to give (S)-N-[2-(l, 6, 7, 8-tetrahydro-2H-indeno [5, 4-b] furan- 8-yl) ethyl] propionamide (compound 1) having a chiral purity of 99% or greater enantioselectivity.

…………

INTERMEDIATES

WO2010115897A2

The intermediate compound of formula Vl, 6,7-dihydro-1 H-indeno[5,4-b]furan-8(2H)-one, can then be subjected to further synthesis steps to yield ramelteon by synthesis route known to or readily devisable by a person skilled in the art, suitably involving the introduction of the side chain having chirality and amide function. The documents mentioned infra are incorporated herein by way of reference. For example, the following synthesis route may be applied:

1 ) NaOH

Vl 2) H₂, Ru-BI NAP

3) HCI

4) H₂, Pd/C

Experimental Procedures

Example 1 :

Preparation of 4-(2-chloroethyl)-2,3-dihydrobenzofuran (II)

intermediate FTIR spectra of MeCN (140 ml) was recorded as reference. MeCN was cooled to -20⁰C, oxalyl chloride (16.5 ml) was added at once and waited until temperature re-stabilized at – 20⁰C. DMF (16.6 ml) was then added drop-wise (temperature between -18°C and -22°C, 0.5 ml/min). Reaction was stirred until no oxalyl chloride was visible and DMF level was stable by FTIR. Vilsmeier reagent is thereby formed in situ according to the following reaction:

Product I was then added portion wise (temperature between -18°C and -21⁰C, about 30 min). Formation of intermediate was immediately observed by FTIR. Reaction was stirred for one hour. Et₃N was then added drop-wise (temperature between -18°C and -22°C, 50 ml/h). At the end of addition, reaction was stirred 15 min at -20⁰C and temperature was slowly raised to 50⁰C (within about 15 min). Disappearance of intermediate and formation of DMF and product Il was monitored by FTIR. When reaction looked completed by FTIR (about 2h at 50°C), the reaction was cooled down to 20⁰C and quenched with water (45 ml). Solution was transferred to a round bottom flask and MeCN was removed under reduced pressure. Solution was then diluted with MTBE (100 ml) and water (50 ml). Phases were separated and aqueous phase was re-extracted twice with MTBE (50 ml). Combined organic phases were washed twice with 10% H₃Pθ₄/10% NaCI solution and stored at 4°C until next step.

List of FTIR bands used to follow the reaction (using 2^nd derivative and solvent subtraction): Oxalyl chloride (reactant): Height to two point baseline, peak from 1800 cm^“1 to 1770 cm^“1, baseline 1800 cm^“1 to 1770 cm^“1.

Intermediate: Height to single point baseline, peak from 1722 cm^“1 to 1712 cm^“1, baseline

1722 cm^“1.

Compound Il (product): Area to two point baseline, peak from 993 cm^“1 to 981 cm^“1, baseline 993 cm^“1 to 981 cm^“1.

DMF: Height to single point baseline, peak from 1694 cm^“1 to 1680 cm^“1, baseline 1694 cm^“1.

Example 2:

Preparation of 4-vinyl-2,3-dihydrobenzofuran (III)

M FTIR spectra of MTBE was recorded prior to the reaction as reference. To the solution of 4- (2-chloroethyl)-2,3-dihydrobenzofuran (II) in MTBE (150 ml) obtained at the previous step, was added, water (38 ml), Kl (1.37 g), Bu₄NOH 40% (19 ml) and NaOH 50% solution (66 ml). Reaction was vigorously stirred and heated at 50⁰C until reaction looked completed by FTIR (4 to 5 h). Warm reaction mixture was then transferred into an extraction funnel to give three phases. Water phase (bottom) was removed and did not contain product. Medium phase (colored black) was diluted with water (120 ml) and was extracted three times with MTBE. Combined organic phases were washed twice with water, once with 0.5M NaHSO₃/10% NaCI solution and once with 1 N NaOH/10% NaCI solution. MTBE solution was dried using MgSO₄, filtered, concentrated and used immediately for next step.List of FTIR bands used to follow the reaction (using 2^nd derivative and solvent subtraction) Compound Il (reactant): Area to zero, peak from 1440 cm^“1 to 1437 cm^“1 Compound III (product): Area to zero, peak from 1417 cm^“1 to 1412 cm^“1. Compound III (product): Area to zero, peak from 1565 cm^“1 to 1562 cm^“1.

Example 3:

Preparation of 1-(2,3-dihydrobenzofuran-4-yl)ethanone (V)

4-vinyl-2,3-dihydrobenzofuran (I I I ) (2.4 g) was dissolved in toluene (2 ml) and were successively added (ITC) (51 mg), PdCI₂(30 mg) and H₂O₂ 30% (2 ml). Reaction was vigorously stirred at 55°C until reaction looked completed by FTIR. (for around 24 h). Reaction was cooled down to room temperature, diluted with EtOAc (50 ml) and water (50 ml). Phases were separated and organic phase was washed with 0.5M NaHSO₃/10% NaCI solution and twice with 1 M NaHCO₃, dried over MgSO₄ and concentrated. Purification by flash chromatography gave 1-(2,3-dihydrobenzofuran-4-yl)ethanone (V). ¹H NMR δ (CDCI₃) 7.35 (dd, 1 H, J = 0.8 Hz, J = 7.8 Hz), 7.19 (t, 1 H, J = 7.9 Hz), 6.95 (d, 1 H, J = 8.0 Hz), 4.57 (t, 2H, J = 8.8 Hz), 3.52 (t, 2H, J = 8.8 Hz), 2.57 (s, 3H). ¹³C NMR δ (CDCI₃) 198.8, 161.0, 133.8, 128.2, 127.9, 121.4, 1 13.4, 71.6, 31.0, 27.6.

List of FTIR bands used to follow the reaction (using 2^nd derivative and solvent subtraction) Compound III (reactant): Area to single point baseline, peak from 925 cm^“1 to 915 cm^“1, baseline 915 cm^“1.

Compound V (product): Area to zero, peak from 1730 cm^“1 to 1724 cm^“1.

Example 4:

Preparation of 6,7-dihydro-1 H-indeno[5,4-b]furan-8(2H)-one (Vl)

V Vl1 -(2,3-dihydrobenzofuran-4-yl)ethanone (V) (1 g, 6.2 mmol) was dissolved in dioxane (9 ml). TADCA (dicyclohexylammonium 2,2,2-trifluoroacetate) (1 .82 g, 1 eq) and paraformaldehyde (0.61 1 g, 1.1 eq) were added. The reaction was heated at 100⁰C for 2 h. A second portion of TADCA (0.91 g, 0.5 eq) and paraformaldehyde (0.333 g, 0.6 eq) were added and the reaction was heated at 100⁰C for 2 h. Reaction was partitioned between water (20 ml) and pentane (30 ml). Aqueous phase was re-extracted 4 times with pentane (10 ml). Combined pentane phases were washed with water and brine, dried over MgSO₄. Solution was diluted to 100 ml with pentane. This solution was added dropwise to a pre-heated solution of sulfuric acid at 67°C (10 ml) under nitrogen stream. At the end of addition, the reaction was stirred for 30 min. Reaction was cooled down to room temperature and poured on iced water (50 ml). Solution was extracted 5 times with MTBE. Combined organic phases were washed with water, NaHCO₃ 1 M and brine, dried over MgSO₄ and concentrated. Purification by flash chromatography furnished pure 6,7-dihydro-1 H-indeno[5,4-b]furan-8(2H)-one (Vl). ¹H NMR δ (CDCI₃) 7.21 (dd, 1 H, J = 0.9 Hz, J = 9.0 Hz), 7.02 (d, 1 H, J = 8.2 Hz), 4.66 (t, 2H, J = 8.9 Hz), 3.48 (t, 2H, J = 8.9 Hz), 3.08 (dd, 2H, J = 4.9 Hz, J = 6.0 Hz), 2.69 (m, 2H). ¹³C NMR δ (CDCI₃) 207.5, 160.2, 147.1 , 133.6, 125.6, 123.9, 1 15.6, 72.3, 37.1 , 28.4, 25.4.

…………………………………

SYNTHESIS

Improve the synthesis and flow properties of an insomnia drug.

Ramelteon (1), marketed as Rozerem by Takeda Pharmaceuticals, is used to treat insomnia. V. K. Kansal and co-inventors describe several processes that are used to prepare it, all of which require many steps. The inventors offer no comments about the relative merits of the processes, but they state that a new industrial-scale process is needed. Their main claims are to intermediate acid 2 as a racemic mixture and individual enantiomers; one enantiomer is converted to 1 by the route shown in Figure 1.

The inventors use diastereomeric crystallization to resolve the racemic mixture by forming its (S)-1-phenylethylamine salt. The salt of the (R)-isomer of 2 is recovered first; then the salt of (S)-2 is isolated from the solution and acidified to give the free acid, which is purified by using (R)-1-phenylethylamine. Both enantiomers are isolated with >99.0% purity and >99.0% ee.

The (S)-acid is converted to acid chloride 3 and then to amide 4 by reactions with SOCl2and NH3 gas, respectively. The chloride is not isolated; the amide is recovered in 85–90% yield with 95–98% purity. When aq NH4OH is used instead of NH3 gas, the purity of 4 is slightly lower (93–96%). An alternative method for preparing 4 is to treat 2 with Et3N and ClCO2Et, followed by NH3. This method produces 4 in yields of 80–95% and 97–99% purity.

Amide 4 is reduced to amine 5 with NaBH4 and BF3·Et2O. The amine is purified by forming its chloride or oxalate salt in yields as high as 85% and 96–98% purity. The salts are used to prepare 1 by treating them with EtCOCl in the presence of base: NaOH for the chloride salt and Na2CO3 for the oxalate. In both cases, the yield of 1 is >92%, and the purity is as high as 99.9% after recrystallization from EtOH.

The inventors also recrystallized 1 from toluene to produce what they describe as a “nonelectrostatic” crystalline form, designated as form A. They describe the measurement of the electrostatic charge of the crystals in one of the patent’s examples. The measurements show that the average charge density of form A is ≈15 times lower than crystals obtained from EtOAc. Low electrostatic charge improves the flow characteristics of the solid, which is important in preparing drug formulations.

The inventors report the details of preparing rac-2 by a multistep procedure shown in Figure 2.

In most of the reaction steps, the product is isolated in crude form; the inventors do not indicate whether the product is purified before it is used in the next stage. The synthesis of rac-2 begins with the conversion of benzofuran (6) to aldehyde7 by treatment with POCl3 followed by hydrolysis. The crude product is isolated as a liquid in 85–90% yield and 90–92% purity.

In the next step, 7 is condensed with malonic acid (8) in the presence of piperidine and HOAc; acid 9 is isolated in 92–95% yield and 95% purity. Catalytic hydrogenation of 9produces 10 in 95% yield and 94–96% purity. The hydrogenation also can be carried out in the presence of NaOH and HCO2NH4; the yield and purity of 10 are the same, but the reaction takes 6 h instead of 2 h. [The patent does not state why NaOH and HCO2NH4 would be used.—Ed.] Acid 10 is brominated to produce acid 11, isolated in 50–60% yield and 92–95% purity.

The next stage begins with treating 11 with SOCl2 to activate the carboxyl group by forming acid chloride 12. The chloride is not isolated but cyclized under Friedel–Crafts conditions to give tricyclic compound 13, isolated in yields of 85–92% and 90-95% purity. This reaction also produces two impurities, 14 and 15, but the amounts are not reported. Removing the impurities gives 13 in good yield, but the inventors do not describe how this is done. They do report that the impurities can be isolated, and 1H and 13C NMR data are provided for both.

In the next step, the bromine atoms in 13 are replaced by hydrogen to give 16 in 85–90% yield and 96–97%purity. This reaction produces two impurities, 17 and 18; again, the amounts are not reported, but 1H and 13C NMR data are. After MeOH reflux in the presence active carbon, 16 is isolated in 80–85% yield with 99.3–99.8% purity. It is then converted to ester 20 by treating it with a solution of phosphonate 19 that contains suspended NaH. Crude product 20 is isolated in 80–85% yield and 92–95% purity as a mixture of (E)- and (Z)-isomers. The isomer mixture is hydrogenated, and base hydrolysis gives rac-2 in 90–95% isolated yield and 95–98% purity.

The inventors claim that the overall process is suitable for producing ramelteon on an industrial scale in a crystalline form that has improved flow characteristics. (Teva Pharmaceutical Industries [Petah Tiqva, Israel]. US Patent 8,084,630, Dec. 27, 2011;

RAMELTEON

…………………….

SYNTHESIS

http://sat.ecnu.edu.cn/Uploadnews/20120213113859628.pdf

CHINESE CHEMICAL LETTERS 22, 2011, 264 SEE SYN OF KEY INTERMEDIATE

1:(S)-N-(2-(6-Methoxy-2,3-dihydro-1H-inden-1-yl)ethyl)propionamide 1======KEY INTERMEDIATE

[a]D20 10.0 (c, 0.20, EtOH); mp 76–77 8C;

1H NMR (500 MHz, CDCl3): d1.15 (t, 3H, J = 7.5 Hz), 1.60 (m, 1H), 1.70 (m, 1H), 2.02 (m, 1H), 2.19 (q, 2H, J = 7.5 Hz), 2.32 (m, 1H), 2.76 (m, 1H), 2.85 (m, 1H), 3.11 (m,1H), 3.41 (m, 2H), 3.79 (s, 3H), 5.48 (s, 1H), 6.71 (dd, 1H, J = 2.0 Hz, 8.5 Hz), 6.75 (s, 1H), 7.11 (d, 1H, J = 8.0 Hz).

13C NMR (100 MHz,DMSO–d6): d173.7, 158.7, 148.1, 135.8, 124.9, 112.3, 109.2, 55.5, 42.7, 37.9, 34.8, 32.5, 30.6, 29.8, 9.9. EI-MS: 247 ([M]+); HR-MS 247.1572([M]+
, C15H21NO2; Calcd. 247.1571). The enantiomeric excess of (S)-1 was determined by HPLC as >99.9% [column, CHIRALPAK AS-H
(4.6 mm 250 mm), room temperature; eluent, hexane-2-propanol-triﬂuoroacetic acid (90:10:0.1); ﬂow rate, 1.0 mL/min; detect, 290 nm; tRof (S)-1, 30.7 min; tR of (R)-1 (enantiomer of (S)-1), 37.1 min].

…………………….

NMR

[PPTData Supplement – Drug Metabolism and Disposition

dmd.aspetjournals.org/content/suppl/…/Supplemental_Information.pptx‎

May 17, 2010 – Ramelteon NMR Assignments. COSY: Black Arrows. HMBC: Red Arrows. Figure S-1b. 1H NMR Spectrum of Ramelteon. Figure S-1c.

References

Owen RT (April 2006). “Ramelteon: profile of a new sleep-promoting medication”. Drugs Today 42 (4): 255–63. doi:10.1358/dot.2006.42.4.970842. PMID 16703122.
Miyamoto M, Nishikawa H, Doken Y, Hirai K, Uchikawa O, Ohkawa S (November 2004). “The sleep-promoting action of ramelteon (TAK-375) in freely moving cats”. Sleep 27 (7): 1319–25.PMID 15586784.
Zammit G, Erman M, Wang-Weigand S, Sainati S, Zhang J, Roth T (August 2007). “Evaluation of the Efficacy and Safety of Ramelteon in Subjects with Chronic Insomnia”. J Clin Sleep Med 3 (5): 495–504. PMC 1978328. PMID 17803013.
Daniel F. Kipke, MD |title=Evidence That New Hypnotics Cause Cancer |journal=University of California |date=March 2008 |url=http://escholarship.org/uc/item/12r2f32g#page-2
http://db.wdc-jp.com/cgi-bin/psj/data/cpb/pdf/201108/c08_1062.pdf
http://sat.ecnu.edu.cn/Uploadnews/20120213113859628.pdf
https://docs.google.com/viewer?url=http%3A%2F%2Fdmd.aspetjournals.org%2Fcontent%2Fsuppl%2F2010%2F05%2F17%2Fdmd.110.034009.DC1%2FSupplemental_Information.pptx
Full-Text PDF – MDPI.com

WO2008106179A1	Feb 26, 2008	Sep 4, 2008	Teva Pharma	Intermediates and processes for the synthesis of ramelteon
WO2008151170A2	Jun 2, 2008	Dec 11, 2008	Teva Pharma	Process for the synthesis of ramelteon and its intermediates
EP0885210B1	Mar 5, 1997	Jun 12, 2002	Takeda Chemical Industries, Ltd.	Tricylic compounds having binding affinity for melatonin receptors, their production and use
EP1792899A1	Sep 12, 2005	Jun 6, 2007	Takeda Pharmaceutical Company Limited	Process for production of optically active amine derivatives
US6034239 *	Mar 6, 1997	Mar 7, 2000	Takeda Chemical Industries, Ltd.	Tricyclic compounds, their production and use
US20010039286 *	Feb 13, 2001	Nov 8, 2001	Kevin Dinnell	2-aryl indole derivatives and their use as therapeutic agents

WO2006030739A1	Sep 12, 2005	Mar 23, 2006	Takeda Pharmaceutical	Process for production of optically active amine derivatives
WO2008062468A2	Oct 15, 2007	May 29, 2008	Cadila Healthcare Ltd	Process for the preparation of optically pure indeno [5,4-b] furan derivatives
WO2008106179A1	Feb 26, 2008	Sep 4, 2008	Teva Pharma	Intermediates and processes for the synthesis of ramelteon
WO2009106966A1	Feb 27, 2009	Sep 3, 2009	Medichem, S.A.	Process for preparing ramelteon.
WO2010055481A1	Nov 12, 2009	May 20, 2010	Watson Pharma Private Limited	Process for the preparation of ramelteon
WO2010092107A1 *	Feb 11, 2010	Aug 19, 2010	Lek Pharmaceuticals D.D.	Synthesis of (s)-n-[2-(1,6,7,8-tetrahydro-2h-indeno-[5,4-b]furan-8-yl)ethyl]propionamide
WO2010103553A1 *	Mar 10, 2009	Sep 16, 2010	Industriale Chimica S.R.L.	Process for the preparation of ramelteon
EP0885210A1	Mar 5, 1997	Dec 23, 1998	Takeda Chemical Industries, Ltd.	Tricyclic compounds, their production and use
US6034239	Mar 6, 1997	Mar 7, 2000	Takeda Chemical Industries, Ltd.	Tricyclic compounds, their production and use
US20100152468	Oct 16, 2009	Jun 17, 2010	Teva Pharmaceutical Industries Ltd.	Process for the synthesis of ramelteon and its intermediates

WO2008062468A2 *	Oct 15, 2007	May 29, 2008	Cadila Healthcare Ltd	Process for the preparation of optically pure indeno [5,4-b] furan derivatives
US5321154 *	Jul 31, 1992	Jun 14, 1994	Nagase & Company, Ltd.	Optical resolution of (.+-.)-2-(4-isobutylphenyl)-propionic acid
US6218429 *	May 10, 1999	Apr 17, 2001	Takeda Chemical Industries, Ltd.	Tricyclic compounds, their production and use
US6348485 *	Jun 8, 1999	Feb 19, 2002	Takeda Chemical Industries, Ltd.	Method for treating or preventing sleep disorders
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US20080242877 *	Feb 26, 2008	Oct 2, 2008	Vinod Kumar Kansal	Intermediates and processes for the synthesis of Ramelteon

WO2012035303A2

Sep 16, 2011

Mar 22, 2012

Cipla Limited Et Al

A novel process for synthesis of ramelteon, and key intermediates for the synthesis of ramelteon

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Sonidegib/Erismodegib..Novartis Cancer Drug LDE225 Meets Primary Endpoint in Phase 2

Phase 3 drug Comments Off

Feb 202014

Sonidegib/Erismodegib

CODE DESIGNATION ..LDE225, NVP-LDE-225

Treatment of medulloblastoma PHASE3 2014 FDA FILING

Treatment of advanced basal cell carcinoma PHASE3 2014 FDA FILING

Treatment of SOLID TUMORS..PHASE1 2017 FDA FILING

READMalignant Solid Tumors of Childhood

THERAPEUTIC CLAIM Oncology, Antineoplastics & Adjunctive Therapies

CHEMICAL NAMES

1. [1,1′-Biphenyl]-3-carboxamide, N-[6-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-3-pyridinyl]-2-
methyl-4′-(trifluoromethoxy)-, rel-

2. N-{6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl}-2-methyl-4′-
(trifluoromethoxy)biphenyl-3-carboxamide

N-[6-[(2S,6R)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl]-2-methyl-3-[4-(trifluoromethoxy)phenyl]benzamide

N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-(trifluoromethoxy)biphenyl-3-carboxamide

MOLECULAR FORMULA C26H26F3N3O3

MOLECULAR WEIGHT 485.5

SPONSOR Novartis Pharma AG

CAS REGISTRY NUMBER 956697-53-3 free form

NOTE… DIPHOSPHATE SALT IS THE DRUG WITH CAS 1218778-77-8

sonidegib – European Medicines Agency READ THIS..

Summary EudraCT Number: 2012-004022-21 Sponsor’s Protocol … READ THIS

Novartis announced that the pivotal trial of the investigational oral compound LDE225 (sonidegib) in advanced basal cell carcinoma met its primary endpoint of demonstrating an objective response rate among patients within six months of treatment. Objective response included complete response (clinically significant tumor response with complete absence of disease) and partial response (clinically significant tumor shrinkage).

Basal cell carcinoma is the most common form of skin cancer, accounting for more than 80% of non-melanoma skin cancers, and can be highly disfiguring and life-threatening if it grows. Worldwide incidence of basal cell carcinoma is rising by 10% each year due to factors such as an aging population and increased ultraviolet exposure. Although basal cell carcinoma rarely metastasizes, once it does, it can be associated with significant morbidity.

“For people living with advanced basal cell carcinoma there are currently limited treatment options,” said Alessandro Riva, president, Novartis Oncology ad interim and global head, Oncology Development and Medical Affairs. “These results demonstrate the potential for LDE225 to offer a treatment option for this patient population, and we look forward to sharing these data with regulatory authorities worldwide.”

Full study results will be presented at a future scientific meeting.

About the Study

The Phase II, randomized, double-blind BOLT (Basal cell carcinoma Outcomes in LDE225 Trial) study was designed to assess the safety and efficacy of two oral dose levels of LDE225 (200 mg and 800 mg) in patients with locally advanced or metastatic basal cell carcinoma[4], which are subtypes of advanced basal cell carcinoma.

The primary endpoint was the proportion of patients achieving an objective response rate, defined as a confirmed complete response and partial response as their best overall response per modified RECIST criteria, within six months of starting treatment with LDE225. Key secondary endpoints of the study included assessing the duration of tumor responseand the rate of complete response. Other secondary endpoints included progression-free survival, time to tumor response and overall surviva

Date: February 19, 2013

Source: Novartis

MORE ABOUT SONIDEGIB

Sonidegib (INN) or Erismodegib (USAN), also known as LDE225 is a Hedgehog signalling pathway inhibitor (via smoothened antagonism) being developed as an anticancer agent by Novartis.[1][2] It has been investigated as a potential treatment for:

Pancreatic cancer [3][4][5][6]
Breast cancer [7][8]
Basal cell carcinoma of the skin[9][10][11]
Small cell lung cancer [12]
Medulloblastoma [13][14]
Advanced solid tumours (including ovarian, breast, pancreatic, stomach, oesophageal cancers and glioblastoma multiforme)[15][16][17]
Acute leukaemia [18]
Chronic myeloid leukaemia [19]
Myelofibrosis and Essential thrombocythaemia [20]

NVP-LDE-225, a product candidate developed by Novartis, is in phase III clinical trials for the treatment of medulloblastoma and basal cell carcinoma. Phase II trials are in progress for the treatment of adult patients with relapsed or refractory or untreated elderly patients with acute leukemia.

Early clinical trials are ongoing for the oral treatment of advanced solid tumors, for the treatment of myelofibrosis in combination with ruxolitinib and for the treatment of small cell lung cancer. A phase II clinical trial for the treatment of basal cell carcinomas in Gorlin’s syndrome patients with a cream formulation of NVP-LDE-225 was discontinued in 2011 since the formulation did not demonstrate tumor clearance rate sufficient to support further development.

Dana-Farber Cancer Institute and the Massachusetts General Hospital are conducting phase I clinical trials for the treatment of locally advanced or metastatic pancreatic cancer in combination with chemotherapy. In 2009, orphan drug designation was assigned in the E.U. for the treatment of Gorlin syndrome.

It has demonstrated significant efficacy against melanoma in vitro and in vivo.[21] It also demonstrated efficacy in a mouse model of pancreatic cancer.[22]

NVP-LDE225 Diphosphate salt (Erismodegib, Sonidegib)

Formula Image

Synonym:Erismodegib, Sonidegib

CAS Number:1218778-77-8

Mol. Formula:C26H26F3N3O3 ∙ 2H3PO4

MW:681.5
nmr.http://www.chemietek.com/Files/Line2/Chemietek,%20NVP-LDE225%20[02],%20NMR.pdf
hplc–http://www.chemietek.com/Files/Line3/Chemietek,%20NVP-LDE225%20[02],%20HPLC.pdf

Brief Description:

A potent, selective, and orally bioavailable Smoothened (Hedgehog Signaling Pathway) antagonist, currently in clinical trials. Diphosphate salt offers a much better bioavailability than free base (Ref. a)

a. Pan, S., et al, Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist, ACS Med. Chem. Lett., 2010, 1 (3), pp 130–134.

About LDE225

LDE225 (sonidegib) is an oral, investigational, selective smoothened inhibitor being studied in a variety of cancers. Smoothened (SMO) is a molecule that regulates the hedgehog (Hh) signaling pathway, which plays a critical role in stem cell maintenance and tissue repair. LDE225 is currently in clinical development for a variety of diseases including myelofibrosis, leukemia and solid tumors.

Given that LDE225 is an investigational compound, the safety and efficacy profile has not yet been fully established. Access to this investigational compound is available only through carefully controlled and monitored clinical trials. These trials are designed to better understand the potential benefits and risks of the compound. Given the uncertainty of clinical trials, there is no guarantee that LDE225 will ever be commercially available anywhere in the world.

Possibility (LDE225) is effective in medulloblastoma relapsed or refractory hedgehog pathway inhibitor sonidegib has been revealed. That the anti-tumor effect was observed in some patients and tolerability in 1/2 test phase.

4th Quadrennial Meeting of the World Federation of Neuro-Oncology in conjunction with the 18th Annual Meeting of the Society for Neuro-Oncology, which was held in San Francisco November 21 to 24 in (WFNO-SNO2013), rice Dana-Farber It was announced by Mark Kieran Mr. Children’s Hospital Cancer Center.

The research group, announced the final results of the Phase 1 trial that target advanced solid cancer in children of sonidegib. 　1 dose increased multi-test phase, was initiated from 372mg/m2 once-daily dosing to target children under the age of 18 more than 12 months. (233mg/m2 group 11 people, 16 people 372mg/m2 group, 11 people group 425mg/m2, 680mg/m2 group 21 women) who participated 59 people, including medulloblastoma 38 patients. 12 median age was (2-17).

Creatine phosphokinase elevation of grade 4 only were seen at 372mg/m2 as dose-limiting toxicity only, and became two recommended dose phase and 680mg/m2. 　Nausea muscle pain creatine kinase rise malaise (22.0%) (15.3%) (15.3%), (13.6%), vomiting side effects were many, was (13.6%). Hypersensitivity vomiting creatine kinase increased (3.4%) (1.7%) (1.7%), rhabdomyolysis side effects of grade 3/4 was (1.7%). 　(One group 372mg/m2, 425mg/m2 group one) complete response was obtained in two people, a strong correlation was found between the activation of the hedgehog pathway and effect.

Phase III clinical trials that target medulloblastoma the activated hedgehog pathway currently are underway.

About Novartis

Novartis provides innovative healthcare solutions that address the evolving needs of patients and societies. Headquartered in Basel, Switzerland, Novartis offers a diversified portfolio to best meet these needs: innovative medicines, eye care, cost-saving generic pharmaceuticals, preventive vaccines and diagnostic tools, over-the-counter and animal health products. Novartis is the only global company with leading positions in these areas. In 2013, the Group achieved net sales of USD 57.9 billion, while R&D throughout the Group amounted to approximately USD 9.9 billion (USD 9.6 billion excluding impairment and amortization charges). Novartis Group companies employ approximately 136,000 full-time-equivalent associates and operate in more than 140 countries around the world.

Increased levels of Hedgehog signaling are sufficient to initiate cancer formation and are required for tumor survival.

These cancers include, but are not limited to, prostate cancer (“Hedgehog signalling in prostate regeneration, neoplasia and metastasis”, Karhadkar S S, Bova G S, Abdallah N, Dhara S, Gardner D, Maitra A, Isaacs J T, Berman D M, Beachy P A., Nature. 2004 Oct. 7; 431(7009):707-12;

“Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling”, Sanchez P, Hernandez A M, Stecca B, Kahler A J, DeGueme A M, Barrett A, Beyna M, Datta M W, Datta S, Ruiz i Altaba A., Proc Natl Acad Sci USA. 2004 Aug. 24; 101(34):12561-6),

breast cancer (“Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer”, Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H, Nomura M, Kuroki S, Katano M., Cancer Res. 2004 Sep. 1; 64(17):6071-4),

medulloblastoma (“Medulloblastoma growth inhibition by hedgehog pathway blockade”, Berman D M, Karhadkar S S, Hallahan A R, Pritchard J I, Eberhart C G, Watkins D N, Chen J K, Cooper M K, Taipale J, Olson J M, Beachy P A., Science. 2002 Aug. 30; 297(5586):1559-61),

basal cell carcinoma (“Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions”, Williams J A, Guicherit O M, Zaharian B I, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter J A, Rubin L L, Wang F Y., Proc Natl Acad Sci USA. 2003 Apr. 15; 100(8):4616-21;

“Activating Smoothened mutations in sporadic basal-cell carcinoma”, Xie J, Murone M, Luoh S M, Ryan A, Gu Q, Zhang C, Bonifas J M, Lam C W, Hynes M, Goddard A, Rosenthal A, Epstein E H Jr, de Sauvage F J., Nature. 1998 Jan. 1; 391(6662):90-2),

pancreatic cancer (“Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis”, Thayer S P, di Magliano M P, Heiser P W, Nielsen C M, Roberts D J, Lauwers G Y, Qi Y P, Gysin S, Fernandez-del Castillo C, Yajnik V, Antoniu B, McMahon M, Warshaw A L, Hebrok M., Nature. 2003 Oct. 23; 425(6960):851-6;

“Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours”, Berman D M, Karhadkar S S, Maitra A, Montes De Oca R, Gerstenblith M R, Briggs K, Parker A R, Shimada Y, Eshleman J R, Watkins D N, Beachy P A., Nature. 2003 Oct. 23; 425(6960):846-51),

and small-cell lung cancer (“Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer”, Watkins D N, Berman D M, Burkholder S G, Wang B, Beachy P A, Baylin S B., Nature. 2003 Mar. 20; 422(6929):313-7).

PATENTS

1 WO 2007131201

2 WO 2008154259

3 WO 2010033481

4 WO 2011009852

5 WO 2011062939

………………………………………

SYNTHESIS

WO2011009852A2

2-Methyl-4′-tr{fluoromethoxy-biphenyl-3-carboxylic acid {6-(cis-2,6-dimethyl- morpholin-4-yl)-pyrid»n-3-yl|-amide:

The following Examples serve to illustrate the invention without limiting the scope thereof, it is understood that the invention is not limited to the embodiments set forth herein, but embraces ali such forms thereof as come within the scope of the disclosure,

Step 1:

To a solution of 2-chloro-5-nitro-pyridine 1 (5.58 g, 35.2 mmoL) and c/s-2,6- dimethylmorpholine (4.05 g, 35.2 mmoL) in anhydrous DMF (30 mi.) was added K2CO3 (9.71 g, 70.4 mnrtoL). The mixture was heated at 50ºC overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na2SO4 and concentrated to give crude product 3 as a yellow solid, after purification by silica gel chromatography, obtained pure product (7.80 g, 93.2%). LC-MS m/z: 238.2 (M+ 1).

Step 2:

The above material 3 (7.3Og. 30.8 mmoL) was hydrogenated in the presence of 10% Pd-C (1.0 g) in MeOH (120 ml) under hydrogen overnight. The suspension was filtered through celite and the filtrate was concentrated to give the crude product 4 (5.92 g) as a dark brown oil which was used directly in the next step without further purification. LC-MS m/z. 208.2 (M+1).

Step 3:

To a solution of 3-bromo-2-methyl benzoic acid (2.71 g, 12.6 mmoL), 6-((2S,6R)-2,6- dimethylmorpholino)pyridin-3-arnine 4 (2.61 g, 12.6 mmoL), and HATU (4.80 g, 12.6 mmoL) in anhydrous DMF (30 mL) was added diisopropylethylamine (6.58 mL, 37.8 mmoL) dropwise. The resulting mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (50 mL), and then extracted with EtOAc (3×120 mL). The organic layer was dried and concentrated to give the crude product. This crude product was then purified by flash column chromatography using 30% EtOAc in hexane as eiuent to give 5 as a white solid (4.23 g, 83.0%). LC-MS m/z: 404.1 (M+1).

Step 4:

A mixture of 4-(trif!uoromethoxy)phenylboronic acid (254 mg, 1.24 mmol), 3-bromo- N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-ylJ-4-methyl-benzamide 5 (250 mg, 0.62mmol), Pd(PPh3)4 (36 mg, 0.03 mmol), Na2CO3 (2.0M aqueous solution, 1.23 mL, 2.4 mmol) and DME (4.5 mL) in a sealed tube was heated at 130ºC overnight. The reaction mixture was diluted with EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine and concentrated to give the crude product which was then purified by preparative mass triggered HPLC (C18 column, etuted with CH3CN-H2O containing 0.05% TFA) to give N-(6-((2S,6R)-2,6-dimethyfmorpholino)pyridin-3-yl)-2-rnethyl- 4′-(trifluoromethoxy)biphenyi-3-carboxamide (183.5 mg, 61.1% yield). LC-MS m/z: 486.2 (M+1).

The resultant crystalline product (Form A) was converted to the amorphous form by dissolving in 3% w/w aqueous ethanol, and the resultant solution spray dried at about 150ºC.

Form B was prepared by heating the amorphous form in an oven at 110ºC for 2 hours. In a further embodiment, the invention relates to a process step or steps, or an intermediate as described herein.

……………………

PAPER

ChemMedChem, 2013 , vol. 8, 8 p. 1261 – 1265

http://onlinelibrary.wiley.com/doi/10.1002/cmdc.201300217/full

http://onlinelibrary.wiley.com/store/10.1002/cmdc.201300217/asset/supinfo/cmdc_201300217_sm_miscellaneous_information.pdf?v=1&s=702e175bfb44b5a61c41e750695f7f7cd20be83d

Continued optimization provided a novel type of Smoothened (Smo) antagonist based on a pyridazine core. The compound, NVP-LEQ506, currently in phase I clinical trials, combines high intrinsic potency and good pharmacokinetic properties resulting in excellent efficacy in rodent tumor models of medulloblastoma. Activity against a Smo mutant conferring resistance observed in a previous clinical trial with a competitor compound suggests additional therapeutic potential.

…………………………..

Links

SYNTHESIS

US20120196849, ENTRY…..95

LC-MS m/z 486.2 (M + 1)

USE SIMILAR METHODOLOGY

EXAMPLESThe present invention is further exemplified, but not limited, by the following example that illustrates the preparation of compounds of Formula I according to the invention.Example 1 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide

Step 1: To a solution of 3-iodo-4-methyl-benzoic acid (10.0 g, 38.2 mmol) in methanol (70 ml) is added concentrated sulfuric acid (0.5 ml). The reaction mixture is heated at 70° C. for 48 hours, cooled to room ambient temperature and then concentrated. After that, ethyl acetate (100 ml) and aqueous NaHCO3 (saturated, 100 ml) solution are added to the residue. The organic layer is separated and washed again with aqueous NaHCO3 (saturated, 100 ml) solution. The organic layer is separated, dried over anhydrous Na2SO4 and concentrated to yield 3-iodo-4-methyl-benzoic acid methyl ester 1. It is used without further purification in the next step. 1H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.87 (d, 1H, J=8.4 Hz), 7.48 (d, 1H, J=8.4 Hz), 3.85 (s, 3H), 3.35 (s, 3H); LC-MS m/z: 277.0 (M+1).

Step 2: To a round-bottom flask containing 3-iodo-4-methyl-benzoic acid methyl ester (1.38 g, 5.00 mmol), 4-cyanophenylboronic acid (1.10 g, 7.48 mmol), palladium acetate (168 mg, 0.748 mmol), 2-(dicyclohexylphosphino)biphenyl (0.526 g, 1.50 mmol) and potassium fluoride (0.870 g, 15.0 mmol) is added anhydrous 1,4-dioxane (15 ml). The flask is purged with argon and sealed. The mixture is stirred at 130° C. for 18 hours, cooled to ambient temperature and then water (20 ml) and ethyl acetate (20 ml) are added. Solid is removed under vacuum filtration. The filtrate is extracted with EtOAc (20 ml×2). The organic layers are combined, washed with aqueous HCl (5%, 20 ml) and saturated NaHCO3 (20 ml). It is dried over MgSO4, and concentrated. The residue is purified by silica gel column chromatography (EtOAc/Hexane, gradient) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2; LC-MS m/z: 252.1 (M+1).

Step 3: To a solution of 4′-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2 (2.56 g, 10.3 mmol) in 1,4-dioxane-H2O (1:1 mixture, 20 ml) is added NaOH (1.22 g, 30.2 mmol)). The reaction is stirred at ambient temperature for 24 hours. To this mixture is added aqueous HCl (1 N, 36 ml) and it is then extracted with ethyl acetate (40 ml×3). The organic layers are combined, dried over anhydrous Na2SO4. The solver is removed. The solid obtained is washed with small amount of acetonitrile and air dried to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid 3: 1H NMR (DMSO-d6) δ 7.94 (d, 2H, J=8.0 Hz), 7.84 (dd, 1H, J1=8.4 Hz, J2=1.2 Hz), 7.75 (d, 1H, J=1.2 Hz), 7.61 (d, 2H, J=8.0 Hz), 7.48 (d, 1H, J=8.4 Hz), 2.29 (s, 3 H); LC-MS m/z 238.1 (M+1).

Step 4: To a suspension of 4′-cyano-6-methyl-biphenyl-3-carboxylic acid 3 (40 mg, 0.17 mmol) in anhydrous methylene chloride (5 ml) is added 2 drops of DMF. Then oxalyl chloride (32 mg, 22 μl, 0.25 mmol) is added. The mixture is stirred at ambient temperature until it turns clear. After that, it is concentrated, re-dissolved in anhydrous methylene chloride (3 ml), and added to a solution of 4-(morpholine-4-sulfonyl)-phenylamine (61 mg, 0.25 mmol) and triethylamine (34 mg, 47 μl, 0.33 mmol) in methylene chloride (2 ml). The mixture is stirred for 2 hours, concentrated and the residue is purified by preparative mass triggered HPLC (C18 column, eluted with CH3CN—H2O containing 0.05% TFA) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide: 1H NMR (DMSO-d6) δ 10.64 (s, 1H), 8.07 (d, 2H, J=8.8 Hz), 7.97 (d, 2H, J=8.4 Hz), 7.95 (d, 1H, J=8.8 Hz), 7.89 (s, 1H), 7.43 (d, 2H, J=8.4 Hz), 7.67 (d, 2H, J=8.8 Hz), 7.53 (d, 2H, J=8.8 Hz), 3.63 (m, 4H), 2.84 (m, 4H) 2.32 (s, 3H); LC-MS m/z: 462.1 (M+1).

Example 2 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide

Step 1: To a solution of 2-chloro-5-nitro-pyridine 4 (2.38 g, 15 mmol.) and cis-2,6-dimethylmorpholine (1.73 g, 15 mmol.) is added K2CO3 (4.14 g, 30 mmol.). The mixture was heated at 50° C. overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na2SO4 and concentrated to give crude product 6 as a yellow solid. The crude product is used directly in next step without further purification. LC-MS m/z: 238.1 (M+1).

Step 2: The above crude material 6 is hydrogenated in the presence of Pd—C (0.2 g) in MeOH (100 mL) under hydrogen over 10 h. The suspension is filtered through celite and the filtrate is concentrated to give the crude product 7 as a dark brown oil which is used directly in the next step without further purification. LC-MS m/z: 208.1 (M+1).

Step 3: To a solution of 3-bromo-4-methyl benzoic acid (108 mg, 0.5 mmol.), 6-(2,6-Dimethyl-morpholin-4-yl)-pyridin-3-ylamine 7 (104 mg, 0.5 mmol.), amd HATU (190 mg, 0.5 mmol.) in dry DMF (5 mL) is added triethylamine (139 uL, 1.0 mmol.) dropwise. The resulting mixture is stirred at room temperature for 2 h. After concentration, the residue is partitioned between EtOAc and water. The organic layer is dried and concentrated to give the crude product. The final compound is purified by flash column chromatography using 50% EtOAc in hexane as eluent to give 8 as a white solid. LC-MS m/z: 404.1 (M+1).

Step 4: A mixture of 4-cyanophenyl boronic acid (18 mg, 0.12 mmol), 3-bromo-N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamide 8 (40 mg, 0.1 mmol), Pd(PPh3)4 (11 mg, 0.01 mmol), and Na2CO3 (42 mg, 0.4 mmol) in a combined solvent system of toluene (0.2 mL) and water (0.2 mL) and ethanol (0.05 mL) is heated at 140° C. under microwave irradiation for 30 min. The reaction mixture is diluted with EtOAc and water. The aqueous layer is extracted with EtOAc. The combined organic layer is washed with brine and concentrated to give the crude product which is purified by preparative mass triggered HPLC (C18 column, eluted with CH3CN—H2O containing 0.05% TFA) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide. LC-MS m/z: 427.2 (M+1).

USE THIS COMPD IN ABOPVE AND YOU WILL GET SONIDEGIB

4-(Trifluoromethoxy)phenylboronic acid

CAS Number 139301-27-2
Linear Formula CF3OC6H4B(OH)2
Molecular Weight 205.93

CONDENSE WITH …3-bromo-N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamideACS Medicinal Chemistry Letters, 2010 , vol. 1, 3 p. 130 – 134

……………………………………………….

PAPER

ACS Medicinal Chemistry Letters, 2010 , vol. 1, 3 p. 130 – 134

http://pubs.acs.org/doi/full/10.1021/ml1000307

ENTRY 5m

A mixture of 4-(trifluoromethoxy)phenylboronic acid (254 mg, 1.24 mmol), 3-bromo-N-[6-(2,6-
dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamide E (250 mg, 0.62mmol), Pd(PPh3)4
(36 mg, 0.03 mmol), Na2CO3 (2.0M aqueous solution, 1.23 mL, 2.4 mmol) and DME (4.5 mL)
in a sealed tube was heated at 1300C overnight. The reaction mixture was diluted with EtOAc
and water. The aqueous layer was extracted with EtOAc. The combined organic layer was
washed with brine and concentrated to give the crude product which was then purified by
preparative mass triggered HPLC (C18 column, eluted with CH3CN-H2O containing 0.05% TFA)
to give N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-
(trifluoromethoxy)biphenyl-3-carboxamide (5m, 183.5 mg, 61.1% yield). LC-MS m/z: 486.2 (M+1).
HRMS (m/z): [M+H]+
calcd for C26H27N3O3F3 486.2005; found 486.1986,
1H-NMR (500 MHz, DMSO-d6): δ (ppm) 10.15 (s, 1H), 8.43 (d, 1H), 7.94 (dd, 1H), 7.52-7.43
(m, 5H), 7.38 (m, 1H), 7.33 (m, 1H), 6.86 (d, 1H), 4.06 (d, 2H), 3.62 (m, 2H), 2,34 (m, 2H), 2.22
(s, 3H), 1.16 (d, 6H).

http://pubs.acs.org/doi/suppl/10.1021/ml1000307/suppl_file/ml1000307_si_001.pdf

Reference

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“A Biomarker Study to Identify Predictive Signatures of Response to LDE225 (Hedgehog Inhibitor) In Patients With Resectable Pancreatic Cancer”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Gemcitabine + Nab-paclitaxel With LDE-225 (Hedgehog Inhibitor) as Neoadjuvant Therapy for Pancreatic Adenocarcinoma”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Dose-escalation, and Safety Study of LDE225 and Gemcitabine in Locally Advanced or Metastatic Pancreatic Cancer Patients”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Pilot Study of a Hedgehog Pathway Inhibitor (LDE-225) in Surgically Resectable Pancreas Cancer”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Study With LDE225 in Combination With Docetaxel in Triple Negative (TN) Advanced Breast Cancer (ABC) Patients (EDALINE)”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014.
“LDE225 in Treating Patients With Stage II-III Estrogen Receptor- and HER2-Negative Breast Cancer”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase II Study of Efficacy and Safety in Patients With Locally Advanced or Metastatic Basal Cell Carcinoma (BOLT)”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“To Evaluate the Safety, Local Tolerability, PK and PD of LDE225 on Sporadic Superficial and Nodular Skin Basal Cell Carcinomas(sBCC)”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Trial to Evaluate the Safety, Local Tolerability, Pharmacokinetics and Pharmacodynamics of LDE225 on Skin Basal Cell Carcinomas in Gorlin Syndrome Patients”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Combination of the Hedgehog Inhibitor, LDE225, With Etoposide and Cisplatin in the First-Line Treatment of Patients With Extensive Stage Small Cell Lung Cancer (ES-SCLC)”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase III Study of Oral LDE225 Versus (vs) Temozolomide (TMZ) in Patients With Hedge-Hog (Hh)-Pathway Activated Relapsed Medulloblastoma (MB)”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase I Dose Finding and Safety Study of Oral LDE225 in Children and a Phase II Portion to Assess Preliminary Efficacy in Recurrent or Refractory MB”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Phase Ib, Dose Escalation Study of Oral LDE225 in Combination With BKM120 in Patients With Advanced Solid Tumors”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Dose Finding and Safety of Oral LDE225 in Patients With Advanced Solid Tumors”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“LDE225 and Paclitaxel in Solid Tumors”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Study of Efficacy and Safety of LDE225 in Adult Patients With Relapsed/Refractory Acute Leukemia”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Nilotinib and LDE225 in the Treatment of Chronic or Accelerated Phase Myeloid Leukemia in Patients Who Developed Resistance to Prior Therapy”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase Ib/II Dose-finding Study to Assess the Safety and Efficacy of LDE225 + INC424 in Patients With MF”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
Jalili, A; Mertz, KD; Romanov, J; Wagner, C; Kalthoff, F; Stuetz, A; Pathria, G; Gschaider, M; Stingl, G; Wagner, SN (30 July 2013). “NVP-LDE225, a potent and selective SMOOTHENED antagonist reduces melanoma growth in vitro and in vivo.” (PDF). PloS one 8 (7): e69064. doi:10.1371/journal.pone.0069064. PMC 3728309.PMID 23935925.
Fendrich, V; Wiese, D; Waldmann, J; Lauth, M; Heverhagen, AE; Rehm, J; Bartsch, DK (November 2011). “Hedgehog inhibition with the orally bioavailable Smo antagonist LDE225 represses tumor growth and prolongs survival in a transgenic mouse model of islet cell neoplasms.”. Annals of Surgery 254 (5): 818–23.doi:10.1097/SLA.0b013e318236bc0f. PMID 22042473.
ChemMedChem, 2013 , vol. 8, 8 p. 1261 – 1265
ACS Med. Chem. Lett., 2010, 1 (3), pp 130–134.
MORE REF

sonidegib

Skin Cancer Foundation. “Skin Cancer Facts.” Available at:http://www.skincancer.org/skin-cancer-information/skin-cancer-facts . Accessed on February 14, 2014.

Rubin AI, Chen EH, Ratner D (2005). Current Concepts: Basal-Cell Carcinoma. N Engl J Med; 353:2262-9.

ClinicalTrials.gov. “A Phase II Study of Efficacy and Safety in Patients With Locally Advanced or Metastatic Basal Cell Carcinoma (BOLT)” Available at:http://clinicaltrials.gov/ct2/show/NCT01327053?term=%22LDE225%22+and+%22BOLT%22&rank=1. Accessed on February 14, 2014.

National Cancer Institute Dictionary of Cancer Terms. “Complete Response.” Available at: http://www.cancer.gov/dictionary?CdrID=45652 . Accessed on February 14, 2014.

National Cancer Institute Dictionary of Cancer Terms. “Partial Response.” Available at: http://www.cancer.gov/dictionary?CdrID=45819 . Accessed on February 14, 2014.

Wong C S M, Strange R C, Lear J T (2003). Basal cell carcinoma. BMJ; 327:794-798.

Copcu E, Aktas A. Simultaneous two organ metastases of the giant basal cell carcinoma of the skin. Int Semin Surg Oncol. 2005;2:1-6. Available at:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC544837/ . Accessed on February 14, 2014.

Skin Cancer Foundation. “Basal Cell Carcinoma Treatment Options.” Available athttp://www.skincancer.org/skin-cancer-information/basal-cell-carcinoma/bcc-treatment-options . Accessed on February 14, 2014.

Stuetz A, et al. LDE225, a specific smoothened inhibitor, for the topical treatment of nevoid basal cell carcinoma syndrome (Gorlin’s syndrome). Melanoma Research. 2010; 20:e40. Available at:http://journals.lww.com/melanomaresearch/Fulltext/2010/06001/FC24_LDE225,_a_specific_smoothened_inhibitor,_for.87.aspx#FC24_LDE225%2C_a_specific_smoothened_inhibitor%2C_for.87.aspx?s=2&_suid=139234380607909969110518506816.

Novartis.com. “The Pipeline of Novartis Oncology: LDE225.” Available at:http://www.novartisoncology.com/research-innovation/pipeline.jsp #. Accessed on February 14, 2014.

Children’s Medical Research Center, Children’s Memorial Hospital/Northwestern University Feinberg School of Medicine. “The Sonic hedgehog/patched/gli signal transduction pathway.” Available at http://www.childrensmrc.org/iannaccone/gli/ . Accessed on February 14, 2014.

Gupta S, Takebe N, LoRusso P. Targeting the Hedgehog pathway in cancer. Ther Adv Med Oncol. 2010 July; 2(4): 237-250. Available at:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126020/ . Accessed on February 14, 2014.

SONIDEGIB

Links

WO2004078163A2	Feb 26, 2004	Sep 16, 2004	Oern Almarsson	Pharmaceutical co-crystal compositions of drugs such as carbamazepine, celecoxib, olanzapine, itraconazole, topiramate, modafinil, 5-fluorouracil, hydrochlorothiazide, acetaminophen, aspirin, flurbiprofen, phenytoin and ibuprofen
WO2007113120A1	Mar 22, 2007	Oct 11, 2007	Frank Hoffmann	Stamping apparatus with feed device
WO2007131201A2 *	May 4, 2007	Nov 15, 2007	Irm Llc	Compounds and compositions as hedgehog pathway modulators
WO2008154259A1	Jun 4, 2008	Dec 18, 2008	Irm Llc	Biphenylcarboxamide derivatives as hedgehog pathway modulators

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The US FDA has issued full approval for Israeli drugmaker Teva’s Synribo (omacetaxine mepesuccinate)高三尖杉酯碱 for chronic myeloid leukaemia (CML).

drugs Comments Off

Feb 182014

Omacetaxine mepesuccinate 高三尖杉酯碱

Alkaloid from Cephalotaxus harringtonia; FDA approved orphan drug status for Ceflatonin in the treatment of chronic myeloid leukemia due to being an inducer of apoptosis in myeloid cells and inhibitor of angiogenesis.
26833-87-4 CAS NO

1-((1S,3aR,14bS)-2-Methoxy-1,5,6,8,9,14b-hexahydro-4H-cyclopenta(a)(1,3)dioxolo(4,5-h)pyrrolo(2,1-b)(3)benzazepin-1-yl) 4-methyl (2R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl)butanedioate

1-((11bS,12S,14aR)-13-methoxy-2,3,5,6,11b,12-hexahydro-1H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]cyclopenta[b]pyrrolo[1,2-a]azepin-12-yl) 4-methyl 2-hydroxy-2-(4-hydroxy-4-methylpentyl)succinate

Also known as: NSC-141633,

BRN 5687925
Ceflatonin
CGX-635
Homoharringtonine
Myelostat
NSC 141633
Omacetaxine mepesuccinate
Omapro
Synribo
UNII-6FG8041S5B
高三尖杉酯碱

CGX-635-14 (formulation), CGX-635, HHT, ZJ-C, Myelostat, Ceflatonin

USFDA on 26th October 2012 APPROVED

US FDA: link

Formula	C₂₉H₃₉N O₉
Mol. mass	545.62 g/mol

Melting Point: 144-146 °C

FEBRUARY 17, 2014

The US Food and Drug Administration has now issued full approval for Israeli drugmaker Teva’s Synribo (omacetaxine mepesuccinate) for chronic myeloid leukaemia (CML).

Synribo is indicated for adult patients with chronic phase (CP) or accelerated phase (AP) CML with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs).

Homoharringtonine is an angiogenesis-inhibiting and apoptosis-inducing alkaloid which was approved in October 2012 by the FDA for the treatment of adult patients with chronic or accelerated phase chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKI). In November 2012, the product was commercialized as Synribo(R) on the U.S. market by Teva.

The original developer, ChemGenex, selected homoharringtonine for the combination trials due to its complementary mechanism of action that can reduce Bcr-Abl protein expression associated with resistance to imatinib mesylate.

In 2004, the compound received orphan drug designation from the EMEA for the treatment of AML and CML. Orphan drug designation was granted by the FDA for the treatment of CML in 2006 and for the treatment of myelodysplasia in 2009. Fast track designation was assigned to homoharringtonine for CML in 2006. In 2009, the product was licensed to Hospira by ChemGenex Pharmaceuticals for development and marketing in Europe, the Middle East and parts of Africa.

Homoharringtonine, AKA HHT or omacetaxine mepesuccinate, is a cephalotaxine ester and protein synthesis inhibitor with established clinical activity as a single agent in hematological malignancies. Homoharringtonine is synthesized from cephalotaxine, which is an extract from the leaves of the plant, Cephalotaxus species. In October 2005, homoharringtonine received Orphan Drug designation from the EMEA for the treatment of chronic myeloid leukemia (CML). Then in March 2006, homoharringtonine received Orphan Drug status from the FDA for the treatment of CML. In November 2006, homoharringtonine, for the treatment of CML, was granted Fast Track designation by the FDA. Most recently, in October 2012, homoharringtonine was marketed under the brand name Synribo” and FDA approved for patients who are intolerant and/or resistant to two or more tyrosine kinase inhibitors used to treat accelerated or chronic phase CML

Omacetaxine mepesuccinate is administered subcutaneously and acts differently from TKIs. It may have a therapeutic advantage for patients who have failed TKIs. Omacetaxine is currently in global phase 2/3 clinical trials for CML and has been granted Orphan Drug designations by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMEA) as well as Fast Track status by the FDA. In vitro and animal model trails are promising and recent results showed that omacetaxine has potential to treat resistant leukemia mainly CML and ALL.

PATENT
US2011071097	3-25-2011	CEPHALOTAXUS ESTERS, METHODS OF SYNTHESIS, AND USES THEREOF

Tetrahedron Letters,Vo1.23,No.34,pp 3431-3434 … – Brock University

Omacetaxine mepesuccinate

Omacetaxine mepesuccinate (INN, trade name Synribo) is a semi-synthetic analogue of an alkaloid from Cephalotaxus harringtonia that is indicated for treatment of chronic myelogenous leukemia (CML). It was approved by the US FDA in October 2012 for the treatment of adult patients with CML with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs).^[1]

Omacetaxine mepesuccinate is a semisynthetic derivative of the cytotoxic plant alkaloid homoharringtonine isolated from the evergreen tree Cephalotaxus with potential antineoplastic activity. Omacetaxine mepesuccinate binds to the 80S ribosome in eukaryotic cells and inhibits protein synthesis by interfering with chain elongation. This agent also induces differentiation and apoptosis in some cancer cell types. Omacetaxine mepesuccinate (INN, or homoharringtonine, trade name Synribo) is an alkaloid from Cephalotaxus harringtonia that is indicated for treatment of Chronic Myelogenous Leukemia. It was approved by the USFDA on 26th October 2012 for the treatment of adult patients with chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs)

Omacetaxine is indicated for use as a treatment for patients with chronic myeloid leukaemia who are intolerant of tyrosine kinase inhibitors.^[2]^[3]

In June 2009, results of a long-term open label Phase II study were published, which investigated the use of omacetaxine infusions in CML patients. After twelve months of treatment, about one third of patients showed a cytogenetic response.^[4] A study in patients who had failed imatinib and who had the drug resistant T315I mutation achieved cytogenetic response in 28% of patients and haematological response in 80% of patients, according to preliminary data.^[5]

Phase I studies including a small number of patients have shown benefit in treating myelodysplastic syndrome (MDS, 25 patients)^[6] and acute myelogenous leukaemia (AML, 76 patients).^[7] Patients with solid tumors did not benefit from omacetaxine.^[8]

Omacetaxine is a protein translation inhibitor. It inhibits protein translation by preventing the initial elongation step of protein synthesis. It interacts with the ribosomal A-site and prevents the correct positioning of amino acid side chains of incoming aminoacyl-tRNAs. Omacetaxine acts only on the initial step of protein translation and does not inhibit protein synthesis from mRNAs that have already commenced translation.^[9]

Omacetaxine mepesuccinate

SYNRIBO contains the active ingredient omacetaxine mepesuccinate, a cephalotaxine ester. It is a protein synthesis inhibitor. Omacetaxine mepesuccinate is prepared by a semi-synthetic process from cephalotaxine, an extract from the leaves of Cephalotaxus sp. The chemical name of omacetaxine mepesuccinate is cephalotaxine, 4-methyl (2R)-hydroxyl-2-(4-hydroxyl-4-methylpentyl) butanedioate (ester).

Omacetaxine mepesuccinate has the following chemical structure:

SYNRIBO™ (omacetaxine mepesuccinate) Structural Formula Illustration

The molecular formula is C₂₉H₃₉NO₉ with a molecular weight of 545.6 g/mol. SYNRIBO for injection is a sterile, preservative-free, white to off-white, lyophilized powder in a single-use vial. Each vial contains 3.5 mg omacetaxine mepesuccinate and mannitol.

SYNRIBO is intended for subcutaneous administration after reconstitution with 1.0 mL of 0.9% Sodium Chloride Injection, USP. The pH of the reconstituted solution is between 5.5 and 7.0.

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INTRODUCTION

Harringtonines 3 are particular cephalotaxanes formed by attachement of a branched hydroxyacyloxy side-chain at the 3-position of various cephalotaxines moieties. Harringtoriines are natural esters of cephalotaxines exhibiting generally a strong cytotoxic activity. However the lost only one atom of this minimal structure lead to a dramatic lost of activity (see below). Some example of harringtonines are harringtonine

3a, homoharringtonine 3b, drupangtonine 3c, anhydroharringtonine 3d and neoharringtonine 3e.

SCHEME 1 DEFINITION NOMENCLATURE AND NUMBERING OF CEPHALOTAXANES

Examples of harringtonines

Examples of cephalotaxines

Harringtonine 3a (n = 2) Anhydroharringtonine 3d Homoharringtonine 3b (n = 3)

(-)-Cephalotaxine 2a

Drupacine 2b Drupangtonine 3c Neoharringtonine 3e (n = 2)

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The term “cephalotaxanes” refers to compounds or salts thereof which have a basic skeleton of formula

where p is equal to 1 or 2 (it being possible for the two units to be identical or different and linked via a single bond or an oxygen atom), which can contain various oxygenated substituents (aliphatic or aromatic ethers, free or esterified alcohols, substituted or free enols and/or phenols, bridged ethers, and more generally any substituent usually encountered in the natural state on compounds of this type).

Harringtonines are alkaloids which are of high interest in anticancer chemotherapy, in particular on certain haematosarcomas which are multi-resistant to the existing therapies. The selectivity of harringtonines, which is based on a novel mechanism of action relating to protein synthesis, is such that this series is favoured with a great future in anticancer therapy.

Several literature compilations give a seemingly exhaustive review of all of the knowledge relating to cephalotaxanes, these compilations being, chronologically: [C. R. Smith, Jr, R. G. Powell and K. L. Mikolajczack, Cancer Treat. Rep., Vol. 60, 1157 (1976); C. R. Smith, Jr, L. Kenneth, K. L. Mikolajczack and R. G. Powell in “Anticancer Agent Based on Natural Product Model”, 391 (1980); Liang Huang and Zhi Xue in “The Alkaloids”, Vol. XXIII (A. Brossi Ed.), 157 (1984); M. Suffness and G. A. Cordell in “The Alkaloids, Chemistry and Pharmacology” (A. Brossi Ed.), Vol. 25, 57-69, 295-298 (1’987); P. J. O’Dwyer, S. A. King, D. F. Hoth, M. Suffness and B. Leyland-Jones, Journal of Clinical Oncology, 1563 (1986); T. Hudlicky, L. D. Kwart and J. W. Reed, in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, 199 (1998)].

Antiparasitic activities, in particular on the haematozoon of malaria, have also been recognized [J. M. Whaun and N. D. Brown, Ann Trop. Med. Par., Vol. 84, 229 (1990)].

Homo-harringtonine (HHT), the most active member of the series, is active at and above daily doses of 2.5 mg/m²of body area per 24 hours, i.e., as a guide, at doses twenty times lower than that for Taxol. HHT has already undergone fourteen phase I and II clinical trials and it is the only known product capable of a 70% reinduction of full haematological remissions in patients suffering from chronic myeloid leukaemias that have become resistant to alpha-interferon [S. O’Brien, H. Kantarjian, M. Keating, M. Beran, C. Koler, L. E. Robertson, J. Hester, M. Rios, M. Andreeff and M. Talpaz, Blood, 332 (1995); Leukemia Insights, Vol. 3, No. 1 (1998)].

Harringtonines were extracted over 35 years ago from an exclusively Asiatic cephalotaxacea known as Cephalotaxus harringtonia, following the programme of research into novel anticancer agents in the plant kingdom developed by the National Cancer Institute. In fact, the Cephalotaxus alkaloids consist essentially (at least 50%) of cephalotaxine, a biosynthetic precursor of the harringtonines, the latter individually representing only a few percent of the total alkaloids.

Besides their low concentration in the natural state in plant starting material, harringtonines are mixed with many congeners which have very similar chemical structures. Thus, in a high resolution high performance liquid chromatography (HPLC) chromatogram of a semi-purified alkaloid extract, no less than several tens of cephalotaxine esters are counted.

Numerous antileukemia drugs have been investigated but so far, there is no single drug that is effective and safe. As discussed in U.S. 3,497,593, an alkaloid from Tylophora plant is said to have antitumor activity against mouse leukemia (L-1210). U.S. 3,928,584 discloses an organic composition derived from tree sap and is said to have activity against mouse leukemia P-388. Also U.S. 4,431,639 discloses that an extract of Rhisoma Stractylis promotes the production of lymphocytes in the circulating blood, consequently eliminating cancer growth

Harringtonine or Homoharringtonine, hereinafter referred to as HH, has been known to be effective against acute chronic granulocytic and monocytic leukemia (Journal of Chinese Internal Medicine 3:162-164, 1978). However, it is highly toxic and causes damage to heart and hematopoietic organs. The results of experiments in animals, such as mice, rabbits and dogs, indicate that most of them die from cardiotoxicity after receiving the drug. Therefore, there is a need to improve the HH drug for safe use against leukemia. This drug is of special importance in that all known antileukemia drugs are effective against lymphatic leukemia and there are no effective drugs for treating nonlymphatic leukemia

All the literature from 1972 to the present date [Mikolajczack et al., Tetrahedron, 1995 (1972); T. Hudlicky, L. D. Kwart and J. W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxane 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3 totally preformed to give a harringtonine 4b, i.e. the conversion 2a+3e(4b as described in the example featured in the scheme below

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SYNTHESIS

Tetrahedron Lett 1982,23(34),3431, J Org Chem 1983,48(26),5321

The oxidation of 2-methyl-1-cyclopentene-1-carbaldehyde (I) with O3 and Ag2O gives 2,6-dioxoheptanoic acid (II), which is esterified with cephalotaxine (III) by means of (COCl)2, yielding the ester (IV). Reformatsky reaction of (IV) with methyl bromoacetate (V) and Zn affords the adduct (VI), which is treated with an excess of methylmagnesium iodide to provide the target homoharringtonine (as a single diastereomer), along with some starting cephalotaxine that is separated by chromatography.

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SYNTHESIS

EP 1064285; FR 2776292; WO 9948894, Tetrahedron Lett 1999,402931

The intermediate (racemic)-2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid (VIII) has been obtained by several related methods: 1. The Grignard condensation of 4-methyl-3-pentenyl bromide (I) with diethyl oxalate (II) in HF gives the 2-oxoheptenoate (III), which is condensed with methyl acetate (IV) by means of LiHMDS in THF to yield 3-(ethoxycarbonyl)-3-hydroxy-7-methyl-6-octenoic acid methyl ester (V).

The cyclization of (V) by means of Ts-OH in hot toluene or by means of hot aqueous formic acid affords 2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid ethyl ester (VI), which is hydrolyzed with KOH in boiling water to provide the corresponding dicarboxylic acid (VII). Finally, this compound is regioselectively monoesterified by means of BF3/MeOH in methanol to furnish the intermediate (racemic)-2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid (VIII). 2.

The reaction of 3-(ethoxycarbonyl)-3-hydroxy-7-methyl-6-octenoic acid methyl ester (V) with HCl in hot methanol gives 3-(ethoxycarbonyl)-3,7-dihydroxy-7-methyloctanoic acid methyl ester (IX), which is then cyclized by means of ZnCl2 in hot dichloroethane to yield the previously described intermediate (VIII). 3. The hydrolysis of 3-(ethoxycarbonyl)-3-hydroxy-7-methyl-6-octenoic acid methyl ester (V) with KOH in refluxing methanol/water gives the corresponding diacid (X), which is regioselectively monoesterified by means of BF3/MeOH in methanol to yield 3-carboxy-3-hydroxy-7-methyl-6-octenoic acid methyl ester (XI).

Finally, this compound is cyclized by means of Ts-OH in hot toluene to afford the previously described carboxylic intermediate (VIII). The racemic acid (VIII) is submitted to optical resolution by esterification with quinine (XII) by means of 2,4,6-trichlorobenzoyl chloride and TEA or DCC to give a diastereomeric mixture of esters (XIII) that is separated by preparative HPLC to obtain the desired diastereomer (XIV).

The hydrolysis of (XIV) with KOH in refluxing ethanol/water gives the corresponding chiral dicarboxylic acid (XV), which is regioselectively monoesterified with BF3/MeOH in methanol to yield the chiral (R)-2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid (XVI).

The esterification of (XVI) with cephalotaxine (XVII) by means of 2,4,6-trichlorobenzoyl chloride and TEA in toluene affords the corresponding ester (XVIII), which is treated with HBr in dichloromethane/HOAc, providing the bromoester (XIX). Finally, this compound is treated with NaHCO3, CaCO3 or BaCO3 in acetone/water to give the target hydroxyester.

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EXTRACTION

EP0203386B1

Throughout the specification, the concentration of the solvent is the same as first given unless stated otherwise. Redeuced pressure means about 2,27 kPa (17 mm Hg. abs), l is liter, kg is kilogram. ml is milliliter. Yield in weight %.
Example 1. HH is extracted from the skins, stems, leaves and seeds of Cephalotaxus fortunel Hook and other related species, such as Cephalotaxus sinensis Li, C. hainanensis, and C. wilsoniana, including C.oliveri mast and C.harringtonia.
1 kg of finely ground Cephalotaxus fortunel Hook is extracted with 8 l of 90% ethanol at room temperature for 24 hrs. The solution is filtered to yield a filtrate A and filtercake. The filtercake is percolated with ethanol and filtered again to yield filtrate B. A and B are combined and distilled under reduced pressure to recover ethanol and an aqueous residue. To this residue, 2% HCl is added to adjust the pH to 2.5. The solids are separated from the solution by filtration to yield a filtrate C. The solids are washed once with 2% HCl and filtered to yield a filtrate D. C and D are combined and the pH adjusted to 9.5 by adding saturated sodium carbonate solution. The alkaline filtrate is extracted with chloroform and the chloroform layer separated from the aqueous layer. This extration process is repeated five times. All the chloroform extracts are combined and distilled at reduced pressure to recover chloroform and alkaloid as a solid residue respectively.
The solid alkaloid is then dissolved in 20 ml. of 6% citric acid in water. The solution is divided into three equal portions. These are adjusted to pH 7,8 and 9 by adding saturated sodium carbonate solution.
The portions having pH 8 and 9 are combined and extracted with chloroform. The chloroform extracts are distilled under reduced pressure, whereby chloroform is removed and recovered and a solid residue of crude Harringtonine is obtained.
The crude Harringtonine is dissolved in pure ethanol i.e. alkaloid : anhydrous ethanol 1:10 , and crystallized. The crystals are refined by recrystalliation in diethyl ether. Overall yield of Harringtonine is about 0.1% including yield from mixed HH from the subsequent process.
Harringtonine has the following chemical structure:

wherein R is

melting point:

135° – 137°C

crystal:

colorless

infrared spectrum:

3750, 1660, 1505, 1490, 1050, and 945 cm⁻¹.
The portion having a pH of 7 and the mother liquors from the foregoing crystallization of Harringtonine are combined and passed through a liquid chromatographic column of diameter to height ratio 1:50 packed with alumina. The column is finally flushed with chloroform and followed by chloroform-methanol of 9:1 mixture. The resulting alkaloids are mixture of HH. The mixed HH is then separated from each other by countercurrent distribution employing chloroform and pH 5 buffer. The first fraction of the countercurrent distribution is Homoharringtonine and the last fraction of the countercurrent distribution is Harringtonine. Homoharringtonine is purified by crystallization in methyl alcohol.
Homoharringtonine has the following chemical structure:

wherein R is

yield:

0.02%

melting point:

144° – 146°C

infrared spectrum:

3500∼3400, 1750, 1665, 1030 and 940 cm⁻¹.

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EXTRACTION

EP1064285B1

All the literature from 1972 to the present date [Mikolajczack et al.,Tetrahedron, 1995 (1972); T. Hudlicky, L.D. Kwart and J.W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S.W. Pelletier Ed.), Vol. 5, 639 (1987); M.A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxine 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3e totally preformed to give a harringtonine 4b , i.e. the conversion 2a + 3e ( 4b as described in the example featured in the scheme below

Example 46

Preparation of purified (-) cephalotaxine from total alkaloidic extract of Cephalotaxus sp

[0319]
Partially racemized cephalotaxine [H. Wenkui; L. Yulin; P. Xinfu, Scientia Sinica,; 23; 7; 835 (1980)]
¹H NMR of two batches of cephalotaxine (extracted in the same conditions as above) with the optically active NMR shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate (1 éq) showed the following results:
- Batch A: ¹H NMR 400 MHz (CDCl₃)(δ ppm): 6.06 (1H, OCH₂O (+)-cephalotaxine) and 5.82 (1H, OCH₂O (+)-cephalotaxine) ; 5.99 (1H, OCH₂O (-)-cephalotaxine) and 5.76 (1H, OCH₂O (-)-cephalotaxine).
  Presence of 11 ± 5 % de (+)-cephalotaxine.
  [α]²² = -134,0° (c = 0,214; CHCl₃) : calculated rate 25 ± 5 %
- Batch B: slightly racemized (1%)
  [α]¹⁹ = -173,3° (c = 0,208; CHCl₃)

Enantiomeric enrichment of the natural cephalotaxine:

Crude chromatographied cephalotaxine (20g) was dissolved at 55°C in dry methanol (100 ml). Crystallization occurs by cooling with rotary evaporator and after filtration the product thus obtained showed 99.9% of HPLC purity.
[α]²⁰ _D =-130° (C1, CHD₃) corresponding to 10 % of racemization. The crystallized product thus obtained (20g) was dissolved again in hot methanol (100 ml).
Slowly cooling the solution allows translucent prisms composed of pure enantiomeric (-)-cephalotaxine [α]²⁰ _D= -185°(C1,CHCl₃).
After filtration, the mother liquors was allowed to slowly evaporate at room temperature and crystals in the form of macled needles exclusively composed of racemic cephalotaxine [α]^D ₂₀ = 0,5° (C1 ; CHCl₃) were obtained.
After filtration, the second mother liquors allowed prisms composed of (-)-cephalotaxine identical to this obtained at the first crystallization.
After filtration, the third mother liquors still allowed macled needles (urchins) composed of (±)-cephalotaxine.
The cycle is repeated three times. The combined prismatic crystals was recrystallized once to give enantiomerically pure (-)-cephalotaxine, while the combined macled needles treated in the same way gives 100% racemic cephalotaxine.

Chemical evaluation of the enantiomeric purity of natural cephalotaxine:

A sample of partially racemized natural cephalotaxine was inserted in the process, which sequence is described in the Examples 1,2,3,4,5,6,15,19 and 21, by using a pure (2R)-homoharrintonic acid resulting from Example 19.
The HPLC analysis of the diastereomeric mixture of anhydro-homoharrintonine thus obtained showed a significant enantio-epi-homoharringtonine rate (11% ± 3%) corresponding to the (+)-cephalotaxine content in the racemic mixture of origin (it has been demonstrated that the two antipodes of the homoharringtonic acid react in a stoechiometric way comparable to the pure enantiomeric cephalotaxine).

Example 47Preparation of homoharringtonine, from anhydro-homoharringtonine:

1)° Method A

A commercial solution of hydrobromic acid in acetic acid (17.4 ml, 86.6 mmol, HBr 30% w/w) was added to a stirred solution of anhydrohomoharringtonine resulting from Example 21 (50.8 g, 9.63 mmol) in anhydrous dichloromethane (25.6 ml) at -10°C. After stirring at -10°C for 3 hours was added water (240 ml) and the reaction mixture was become viscous. The temperature was allowed to rise to room temperature and after stirring for 2.5 hours was added sodium carbonate 0.76M (406 ml) to pH 8. The resulting aqueous layer was saturated with sodium chloride, then was extracted with dichloromethane (3 × 230 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a foam. After phase reverse chromatography below-mentioned were obtained 4.03g of homoharringtonine (77%). The product thus obtained showed identical characteristics to this resulting from Example 25.

2°) Method B

To a stirred solution of anhydrohomoharringtonine resulting from Example 21 (214 mg, 0.406 mmol) in anhydrous dichloromethane (1.1 ml) was added at -10°C a commercial solution of hydrobromic acid in acetic acid (0.728 ml, 3.6 mmol, HBr 30% w/w). After stirring at -10°C for 3 hours, was added water (13 ml) and then the temperature was raised to 20°C. After stirring at 20°C for 3 hours, was added a sodium carbonate solution (0.76M; 31.5 ml) up to pH 8. The resulting aqueous layer, after saturation with sodium chloride, was extracted with dichloromethane (3 × 20 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by phase reverse chromatography below-mentioned to provide homoharringtonine (166 mg, 75%). The product thus obtained showed identical characteristics to this resulting from Example 25.

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SEMISYNTHESIS

US6831180

EXAMPLE 27 Preparation of homoharringtonine as a pharmaceutical use from crude semi-synthetic homoharringtonine resulting from example 25 by preparative high-performance liquid chromatography

1°) Method A

Crude homoharringtonine (35 g) is dissolved in buffer (triethylamine (1.55/1000) in deionised water and orthophosphoric acid to adjust pH to 3. The solution was filtered then injected on a preparative high-performance liquid chromatograph equipped with axial compression and high pressure pump (stationary phase: n-octadecylsilane, 15 μm, porosity 100, 1 kg; mobile phase; buffer/tetrahydrofurane 85/15). Elution was performed at a flow rate of 0.2 l/min. Fractions contain was monitored by U.V. detector and TLC. Retained fraction were finally checked by HPLC then combined, alkalinised with 2.5% aqueous ammonia and extracted with dichloromethane (4×400 ml). After concentration under reduced pressure homoharringtonine is obtained as a pale yellow resin which on trituration in a 8/2 water-methanol mixture gave pure homoharringtonine as a white crystalline solid (mp=127° C.), HPLC purity was higher than 99.8%.

2°) Method B

Same procedure of purification as method A was performed but mobile phase buffer/methanol (68/32) was used instead buffer/tetrahydrofurane.

3°) Method C

Same procedure of purification as method A was performed but mobile phase buffer/acetonitrile (85/15) was used instead buffer/tetrahydrofurane.

EXAMPLE 28 Preparation of homoharringtonine as a pharmaceutical use from semi-purified natural cephalotaxine

Crude homoharringtonine, prepared according to Example 25 from a partially racemized natural cephalotaxine and purified by chromatography and crystallisation according to the method A of Example 27, gave an homoharringtonine showing a non natural enantiomeric epi-homoharringtonine content less than 0.05%.

EXAMPLE 46 Preparation of purified (−) cephalotaxine from total alkaloidic extract of cephatotaxus sp

Partially racemized cephalotaxine [H. Wenkui; L. Yulin; P. Xinfu, Scientia Sinica; 23; 7; 835 (1980)]

¹H NMR of two batches of cephalotaxine (extracted in the same conditions as above) with the optically active NMR shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate (1éq) showed the following results:

Batch A: ¹H NMR 400 MHz (CDCl₃)(δ ppm): 6.06 (1H, OCH₂O (+)-cephalotaxine) and 5.82 (1H, OCH₂O (+)-cephalotaxine); 5.99 (1H, OCH₂O (−)-cephalotaxine) and 5.76 (1H, OCH₂O (−)-cephalotaxine). Presence of 11±5% de (+)-cephalotaxine. [α]²²=−134,0°(c=0,214; CHCl₃): calculated rate 25±5%

Batch B: slightly racemized (1%) [α]¹⁹=−173,3°(c=0,208; CHCl₃)

Enantiomeric Enrichment of the Natural Cephalotaxine:

Crude chromatographied cephalotaxine (20 g) was dissolved at 55° C. in dry methanol (100 ml). Crystallization occurs by cooling with rotary evaporator and after filtration the product thus obtained showed 99.9% of HPLC purity, [α]²⁰ _D=−130°(C1, CHD₃) corresponding to 10% of racemization. The crystallized product thus obtained (20 g) was dissolyed again in hot methanol (100 ml).

Slowly cooling the solution allows translucent prisms composed of pure enantiomeric (-−)-cephalotaxine [α]²⁰ _D=−185°(C1, CHCl₃).

After filtration, the mother liquors was allowed to slowly evaporate at room temperature and crystals in the form of macled needles exclusively composed of racemic cephalotaxine [α]^D ₂₀=0,5°(C1; CHCl₃) were obtained.

After filtration, the second mother liquors allowed prisms composed of (−)-cephalotaxine identical to this obtained at the first crystallization.

After filtration, the third mother liquors still allowed macled needles (urchins) composed of (±)-cephalotaxine.

The cycle is repeated three times. The combined prismatic crystals was recrystallized once to give enantiomerically pure (−)-cephalotaxine, while the combined macled needles treated in the same way gives 100% racemic cephalotaxine.

Chemical Evaluation of the Enantiomeric Purity of Natural Cephalotaxine:

A sample of partially racemized natural cephalotaxine was inserted in the process, which sequence is described in the Examples 1,2,3,4,5,6,15,19 and 21, by using a pure (2R)-homoharrintonic acid resulting from Example 19. The HPLC analysis of the diastereomeric mixture of anhydro-homoharrintonine thus obtained showed a significant enantio-epi-homoharringtonine rate (11%±3%) corresponding to the (+)-cephalotaxine content in the racemic mixture of origin (it has been demonstrated that the two antipodes of the homoharringtonic acid react in a stoechiometric way comparable to the pure enantiomeric cephalotaxine).

EXAMPLE 47

Preparation of homoharringtonine, from anhydro-homoharringtonine

1°) Method A

A commercial solution of hydrobromic acid in acetic acid (17.4 ml, 86.6 mmol, HBr 30% w/w) was added to a stirred solution of anhydrohomoharringtonine resulting from Example 21 (50.8 g, 9.63 mmol) in anhydrous dichloromethane (25.6 ml) at −10° C. After stirring at −10° C. for 3 hours was added water (240 ml) and the reaction mixture was become viscous. The temperature was allowed to rise to room temperature and after stirring for 2.5 hours was added sodium carbonate 0.76M (406 ml) to pH 8. The resulting aqueous layer was saturated with sodium chloride, then was extracted with dichloromethane (3×230 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a foam. After phase reverse chromatography below-mentioned were obtained 4.03 g of homoharringtonine (77%). The product thus obtained showed identical characteristics to this resulting from Example 25.

2°) Method B

To a stirred solution of anhydrohomoharringtonine resulting from Example 21 (21.4 mg, 0.406 mmol) in anhydrous dichloromethane (1.1 ml) was added at −10° C. a commercial solution of hydrobromic acid in acetic acid (0.728 ml, 3.6 mmol, HBr 30% w/w). After stirring at −10° C. for 3 hours, was added water (13 ml) and then the temperature was raised to 20° C. After stirring at 20° C. for 3 hours, was added a sodium carbonate solution (0.76M; 31.5 ml) up to pH 8. The resulting aqueous layer, after saturation with sodium chloride, was extracted with dichloromethane (3×20 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by phase reverse chromatography below-mentioned to provide homoharringtonine (166 mg, 75%). The product thus obtained showed identical characteristics to this resulting from Example 25.

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EXTRACTION

US20100240887

The remarkable clinical efficacy of Homoharringtonine (HHT) resulting in lot of observations of complete remission of leukemia and other solid cancer in human being since 1988. Recently, research articles reported that the HHT efficacy in glaucoma, inhibition of Hepatities B virus replication and using in bone marrow transplantation. For example, the University of Texas M.D. Anderson Cancer Center and National Cancer Institute reported that “Ninety-two percent of patients achieved CHR with HHT.” [Susan O’Brien, at al.; Sequential homoharringtonine and interferon-α in the treatment of early chronic phase chronic myelogenous leukemia; Blood, Vol 93, No 12 (June 15), 1999: pp 4149-4153]. Another article reported that “the median number of days on HHT per month was 2 days with a median follow-up of 26 months; the estimated 2-year survival rate was 90%.” (Susan O’Brien, at al.; Simultaneous homoharringtonine and interferon-α in the treatment of patients with chronic-phase chronic myelogenous leukemia; American Cancer Society; Apr. 1, 2002, Vol 94, No. 7).

On Nov. 8, 1988, U.S. Pat. No. 4,783,454 titled Process for producing harringtonine and homoharringtonine disclosed the technique of isolation of a purified HHT from bark of Cephalotaxus. However, the natural source ofCephalotaxus is very limited. Trees of Cephalotaxus grow slowly. Bark ofCephalotaxus has very low content of HHT. Extracting HHT from bark ofCephalotaxus the yield was about 0.02% only. More important to harvest bark ofCephalotaxus will kill and destroy trees. Supply of HHT is very short now. Therefore, it is necessary to find a new manufacturing method.

DETAILED DESCRIPTION

Great progress has been made in research on Homoharringtonine (HHT) production and on future generation HHT drug since 1988. For example, the University of Texas M.D. Anderson Cancer Center and National Cancer Institute reported that “Ninety-two percent of patients achieved CHR with HHT.” Another article reported that “the median number of days on HHT per month was 2 days with a median follow-up of 26 months; the estimated 2-year survival rate was 90%.”

The good clinical results of HHT in treating cancer brought to the major problem, which is the supply of HHT both short term and long term. It is apparent that a huge amount of bark of Cephalotaxus is needed for collection, extraction and purification of HHT. It is clear that due to the slow growth of the trees ofCephalotaxus, which is a nature source of HHT, and the killing of trees by harvesting bark is not a sustainable resource for HHT production.

Present invention disclosed new methods for producing HHT. The new methods of producing HHT are shown as follows.

1. Tissue Culture (Plant Cell Culture):

Culture manipulation to promote secretion of HHT is a new way for an extracellular product HHT. The biosynthetic methods can yield more HHT through precursor of HHT feeding. The production of HHT increased significantly after the addition of the precursors and special biochemical agents. Content of precursor of HHT abounds in tree and it is very cheap. The present methods include several significant developments in technique of culture plant tissues that are

- (a) yields of HHT selected from rapid growth, resistance to infections organisms; and
- (b) HHT can excrete into media.

Traditional method of plant culture is very difficult to overcome the problem of high cost. Therefore, traditional method appears too long to have commercial value. HHT is secondary metabolite of Cephalotaxus. Secondary compound acts in defense against the harmful effects of toxins, carcinogens or mutagens found in the plant. In fact, traditional method is very difficult to increase HHT contenting in plant tissues. The present new method uses a special biochemical agent for increasing content of HHT and more easily to purify HHT from other metabolites.

More important is that the key of the present new technique for producing high content of HHT in plant cell culture is to increase production of HHT by directed fermentation through precursor of HHT feeding. The present new methods are used special metabolite of Cephalotaxus for markedly enhance production of HHT. Therefore, the present invention disclosed a new source for the long term of producing HHT.

2. Using Precursor of HHT:

Recent research’s results have established that direct production of HHT from its precursor and advances in biosynthetic understanding for HHT metabolism. Biosynthesis or semisynthesis of HHT from major nonactivity ingredients is well established through great advances in special biochemistry reactions. Using precursor of HHT for semisynthesis and increase of production in plant cell culture are new developing methods for producing HHT.

3. Using Leaves:

Our new method use leaves of tree of Cephalotaxus not use the bark. So far, the extraction of HHT is used bark. The leaves are harvested from the trees ofCephalotaxus, which grow in mountains of South China. The natural source of leaves is very abundance. The new methods do not use bark. Therefore, it can avoid destroy trees. The natural source of Cephalotaxus tree is very limited and slow growing. In fact, bark of Cephalotaxus has very low yield of HHT. The yield of HHT from Cephalotaxus bark is about 50-100 mg/kg of dried bark. The present new method, therefore, has a great economic and environmental value.

4. Semisynthesis:

HHT has received important chemical studies particularly in regard to structure and anticancer activity relationship and semisynthesis.

A great progress in biochemistry allows semisynthesis to use precursor of HHT from leaves of Cephalotaxus and to produce HHT. The total chemical synthesis of HHT appears too long to have commercial value too. Semisynthesis method can yield a high efficient conversion of precursor to HHT. It is other better biological source for manufacturing HHT. This new method uses closing chemical analogues to convert to HHT. This analogue is produced from leaves or other organ of Cephalotaxus. The present invention disclosed that new methods and techniques of manufacturing HHT could avoid chopping down Cephalotaxus trees which governmental environmentalists are trying to have declared a threatened species.

5. Using Taxol Residual

The anticancer drug Taxol is the most promising new chemotherapeutic agents that developed for cancer treatment in the past twenty years. Taxol has a unique mechanism of action. It has been shown to promote tubulin polymerization and stabilize microtubules against depolymerization. The FDA approved the clinical use of Taxol for several types of cancer. So far, annual sales of Taxol are more than $2 billion in market. Taxol is extracted from bark or leaves of an evergreen tree named Taxus species including Taxus brevifolia (or called Pacific yew). After Taxol has been extracted from bark or leaves, all residual materials of Taxus brecifolia named Taxus residual, which are waste.

Both taxol and HHT can be extracted from yew tree. The content of taxol is less than 0.01% in yew tree. The content of HHT in yew tree is about 0.01% -0.22%. The content of HHT is much higher than content of Taxol. Taxol extracted from bark of yew is difficult and expensive. One reason is that the presences of closely related congeners are similar to Taxol. A major congener is Cephalomannine (CPM), which is a waster of process in manufacturing of Taxol.

The chemical and physical characters are very close between Taxol and Cephalomannine (CPM).

CPM characterized by the same ring structure as Taxol and distinguishes from them only in C-13 ester structure. The present invention disclosed that CPM and related derivative are used to produce HHT.

The following specific examples will provide detailed illustrations of methods of producing relative drugs, according to the present invention and pharmaceutical dosage units containing demonstrates its effectiveness in treatment of cancer cells. These examples are not intended, however, to limit or restrict the scope of the invention in any way, and should not be construed as providing conditions, parameters, reagents, or

EXAMPLE 1

Production of HHT by Culture Cells

So far, HHT is extracted from bark and skins of Cephalotaxus species. However, growth of Cephalotaxus species is very slow and concentration of HHT in plant is extremely low. Furthermore, it is difficult to harvest the plants because of their low propagation rate and the danger of drastic reduced in plant availability. Also, cost of total chemical synthesis of HHT is very expensive and is not available for commerce now. For the reasons given above it is more difficult to obtainCephalotaxus on a large scale for long time. Therefore, Cephalotaxus cell cultures are one of best methods for obtaining HHT. In this present invention, special elicitation is disclosed and it will significantly increase production of HHT.

The methods of cell and tissue culture are disclosed as below.

Parts of bark, stems, leaves, or roots of Cephalotaxus species were surface disinfected by treatment in 70% ethanol for 10 minutes and followed by 0.1 HgCl₂for 3 minutes. Plant materials were washed five times for 10 minutes each by sterilized water. Parts of plant were cut into small pieces (0.5-1 mm) and put pieces to Murashige and Skoog’s (MS) medium and supplemented with derivative of new active ingredient of phylum mycota (IPM), precursor of HHT which is a derivative of Cephalotaxus (CEP), tyrosine (TYR) naphthaleneacetic acid (NAA), Kinetin (3 mg/L), and 3% sucrose (w/v). PH of medium was adjusted to 5.7˜5.8. Agar (10 g/L) added to medium. Callus tissues are collected from agar media and suspension cultured cells were harvested by filtration and cultured in MS medium.

The cultures were kept in a culture room at 26° C.±1° C. Friable callus tissues were obtained. The callu was inoculated into 4 L of MS liquid medium containing sucrose, derivative of CEP, PHE, TYR, NAA and Kinetin. Then callus tissues were cultivated 26° C. for 35 days on rotary shaker operated at 120 rpm in the dark. Cells were subcultured into fresh medium of same composition every 2 weeks and maintained at 120 rpm at 26°±1° C. Packed cell volume (PCV), fresh weight (FW), dry weight (DW), concentration of HHT and concentration of sugar were determined every 5th day. The cells were harvested and dried.

In general, callus and suspension cultures of cephalotaxus species grow very slow and no production of free or esterified HHT. However, according to the present invention, addition of IPM to cultures cause a drastic increasing in HHT after 30 days of incubation. For example, in control group (no IPM), HHT in cultured cells is 0.020 mg/g dry weight, but in treatment group (addition of IPM) HHT is about 0.050 mg/g dry weight. Therefore, IPM can increase 250% of content of HHT. It has resulted in plant cell culture systems that producing HHT at concentration higher than those produced by the mother plant. The production of HHT increases significantly after the addition of precursors (CEP). Addition of CEP can increase HHT. Obviously, the present invention provided a new commercial and economic method for producing HHT. The IPM and precursors (CEP) play key role in cultured cells.

EXAMPLE 2

Semi-Synthesis of HHT

HHT shows a significant inhibitory activity against leukemia and other cancer. Concentration of HHT, however, has only 0.01% in natural sources. Cephalotazine (CEP) is major alkaloids present in plant extracts and the concentration ofCephalotaxus has about 1%. Therefore, concentration of CEP is about 100 times higher then HHT in nature plant sources. But CEP is inactive. For the reason given above, semisynthesis of HHT from CEP will increase huge natural sources of HHT.

- (1) Extraction of CEP

10 kg of dried stems or leaves or roots of Cephalotaxus species were milled, placed in a percolator, along 80 L of 95% of ethanol, and allowed to stand 24 hours. The ethanol was recovered under reduced pressure (below 40° C.). 20 L of 5% tartaric acid was added to concentrated ethanol solution. The ammonia water was added to the acidic solution and adjusted pH to 9. The solution of pH 9 was filtered and yielded a filtrate. The filtrate was extracted with CHCl₃. CHCl₃was recovered under reduced pressure and residue was obtained. The residue was chromatographed packed with alumna and eluted by CHCl₃-MeOH (9:1). Eluate was concentrated under reduced pressure. Residue was dried under vacuum. The product is CEP.

- (2) Semisynthesized HHT from CEP

Materials and Methods

Melting points were determined on a Fisher-Johns apparatus. Infrared spectra were obtained on a Perkin-Elmer 567 infrared spectrophotometer or on a Beckman 4230 IR spectrophotometer. Peak positions were given in cm⁻¹. The IR spectra of solid samples were measured as potassium bromide dispersions, and the spectra of liquids were determined in chloroform or carbon tetrachloride solutions. NMR spectra were measured on a Varian A-60, Perkin-Elmer R-32, Varian EM-390, or Brüker WH-90 NMR spectrometer. Chemical-shift values were given in parts per million downfield from Me₄Si as an internal standard. Mass spectra were run on an AE1 MS-12 Finnigan 3300, or CEC21-110B mass spectrometer.

Preparative thin-layer chromatography was accomplished using 750-μm layers of aluminum oxide HF-254 (type E), aluminum oxide 60 PF-254 (type E), silica gel HF-254 (type 60 PF-254), or silica gel GF-254. Visualization was by short-wave ultraviolet light. Grace silica gel, Grade 923, and Woelm neutral aluminum oxide, activity III, were used for column chromatography. Analytical thin-layer chromatography was run on plastic sheets precoated with aluminum oxide F-254 neutral (type T), 200-μm thick, and on Polygram Sil G/UV254 (silica gel), 250 μm on plastic sheets. Visualization was usually by short-wave ultraviolet light, phosphomolybdic acid, or iodoplatinate.

Preparation of α-Ketoester-Harringtonine

1 g of Benzene-α-acetone Na was put into 10 L of benzene. Mixture was stirred at room temperature then was dissolved in 10 L of pyridine and stirred at 0° C. Oxalic chloride was added from a dropping funnel to solution of pyridine. Stirring was continued while the solution warmed to room temperature and stand overnight. Excess reagent was removed. This solution was dissolved in CH₂Cl₂and cooled to near 0° C. in an ice water bath. 5 g of CEP, 2.5 L of CH₂Cl₂and 2.5 L of pyridine were added to cold CH₂Cl₂solution. Manipulations were done in a dry N₂atmosphere and all glassware heat-dried just before use. The suspension was stirred at room temperature and overnight. The mixture was washed with 10% Na₂CO₃and saturated aqueous NaCl, then dried with auhydrous magenesium sulfate, and filtered and the solvents were removed in vacuo. Evaporation provided as an amorphous solid α-ketoester-harringtonine (mp 143˜145° C.).

Semi-Synthesis of HHT

10 L of CH₃CHBrCOOEt and activated zin dust and THF were added to the α-ketoester-harringtonine (at −78° C.) for 6 hours followed by slow warming to room temperature with stirred. The reaction mixture was diluted with 10 L CHCl₃and 10 L H₂O and solid Na₂CO₃was added. CHCl₃was evaporated under reduced pressure and residue was obtained.

The residue was purified by chromatography on alumina. The column was flushed with chloroform and followed by chloroform-methanol (9:1). The solvents were recovered under reduced pressure to provide as a solid. Solid was dissolved in pure ethanol and crystallized. The crystals were refined by recrystalization in diethyl ether. The crystals dried under vacuum. The product is HHT, which has the following characters:

[α]_D−119° (C=0.96),

MSm/e (%): 689 (M⁺, 3), 314 (3), 299 (20), 298 (100), 282 (3), 266 (4), 20 (3), 150 (8), 131 (12), 73 (18)

EXAMPLE 3

HHT Extracted from Plant Tissue

Extraction of HHT has several major methods which including extraction by organic solvent, chromatograph and adjust pH.

HHT was extracted from plant tissue culture, plant cells or leaves of Cephalotaxusspecies.

1 kg of ground Cephalotaxus fortunei Hook was extracted with 10 liters of water at room temperature for 24 hrs. To filtered the solution to yield a filtrate. Ten liters of 90% ethanol added to filtrate. The mixture was Centrifugalized to yield a sediment. Percolated the sediment with ethanol and filter again to yield filtrate, combined filtrates, and distilled under reduced pressure to recover ethanol and an aqueous residue. To this residue, added 10% of HCl to adjust the pH to 2.5. To separated the solids from the solution by filtration to yield a filtrate (1). Washed the solids once with 2% HCl and filtered to yield a filtrate (2). Combined (1) and (2) and adjusted the pH to 9.5 by adding saturated sodium carbonate solution. Extracted the alkaline filtrate with chloroform and separated the chloroform layer from the aqueous layer. To repeated this extraction process five times. Combined all the chloroform extracts and distilled at reduced pressure to recover chloroform and alkaloid as a solid residue obtained. The solid alkaloid was then dissolved in 6% citric acid in water. The solution was divided into three equal portions. These were adjusted to pH 7, 8 and 9 by adding saturated sodium carbonate solution. The portions having pH 8 and 9 were combined and extracted with chloroform. The chloroform extracts were distilled under reduced pressure, whereby chloroform was removed and recovered and crude HHT was obtained. The crude HHT was dissolved in pure ethanol and crystallized. The crystals were refined by recrystallization in diethyl ether. The crude HHT obtained.

The portion having a pH of 7 passed through a liquid chromatographic column packed with alumina of diameter to height 1:50. The column was finally flushed with chloroform and followed by chloroform-methanol of 9:1 mixture. The resulting alkaloids were mixture crude of HHT. Combined crude HHT and then separated from each other by countercurrent distribution employing chloroform and pH 5 buffers. The first fraction of the countercurrent distribution was HHT. HHT was purified by crystallization in methyl alcohol. The crystallization was purified by recrystallization in methyl alcohol and dried under vacuum.

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EP1373275A2

Example 1 : Preparation of harringtonine drug substance by purification of commercial natural harringtonine

A. Analytical profile of starting product

By combination of HPLC analysis with UV detection (see Figure 6) and mass spectrometry detection (see figure 7 and 8) a total of 6.5% of related compound (identified as b,c: position isomer of harringtonine = 3.4%; d: homoharringtonine = 3%; e: 4′-demethyl harringtonine = 0.01%; f: drupacine derivative: 0.05%) are found in the starting product.

B. Chromatography of natural harringtonine

Natural harringtonine (5 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 80 mm; length: 1000 mm) containing 1000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi). Unwanted fractions are discarded based upon in-line UV spectrophotometric detection. Kept fractions are collected in 16 separate containers which each are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase). During the development phase, a dual in-line UV-MS detection is used. After discarding of the fractions representing more than 0.5 % of the total content of harringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure. Then crude concentrated solution of harringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 1.5 %. C. Crystallization of raw harringtonine

Under a laminar flow hood, the above raw harringtonine (4.1 grams) is dissolved in methanol (5ml), at 30°C. The resulting alcoholic solution was filtered on a 0.25 μ sterile Millipore filter to remove microparticules and germs and collected in a sterilized rotary flask. Then, desionized water (50mL) is added and methanol is completely removed under vacuum at 30°C in using a decontaminated rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of harringtonine is kept under vacuum and rotation is continued during appearance of white crystals of pure harringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (10 mL x 2). The white translucent crystals are then dried using high vacuum at 40°C for 24 hours. Overall yield is 76%. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied. This clinical batch corresponds to 400 therapeutic units dosed at 10mg.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. Figure 7 and 9 compare HPLC chromatogram before and after purification in using this process. Table II shows the comparison of the corresponding related compound content.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC) thermogravimetry, 2D NMR, solid NMR and X-ray powder diffractometry.

Infrared Spectrometry:

Identical IR spectra were obtained by either the KBr pellet and/or mineral oil mull preparation technique. Figure 5 shows typical infrared spectrum (KBr) for unambiguous identification at the solid state of the crystalline harringtonine obtained by this process. A series of sharp absorption bands are noted at 615, 654, 674, 689, 709, 722, 750, 761 805, 850, 928, 989, 1022, 1033, 1062, 1083, 1112, 1162, 1205, 1224, 1262, 1277, 1308, 1340, 1364, 1382, 1438 1486, 1508, 1625, 1656, 1725, 1745, 2883, 2936, 2972, 3079, 3353, 3552 and 3647 cm^“1

Differential Scanning Calorimetry (DSC) And Thermogravimetry (TG) Measurement of DSC and TG were obtained on a Mettler Toledo STAR System. Approximately 12 mg of harringtonine drug substance were accurately weighed (12.4471 mg) into a DSC pan. The sample was heated from 25°C to 200°C at a rate of 10°C/min. The DSC data were obtained following a standard method in the art. The DSC curve of crystalline harringtonine drug substance ((Figure 4), exhibits a melting endotherm at 79.5 °C . No subsequent decomposition occurred under the upper tested temperature 200°C. Simultaneous TG measurement, indicated a loss on drying of 1.3 % which did not correspond to a lost of structural molecule of solvent or water.

Example 2: Preparation of homoharringtonine drug substance by purification of raw semi- synthetic (hemi-synthetic) homoharringtonine

A. Analytical profile of starting product

Crude reaction mixture of raw homoharringtonine contains a potential of 250 grams of homoharringtonine DS together with process impurities such as catalyst, unchanged starting product (anhydro-homo-harringtonine), and some related side product. HPLC analysis with UV detection (see left-side chromatogram on Figure 10) indicated a total of 9 % of related impurities. B. Chromatography of semi-synthetic homoharringtonine

Raw semi-synthetic homoharringtonine (550 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 450 mm; length: 1000 mm) containing 48,000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi, flow-rate 540 L/hour). Unwanted fractions are discarded based upon by- passed in-line UV spectrophotometric detector. Kept fractions are collected in 30 separate stainless steel containers (20 or 50 L each) which are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase) and equipped with a diode array detector. After discarding of the fractions representing more than 0.5 % of the total content of homoharringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure in using a mechanically stirred thin film evaporator. Then crude concentrated solution of homoharringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 0.5 % (see rigth-side chromatogram on Figure 10)

C. Crystallization of homoharringtonine DS

In a controlled clean room, under a laminar flow hood, the above raw homoharringtonine DS (210 grams) is dissolved in methanol (240 mL), at 30°C. The resulting alcoholic solution is filtered on a 0.25 μ sterile Millipore filter to remove microparticules and germs and collected in a sterilized pilot rotary flask. Then, desionized water (2400mL) is added and methanol is completely removed under vacuum at 30°C in using a decontaminated pilot rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of homoharringtonine DS is kept under vacuum and rotation is continued during appearance of white crystals of pure homoharringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (450 mL x 2). The white cryitals are then dried using high vacuum at 60°C for 48 hours. Overall yield is 88% from potential content of homoharringtonine in raw semi-synthetic homoharringtonine. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied. This clinical batch corresponds to 40,000 therapeutic units dosed at 5mg.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. Figure 11 shows HPLC chromatogram before and after crystallization. Total of related impurities of homoharringtonine DS is 0.03%.

Infrared Spectrometry:

Identical IR spectra were obtained by either the KBr pellet and/or mineral oil mull preparation technique. Figure 3 shows typical infrared spectrum (KBr) for unambiguous identification at the solid state of the crystalline homoharringtonine obtained by this process. A series of sharp absorption bands are noted at 612, 703, 771 , 804, 826, 855, 879, 932, 1029, 1082, 1119,

1135, 1161 , 1191 , 1229, 1274, 1344, 1367, 1436, 1457, 1488, 1505, 1653, 1743, 2814, 2911 ,

2958, 3420, and 3552 cm^“1

Differential Scanning Calorimetry (DSC) And Thermogravimetry (TG)

Measurement of DSC and TG were obtained on a Mettler Toledo STAR System. Approximately 11 mg of homoharringtonine drug substance were accurately weighed (10.6251 mg) into a DSC pan. The sample was heated from 25°C to 250°C at a rate of 5°C/min. The

DSC data were obtained following a standard method in the art. The DSC curve of crystalline homoharringtonine drug substance (Figure 1), exhibits a melting endotherm at 145.6 °C.

Melting range performed by the capillary method (Bucchi Apparatus) gave 143-145°C. Literature indicated 144-146°C [Anonymous, Acta Bot. Sin. 22, 156 (1980) cited by L. Huang and Z. Xue, Cephalotaxus Alkaloids, in “The Alkaloids”, vol. XXIII, pp157, (1988).

Crystallization medium was not published. This is the only literature reference regarding melting point of a crystalline form of HHT] X-Ray Powder Diffraction

X-ray powder diffraction pattern was collected on a INEL microdiffractomer, model

DIFFRACTINEL. Powdered homoharringtonine DS was packed in a glass capillary tube and was analyzed according to a standard method in the art. The X-ray generator was opered at 45 kV and 40 mA, using the copper Kalpha line as the radiation source. The sample was rotated along the chi axis and data was collected between 0 and 120 deg 2-theta. A collection time of 1200 sec was used. As showed on Figure 2, the x-ray powder diffraction for this crystalline form of homoharringtonine shows a typical pattern including major reflection peaks at approximately 7.9, 9.2, 10.9, 14.9 16.0, 17.7, 19.5, 19.7, 21.78, 23.1 , 25.3, 25.4 and 25.7 deg 2-theta.

Example 3: Preparation of homoharringtonine drug substance by purification of a commercial sample of impure homoharringtonine from Chinese source

A. Analytical profile of starting product

Analytical HPLC chromatogram of natural homoharringtonine (China National Pharmaceutical) is displayed on Figure 12 (bottom left).

B. Chromatography of Natural Homoharringtonine

Natural homoharringtonine (25 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 200 mm; length: 1000 mm) containing 12,000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi, flow-rate 120 IJhour). Unwanted fractions are discarded based upon bypassed in-line UV spectrophotometric detector. Kept fractions are collected in 22 separate stainless steel containers which are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase) and equipped with a diode array detector. After discarding of the fractions representing more than 0.5 % of the total content of homoharringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure in using a mechanically stirred thin film evaporator. Then crude concentrated solution of homoharringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 0.5 %.

C. Crystallization of homoharringtonine DS

In a controlled clean room, under a laminar flow hood, the above chromatographied homoharringtonine DS (18 grams) is dissolved in methanol (35 mL), at 30°C. The resulting alcoholic solution is filtered on a 0.25 μ sterile Millipore filter to remove microparticules and germs and collected in a sterilized pilot rotary flask. Then, desionized water (300 mL) is added and methanol is completely removed under vacuum at 30°C in using a decontaminated pilot rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of homoharringtonine DS is kept under vacuum and rotation is continued during appearance of white crystals of pure homoharringtonine. The stirring is continued until no more crystal occurs.

Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water

(50 mL x 2). The white crystals are then dried using high vacuum at 60°C for 48 hours. Overall yield is 84% from potential content of homoharringtonine in raw semi-synthetic homoharringtonine. All operations were documented prior to start the process and full current

Good Manufacturing Practices were applied.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. Figure 12 (bottom right) shows HPLC chromatogram after crystallization. Total of related impurities of homoharringtonine DS is 0.05%.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC), thermogravimetry (TD), 2D NMR, solid NMR and X-ray powder diffractometry. Infrared Spectra, Differential Scanning Calorimetry (DSC) and X-Ray Powder Diffraction gave patterns strictly superimposable to the one of example 2 obtained from semi-synthetic homoharringtonine (Figure 3, 1 , and 2, respectively).

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KOREAN PAPER.. LINK

Title: 한국산 개비자(Cephalotaxus koreans)에서의 Harringtonine과 Homoharringtonine의 확인 및 함량 분석
Author: 박호일 ; 이연 (한국생물공학회)
Source: 한국생물공학회지 = Korean journal of biotechnology and bioengineering; ISSN:1225-7117 @ 1225-7117 @ ; VOL.11; NO.6; PAGE.689-695; (1996)
Pub.Country: Korea
Language: Korean
Abstract: Harringtonine and homoharringtonine known as anti-cancer agents were isolated from Korean native plumyew(Cephalotaxus koreana) using column chromatography(CHCl3:MeOH=19:1, Rf=0.28). The structure of the mixture of two compounds was characterized by 1H-NMR. Comparison of our spectra of harringtonine and homoharringtonine with previously reported ones indicated that the two are identical. The contents of harringtonine and homoharringtonine in the needles, stems, and roots of Korean native plumyew were determined by high performance liquid chromatography(HPLC). The contents of both compounds varied with the site of location and the part of plant. The content of harringtonine was higher in needles and roots than in stems, whereas the content of homoharringtonlne was lower than harringtonine. Homoharringtonine contents in needles at Mt. Palgong, Mt. Dukyu, Mt. Baekyang, Mt. Jiri, and Namhae were higher than in stems and roots. But homoharringtonine contents in needles al Mt. Jokye and Jindo were lower than in stems and roots.

http://img.kisti.re.kr/originalView/originalView.jsp

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《Acta Chimica Sinica》 1985-02

http://en.cnki.com.cn/Article_en/CJFDTOTAL-HXXB198502009.htm

SYNTHESIS OF HOMOHARRINGTONINE AND SEPARATION OF ITS STEREOMERS

WANG YONG-KENG LI YU-LIN PAN XIN-FU LI SHAO-BAI HUANG WEN-KUI (Institute of Organic Chemistry,Lanzhou University)

Ethyl 2-oxo-6-ethylene dioxy-heptanoate(2),an important intermediate in the preparation of homoharringtonine(8a),was prepared by the reaction of Grignard reagent made from 1-bromo-4-ethylene dioxy pentane with ethyl oxalate in THF. Compound 2 was converted into α-keto-acyl-cephalotaxine(5)via sodium carboxylate 3 and acyl chloride 4.Reformatsky reaction of 5 with methyl bromoacetate in the presence of freshly prepared active zinc affords 6.Acid treatment of 6 gave 7.Reaction of 7 with methyl magnesium iodide provided a mixture of homoharringtonine(8a) and its epimer 8b.Their separation is effected by fractional crystallization of their picrates and subsequent recovery of the free alkaloids 8a and 8b.The TLC,IR,~1H NMR and MS data of 8a are identical with those of natural homoharringtonine.The IR and MS of 8a and 8b are quite similar,but their ~1H NMR are markedly different

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Chapter 1 Drug Discovery from Plants – Springer

www.springer.com/cda/content/…/cda…/9783540746003-c1.pdf?…0…‎

LC-NMR-MS and LC-SPE-NMR to accelerate their future discovery. Keywords …..Ceflatonine (34), a synthetic version of homoharringtonine produced by.

…………………………………………………………………………….

References

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“SYNRIBO (omacetaxine mepesuccinate) injection, powder, lyophilized, for solution [Cephalon, Inc.]”. DailyMed. Cephalon, Inc. October 2012. Retrieved 18 February 2014.
Sweetman, S, ed. (14 November 2012). Omacetaxine Mepesuccinate. “Martindale: The Complete Drug Reference”. Medicines Complete(Pharmaceutical Press).
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Quintás-Cardama, A.; Kantarjian, H.; Cortes, J. (2009). “Homoharringtonine, omacetaxine mepesuccinate, and chronic myeloid leukemia circa 2009”. Cancer 115 (23): 5382–5393.doi:10.1002/cncr.24601. PMID 19739234. edit
Wu, L.; Li, X.; Su, J.; Chang, C.; He, Q.; Zhang, X.; Xu, L.; Song, L.; Pu, Q. (2009). “Effect of low-dose cytarabine, homoharringtonine and granulocyte colony-stimulating factor priming regimen on patients with advanced myelodysplastic syndrome or acute myeloid leukemia transformed from myelodysplastic syndrome”. Leukemia & Lymphoma50 (9): 1461. doi:10.1080/10428190903096719. edit
Gu, L. F.; Zhang, W. G.; Wang, F. X.; Cao, X. M.; Chen, Y. X.; He, A. L.; Liu, J.; Ma, X. R. (2010). “Low dose of homoharringtonine and cytarabine combined with granulocyte colony-stimulating factor priming on the outcome of relapsed or refractory acute myeloid leukemia”.Journal of Cancer Research and Clinical Oncology 137 (6): 997–1003.doi:10.1007/s00432-010-0947-z. PMID 21152934. edit
Kantarjian, H. M.; Talpaz, M.; Santini, V.; Murgo, A.; Cheson, B.; O’Brien, S. M. (2001). “Homoharringtonine”. Cancer 92 (6): 1591–1605.doi:10.1002/1097-0142(20010915)92:6<1591::AID-CNCR1485>3.0.CO;2-U. PMID 11745238. edit
Wetzler M, Segal D. Omacetaxine as an Anticancer Therapeutic: What is Old is New Again. Current Pharmaceutical Design 2011;17:59-64
Concise total synthesis of (±)-cephalotaxine via a transannulation strategy: Development of a facile reductive oxy-nazarov cyclization
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The first semi-synthesis of enantiopure homoharringtonine via anhydrohomoharringtonine from a preformed chiral acyl moiety
Tetrahedron Lett 1999, 40: 2931
Synthesis of homoharringtonine and its derivative by partial esterification of cephalotaxine
Tetrahedron Lett 1982, 23(34): 3431
Construction of chiral tertiary alcohol stereocenters via the (2,3)-Meisenheimer rearrangement: Enantioselective synthesis of the side-chain acids of homoharringtonine and harringtonine
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WO2002032904A1 *	Oct 17, 2000	Apr 25, 2002	Oncopharm Corp	New cephalotaxanes, their method of preparation and their use in treatment of cancers, leukemias, parasites including thus resistant to usual chemotherapeutic agents and as reversal agents
EP0393575A1 *	Apr 17, 1990	Oct 24, 1990	G.D. Searle & Co.	Neoplasia treatment compositions containing antineoplastic agent and side-effect reducing protective agent
USH271 *	Dec 18, 1985	May 5, 1987	The United States Of America As Represented By The Secretary Of The Army	Treatment of malaria with esters of cephalotaxine

US7169774	Jun 25, 2004	Jan 30, 2007	Stragen Pharma S.A.	Cephalotaxane derivatives and their processes of preparation and purification
US7842687	May 25, 2006	Nov 30, 2010	Chemgenex Pharmaceuticals, Inc.	Cephalotaxane derivatives and their processes of preparation and purification
US8466142	Mar 3, 2009	Jun 18, 2013	Sloan-Kettering Institute For Cancer Research	Cephalotaxus esters, methods of synthesis, and uses thereof

Reference
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2	*	LEVY, VINCENT (1) ET AL: “Subcutaneous homoharringtonine (SQ HHT ): 1. Pharmacokinetic study in dogs and HHT determination in blood in using LC-MS method.” BLOOD, (NOVEMBER 16, 2001) VOL. 98, NO. 11 PART 2, PP. 179B. HTTP://WWW.BLOODJOURNAL.ORG/. PRINT. MEETING INFO.: 43RD ANNUAL MEETING OF THE AMERICAN SOCIETY OF HEMATOLOGY, PART 2 ORLANDO, FLORIDA, USA DECEMBER 07-11, 2001 , XP001095449
3	*	LEVY, VINCENT (1) ET AL: “Subcutaneous homoharringtonine (SQ HHT ): 2. Tolerance in humans and case report of a refractory patient with AML treated by very small dose of SQ HHT.” BLOOD, (NOVEMBER 16, 2001) VOL. 98, NO. 11 PART 2, PP. 202B. HTTP://WWW.BLOODJOURNAL.ORG/. PRINT. MEETING INFO.: 43RD ANNUAL MEETING OF THE AMERICAN SOCIETY OF HEMATOLOGY, PART 2 ORLANDO, FLORIDA, USA DECEMBER 07-11, 2001 , XP001095450
4	*	WHAUN J M ET AL: “TREATMENT OF CHLOROQUINE -RESISTANT MALARIA WITH ESTERS OF CEPHALOTAXINE HOMOHARRINGTONINE.” ANN TROP MED PARASITOL(1990) 84(3), 229-237, XP008006193

1H NMR

13 CNMR

HPLC

TADALAFIL.. cialis

GENERIC Comments Off

Feb 162014

Tadalafil

GF-196960, IC-351, Cialis

6R–trans)-6-(1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino [1′, 2′:1,6] pyrido[3,4-b]indole-1,4-dione

Pyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-, (6R-trans)-; (6R,12aR)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-ethylpyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione; GF 196960; Adcirca;

171596-29-5^casno

Molecular Weight:
389.40

Molecular Formula:C₂₂H₁₉N₃O₄

GlaxoSmithKline (Originator), Lilly Icos (Marketer), Lilly (Licensee), Lilly Icos (Licensee)

Launched-2003

Tadalafil is currently marketed as Cialis. Cialis was developed by Eli Lilly as a treatment for impotence. In this capacity, it is reported that tadalafil functions by inhibiting the formation of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5). The inhibition of PDE5 presumably lessens impotence by increasing the amount ot c(iMP, resulting in smooth muscle relaxation and increased blood flow.

Tadalafil is a PDE5 inhibitor marketed in pill form for treating erectile dysfunction (ED) under the name Cialis, and under the name Adcirca for the treatment of pulmonary arterial hypertension. In October 2011 the U.S. Food and Drug Administration (FDA) approved Cialis for treating the signs and symptoms of benign prostatic hyperplasia (BPH) as well as a combination of BPH and erectile dysfunction (ED) when the conditions coincide. It initially was developed by the biotechnology company ICOS, and then again developed and marketed world-wide by Lilly ICOS, LLC, the joint venture of ICOS Corporation and Eli Lilly and Company. Cialis tablets, in 2.5 mg, 5 mg, 10 mg, and 20 mg doses, are yellow, film-coated, and almond-shaped. The approved dose for pulmonary arterial hypertension is 40 mg (two 20-mg tablets) once daily.

Tadalafil can be prepared via a series of intermediates. One synthesis scheme is illustrated in Scheme 1: Scheme 1

U.S. Patent No. 5,859,006 describes the synthesis of the tadalafil intermediate (Compound III) from D-tryptophan methyl ester (Compound II) and piperonal (Compound I) using trifluoroacetic acid and dichloromethane, a halogenated solvent. Compound III is then reacted with chloroacetyl chloride (Compound IV) and chloroform, providing another intermediate of tadalafil (Compound V). WO 04/011463 describes a process of preparing tadalafil intermediates from D-tryptophan methyl ester HCl salt and piperonal by refluxing the reagents in isopropyl alcohol; the obtained intermediate is reacted with chloroacetyl chloride and THF, resulting in another intermediate of tadalafil.

Tadalafil is also manufactured and sold under the name of Tadacip by the Indian pharmaceutical company Cipla in doses of 10 mg and 20 mg.

On November 21, 2003 the FDA approved tadalafil (as Cialis) for sale in the United States as the third ED prescription drug pill (after sildenafil citrate(Viagra) and vardenafil (Levitra)). Like sildenafil and vardenafil, tadalafil is recommended as an ‘as needed’ medication. Cialis is the only one of the three that is also offered as a once-daily medication.

Moreover, tadalafil was approved in May 2009 in the United States for the treatment of pulmonary arterial hypertension and is under regulatory review in other regions for this condition. In late November 2008, Eli Lilly sold the exclusive rights to commercialize tadalafil for pulmonary arterial hypertension in the United States to United Therapeutics for an upfront payment of $150 million.

The FDA’s approval of Viagra (Sildenafil) on March 27, 1998 was a ground-breaking commercial event for the treatment of ED, with sales exceedingUS$1 billion. Subsequently, the FDA approved Levitra (vardenafil) on August 19, 2003, and Cialis (tadalafil) on November 21, 2003.

Cialis was discovered by Glaxo Wellcome (now GlaxoSmithKline) under a partnership between Glaxo and ICOS to develop new drugs that began in August 1991. ^[1]^[2] In 1993, the Bothell, Washington biotechnology company ICOS Corporation began studying compound IC351, a phosphodiesterase type 5 (PDE5) enzyme inhibitor. In 1994, Pfizer scientists discovered that sildenafil, which also inhibits the PDE5 enzyme, caused penile erection in men participating in a clinical study of a heart medicine. Although ICOS scientists were not testing compound IC351 for treating ED, they recognized its potential usefulness for treating that disorder. Soon, in 1994, ICOS received a patent for compound IC351 (structurally unlike sildenafil and vardenafil), and Phase 1 clinical trials began in 1995. In 1997, the Phase 2 clinical studies were initiated for men experiencing ED, then progressed to the Phase 3 trials that supported the drug’s FDA approval. Although Glaxo had an agreement with ICOS to share profits 50/50 for drugs resulting from the partnership, Glaxo let the agreement lapse in 1996 as the drugs developed were not in the company’s core markets.^[3]

In 1998, ICOS Corporation and Eli Lilly and Company formed the Lilly ICOS, LLC, joint venture company to further develop and commercialize tadalafil as a treatment for ED. Two years later, Lilly ICOS, LLC, filed a new drug application with the FDA for compound IC351 (under the tadalafil generic name, and the Cialis brand name). In May 2002, Lilly ICOS reported to the American Urological Association that clinical trial testing demonstrated that tadalafil was effective for up to 36 hours, and one year later, the FDA approved tadalafil. One advantage Cialis has over Viagra and Levitra is its 17.5-hour half-life (thus Cialis is advertised to work for up to 36 hours, after which time there remains approximately 25 percent of the absorbed dose in the body) when compared to the four-hour half–life of sildenafil (Viagra).

In 2007, Eli Lilly and Company bought the ICOS Corporation for $2.3 billion. As a result, Eli Lilly owned Cialis and then closed the ICOS operations, ending the joint venture and firing most of ICOS’s approximately 500 employees, except for 127 employees of the ICOS biologics facility, which subsequently was bought by CMC Biopharmaceuticals A/S (CMC).

Tadalafil Molecule

Persons surnamed “Cialis” objected to Eli Lilly and Company’s so naming the drug, but the company has maintained that the drug’s trade name is unrelated to the surname.^[4]

On October 6, 2011, the U.S. FDA approved tadalafil ^[5] to treat the signs and symptoms of benign prostatic hyperplasia (BPH). BPH is a condition in males in which the prostate gland becomes enlarged, obstructing the free flow of urine. Symptoms may include sudden urges to urinate (urgency), difficulty in starting urination (hesitancy), a weak urine stream, and more frequent urination- especially at night. The FDA has also approved tadalafil for treatment of both BPH and erectile dysfunction (ED) where the two conditions co-exist.

Although available since 2003 in 5, 10, 20 mg dosage, in late 2008/early 2009, the U.S. FDA approved the commercial sale of Cialis in 2.5 mg dosage as a one-a-day treatment for ED. The 2.5 mg dose avoids earlier dispensing restrictions on higher dosages. The price of the 5 mg and 2.5 mg are often similar, so some people score and split the pill.^[6] The manufacturer does not recommend splitting.

Moreover, tadalafil (Adcirca) 40 mg was approved in 2009 in the United States and Europe (and 2010 in Canada and Japan) as a once-daily therapy to improve exercise ability in patients withpulmonary arterial hypertension. In patients with pulmonary arterial hypertension, the pulmonary vascular lumen is decreased as a result of vasoconstriction and vascular remodeling, resulting in increased pulmonary artery pressure and pulmonary vascular resistance. Tadalafil is believed to increase pulmonary artery vasodilation, and inhibit vascular remodeling, thus lowering pulmonary arterial pressure and pulmonary vascular resistance. Right heart failure is the principal consequence of pulmonary arterial hypertension.

On October 6, 2011, the U.S. FDA approved tadalafil [6] to treat the signs and symptoms of benign prostatic hyperplasia (BPH). BPH is a condition in males in which the prostate gland becomes enlarged, obstructing the free flow of urine. Symptoms may include sudden urges to urinate (urgency), difficulty in starting urination (hesitancy), a weak urine stream, and more frequent urination- especially at night. The FDA has also approved tadalafil for treatment of both BPH and erectile dysfunction (ED) where the two conditions co-exist.

Tadalafil has been used in approximately 15,000 men participating in clinical trials, and over eight million men worldwide (primarily in the post-approval/post-marketing setting). The most commonside effects when using tadalafil are headache, stomach discomfort or pain, indigestion, burping, acid reflux. back pain, muscle aches, flushing, and stuffy or runny nose. These side effects reflect the ability of PDE5 inhibition to cause vasodilation (cause blood vessels to widen), and usually go away after a few hours. Back pain and muscle aches can occur 12 to 24 hours after taking the drug, and the symptom usually disappears after 48 hours.
In May 2005, the U.S. Food and Drug Administration found that tadalafil (along with other PDE5 inhibitors) was associated with vision impairment related to NAION (nonarteritic anterior ischemic optic neuropathy) in certain patients taking these drugs in the post-marketing (outside of clinical trials) setting. Most, but not all, of these patients had underlying anatomic or vascular risk factors for development of NAION unrelated to PDE5 use, including: low cup to disc ratio (“crowded disc”), age over 50, diabetes, hypertension, coronary artery disease, hyperlipidemia and smoking. Given the small number of NAION events with PDE5 use (fewer than one in one million), the large number of users of PDE5 inhibitors (millions) and the fact that this event occurs in a similar population to those who do not take these medicines, the FDA concluded that they were not able to draw a cause and effect relationship, given these patients underlying vascular risk factors or anatomical defects. However, the label of all three PDE5 inhibitors was changed to alert clinicians to a possible association.

In October 2007, the FDA announced that the labeling for all PDE5 inhibitors, including tadalafil, requires a more prominent warning of the potential risk of sudden hearing loss as the result of postmarketing reports of deafness associated with use of PDE5 inhibitors.^[7]

Selectivity compared with other PDE5 inhibitors

Tadalafil, sildenafil, and vardenafil all act by inhibiting the PDE5 enzyme. These drugs also inhibit other PDE enzymes. Sildenafil and vardenafil inhibit PDE6, an enzyme found in the eye, more than tadalafil.^[9] Some sildenafil users see a bluish tinge and have a heightened sensitivity to light because of PDE6 inhibition.^[3] Sildenafil and vardenafil also inhibit PDE1 more than tadalafil.^[9]PDE1 is found in the brain, heart, and vascular smooth muscle.^[9] It is thought that the inhibition of PDE1 by sildenafil and vardenafil leads to vasodilation, flushing, and tachycardia.^[9] Tadalafil inhibits PDE11 more than sildenafil or vardenafil.^[9] PDE11 is expressed in skeletal muscle, the prostate, the liver, the kidney, the pituitary gland, and the testes.^[9] The effects on the body of inhibiting PDE11 are not known.^[9]

20 mg Cialis tablet

In the United States, the FDA relaxed rules on prescription drug marketing in 1997, allowing advertisements targeted directly to consumers.^[10] Lilly-ICOS hired the Grey Worldwide Agency in New York, part of the Grey Global Group, to run the Cialis advertising campaign.^[11] Marketers for Cialis has taken advantage of its greater duration compared to its competitors in advertisements for the drug; Stuart Elliot of The New York Times opined: “The continuous presence of women in Cialis ads is a subtle signal that the drug makes it easier for them to set the pace with their men, in contrast to the primarily male-driven imagery for Levitra and Viagra.”^[11] Iconic themes in Cialis ads include couples in bathtubs and the slogan “When the moment is right, will you be ready?”^[11] Cialis ads were unique among the ED drugs in mentioning specifics of the drug.^[12] As a result, Cialis ads were also the first to describe the side effects in an advertisement, as the FDA requires advertisements with specifics to mention side effects. One of the first Cialis ads aired at the 2004 Super Bowl.^[12] Just weeks before the Super Bowl, the FDA required more possible side effects to be listed in the advertisement, including priapism.^[12] Although many parents objected to the Cialis ad being aired during the Super Bowl, Janet Jackson‘s halftime “wardrobe malfunction” overshadowed Cialis.^[12] In January 2006, the Cialis ads were tweaked, adding a doctor on screen to describe side effects and only running ads where more than 90 percent of the audience are adults, effectively ending Super Bowl ads.^[10] In 2004, Lilly-ICOS, Pfizer, and GlaxoSmithKline spent a combined $373.1 million to advertise Cialis, Viagra, and Levitra respectively.^[12] Cialis has sponsored many golf events, including the America’s Cup and the PGA Tour, once being title sponsor of the PGA Tour Western Open tournament.^[13]

CIALIS (tadalafil) is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5). Tadalafil has the empirical formula C22H19N3O4 representing a molecular weight of 389.41. The structural formula is:

CIALIS (tadalafil) Structural Formula Illustration

The chemical designation is pyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione, 6-(1,3-benzodioxol-5-yl)2,3,6,7,12,12a-hexahydro-2-methyl-, (6R,12aR)-. It is a crystalline solid that is practically insoluble in water and very slightly soluble in ethanol.

CIALIS is available as almond-shaped tablets for oral administration. Each tablet contains 2.5, 5, 10, or 20 mg of tadalafil and the following inactive ingredients: croscarmellose sodium, hydroxypropyl cellulose, hypromellose, iron oxide, lactose monohydrate, magnesium stearate, microcrystalline cellulose, sodium lauryl sulfate, talc, titanium dioxide, and triacetin.

Tadalafil, (6R-trans)-6-(l,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2- methyl-pyrazino[r,2′:l,6]pyrido[3,4-b]indole-l,4-dione, with the structural formula shown below, is a white crystalline powder. (CAS# 171596-29-5). Tadalafil is a potent and selective inhibitor of the cyclic guanosine monophosphate (cGMP) – specific phosphodiesterase enzyme, PDE5. The inhibition of PDE5 increases the amount of cGMP, resulting in smooth muscle relaxation and increased blood flow. Tadalafil is therefore currently used in the treatment of male erectile dysfunction, and is commercially available as CIALIS ^®.

Tadalafil U.S. Patent No. 5,859,006 describes the synthesis of tadalafil via the cyclization of TDCL (i.e., cis-methyl l,2,3,4-tetrahydro-2-chloroacetyl-l-(3,4- methylenedioxyphenyl)-9H-pyrido[3,4-b]mdole-3-carboxylate) using methylamine by purification by flash chromatography, followed by subsequent crystallization from methanol. Crude tadalafil typically requires additional purification steps, such as multiple extractions, crystallization, and/or flash chromatography, to remove the impurities present in the compound after synthesis is complete. Such purification processes increase the cost of producing tadalafil. Also, when repeating the US ‘006 process, about 250 volumes of methanol were necessary for the crystallization step

Tadalafil can be prepared via a series of intermediates. One synthesis for preparing tadalafil is illustrated below in Scheme I:

SCHEME I

U.S. Patent No. 5,859,006 discloses the synthesis of a tadalafil intermediate

(Compound III) from D-tryptophan methyl ester (Compound II) and piperonal (Compound

I) using trifluoroacetic acid and dichloromethane, a halogenated solvent. Compound III is then reacted with chloroacetyl chloride (Compound IV) and chloroform to provide another intermediate of tadalafil (Compound V).

WO 2004/011463 discloses a process of preparing tadalafil intermediates from D-tryptophan methyl ester HCl salt and piperonal by refluxing the reagents in isopropyl alcohol, reacting the intermediate thus obtained with chloroacetyl chloride and tetrahydrofuran (THF) to provide another intermediate of tadalafil.

WO 2006/110893 discloses a process for the preparation of methyl ester intermediate (Compound III), and tadalafil using the methyl ester intermediate (CompoundII).

U.S. Patent Application Publication No. 2006/0258865 Al discloses a synthesis of the tadalafil intermediate (Compound III) from D-tryptophan methyl ester

(Compound II) and piperonal (Compound I) using a dehydrating agent selected from Na₂SO₄, K₂SO₄, MgSO₄, CaSO₄, CaCl_2, molecular sieve or mixtures thereof and a high boiling solvent such as N,N-Dimethyl acetamide. Compound III is then reacted with chloroacetyl chloride (Compound IV) in the presence of a base such as NaHCO₃ and an organic solvent such as dichloromethane, providing another intermediate of tadalafil (Compound V), which is further reacted with aqueous methyl amine solution to provide tadalafil.

………………………………………….

EP2004644A1

WO2007110734A1

Scheme II and III.

……………………………………………………………………………………………………a compound of .Formula I

SCHEME III

SCHEME IV

Compound – 1 Compound – II

EXAMPLE l

The reaction scheme of this example is generally shown below in SchemeIV.

SCHEME IV

Compound – 1 Compound – II

Into a clean dry glass flask charged with ethanol (250 ml) under a nitrogen atmosphere was added Compound 1 (25 g) under stirring. The reaction mass was cooled to 0 to 5⁰C and monomethylamine gas was purged into the reaction mixture for about 2 hours while maintaining the temperature between 0 to 5⁰C. The temperature was raised to 75 to 😯⁰C and the reaction mixture was stirred under reflux for 2 hours. The reaction mixture was then cooled to 0 to 5°C and monomethylamine gas was again purged into the reaction mixture at 0 to 5°C. The temperature was again raised to 75 to 80⁰C and stirred for about 1 hour. The reaction mixture was concentrated under vacuum to about 1/3 its original volume, cooled to 5 to 1O⁰C and stirred for 1 hour at this temperature. The solids were filtered and washed with chilled ethanol (50 ml). The wet solids were dried under vacuum for 6 hours.

Yield: 25g; Mp: 202-206.7⁰C

Specific rotation (25°C) :+44.0 ( C=l% in DMSO)

¹³C NMR, DMSO-D6 : 25.78, 25.92, 57.89, 57.98, 101.17, 108.09, 108.32,

109.08, 111.48, 117.82, 118.62, 122.23, 122.97, 126.97, 135.97, 136.22, 136.55, 146.99,

147.48, 173.13

¹H NMR, DMSO-D6, 300 MHz, Delta values: 2.6(m,lH), 2.7(m,3H),

2.8(d,lH), 3.0(d,lH), 3.6(bs,lH), 5.1(m,lH), 6.0(s,3H), 6.9-7.1(m, 5H), 7.2(d,lH),

7.4(d,lH), 7.8(bs, IH), 10.3(s, IH)

EXAMPLE 2

The reaction scheme of this example is generally shown below in SchemeV.

SCHEME V

Formula III Formula II

Into a clean dry flask charged with dichloromethane (200 ml) was added

Compound II (25 g) obtained in Example 1 under stirring at 25 to 30⁰C. Next, triethylamine (16.11 g) was added to the reaction mixture and stirred for 30 minutes at 20 to 30⁰C. The reaction mixture was cooled to 0 to 5°C and a solution of chloroacetyl chloride (12.93 g) in chloroform (50 ml) was added to the reaction mixture while maintaining temperature between -5 to 5⁰C. The reaction mixture was stirred at -5 to 5°C for about 2 hours. Saturated aqueous sodium bicarbonate solution (50 ml) was added to the reaction mass slowly and the temperature of the reaction mixture was raised to 25 to 30⁰C. The lower organic layer was separated and washed twice with water (75 ml). The chloroform extract was dried over anhydrous sodium sulfate. The organic layer was concentrated under vacuum until a thick yellow slurry was obtained. The slurry was cooled to 0 to5°C. The solids obtained were filtered and washed with 50 ml chilled chloroform. The wet product was dried at 75⁰C under vacuum for 6 hours.

Yield: 22.5 g; HPLC Purity: 97%; Mp: 180-182⁰C

Specific rotation(25°C): -154.3(C=1% in DMSO)

¹³C. NMR(DMSO-Do, 300 MHZ)= 21.11, 25.88, 44.207, 51.60, 53.95,

101.16,107.66 109.56, 111.38, 118.36, 118.75, 121.58,122.74, 126.30, 130.31, 134.13,

136.57, 146.66, 147.03,167.43, 168.45

¹H. NMR (CDC13, 300 MHZ):2.4(bs,3H), 3.1(m,lH), 3.8(m,lH),

4.3(bs,2H), 4.9(m,lH), 5.4(m,lH), 5.9(s,2H), 6.6-6.8(m,3H), 6.9(bs,lH), 7.1-7.3(m,3H),

7.6(d, IH), 7.7(bs,lH)

¹H. NMR (DMSO-D6, 300 MHZ): 2.0 (bs,3H), 2.9(m,lH), 3.4(m,lH),

4.5(m,lH), 4.8(m,lH), 4.9(m,lH), 6.0(m,2H), 6.4-6.8(m,4H), 6.9-7.2(m,2H), 7.3(d, IH),

7.4(bs,lH), 7.5(d,lH), 10.8(s,lH)

EXAMPLE 3

The reaction scheme of this example is generally shown below in SchemeVI.

SCHEME VI

Formula II Formula I

Into a clean dry round bottom (RB) flask was charged tetrahydrofuran

(THF) (175 ml) under a nitrogen blanket and then cooled to -35 to -40⁰C. Next 92 ml n- butyllithium (1.6 m solution in hexane) was added while maintaining the temperature between -35 to -40⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -40⁰C for 15 minutes. A solution of compound of formula II (22.5 g) obtained in Example 2 in THF (75 ml) was prepared and slowly added to the reaction mixture while maintaining the temperature between -35 to -40⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -40⁰C for 2.5 hours. Saturated aqueous ammonium chloride solution (25 ml) and 50 ml ethyl acetate was added to the reaction mixture at -35 to -40⁰C. The temperature was raised to 25 to 30⁰C and the two layers formed were separated. The upper organic layer was collected. The lower aqueous layer was thrice extracted with ethyl acetate (25 ml). The organic layers were combined together and washed with water (50 ml). The organic extract was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain crude tadalafil as a dark brown solid. [0058] Yield: 22 g; HPLC Purity: 50%.

EXAMPLE 4

Purification of crude tadalafil

The crude tadalafil (22 g) obtained in Example 3 was suspended in 110 ml methanol and stirred for 1 hour at 25 to 30⁰C. The solids obtained were filtered and washed with 25 ml chilled methanol. The wet product was dried at 60⁰C under vacuum for 6 hours. This was further purified by using isopropyl alcohol. Yield: 9 g; HPLC Purity: >99.5%.

EXAMPLE 5

The reaction scheme of this example is generally shown below in SchemeVII.

Scheme VII

Formula VI where R = -OCH3 Formula VIA [0062] Into a clean dry RB flask charged with methanol (1900 ml) was added D- tryptophan methyl ester (190 g) under stirring at 25 to 30⁰C. The reaction mixture was cooled to 0 to 5⁰C. Monomethylamine gas was purged into the reaction mixture at 0 to 5°C for about 5-7 hours under stirring. The temperature of the reaction mixture was slowly raised to about 25 to 3O⁰C and stirred at this temperature for 5-7 hours. The reaction mixture was concentrated under vacuum to distill out the solvent. Diisopropyl ether (950 ml) was added and cooled to 25 to 3O⁰C under stirring for 1-2 hrs. The solids obtained were filtered, washed with Diisopropyl ether and dried under vacuum. [0063] MP: 122.4-124⁰C; Yield: 150 g (78.9 % w/w).

Specific rotation(25°C): +12.5 (C=I % in DMSO)

13 C NMR (300 MHZ,DMSO-D6): 25.71, 31.40, 55.67, 110.93, 111.55,

118.42, 118.73, 121.09, 123.95, 127.66,136.44, 175.39.

¹H NMR (300 MHZ,DMSO-D6): 1.6(bs,2H), 2.5(m,3H), 2.8(m,lH),

3.1(m,lH), 3.4(m, IH), 6.9-7.2(m,3H), 7.3(d,lH), 7.5(d,lH), 7.8(bs,lH), 10.8(bs,lH)

EXAMPLE 6

The reaction scheme of this example is generally shown below in Scheme VIII.

SCHEME VIII

Formula VIA Formula VII

Into a clean, dry flask charged with methylene dichloride (MDC) (1000 ml) was added D-tryptophan methyl amide, the compound of Formula VIA (50 g), and piperonal, the compound of Formula VII (31.09 g), under stirring at 25 to 30⁰C. The reaction mixture was cooled to 0 to 5°Cunder nitrogen atmosphere. Trifluoroacetic acid (85.3 g) was dissolved in MDC (250 ml) and the solution was slowly added to the reaction mixture at 0 to 5°C. The temperature of the reaction mixture was raised to 20 to 30⁰C and stirred at this temperature for 14-16 hours. The reaction was monitored by TLC, workup was done as follows, the pH of the reaction mixture was adjusted to 8-9 using sodium carbonate solution under stirring, the two layers were settled, separated and the lower MDC layer was washed with water. The MDC layer was then dried over anhydrous sodium sulfate. The reaction mass was concentrated under vacuum at 40 to 5O⁰C to remove the solvent. The compound was precipitated using ethyl acetate, the solids were filtered, washed with ethyl acetate and dried.

Yield: 52.5 g; Yield: 105% w/w, HPLC Purity: 71% cis and 27% trans isomer (HPLC).

EXAMPLE 7

The reaction scheme of this example is generally shown below in Scheme IX.

SCHEME IX

1]CICOCH2C1 2]crystn

Formula m

Formula H

Into a clean dry flask charge with dichloromethane (400 ml) under a nitrogen atmosphere was added the compound of Formula III obtained in Example 6 and triethylamine (28.96 g) under stirring at 20 to 3O⁰C. The reaction mixture was then cooled to 0 to 5⁰C. A mixture of chloroacetyl chloride (25.85 g) in dichloromethane (100 ml) was prepared and slowly added to the reaction mixture while maintaining the temperature between -5 to 5⁰C in 1-2 hrs. The reaction mixture was stirred at 0 to 5⁰C for 30 min and then saturated sodium bicarbonate solution (100 ml) was added at 5 to 10⁰C under stirring. The temperature of the reaction mixture was raised to 25 to 30⁰C and stirred at this temperature for 15 minutes. The layers were then separated. The lower MDC layer was collected, washed twice with 100 ml water and dried over anhydrous sodium sulfate. The

MDC layer was concentrated to distill out MDC until a stirrable mass was left behind. The mass was cooled to 25-3O⁰C and filtered, washed, to yield off-white to light yellow colored solids. The resulted product was the cis isomer, the trans isomer left behind in the mother liquor.

Yield = 25.5 g (50%w/w); HPLC Purity: > 97%.

The physical and spectral data was similar to that obtained in Example 2.

EXAMPLE 8

The reaction scheme of this example is generally shown below in SchemeX.

SCHEME X

Formula II

Into a clean dry round bottom (RB) flask was charged THF (1625 ml) under a nitrogen blanket and then cooled to -35 to -40⁰C. Next, 505 ml n-butyllithium (1.6 m solution in hexane) was added while maintaining the temperature between -35 to -4O⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -4O⁰C for 15 minutes. 72 ml diisopropyl amine was then added at -35 to -40⁰C and then stirred at 0-5⁰C for 1 hr. A solution of Compound of formula II (125 g) obtained in Example 7 in THF (625 ml) was prepared and slowly added to the reaction mixture while maintaining the temperature between -40 to -5O⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -40⁰C for 2-6 hours. Saturated aqueous ammonium chloride solution (250 ml) and ethyl acetate (125 ml) was added to the reaction mixture at -35 to -40⁰C. The temperature was raised to 25 to 30⁰C and the two layers formed were separated. The upper organic layer was collected. The lower aqueous layer was extracted with ethyl acetate (65 ml). The organic layers were combined together and distilled. Isopropyl alcohol (1250 ml) was added and the distillation was continued. A mixture of methanol (250 ml) and isopropanol (375 ml) were added and crude tadalafϊl was obtained upon cooling. The crude product was filtered, washed with water and dried. [0076] Yield: 60 g; (48% w/w); HPLC Purity: >99%.

EXAMPLE 9

Purification of crude Tadalafil

The crude tadalafil obtained in Example 8 was suspended in methanol (600 ml) and stirred for 1 hour at reflux. The mixture was cooled and the solids obtained were filtered and washed with chilled methanol (60 ml). The wet product was dried at under vacuum.

Yield: 56 g; HPLC Purity: 99.8%.

……………………………………………………………………………………

Beilstein J. Org. Chem. 2011, 7, 442–495.

http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-7-57#S28

A different approach was used in the synthesis of the phosphodiesterase inhibitor tadalafil (132, Cialis) starting from commercially available (D)-tryptophan methyl ester to form the indolopiperidine motif 135 via a Pictet–Spengler reaction followed by a double condensation to install the additional diketopiperazine ring (Scheme 28) [38,39].

Scheme 28: Optimised Pictet–Spengler reaction towards tadalafil.

To achieve the high levels of cis selectivity required from the Pictet–Spengler reaction, an extensive investigation of solvents and the influence of additives was undertaken [40]. It was identified that the use of a specific 23 mol % of benzoic acid significantly increased the cis/trans ratio from a base level of 55:45 to 92:8 (16 h reaction time at ambient temperature) in an overall yield of 86%. It was also determined that more polar solvents such as acetonitrile and nitromethane preferentially solvated the trans product and thereby allowed the isolation of the ciscompound by precipitation. It was also shown that by heating the reaction mixture under reflux the product distribution could be driven to the thermodynamically more favoured cis isomer having both the ester and the piperonyl moiety in equatorial positions. Hence, after heating under reflux for 8 h the cis/trans ratio was found to be 99:1 and the product could be isolated in an overall yield of 91%. This work represents an impressive example of a well considered and executed process optimisation study.

………………………………

The process disclosed in the patent US 5 859 006 (Scheme 1) involves condensation of D-tryptophan methyl ester with a piperonal derivative to yield a compound of formula (II). After conversion into a thioamide derivative of formula (III), cyclization occurs in presence of both an alkylating and reducing agents to provide a tetrahydro-β-carboline derivative of formula (IV), which on treatment with chloroacetyl chloride and methyl amine, gives Tadalafil. The compound of formula (IV) can also be obtained in one step, after separation of the other diastereoisomer, by a Pictet Spengler reaction between D-tryptophan methyl ester and piperonal in presence of an acid, such as trifluoroacetic acid.

The patent application WO2007/10038 discloses the reaction of D-tryptophan with piperonal to provide a tetrahydro-β-carboline acid that was cyclised to Tadalafil in presence of a sarcosine derivative.
The patent application WO2007/1107 discloses the reaction of D-tryptophan methyl amide with piperonal, to provide an intermediate that after reaction with chloroacetyl chloride cyclises to Tadalafil in presence of butyllithium.
Thus, the active substance prepared by the processes known up till now can only be obtained in a satisfactory quality after running through a large number of process steps. Moreover a toxic alkylating agent, such as methylamine, is often used.

EP2181997A1

Example 1

(1R,3R)-methyl-1,2,3,4-tetrahydro-2-(2-(benzyl(methyl)amino)acetyl)-1-(3,4-methylenedioxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (VII)
A 50 mL three-necked flask fitted with thermometer and reflux condenser was charged with (1R,3R)-methyl 1,2,3,4-tetrahydro-2-chloroacetyl-1-(3,4-methylenedioxyphenyl)-9H-pyrido [3,4-b] indole-3-carboxylate (VI) (1.39 g, 3.26 mmol), DMA (5.33 mL), K₂CO₃ (0.5 g, 3.6 mmol) and N-benzylmethylamine (0.41 mL, 3.26 mmol). The resultant solution was stirred at room temperature. After 2 hours, the mixture was poured in brine (20 mL) and extracted with isopropyl acetate. The combined organic phases were washed with brine (3 x 5 mL), dried over sodium sulfate and concentrated to a residue under reduced pressure, affording 1.5 g of the desired product (VII), as a white solid. Yield: 70%.
¹H NMR (d₆-DMSO 300 MHz, 298K) 2.24 (s, 3H), 2.94-3.00 (m, 5H), 3.44-3.68 (m, 3H), 5.56 (bd, J = 6.4, 1H), 5.95 (s, 1H), 5.96 (s, 1H), 6.55 (bd, J = 7.4, 1H), 6.75 (bs, 1H), 6.77 (d, J = 8.0, 1H), 6.84 (bs, 1H), 7.05 (td, J = 7.4, 0.9, 1H), 7.12 (td, J = 7.5, 1.2, 1H), 7.17-7.32 (m, 6H), 7.56 (d, J = 7.7, 1H), 10.76 (bs, 1H)
¹³C NMR (d₆-DMSO 75.4 MHz, 298K) 21.9, 42.5, 51.3, 51.9, 52.4, 61.0, 61.7, 101.5, 107.0, 108.0, 109.8, 111.8, 118.5, 119.2, 122.0, 123.0, 126.7, 127.7, 128.7, 129.6, 131.1, 134.7, 137.1, 138.6, 147.1, 147.5, 170.6, 171.5

Example 2

(6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino [1′,2′:1,6] pyrido [3,4-b] indole-1,4-dione (Tadalafil) (I)

Under H₂ atmosphere (3 atm) and magnetic stirring, Raney^® Ni (2800 slurry in water, 0.0276 g, 0.47 mmol), previously washed with methanol (3 times), was added to a solution of (1R,3R)-methyl-1,2,3,4-tetrahydro-2-(2-(benzyl(methyl)amino)acetyl)-1-(3,4-methylenedioxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (VII) (3.00 g, 4.70 mmol) in DMA (21.3 mL). The mixture was heated at 90°C for 17 hours and then cooled to room temperature. The suspension was filtered over a pad of Celite^® and the resulting solutionand the resulting solution was concentrated until 6 mL. Methanol (12 mL) was added and the solid which was so obtained was filtered over Buchner, washed with methanol (4 mL) and oven-dried under reduced pressure for 2 hours, affording 1.3 g of the title compound, as a white solid. Yield: 70%
¹H NMR (d₆-DMSO 300 MHz, 298K): 2.91-3.00 (m, 4H), 3.32 (s, 1H), 3.47-3.54 (dd, J = 4.6, 11.3, 1H), 3.93 (d, J = 17.1, 1H), 4.17 (d, J = 17.1, 1H), 4.35-4.40 (dd, J = 4.27, 11.6, 1H), 5.91 (s, 2H), 6.11 (s, 1H), 6.76 (s, 2H), 6.85 (s, 1H), 6.98-7.06 (m, 2H), 7.28 (d, J = 7.9, 1H), 7.52 (d, J = 7.3, 1H), 11.0 (s, 1H)
¹³C NMR (d₆-DMSO 75.4 MHz, 298K) 23.8, 33.4, 52.0, 55.9, 56.1, 101.5, 105.3, 107.6, 108.6, 111.9, 118.7, 119.5, 119.9, 121.8, 126.4, 134.5, 136.8, 137.6, 146.7, 147.6, 167.1, 167.5

References

Daugan, A; Grondin P, Ruault C, Le Monnier de Gouville AC, Coste H, Kirilovsky J, Hyafil F, Labaudinière R (October 9, 2003). “The discovery of tadalafil: a novel and highly selective PDE5 inhibitor. 1: 5,6,11,11a-tetrahydro-1H-imidazo[1′,5′:1,6]pyrido[3,4-b]indole-1,3(2H)-dione analogues”. Journal of Medicinal Chemistry 46 (21): 4525–32. doi:10.1021/jm030056e . PMID 14521414.
Richards, Rhonda (September 17, 1991). “ICOS At A Crest On Roller Coaster”. USA Today. p. 3B.
Ervin, Keith (June 21, 1998). “Deep Pockets + Intense Research + Total Control = The Formula — Bothell Biotech Icos Keeps The Pipeline Full Of Promise”. The Seattle Times. p. F1. Retrieved January 10, 2009.
Revill, Jo (February 2, 2003). “Drugs giant says its new pill will pack more punch than rival Viagra”. The Observer. Retrieved 2007-04-06.
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm274642.htm
https://www.consumerreports.org/health/resources/pdf/best-buy-drugs/money-saving-guides/english/PillSplitting-FINAL.pdf
“FDA Announces Revisions to Labels for Cialis, Levitra and Viagra”. Food and Drug Administration. 2007-10-18. Retrieved 2009-09-28.
“Cialis: Warnings, Precautions, Pregnancy, Nursing, Abuse”. RxList. 2007. Retrieved 2007-04-06.
Bischoff, E (June 2004). “Potency, selectivity, and consequences of nonselectivity of PDE inhibition”. International Journal of Impotence Research 16: S11–4.doi:10.1038/sj.ijir.3901208 . PMID 15224129. Retrieved January 19, 2009.
Elliott, Stuart (January 10, 2006). “For Impotence Drugs, Less Wink-Wink”. The New York Times. p. C2. Retrieved January 15, 2009.
Elliott, Stuart (April 25, 2004). “Viagra and the Battle of the Awkward Ads”. The New York Times. p. 1. Retrieved January 15, 2009.
McCarthy, Shawn (March 5, 2005). “First they tried to play it safe; Ads for erectile dysfunction drug Cialis bared all – including a scary potential side effect. It was risky but it has paid off”. The Globe and Mail. p. B4.
Loyd, Linda (July 6, 2003). “Two Pills Look to Topple Viagra’s Reign in Market; Levitra Expects Approval Next Month, Cialis Later This Year”. The Philadelphia Inquirer. p. E01.
38 is 1 below
39 is 2 below
40 is 3 below
1. daugan, A. C.-M. Tetracyclic Derivatives; Process of Preparation and Use. U.S. Patent 5,859,006, Jan 12, 1999.
2. Daugan, A. C.-M. Tetracyclic Derivatives, Process of Preparation and Use. U.S. Patent 6,025,494, Feb 15, 2000.
3. Shi, X.-X.; Liu, S.-L.; Xu, W.; Xu, Y.-L. Tetrahedron: Asymmetry 2008, 19, 435–442.doi:10.1016/j.tetasy.2007.12.017

DAUGAN A ET AL: “THE DISCOVERY OF TADALAFIL: A NOVEL AND HIGHLY SELECTIVE PDE5 INHIBITOR. 2: 2,3,6,7,12,12A-HEXAHYDROPYRAZINO[1′,2′:1,6 ÜPYRIDO[3,4-B ÜINDOLE-1,4-DIONE” JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 46, no. 21, 2003, pages 4533-4542, XP008052656 ISSN: 0022-2623

13 C NMR OF TADALAFIL

COSY NMR OF TADALAFIL

DEPT NMR OF TADALAFIL

HSQC NMR OF TADALAFIL

HMBC NMR OF TADALAFIL

MASS SPECTRUM OF TADALAFIL

UV OF TADALAFIL

RAMAN SPEC OF TADALAFIL

WO2009004557A2 *	Jun 28, 2008	Jan 8, 2009	Ranbaxy Lab Ltd	A process for the preparation of intermediates of tetracyclic compounds
WO2009148341A1	Jun 3, 2009	Dec 10, 2009	Zaklady Farmaceutyczne Polpharma Sa	Process for preparation of tadalafil
WO2012107549A1	Feb 10, 2012	Aug 16, 2012	Interquim, S.A.	PROCESS FOR OBTAINING COMPOUNDS DERIVED FROM TETRAHYDRO-ß-CARBOLINE
EP2107059A1	Mar 31, 2008	Oct 7, 2009	LEK Pharmaceuticals D.D.	Conversion of tryptophan into ß-carboline derivatives
US8445698	Jun 28, 2008	May 21, 2013	Ranbaxy Laboratories Limited	Process for the preparation of an intermediate of tadalafil

Temozolomide 替莫唑胺

GENERIC Comments Off

Feb 152014

Temozolomide 替莫唑胺

Temozolomide is a DNA damage inducer.

4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide

3,4-dihydro-3-methyl-4-oxoimidazo(5,1-d)-1,2,3,5-tetrazine-8-carboxamide

Methazolastone, Temodar, Temodal

CAS NO 85622-93-1

Molecular Weight: 194.15

MF C₆H₆N₆O₂

Cancer Research UK (Originator), Schering-Plough (Licensee), National Cancer Institute (Codevelopment)

NMR..http://file.selleckchem.com/downloads/nmr/S123702-Methazolastone-NMR-Selleck.pdf

HPLC.http://file.selleckchem.com/downloads/hplc/S123702-Methazolastone-HPLC-Selleck.pdf

Temozolomide is an antitumor agent indicated for treating patients with malignant glioma such as cancer, breast cancer, refractory anaplastic astrocytoma, i.e., patients at first relapse who have experienced disease progression in malignant glioma, glioblastoma multiform and anaplastic astrocytoma, on a drug regimen containing a nitrosourea and procarbazine.

Temozolomide preparations are sold on the US market as hard capsules containing 5 mg, 20 mg, 100 mg or 250 mg Temozolomide (marketed as Temodar® by Schering Corporation, Kenilworth, N.J., USA). In other markets it is sold as Temodal®.

Temozolomide (brand names Temodar and Temodal and Temcad) is an oral chemotherapy drug. It is an alkylating agent used for the treatment of Grade IV astrocytoma — an aggressive brain tumor, also known as glioblastoma multiforme — as well as for treating melanoma, a form of skin cancer.

Temozolomide is also indicated for relapsed Grade III anaplastic astrocytoma and not indicated for, but as of 2011 used to treatoligodendroglioma brain tumors in some countries, replacing the older (and less well tolerated) PCV (Procarbazine–Lomustine–Vincristine) regimen.

Temozolomide, 3-methyl-8-aminocarbonyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one, is a known antitumor drug; see for example Stevens et al., J. Med. Chem. 1984, 27, 196-201, and Wang et al., J. Chem. Soc., Chem. Commun.,1994,1687-1688. Temozolomide, the compound of formula 1:

is described in U.S. Pat. No. 5,260,291 (Lunt et al.).

The synthesis of 1 by the process described in J. Med. Chem. 1984, 27, 196-201 is depicted in the scheme I below.

In this process, 5-amino-1H-imidazole-4-carboxamide (A) is converted into 5-diazo-1H-imidazole-4-carboxamide (B), which is then cyclized with methylisocyanate in dichloromethane to provide a high yield of temozolomide. However, this process requires isolation of the unstable and potentially dangerous 5-diazo-1H-imidazole-4-carboxamide (B). Moreover, methylisocyanate is a difficult reagent to handle and ship, especially on the industrial scale, and indeed is better avoided in industrial manufacture. Furthermore, the cycloaddition of methylisocyanate requires a very long reaction time: Table I in J. Med Chem.1984, 27,196-201, suggests 20 days. Additionally, Stevens et al mention that the cycloaddition of the methylisocyanate to the compound of the formula (B) can proceed through two different intermediates:

The production of I by the two processes described in J. Chem. Soc., Chem. Commun., 1994, 1687-1688 provides a low overall yield from 5-amino-1H-imidazole-4-carboxamide (A): less than 20% (unoptimized—about 17% through 5-diazo-1H-imidazole-4-carboxamide (B) and about 15% through 5-amino-N¹-(ethoxycarbonylmethyl)-1H-imidazole-1,4-dicarboxamide (C)); Scheme II below

The agent was developed by Malcolm Stevens^[1] and his team at Aston University in Birmingham,^[2]^[3] Temozolomide is a prodrug and animidazotetrazine derivative of the alkylating agent dacarbazine. It has been available in the US since August 1999, and in other countries since the early 2000s.

The therapeutic benefit of temozolomide depends on its ability to alkylate/methylate DNA, which most often occurs at the N-7 or O-6 positions ofguanine residues. This methylation damages the DNA and triggers the death of tumor cells. However, some tumor cells are able to repair this type of DNA damage, and therefore diminish the therapeutic efficacy of temozolomide, by expressing a protein O⁶-alkylguanine DNA alkyltransferase (AGT) encoded in humans by the O-6-methylguanine-DNA methyltransferase (MGMT) gene.^[4] In some tumors, epigenetic silencing of the MGMT gene prevents the synthesis of this enzyme, and as a consequence such tumors are more sensitive to killing by temozolomide.^[5] Conversely, the presence of AGT protein in brain tumors predicts poor response to temozolomide and these patients receive little benefit from chemotherapy with temozolomide.^[6]

Nitrosourea- and procarbazine-refractory anaplastic astrocytoma
Newly diagnosed glioblastoma multiforme
Malignant prolactinoma

Temozolomide (sometimes referred to as TMZ) is an imidazotetrazine derivative of the alkylating agent dacarbazine. It undergoes rapid chemical conversion in the systemic circulation at physiological pH to the active compound, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC). Temozolomide exhibits schedule-dependent antineoplastic activity by interfering with DNA replication. Temozolomide has demonstrated activity against recurrent glioma. In a recent randomized trial, concomitant and adjuvant temozolomide chemotherapy with radiation significantly improves, from 12.1 months to 14.6 months, progression free survival and overall survival in glioblastoma multiforme patients.

Formulations

Temozolomide is available in the United States in 5 mg, 20 mg, 100 mg, 140 mg, 180 mg & 250 mg capsules. Now also available in an IV form for people who can not swallow capsules or who have insurance that does not cover oral cancer agents.

A generic version is available in the UK.

Further improvement of anticancer potency

Laboratory studies and clinical trials are investigating whether it might be possible to further increase the anticancer potency of temozolomide by combining it with other pharmacologic agents. For example, clinical trials have indicated that the addition of chloroquine might be beneficial for the treatment of glioma patients.^[8] In laboratory studies, it was found that temozolomide killed brain tumor cells more efficiently when epigallocatechin gallate (EGCG), a component of green tea, was added; however, the efficacy of this effect has not yet been confirmed in brain tumor patients.^[9]More recently, use of the novel oxygen diffusion-enhancing compound trans sodium crocetinate (TSC) when combined with temozolomide and radiation therapy has been investigated in preclinical studies ^[10] and a clinical trial is currently underway.^[11]

Because tumor cells that express the MGMT gene are more resistant to killing by temozolomide, it was investigated^{[according to whom?]} whether the inclusion of [[O⁶-benzylguanine]] (O⁶-BG), an AGT inhibitor, would be able to overcome this resistance and improve the drug’s therapeutic effectiveness. In the laboratory, this combination indeed showed increased temozolomide activity in tumor cell culture in vitro and in animal models in vivo.^[12] However, a recently completed phase-II clinical trial with brain tumor patients yielded mixed outcomes; while there was some improved therapeutic activity when O⁶-BG and temozolomide were given to patients with temozolomide-resistant anaplastic glioma, there seemed to be no significant restoration of temozolomide sensitivity in patients with temozolomide-resistant glioblastoma multiforme.^[13]

There are also efforts to engineer hematopoietic stem cells expressing the MGMT gene prior to transplanting them into brain tumor patients. This would allow for the patients to receive stronger doses of temozolomide, since the patient’s hematopoietic cells would be resistant to the drug.^[14]

High doses of temozolomide in high grade gliomas have low toxicity, but the results are comparable to the standard doses.^[15]

A case report suggests that temozolomide may be of use in relapsed primary CNS lymphoma.^[16] Confirmation of this possible use seems indicated.

Temozolomide, 3-methyl-8-aminocarbonyl-imidazo[5,1-d]- 1 ,2,3,5-tetrazin- 4(3H)-one, is a known antitumor drug; see for example Stevens et al., J. Med. Chem. 1984, 27, 196-201 , and Wang et al., J. Chem. Soc, Chem. Commυn., 1994, 1687-1688. Temozolomide, the compound of formula 1 :

1 is described in U.S. Patent No. 5,260,291 (Lunt et al.).

The synthesis of 1 by the process described in J. Med. Chem. 1984, 27, 196- 201 is depicted in the scheme I below. Scheme I:

However, this process requires isolation of the unstable and potentially dangerous 5-diazo-1 H-imidazole-4-carboxamide (B). Moreover, methylisocyanate is a difficult reagent to handle and ship, especially on the industrial scale, and indeed is better avoided in industrial manufacture.

Furthermore, the cycloaddition of methylisocyanate requires a very long reaction time: Table I in J. Med Chem. 1984, 27,196-201 , suggests 20 days. Additionally, Stevens et al mention that the cycloaddition of the methylisocyanate to the compound of the formula (B) can proceed through two different intermediates:

The production of I by the two processes described in J. Chem. Soc, Chem.

Commun., 1994, 1687-1688 provides a low overall yield from 5-amino-1 H- imidazole-4-carboxamide (A): less than 20% (unoptimized – about 17% through 5- diazo-1 H-imidazole-4-carboxamide (B) and about 15% through 5-amino-N¹– (ethoxycarbonylmethyl)- 1 H-imidazole- 1 ,4-dicarboxamide (C)); Scheme II below

Scheme II:

Moreover, the unstable 5-diazo-1 H-imidazole-4-carboxamide (B) still has to be isolated in the branch of this process that uses it as an intermediate. Clearly, therefore, there is a need for synthetic methods that: a) are more convenient and higher yielding, especially on commercial scale; b) approach the synthesis of the temozolomide nucleus in novel ways; or c) improve the preparation or use of intermediates for the processes.

Temozolomide of formula I, is an antitumor drag and is chemically known as 3-methyl-8- aminocarbonyl-imidazole[5,l-d]-l,2,3,5-tetrazin-4(3H)-one.

Formula I

It is indicated for treating patients with malignant glioma such as cancer, breast cancer, refractory anaplastic, astrocytoma, i.e. patient at first relapse who have experienced disease progression in malignant glioma, glioblastoma multiform and anaplastic astrocytoma, on a drug containing a nitrosourea and procarbazine. It is sold in the US market as hard capsules containing 5 mg, 20 mg, 100 mg or 250 mg as Temodar® by Schering corporation.

Temozolomide and compounds having similar activity (higher alkyl analogues at the 3 -position) were first disclosed in US patent 5,260,291. According to said patent, temozolomide is prepared by the reaction of 5-diazoimidazole-4-carboxamide with methyl isocyanate in the presence of N- methylpyrrolid-2-one in dichloromethane at room temperature for three to four weeks. Melting point of temozolomide reported in above patent is 200 ⁰C (recrystallized from acetonitrile); 21O⁰C with effervescence (recrystallized from acetone and water), and 215⁰C with effervescence and darkening (recrystallized from hot water). Major drawback of process is the longer reaction duration of three to four weeks for completion of reaction.

Further, the process described in the patent involves use of low boiling and extremely toxic, methyl isocyanate, which is very difficult to handle, especially on industrial scale, as its use should be avoided in the industrial synthesis. Further, cycloaddition reaction requires a very long period of 21 to 28 days, which makes the process unattractive for industrial scale.

US patent 5,003,099 discloses a process for preparation of aminocyanoacetamide, a key intermediate for the synthesis of temozolomide. According to the patent, aminocyanoacetamide is synthesized in two steps by the reaction of cyanoacetic acid alkyl ester using sodium nitrite in the presence of glacial acetic acid to form a hydroxyimino intermediate, which is then reduced in the presence of platinum on carbon to yield aminocyanoacetic acid alkyl ester, which is unstable.

The alkyl ester intermediate is then in situ reacted with aqueous ammonia to give the desired product. The main drawback of the above mentioned process is the use of aqueous ammonia, since aminocyanoacetamide, generated in reaction, is soluble in aqueous solution and hence difficult to extract from the reaction mass which results in lower yields. The patent is silent about the purity of intermediate and process needs extraction of the above mentioned intermediate from filtrate.

US patent 6,844,434 describes synthesis of temozolomide by cyclization of 5-amino-l-(N-rnethyl- hydrazinocarbonyl)-lH-imidazole-4-carboxylic acid in the presence of tetrabutyl nickel and periodic acid to form a reaction mixture which is concentrated under reduce pressure and resulting residue was treated with acetonitrile and filtered. The filtrate was concentrated and chromatographed on a column of silica gel to give temozolomide.

Use of time consuming and cumbersome technique i.e. column chromatography for isolation of product makes the process not suitable to employ at industrial level. US patent 7,087,751 discloses a process for the preparation of temozolomide from protected imidazole intermediate.

The process involves reaction of l-methyl-3-carbamoyliminomethyl-urea with JV- protected aminocyanoacetamide in the presence of acetic acid in a suitable solvent to form an JV- protected imidazole intermediate which is then cyclized in the presence of lithium chloride to minimize undesired cyclisation product. After cyclisation, the protected group has to be removed which makes the process more laborious with more number of steps.

As exemplified in example 1 of the above patent, yield of the JV-protected imidazole intermediate obtained is very low, almost half of the product goes in the filtrate which further needs extraction from the filtrate. After extraction of inteπnediate from the filtrate, the combined yield is only 67 %. The intermediate obtained is only 93 to 94% pure and requires additional purifications, crystallization using ethyl acetate and slurry wash with mixture of methyl tertiary butyl ether and isopropanol. These additional purification further takes away around 20 % yield of the inteπnediate thus yield of the pure intermediate, which is suitable for the further reaction, remains around 53 % which is very low from commercial point of view.

The patent also describes condensation of l-methyl-3-carbamoyliminomethyl-urea with unprotected aminocyanoacetamide in presence of acetic acid to give an imidazole intermediate. This patent fails to disclose the process of conversion of above imidazole intermediate to temozolomide, but only up to hydrolysis to prepare 5-amino-lH-imidazole-4-carboxamide hydrochloride is reported.

Another US patent no. 6,844,434 of same applicant (Schering) discloses a process for the conversion of 5-amino- lH-imidazole-4-carboxamide hydrochloride, which is prepared by the hydrolysis of above imidazole intermediate, to temozolomide. By combining the above two processes, this adds further four additional steps to the synthesis of temozolomide. The process of preparation of temozolomide is described by the following scheme:

It has been observed that for the preparation of unprotected imidazole intermediate as exemplified in US 7,087,751, use of excess amount of the acetic acid (around 21 times with respect to aminocyanoacetamide) is reported. Thereafter acetic acid is removed by distillation.

The inventors of the present invention have repeated example 2 as described in US 7,087,751 for the preparation of unprotected imidazole intermediate. As per the process, after the completion of the reaction, acetic acid has to be removed from the reaction mixture. It is noticed that removal of acetic acid is a very tedious move so as on commercial scale and leads to decomposition.

In a publication namely, Journal of Organic Chemistry, volume 62, no. 21, 7288-7294, a process is disclosed for the preparation of temozolomide by the hydrolysis of 8-cyano-3-methyl-[3H]-imidazole~ [5,l-d]-tetrazin-4-one in the presence of hydrochloric acid to give hydrochloride salt of temozolomide, which has to be neutralized to obtain temozolomide. In the same Journal, another process for the preparation of temozolomide is also described. Temozolomide is prepared by the nitrosative cyclization of imidazole intermediate using aqueous solution of sodium nitrite and tartaric acid to give temozolomide in 45 % yield in solution.

US patent publication 2007/0225496 exemplified a process for preparation of temozolomide by pyrolising N’-methyl-N,N-diphenyl urea to form vapor of methyl isocyanate which is then reacted with 5-diazo-5H-imidazole-4-carboxylic acid amide to form temozolomide.

The above described process involves use of methyl isocyanate, which is highly flammable and makes the process unsuitable for industrial synthesis, hi addition to this, isolation of temozolomide from the reaction mixture requires addition of large amount of ethyl acetate followed by addition of hexane and again ethyl acetate to isolate compound.

US patent publication 2009/0326028 describes a process for preparation of temozolomide by diazotization of imidazole intermediate in the presence of at least one metal halide, a source of nitrous acid and an acid to form acidic solution of temozolomide, wherein temozolomide forms a salt with acid. The desired product i.e. temozolomide is then isolated from the acidic solution by extraction with a solvent.

The process requires very strict reaction parameters including the addition of metal halide during diazotization as well as addition of pre-cooled reaction mixture to sodium nitrite solution to achieve desired level of selective cyclization. Patent application also describes two methods for the extraction of temozolomide.

US patent publication 2010/0036121 discloses a process for the preparation of temozolomide by reaction of 5-aminoimidazole-4-carboxamide with N-succinimidyl-N’-methylcarbamate to form carbamoyl 5~aminoimidazole-4-carboxamide which is then reacted with alkali or alkaline earth nitrile to give reaction mass containing temozolomide

Temozolomide, is a known antitumour drug, and is represented by formula I:

3-methyl-8-aminocarbonyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one
It is described in US 5,260,291 together with compounds of broadly similar activity such as higher alkyl analogs at the 3-position.
J.Med.Chem. 1984, 27, 196-201 describes a process wherein 5-amino-1H-imidazole-4-carboxamide is converted into 5-diazo-1H-imidazole-4-carboxamide, which is then cyclised with methylisocyanate in dichloromethane to provide a high yield of temozolomide.
This process requires isolation of the unstable and potentially dangerous 5-diazo-1H-imidazole-4-carboxamide, methyl isocyanate is a difficult reagent to handle and ship, especially on the industrial scale. Furthermore, the cycloaddition of methylisocyanate requires a long reaction time (Table I in J.Med.Chem. 1984, 27, 196-201, suggests 20 days).
The product obtained by this process contains, high residual dichloromethane. It is essential to limit dichloromethane content in the final API below 600 ppm as per ICH guideline. Dichloromethane content can be reduced if one follows technique of US 5,260,291 .
US 5,260,291 discloses acetone-water recrystallisation of temozolomide, which results in low yield (60% recovery) due to decomposition of temozolomide to impurities like 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, compound of formula V

and 5-amino-1H-imidazole-4-carboxamide.
The production of compound of formula I by the two processes described in J.Chem.Soc., Chem.Commun., 1994, 1687-1688 provides a low overall yield from 5-amino-1H-imidazole-4-carboxamide: less than 20% (about 17% through 5-diazo-1H-imidazole-4-carboxamide and about 15% through 5-amino-N¹-(ethoxy carbonylmethyl)-1H-imidazole-1,4-dicarboxamide).
The unstable 5-diazo-1H-imidazole-4-carboxamide has to be isolated in the branch of this process that uses it as an intermediate.
US 2002/0133006 discloses a process for the preparation of compound of formula I using methyl hydrazine which is a toxic and flammable liquid, hence not feasible on industrial scale and the final isolation involves tedious workup including column chromatography.
J.Org.Chem. 1997, 62, 7288-7294 describes a process wherein the final step of diazotization provides equi-formation of aza-hypoxanthine and temozolomide, resulting in low yield. This literature does not provide the experimental procedure for work up.
US 2005/0131227 describes a process involving the use of a bulky protecting group on nitrogen of the primary amide for cyclisation in presence of LiCl to minimize the undesired cyclization product. After cyclization the protecting group has to be removed which makes the process more laborious with more number of steps (Scheme I).

U.S. Pat. No. 6,844,434 describes the preparation of Temozolomide, alkyl analogs and intermediates thereof. The process, which is depicted in Scheme 3 below, comprises reacting 5-amino-1H-imidazole-4-carboxamide hydrochloride (II) with 4-nitrophenyl chloroformate to afford compound (III), which is subsequently reacted with methyl hydrazine to obtain the corresponding compound (IV), which is cyclized to yield Temozolomide.

Another process for preparing Temozolomide is described in U.S. patent application having the Publication No. 2002/0095036 (see Scheme 4 below). In this process, the imine (V) is converted to 2-cyano-N-(1,1-dimethylethyl)-2-[(diphenyl-methylene)amino]-acetamide, which is converted to 2-amino-2-cyano-N-(1,1-dimethyl-ethyl)-acetamide hydrochloride.

The latter is reacted with compound (VI) to obtain 5-amino-N⁴-(1,1-dimethylethyl)-N¹-methyl-1H-imidazole-1,4-dicarboxamide, which is converted to 3,4-dihydro-N-(1,1-dimethylethyl)-3-methyl-imidazo-[5,1-d]-1,2,3,5-tetrazine-8-carboxamide (tert-butyl-Temozolomide), which yields Temozolomide under acidic treatment with concentrated sulfuric acid.

Yet another synthesis of Temozolomide is described by Stevens et al. in J. Org. Chem., Vol. 62, No. 21, 7288-7294, 1997, wherein Temozolomide hydrochloride salt is obtained in 65% yield by the hydrolysis of 8-cyano-3-methyl-[3H]-imidazo-[5,1-d]-tetrazin-4-one with hydrochloric acid, as shown in Scheme 5.

The main disadvantage of this process is the low yield in which Temozolomide hydrochloride is obtained (65%). It is assumed that the relatively elevated temperature of 60° C. used in the process increases the content of decomposition products.

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Synthesis

US Patent 8,232,392

Temozolomide (1) is a drug that was discovered more than 30 years ago. In the past 10 years, it has been used to treat aggressive brain tumors. S. Turchetta and co-inventors summarize several processes for preparing temozolomide, all of which use toxic reagents such as MeNCO or MeNHNH2or generate large amounts of chemical waste. They describe a safer route to 1.

The inventors’ method starts with the preparation of carbamoyl compound 4 from amide 2 by treating it with succinimidyl reagent 3 in the presence of a base. The product is isolated in 88% yield and 96.9% purity by HPLC. Reagent 3 is a nonexplosive, crystalline solid with comparatively low toxicity and is much safer than MeNCO for this reaction.

In the next stage, the amine group in 4 is converted to diazonium salt 5 via a diazotization reaction. The details of this reaction are not described, but reference is made to a method reported in 1997 (Wang, Y., et al. J. Org. Chem. 1997, 62, 7288–7294). Compound 5 is not isolated; when acid is added, it cyclizes by the reaction of the diazonium group with one of the two amide groups to give products 1 and 6 in approximately equal amounts. The desired product 1 is formed by the reaction of the secondary amide group; when the primary amide reacts, the product is its isomer, 6.

Products 1 and 6 are separated by passing the acidified reaction mixture from the diazotization reaction over a column of a polymeric adsorbent resin. The material used in the example is XAD 1600 from Rohm & Haas; other resins are covered in the claims. Compound 6 elutes from the column first; then 1 is eluted with acidified aq EtOH. After separation, 1 is recrystallized from acidified acetone and isolated in 30% yield with 99.9% purity.

The process provides an alternative, safer route to temozolomide, but half of intermediate 4 is lost as unwanted product 6. [Chemi S.p.A. [Cinisello Balsamo, Italy]. US Patent 8,232,392, July 31, 2012; )

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SYNTHESIS

http://www.google.com/patents/WO2002057268A1?cl=en

EXAMPLE 1

Preparation of Temozolomide (1 ) Step A Preparation compound (3)

5-Amino-1 H-imidazole-4-carboxamide*HCI (4) (25 g, 0.154 mol) (Aldrich 16,496-8), CH₂CI₂ (0.6 L) and Et₃N (45 mL) (Aldrich, 13,206-3) were placed into a dry 2-liter, three-necked flask equipped with dropping funnel, a gas inlet tube, a gas outlet tube, reflux condenser and mechanical stirrer, and maintained under a positive pressure of nitrogen at ambient temperature. The mixture was stirred, and a solution of 400 mL of 4-nitrophenyl chloroformate (34 g, 0.169 mol) (Aldrich, 16,021-0) in CH₂CI₂was added dropwise.

The reaction mixture was stirred vigorously for 4 hours and then left to stand for 18 hours at room temperature. The precipitate was collected by vacuum filtration and washed with H₂0 (1.5 L) to afford the product (3) as a pale yellow solid (42 g, 0.144 mol). ¹H NMR (400MHz, DMSO-d₆, δ): 8.40 (d, 2H), 7.83 (s, 1 H), 7.74 (d, 2H), 7.08 (bs, 1 H), 6.95 (bs, 1 H), 6.52 (s, 2H). Step B Preparation of compound (2)

Compound (3) (42 g, 0.144 mol) and DMF (0.27 L) were placed into a dry

1 -liter, three-necked flask equipped with dropping funnel, a gas inlet tube, a gas outlet tube, reflux condenser and mechanical stirrer, and maintained under a positive pressure of nitrogen. The reaction mixture was cooled to 0°C, and methylhydrazine (10 mL, 0.188 mol) (Aldrich, M5.000-1 ) was added dropwise.

The reaction mixture was stirred vigorously for 1 hour at 0°C and was then poured into EtOAc (2.1 L). The precipitate was collected by vacuum filtration and was dried under vacuum (20 mm Hg, room temperature, 18 hours) to afford (2) as a tan solid (27.1 g, 0.137 mol). ¹H NMR (400MHz, DMSO-d₆, δ): 7.62 (s, 1 H), 6.85 (bs, 1 H), 6.75 (bs,1 H), 6.00 (s, 2H), 5.10 (s, 2H), 3.15, s, 3H).mp: 188°C (dec).

Analysis: Calcd for C₆H₁₀N₆O₂: C, 36.36; H, 5.09; N, 42.41.

Found: C, 36.46; H, 4.99; N, 42.12.

Step C Preparation of Temozolomide (1 )

2 1 (Temozolomide)

The reaction mixture was heated at 60°C for 20 mm and then cooled to room temperature. H₅lθ₆ (1.14 g, 5 mmol) was added and the reaction mixture was stirred vigorously at room temperature for 1 hour. The resulting solution was treated with saturated aqueous Na₂S₂O₃ (5 mL) and was then concentrated under reduced pressure to dryness. The residue was treated with CH₃CN (200 mL) and was filtered. The filtrate was concentrated and chromatographed on a column of silica gel (1.5% to 2% AcOH/EtOAc) to afford temozolomide (1 ) (280 mg). ¹H NMR (400MHz, DMSO-d₆, δ): 8.80 (s, 1 H), 7.80 (bs, 1 H), 7.66 (bs, 1 H), 3.43 (s,3H).

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SYNTHESIS

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SYNTHESIS

http://www.google.com/patents/WO2010140168A1?cl=en

Accordingly, the present invention provides an improved process for the preparation of temozolomide of formula I,

Formula I which proves to be efficient and industrially advantageous.

The process comprises the step of: a), condensing compound of formula II,

Formula II with compound of formula III,

CH₃ H CH₃ Formula III in the presence of an acid in an alcoholic solvent to form a compound of formula IV;

Formula IV b). isolating the compound of formula IV from the reaction mixture by filtration; c). diazotizing and cyclizing the compound of formula IV in the presence of source of nitrous acid and a suitable acid; d). isolating temozolomide therefrom; and e). optionally purifying temozolomide of formula I.

Accordingly, the present invention provides an improved process for the preparation of temozolomide of formula I, process comprises the steps of: a), diazotizing and cyclizing the compound of formula IV in the presence of a source of nitrous acid and a suitable acid; b). optionally, cooling the reaction mixture; c). isolating precipitate of temozolomide from the reaction mixture; and d). purifying temozolomide of formula I with a suitable solvent

REFERENCE EXAMPLE:

Preparation* of S-Aøiino-N’-methyl-lH-imidazole-ljΦdicarboxamide (US 7,087,751) 2-Amino-2-cyanoacetamide (10 g), l-methyl-3-methylcarbamoyliminomethyl urea (19 g) and acetic acid (120 ml) were stirred together at ambient temperature under the positive pressure of nitrogen for 2 hours. Excess acetic acid was removed under reduced pressure and methyl tertiary butyl ether (25 ml) was added to the concentrated reaction mass, cooled to obtained crude solid.

The mixture was stirred for 30 minutes and the precipitate was collected by vacuum filtration. The solid was dried under vacuum at 20-25⁰C for 18 hours to obtain 13 g of title compound as grayish solid. The crude product was stirred with water (66 ml) for 1 hour at 20-25⁰C, filtered, suck dried and dried under vacuum at2O⁰C for 18 hours to obtain 11.2 g of title compound as greyish solid.

EXAMPLES

Example 1: Preparation of hydroxylirainocyano acetic acid ethyl ester

To a suspension of ethyl cyanoacetate (1.0 Kg, 8.84 mol) and sodium nitrite (0.735 kg, 10.65 mol) in water (0.80 L), acetic acid (0.70 kg, 11.66 mol) was added at 0-5⁰C over a period of one hour.

Temperature was slowly raised to 23-27⁰C and the reaction mixture was stirred for one hour at that temperature. After the complete consumption of ethyl cyanoacetate (monitored by TLC/GC), the reaction mixture was extracted with ethyl acetate (5 x 1.5 L). The combined organic layer was successively washed with 10% sodium bicarbonate (2 x 1.25 L) and brine solution (1.25 L), dried over sodium sulfate and filtered through hyflow bed. Solvent was removed under reduced pressure at 40-

45⁰C. The resulting solid was stirred with cyclohexane (3.0 L) for 30 minutes at 25-30⁰C, filtered and dried at 40-45⁰C under vacuum to afford 1.14 kg (91.2 %) of title compound having purity 99.82% by

HPLC.

Example 2: Preparation of aminocyanoacetic acid ethyl ester

To a solution hydroxyliminocyano acetic acid ethyl ester (1.14 Kg, 8.02 mol) in methanol (11.4 L) was added 5% platinum on carbon (91.2 g, 50 % wet) and the mixture was hydrogenated at hydrogen gas pressure of 6.2-6.4 kg/cm² over a period of 12 hours and the completion of reaction was checked by

TLC. The reaction mixture was filtered under nitrogen atmosphere to recover the catalyst. The filtrate was used as such for the next stage.

Example 3: Preparation of amimøcyanoacetamide

The solution of aminocyanoacetic acid ethyl ester (as prepared above) in methanol was cooled to 0-5

⁰C and ammonia gas was purged into it approximately for 1 hour. After the completion of the reaction

(monitored by TLC), the reaction mass was concentrated to 2.5-3.0 L under reduced pressure at 40-

45°C, cooled to 0-5⁰C and stirred for 1 hour. The precipitated solid was filtered, washed with chilled methanol (200 ml) and dried at 35-40⁰C under vacuum for 6 hours to obtain 572 g of title compound.

The resulting product was added to methanol (4.57 L) and heated to reflux till the solution become clear. Activated charcoal (25g) was added to the reaction mixture and refluxed for 15 minutes. The solution was filtered through hyflow bed, the bed was washed with methanol (500 ml) and the filtrate was concentrated to half of its original volume (approx 2.0 L). The mixture was cooled to 0-5⁰C and stirred for 45 minutes. The resulting solid was filtered, washed with chilled methanol (250 ml) and dried at 40-45⁰C under vacuum to obtain 425g (53.6%) of pure title compound having purity 99.46% by HPLC. Example 4: Preparation of l-methyl-3-methylcarbamoyliminomethyl urea

A suspension of monomethyl urea (1.5 kg, 20.27 mol) in triethyl orthoformate (4.5 L, 30.40 mol) was heated to reflux at 150-160⁰C for 12 hours. The reaction mixture was cooled to 5-10⁰C, and stirred for 1 hour to ensure complete precipitation, of the product. The resulting solid was filtered, washed with ethyl acetate (350ml) and dried under vacuum at 45-5O⁰C to yield 1.08 kg (67.9%) of title compound having purity 93.82% by HPLC.

Exainple-5: Preparation of S-amino-N^methyl-lH-imidazole-l^-dicarboxamide Acetic acid (200 ml, 3.53 mol) was added to a suspension of aminocyanoacetamide (40Og, 4.04 mol) and l-methyl-3-methylcarbamoyliminomethyl urea (76Og, 4.8 mol) in methanol (2.0 L) at 20-25⁰C and the mixture was stirred at 20-25⁰C for 18 hours till completion of the reaction (monitored by HPLC). The reaction mixture was cooled to 0-5⁰C, stirred for 1 hour and the resulting solid was filtered, washed with chilled methanol (450 ml), suck dried and finally dried under vacuum at 30-35⁰C to afford 648 g (88.04%) of title compound as an off white colored solid having purity 99.21 % by HPLC. Example 6: Preparation of temozolomide

Acetic acid (450 ml, 7.95 mol) was added to a suspension of S-amino-N^methyl-lH-imidazole-l^- dicarboxamide (500g, 2.73mol) and sodium nitrite (25Og, 3.62mol) in water (5.0 L) at -5 to 0⁰C at such a rate so that temperature does not rise above 5°C. The reaction mixture was stirred at 0 to 5°C for one hour and absence of starting material was checked by HPLC analysis. Ice bath was removed and powdered calcium chloride (1.25Kg) was added in small lots to the reaction mass and stirred at 25- 30⁰C for 2 hours. The reaction mass was extracted with a 2.5% solution of dimethylsulfoxide in dichloromethane (5 X 50 L). Combined organic layer was dried over sodium sulfate and filtered through a hyflow bed. Solvent was removed under reduced pressure below 4O⁰C and residual dimethylsulfoxide layer was degassed completely. The dimethylsulfoxide layer was cooled to 0 to – 10⁰C and stirred for 1 hour. The resulting solid was filtered, washed with ethyl acetate (25OmL), and suck dried for 2 hours to afford 32Og of the title compound having purity 78.5% by HPLC. Example 7: Preparation of temozolomide

Acetic acid (9ml, 0.159mol) was added to a suspension of 5-ammo-N¹ -methyl- lH-imidazole- 1,4- dicarboxamide (1Og, 0.054mol) and sodium nitrite (5g, 0.072mol) in water (100ml) at -5 to 0⁰C at a rate so that temperature does not rise above 0-5⁰C. The reaction mixture was stirred at 0-5⁰C for one and half hour. Brine (30g) was added to the reaction mixture and stirred at room temperature for two hours to saturate the reaction mixture. The reaction mass was extracted with a 2.5% solution of dimethylsulfoxide in dichloromethane (5 X 1 L). Combined organic layer was dried over sodium sulfate and filtered through a hyflow bed. Solvent was removed under reduced pressure and residual dimethylsulfoxide layer was degassed completely. The dimethylsulfoxide layer was cooled to 0 to -5°C and stirred for 1 hour. The resulting solid was filtered, washed with ethyl acetate (2x 5 ml), and suck dried for 2 hours to afford 5.0 g of the title compound having purity 81.6% by HPLC. Example 8: Preparation of temozolomide

Acetic acid (450ml) was added to a suspension of 5 -amino-N¹ -methyl- lH-imidazole- 1,4- dicarboxamide (500g) and sodium nitrite (25Og) in water (5.0 L) at -5 to O⁰C at a rate so that temperature does not rise above 0-5⁰C. The reaction mixture was stirred at 0-5⁰C for one and half hour and the absence of starting material was checked by HPLC analysis. Ice bath was removed and powdered calcium chloride (1.25 kg) was added to the reaction mixture and stirred at room temperature for two hours. The reaction mass was extracted with a 2.5% solution of dimethylsulfoxide in dichloromethane (5 X 50 L). Combined organic layer was dried over sodium sulfate and filtered through a hyflo bed. Solvent was removed under reduced pressure at below 40⁰C and residue at 35- 40⁰C was filtered through a candle filter to remove suspended particles and the filtrate was then degassed completely. The residual dimethylsulfoxide layer was cooled to 0±2°C and stirred for one hours. The resulting solid was filtered and sucked dried. The solid was then washed with ethyl acetate (2x 250 ml), and suck dried for 1 hours to afford 240 g of the title compound.

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SYNTHESIS

http://www.google.com/patents/US20020133006

Example 1

Preparation of Temozolomide (1)

5-Amino-1H-imidazole-4-carboxamide.HCl (4) (25 g, 0.154 mol) (Aldrich 16,496-8), CH₂Cl₂(0.6 L) and Et₃N (45 mL) (Aldrich, 13,206-3) were placed into a dry 2-liter, three-necked flask equipped with dropping funnel, a gas inlet tube, a gas outlet tube, reflux condenser and mechanical stirrer, and maintained under a positive pressure of nitrogen at ambient temperature. The mixture was stirred, and a solution of 400 mL of 4-nitrophenyl chloroformate (34 g, 0.169 mol) (Aldrich, 16,021-0) in CH₂Cl₂was added dropwise. The reaction mixture was stirred vigorously for 4 hours and then left to stand for 18 hours at room temperature. The precipitate was collected by vacuum filtration and washed with H₂O (1.5 L) to afford the product (3) as a pale yellow solid (42 g, 0.144 mol).

¹H NMR (400 MHz, DMSO-d₆, δ): 8.40 (d, 2H), 7.83 (s, 1H), 7.74 (d, 2H), 7.08 (bs, 1H), 6.95 (bs, 1H), 6.52 (s, 2H).

Compound (3) (42 g, 0.144 mol) and DMF (0.27 L) were placed into a dry 1-liter, three-necked flask equipped with dropping funnel, a gas inlet tube, a gas outlet tube, reflux condenser and mechanical stirrer, and maintained under a positive pressure of nitrogen. The reaction mixture was cooled to 0° C., and methylhydrazine (10 mL, 0.188 mol) (Aldrich, M5,000-1) was added dropwise. The reaction mixture was stirred vigorously for 1 hour at 0° C. and was then poured into EtOAc (2.1 L). The precipitate was collected by vacuum filtration and was dried under vacuum (20 mm Hg, room temperature, 18 hours) to afford (2) as a tan solid (27.1 g, 0.137 mol).

¹H NMR (400 MHz, DMSO-d₆, δ): 7.62 (s, 1H), 6.85 (bs, 1H), 6.75 (bs,1H), 6.00 (s, 2H), 5.10 (s, 2H), 3.15, s, 3H).mp: 188° C. (dec.).

Analysis: Calcd for C₆H₁₀N₆O₂: C, 36.36; H, 5.09; N, 42.41.

Found: C, 36.46; H, 4.99; N, 42.12.

Compound (2) (500 mg, 2.5 mmol), Bu₄NI (95 mg, 0.25 mmol), THF (250 mL) and CH₃CN (250 mL) were placed into a dry 1-liter, three-necked flask equipped with dropping funnel, a gas inlet tube, a gas outlet tube, reflux condenser and mechanical stirrer, and maintained under a positive pressure of nitrogen. The reaction mixture was heated at 60° C. for 20 mm and then cooled to room temperature. H₅I0₆(1.14 g, 5 mmol) was added and the reaction mixture was stirred vigorously at room temperature for 1 hour. The resulting solution was treated with saturated aqueous Na₂S₂O₃(5 mL) and was then concentrated under reduced pressure to dryness. The residue was treated with CH₃CN (200 mL) and was filtered. The filtrate was concentrated and chromatographed on a column of silica gel (1.5% to 2% AcOH/EtOAc) to afford temozolomide (1) (280 mg).

¹H NMR (400 MHz, DMSO-d₆, δ): 8.80 (s, 1H), 7.80 (bs, 1H), 7.66 (bs, 1H), 3.43 (s, 3H).

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EXAMPLES

EP2374807A2

Example 1:

Glacial acetic acid (25 ml), water (250 ml) and LiCl (225 g) were charged and the contents were stirred for 30 minutes and cooled to room temperature. 5-Amino-1-(N-methylcarbamoyl) imidazole-4-carboxamide (II) (25 g) was added and stirred the contents for further 30 minutes. The reaction mixture was cooled to 0°C and then added drop wise to NaNO₂ solution (12.5 g in 50 ml water) at -10 to 5 °C. The reaction mass was stirred for 1 hr at 0-5 °C and then at room temperature for 5 hrs. To this reaction mixture, sodium thiosulphate solution (25 g in 250 ml of water) was added slowly and stirred for 20 minutes (solution A). This process yielded an acidic solution containing temozolomide.

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SYNTHESIS

US20060183898

EXAMPLES Example 1

A 250 ml reaction vessel equipped with a magnetic stirrer and a reflux condenser was charged with 8-cyano-3-methyl-[3H]-imidazo-[5,1-d]-tetrazin-4-one (10 grams, 0.0568 mol) and hydrochloric acid (36.5-38%, 50 ml). The reaction mixture was heated to 32-35° C. and stirring was maintained at this temperature for about 3 hours. A sample was withdrawn and analyzed by HPLC to verify that the high conversion was received. (If the content of the starting material 8-cyano-3-methyl-[3H]-imidazo-[5,1-d]-tetrazin-4-one is more than 2.5% by area according to HPLC, the stirring may be continued for additional one hour).

The reaction mixture was then cooled to 20° C. and 50 ml of acetone were added drop-wise while maintaining the temperature at 20° C. Stirring was continued for 15-30 minutes. The precipitated white crystals were washed with cold acetone (20 ml) and dried at 40° C. in vacuum to obtain 11.7 grams (0.0507 mol) of Temozolomide hydrochloride (89.3% yield). Purity (by HPLC): 99.6%.

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SYNTHESIS

US6844434

EXAMPLES

The following Examples illustrate but do not in any way limit the present invention. Chemicals obtained from Aldrich Chemical Company (Milwaukee, Wis.) are identified by their catalog number. It should be noted that nomenclature may differ slightly between this specification and the Aldrich catalog.

Example 1 Preparation of Temozolomide (1)

Step A Preparation Compound (3)

¹H NMR (400 MHz, DMSO-d₆, δ): 8.40 (d, 2H), 7.83 (s, 1H), 7.74 (d, 2H), 7.08 (bs, 1H), 6.95 (bs, 1H), 6.52 (s, 2H).
Step B Preparation of Compound (2)

¹H NMR (400 MHz, DMSO-d₆, δ): 7.62 (s, 1H), 6.85 (bs, 1H), 6.75 (bs,1H), 6.00 (s, 2H), 5.10 (s, 2H), 3.15, s, 3H).mp: 188° C. (dec.). Analysis: Calcd for C₆H₁₀N₆O₂: C, 36.36; H, 5.09; N, 42.41. Found: C, 36.46; H, 4.99; N, 42.12.
Step C Preparation of Temozolomide (1)

Compound (2) (500 mg, 2.5 mmol), Bu₄NI (95 mg, 0.25 mmol), THF (250 mL) and CH₃CN (250 mL) were placed into a dry 1-liter, three-necked flask equipped with dropping funnel, a gas inlet tube, a gas outlet tube, reflux condenser and mechanical stirrer, and maintained under a positive pressure of nitrogen. The reaction mixture was heated at 60° C. for 20 mm and then cooled to room temperature. H₅IO₆(1.14 g, 5 mmol) was added and the reaction mixture was stirred vigorously at room temperature for 1 hour. The resulting solution was treated with saturated aqueous Na₂S₂O₃(5 mL) and was then concentrated under reduced pressure to dryness. The residue was treated with CH₃CN (200 mL) and was filtered. The filtrate was concentrated and chromatographed on a column of silica gel (1.5% to 2% AcOH/EtOAc) to afford temozolomide (1) (280 mg).

¹H NMR (400 MHz, DMSO-d₆, δ): 8.80 (s, 1H), 7.80 (bs, 1H), 7.66 (bs, 1H), 3.43 (s, 3H).

TEMOZOLOMIDE

References

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US5260291	Oct 18, 1991	Nov 9, 1993	Cancer Research Campaign Technology Limited	Tetrazine derivatives
US20020133006	Jan 16, 2002	Sep 19, 2002	Schering Corporation	Synthesis of temozolomide and analogs
US20050131227	Jan 21, 2005	Jun 16, 2005	Schering Corporation	Synthesis of temozolomide and analogs
US20060183898 *	Feb 16, 2006	Aug 17, 2006	Olga Etlin	Process for preparing temozolomide

CN1487941A *	Jan 16, 2002	Apr 7, 2004	先灵公司	Synthesis of temozolomide and analogs
CN1706843A *	Apr 8, 2005	Dec 14, 2005	江苏天士力帝益药业有限公司	Temozolomide refining process
US20060183898 *	Feb 16, 2006	Aug 17, 2006	Olga Etlin	Process for preparing temozolomide
US20070225496 *	Mar 23, 2007	Sep 27, 2007	Palle Raghavendracharyulu Venk	rocess for preparing temozolomide

US8258294 *	Sep 28, 2007	Sep 4, 2012	Cipla Limited	Process for the preparation of temozolomide and analogs
EP2151442A2	Jul 22, 2009	Feb 10, 2010	Chemi SPA	Process for preparing temozolomide
EP2374807A2 *	Sep 28, 2007	Oct 12, 2011	Cipla Limited	An improved process for the isolation of temozolomide
WO2008038031A1	Sep 28, 2007	Apr 3, 2008	Cipla Ltd	An improved process for the preparation of temozolomide and analogs
WO2010140168A1 *	Jun 2, 2010	Dec 9, 2010	Ind-Swift Laboratories Limited	Improved process for preparing temozolomide
WO2011036676A2	Sep 14, 2010	Mar 31, 2011	Ashwini Nangia	Stable cocrystals of temozolomide

ILOPERIDONE

new drugs Comments Off

Feb 102014

Iloperidone(Fanapt)

Iloperidone (Fanapt), ILO-522, HP-873, Zomaril, 133454-47-4, antipsychotic

1-[4-[3-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]ethanone; 1-[3-(4-Acetyl-2-methoxyphenoxy)propyl]-4-(6-fluoro-1,2-benzisoxazol-3-yl)piperidine; 4′-[3-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3′-methoxyacetophenone

Aventis Pharma (Originator), Novartis (Licensee), Titan (Licensee)Vanda Pharmaceuticals (Licensee)

Iloperidone(Fanapt) is a monoamine directed towards acting upon and antagonizing specific neurotransmitters, particularly multiple dopamine and serotonin receptor subtypes.

Schizophrenia is a chronic, severe, and debilitating mental disorder that affects approximately 2.4 million Americans, around 1.1% of the population. The net cost of this disorder is staggering as estimates from 2002 reveal this disorder to cost $62.7 billion. A major issue with the treatment of schizophrenia is that patients show varying levels of response and tolerance to available therapies. Although the symptoms of the disease are very severe, estimates show that approximately 3 out of 4 patients discontinue medication prior to completing 18 months of treatment, many times due to the severe side effects of the approved medications.

Synthesis

J.T. Strupczewski, K.J. Bordeau, Y. Chiang, E.J. Glamkowski, P.G.
Conway, R. Corbett, H.B. Hartman, M.R. Szewczak, C.A. Wilmot andG.C. Helsley, J. Med. Chem., 38, 1119 (1995).

US 4355037
V. Miklos, WO Patent 031497 (2010).
J.T. Strupczewski, EP Patent 0402644 (1990)

The product is protected by the U.S. Pat. No. 5,364,866, U.S. Pat. No. RE 39198 E and EP 402644 B1.U.S. Pat. No. 5,364,866 and U.S. Pat. No. 5,663,449.EP 542136, EP 612318, EP 730452, JP 95501055, JP 97511215, US 5364866, US 5776963, WO 9309102, WO 9511680.US 4355037,EP 0542136; EP 0612318; EP 0730452; EP 0957102; EP 0959075; EP 0959076; EP 0963984; JP 1995501055; JP 1997511215; US 5364866; US 5776963; WO 9309102; WO 9511680

The first reported synthetic method for Iloperidone is described in patent EP 402644 A1.

In U.S. Patent US5776963 and patent family EP4 (^ 644, there is disclosed a method for preparing iloperidone,

The synthetic method reported(4, 5) for 1 involves two chemical steps: O-alkylation of acetovanillone (2) with 1-bromo-3-chloropropane (3) to obtain chloro derivative 4 followed byN-alkylation of piperidine intermediate 5 with 4. The reported process for 4 comprises O-alkylation of 2 with 3 in acetone in the presence of potassium carbonate for 20 h to provide 4as an oil after usual work up, which was then vacuum (0.1 mmHg) distilled to collect desired product 4 at 141–143 °C with around 85% yield (Scheme 1, Path A). Some of the drawbacks of this process are as follows: longer reaction time (around 20 h), formation of 6–7% of dimer impurity (10, Scheme 2), high-vacuum distillation to achieve the quality, which is always a cumbersome process at industrial scale, requiring special apparatus and skill set, and degradation and charring of some portion of product during high-vacuum distillation. Further, the next step comprises N-alkylation of 4 with 5 in N,N-dimethylformamide (DMF) in the presence of potassium carbonate to provide iloperidone (1) as a crude solid, which was purified by crystallization using ethanol to yield pure 1 with 58% yield (Scheme 1, Path A). Some of the lacunae observed with the above process includes the following: (a) low yields, (b) formation of carbamate impurity 13 (Scheme 2) in the range 15–20% due to the use of potassium carbonate, (c) ineffective purification by crystallization using ethanol to eliminate carbamate impurity below 0.15%, and (d) iloperidone obtained by the above synthetic process was beige in color.

Scheme 1. Reported (Path A) and Improved (Path B) Process for Preparation of 1

Scheme 2. Flow Chart Representing the Formation of Impurities

A few other improved processes reported…(Improved and Efficient Process for the Production of Highly Pure Iloperidone: A Psychotropic Agent)subsequently for 1 follow the same reaction sequence (Scheme 1, Path A) using compounds 4 and 5 as key starting materials with different bases and solvents.(6-13) However, the reported processes do not address a control mechanism for impurities 8, 9, 11, and 13 (Scheme 2) formed during the synthesis of 1. In order to eliminate these impurities, the reported processes involve employment of multiple purifications using a single solvent or mixture of solvents or purification by means of formation of the acid addition salt of 1 followed by converting back to pure 1.(6-13)

The synthetic route is as follows:

The reaction of piperidine-4-carboxylic acid (I) with formic acid and acetic anhydride gives 1-formylpiperidine-4-carboxylic acid (II), which is treated with SOCl2 and acetic anhydride to yield the corresponding acyl chloride (III). The Friedel-Crafts condensation of (III) with refluxing 1,3-difluorobenzene (IV) by means of AlCl3 affords 4-(2,4-difluorobenzoyl)-1-formylpiperidine (V), which is treated with hydroxylamine in refluxing ethanol to give the corresponding oxime (VI). The cyclization of (VI) by means of NaH in hot THF/DMF yields 6-fluoro-3-(1-formylpiperidin-4-yl)-1,2-benzisoxazole (VII), which is treated with HCl in refluxing ethanol to afford 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole (VIII). Finally, this compound is condensed with 4-(3-chloropropoxy)-3-methoxyacetophenone (IX) by means of K2CO3 in hot DMF. The intermediate 4-(3-chloropropoxy)-3-methoxyacetophenone (IX) can be obtained by condensation of 4-hydroxy-3-methoxyacetophenone (IX) with 3-chcloropropyl bromide (X) by means of NaH or K2CO3 in DMF.

Iloperidone, also known as Fanapt, Fanapta, and previously known as Zomaril, is an atypical antipsychotic for the treatment ofschizophrenia.

Accordingly, 6-fluoro-3-(4-piperidyl)-1,2-benzoxazole 1 and 1-[4-(3-chloropropoxy)-3-methoxy-phenyl]ethanone 2 were heated in presence of potassium carbonate using dimethylformamide solvent to afford 1-[4-[3-[4-(6-fluoro-1,2-benzoxazol-3-yl)-1-piperidyl]propoxy]-3-methoxy-phenyl]ethanone also called Iloperidone

It was approved by the U.S. Food and Drug Administration (FDA) for use in the United States on May 6, 2009.

It’s not yet approved in India.

Hoechst Marion Roussel Inc. made initial inquiries into the drug; however, in May 1996, they discontinued research, and in June 1997 gave research rights to Titan Pharmaceuticals. Titan then handed over worldwide development, manufacturing and marketing rights to Novartis in August 1998. On June 9, 2004, Titan Pharmaceuticals announced that the Phase III development rights have been acquired by Vanda Pharmaceuticals. The original launch date was scheduled for 2002. On November 27, 2007, Vanda Pharmaceuticals announced that the U.S. Food and Drug Administration (FDA) had accepted their New Drug Application for iloperidone, confirming the application is ready for FDA review and approval. On July 28, 2008, the FDA issued a “Not Approvable” letter to Vanda Pharmaceuticals concerning the drug, stating that further trials are required before a decision can be made concerning marketed usage of iloperidone.

Chemically designated as 1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]ethanone, is a second generation atypical antipsychotic agent. Iloperidone, also known as Fanapt, Fanapta, and Zomaril, was approved by the U.S. Food and Drug Administration (FDA) for use in the United States on May 6, 2009 and is indicated for the acute treatment of schizophrenia in adults. Iloperidone has been shown to act as an antagonist at all tested receptors. It was found to block the sites of noradrenalin (α2C), dopamine (D2A and D3), and serotonin (5-HT1A and 5-HT6) receptors.(2) In addition, pharmacogenomic studies identified single nucleotide polymorphisms associated with an enhanced response to iloperidone during acute treatment of schizophrenia. It is considered an “atypical” antipsychotic because it displays serotonin receptor antagonism, similar to other atypical antipsychotics. The older typical antipsychotics are primarily dopamine antagonists.(3)

Iloperidone won FDA approval for use treating schizophrenia in the United States on May 6, 2009

Iloperidone (1-[4-[3-[4-(6-fluoro-1,2-benzisoxazole-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]ethanone) is an atypical new-generation antipsychotic medicament belonging to the class of piperidinyl-benzisoxazole derivatives, which is used to treat schizophrenia, bipolar disorder and other psychiatric conditions. Iloperidone acts as a serotonin/dopamine receptor antagonist (5-HT2A/D2).

Iloperidone, also known as Fanapt, Fanapta, and previously known as Zomaril, is an atypical antipsychotic drug used for the treatment of schizophrenia. The chemical name of iloperidone is l-[4-[3-[4-(6-fluoro-l,2-benzisoxazol-3-yl)-l- piperidinyl]propoxy] -3-methoxyphenyl]ethanone.

EP 0402644 patent discloses first synthetic route of synthesis of iloperidone as shown in Scheme I, which consists of alkylation reaction between l-(4-(3-chloropropoxy-3- methoxyphenyl)ethanone of the formula (II) and 6-fluoro-3-piperidin-4-yl-l ,2 benzisoxazole hydrochloride of the formula (III) in presence of potassium carbonate in N,N dimethyl formamide. The reaction has been subsequently worked up and the compound of formula (I) is extracted from water using ethyl acetate. The compound of formula (I) is purified by crystallization using ethanol. The overall yield of compound of formula (I) is 58%.

Formula (I)

SCHEME 1 Further, we have analyzed the reported synthetic route for synthesis of iloperidone; following limitations have been observed and identified in the reported synthetic route:

a) The yield obtained using said synthetic route as reported in US RE39198 is 58%. Hence, this route of synthesis is not cost efficient at commercial scale due to low yield;

b) Use of potassium carbonate as a base in reaction leads to formation of carbon dioxide as one of the side products during the reaction, which further hinders in the manufacturing process by actively participating in manufacturing process and thereby leads to the formation o

Formula (IV)

which is in the range of 15-20%, and thereby resulting in low yield of iloperidone;

c) Purification by crystallization using ethanol as a solvent is not effective in eliminating or controlling carbamate impurity below 0.15% as per the ICH guide lines for the known impurities; and

d) Iloperidone obtained by the above synthetic process is beige in colour.

CN101768154 discloses the synthesis of iloperidone by N-alkylation reaction between l-(4-(3- chloropropoxy-3-methoxyphenyl)ethanone of the formula (II) and 6-fluoro-3-piperidin-4-yl-l,2 benzisoxazole hydrochloride of the formula (III) in inorganic alkaline solution, particularly; alkali metal carbonate solution. We have analyzed the reported synthetic route for synthesis of iloperidone and have observed and identified that the use of alkaline carbonate solution leads to the formation of carbamate impurity in the range of 1 to 1.5%.

Several patents were published after, describing essentially the same synthetic way such as US5364866 and US5663449.

The synthesis of iloperidone is described in USRE39198 (corresponding to EP 0 402 644 example 3) according to the following synthesis scheme:

In agreement with said patent, the intermediate isolated, 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone, is reacted with 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride in N,N-dimethyl formamide at 90° C. for 16 hours. When the reaction is complete, the mixture is poured into water and extracted with ethyl acetate. The crude product thus obtained is crystallised twice from ethanol to give crystallised iloperidone with a total yield of 58%.

The yield of this process is very low; moreover, the process begins with two isolated intermediates, and requires an aqueous extractive work-up step with an increase in volumes and consequent reduction in the productivity and efficiency of the process. Said process also requires a double crystallisation step to obtain a beige product. The quality levels obtained are not described in the text of the example, but a beige color does not suggest a high-quality product, as iloperidone is a white substance.

The synthesis of intermediate 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone is disclosed in U.S. Pat. No. 4,366,162. Example 1 describes the preparation of said intermediate by reacting acetovanillone with 1-bromo-3-chloropropane in acetone with potassium carbonate. At the end of the reaction the resulting product is purified by distillation and obtained as an oily intermediate which is left to stand in order to obtain the solid intermediate.

The synthesis of intermediate 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone is also disclosed in U.S. Pat. No. 4,810,713. Preparation 12 describes the synthesis of said intermediate from acetovanillone and 1-bromo-3-chloropropane in sodium hydroxide alkalinized water. At the end of the reaction the product obtained is extracted in toluene, the organic phases are washed with basic aqueous solutions and finally, the intermediate 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone is crystallised with the aid of diisopropyl ether. The intermediate isolated is then recrystallised twice from cyclohexane and twice from petroleum ether.

An alternative process for the synthesis of iloperidone is reported in CN 102070626.

Scheme 2 shows the synthesis procedure:

The decision to alkylate acetovanillone with 1-chloro-3-propanol requires an extra synthesis step (to convert the OH group to an OR leaving group) compared with the procedure reported by the combination of patents USRE39198 (EP402644) and U.S. Pat. No. 4,366,162/U.S. Pat. No. 4,810,713, making said process less efficient from the economic standpoint.

WO2011061750 discloses an alternative iloperidone synthesis process as reported in Scheme 3:

Said process uses reagents such as methyl magnesium chloride to effect the Grignard reaction to convert the aldehyde group to a secondary alcohol group, which are much more complicated to manage on an industrial scale than the synthesis methods previously described. Moreover, the oxidation reaction of the next step uses reagents such as chromic acid or potassium permanganate, which have a very high environmental impact and very low industrial applicability.

WO2011055188 discloses a process for the synthesis of iloperidone comparable to the one reported in USRE39198 from two isolated intermediates 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride. The same patent application also gives preparation examples of the intermediate 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone isolated as crystalline solid by procedures similar to those known in the literature.

CN 101824030 reports an iloperidone synthesis method similar to that of CN 102070626 which involves the same problems of inefficiency due to the additional step of inserting the leaving group required for alkylation with 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride.

CN 101781243 discloses an alternative iloperidone synthesis process as reported in Scheme 4.

Said process is not advantageous compared with the preceding processes as the intermediate with the oxime group, due to the nature of this functional group, is particularly liable to degradation due to the action of numerous factors such as the presence of metals, acid pHs and basic pHs.

CN101768154 discloses a process for the synthesis of iloperidone comparable to the one reported in USRE39198 from two isolated intermediates, 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride.

CN 101735208 describes a process for the synthesis of iloperidone comparable to the one reported in CN 101781243, namely through the intermediate with the functional oxime group.

IN 2007MU01980 discloses a process for the synthesis of iloperidone comparable to the one reported in USRE39198 from two isolated intermediates, 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride.

WO 2010031497 describes an alternative iloperidone synthesis process as reported in Scheme 5.

The considerable economic disadvantage of the process reported in WO2010031497 is based on the fact that by reversing the order of alkylation and performing that of intermediate 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride first, a greater loss of yield is generated on this intermediate which, according to the literature, is more difficult to synthesise and consequently more expensive than the intermediate 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone, with a globally greater economic inefficiency of the iloperidone preparation process.

CN 102212063 discloses a process for the synthesis of iloperidone with the same arrangement of the synthesis steps as patent application WO 2010031497.

WO2011154860 describes a process for the synthesis of iloperidone wherein a phase transfer catalyst is used to prepare the intermediate 1-[4-(3-chloropropoxy)-3-methoxyphenyl]ethanone which, as in all the other preparation examples previously described, is crystallised, isolated and dried before use in the next step with 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride. Scheme 6 shows the synthesis scheme of the process of WO2011154860.

………………………………

US20100076196

Figure US20100076196A1-20100325-C00003

……………………………………

WO2012123963A2

EXAMPLE 1:

Tetrabutyl ammonium bromide (2.40 gm) was added to a stirred solution of Potassium hydroxide (0.724 kg) in mixture of Heptane (2.0L). and water (10.0L), followed by addition of 1- [4-(3-chloropropoxy)-3-methoxyphenyl]ethanone (2, 1.0kg) and 6-fluoro-3-piperidin-4-yl-l,2- benzisoxazole hydrochloride^, 1.1 1kg) at 30°C. This reaction mass was stirred for 15 to 20 min. The temperature of the reaction mass was raised to 70°C and was maintained for 8 to 10 hours. After completion of reaction (by TLC, Mobile Phase: Toluene/ Acetone/Ethyl acetate = 6:2:2 mL), the mixture was cooled to 30°C, diluted with dichloromethane (2.5 L) and stirred for 30 minutes. The dichloromethane layer was separated. The aqueous layer was re-extracted with dichloromethane (1.0L). The combined dichloromethane layer was washed with water (1.5L) and decolorized with activated charcoal (0.05 kg). The solvent was distilled off completely to obtain the residue. The residue obtained was dissolved in isopropyl alcohol (5.0L) at reflux temperature to obtain the clear solution. The clear solution obtained was cooled to 30°C followed by 0°C and stirred for 60 min to precipitate out crystals. The colorless crystals of compound (I) obtained were filtered. The crystalline solid was dried under vacuum (650-700 mm/Hg) to obtain pure compound (I) as a crystalline solid. HPLC analysis was performed for the crystalline solid obtained. The purity of Iloperidone, impurity profile and yield are shown in table 1 below.

Table 1 : Analysis data of iloperidone i.e. purity, yield and impurity profile.

EXAMPLE 2:

Tetrabutyl ammonium bromide (2.40 gm) was added to a stirred solution of Potassium hydroxide (0.724 kg) in mixture of Heptane (2.0L) and water (10.0L), followed by addition of 1- [4-(3-chloropropoxy)-3-methoxyphenyl]ethanone (2, 1.0kg) and 6-fluoro-3-piperidin-4-yl-l,2- benzisoxazole hydrochloride^, 1.1 1kg) at 30°C. This reaction mass was stirred for 15 to 20 min. The temperature of the reaction mass was raised to 70°C and maintained for 8 to 10 hours. After completion of reaction (by TLC, Mobile Phase: Toluene/ Acetone/Ethyl acetate = 6:2:2 mL), the mixture was cooled to 30°C, the reaction mixture was filtered to obtain wet crude iloperidone. Further, the obtained wet crude was dried at 60-65 °C under vacuum to furnish crude iloperidone (1.72 kg). The dried crude iloperidone was dissolved in isopropyl alcohol (5.0 L) at reflux temperature and decolorized with activated charcoal (0.05 kg). Obtained filtrate was cooled to 30°C followed by 0°C and stirred for 60 min to precipitate out crystals. The colorless crystals of compound (I) obtained were filtered. The crystalline solid was dried under vacuum (650-700 mm/Hg) to obtain pure compound (I) as a crystalline solid. HPLC analysis was performed for the crystalline solid obtained. The purity of Iloperidone, impurity profile and yield are shown in table 2 below.

Table 2: Analysis data of iloperidone i.e. purity, yield and impurity profile.

EXAMPLE-3:

……………………..

US20130261308

UPLC-MS [M+H+]=427

1H-NMR (in DMSO) (chemical shifts expressed in ppm with respect to the TMS signal): 2.06-1.78 (6H, m); 2.13 (2H, m); 2.49 (2H, t); 2.52 (2H, m); 2.97 (2H, m); 3.11 (1H, tt); 3.83 (3H, s); 4.12 (2H, t); 7.06 (1H, d); 7.22 (1H, m); 7.46 (1H, d); 7.61-7.58 (2H, m); 7.94 (1H, dd).

………………………………

.Improved and Efficient Process for the Production of Highly Pure Iloperidone: A Psychotropic Agent

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/op400335p

http://pubs.acs.org/doi/full/10.1021/op400335p?prevSearch=iloperidone&searchHistoryKey=

The present work describes an improved and highly efficient process for the synthesis ofiloperidone (1), an antipsychotic agent, which is free from potential impurities. The synthesis comprises N-alkylation of 1-(4-(3-chloropropoxy)-3-methoxyphenyl)ethanone (4) with 6-fluoro-3-piperidin-4-yl-1,2-benzisoxazole hydrochloride (5) in a mixture of water and heptane as solvent and sodium hydroxide as a base in the presence of tetrabutylammonium bromide as a phase transfer catalyst to yield iloperidone (1) with a yield of around 95% and a purity of 99.80% by HPLC. The present work also describes the optimization details performed to achieve the process attributes responsible for high yield and purity.

FT-IR (KBr, λmax, cm–1): 3031, 2949, 2779, 2746, 2822, 1669, 1614, 1585, 1510, 1462, 1448, 1415, 1380, 1313, 1262, 1221, 1177, 1150, 1123, 1077, 1034, 997, 985, 955, 884, 876, 853, 812, 781, 643, 610, 569, 475.

1H NMR (CDCl3): δ 2.03–2.10 (m, 6H), 2.12–2.18 (m, 2H), 2.55–2.56 (s, 3H), 2.58–2.60 (t, 2H), 3.02–3.09 (m, 3H), 3.91 (s, 3H), 4.10–4.19 (t, 2H), 6.91–6.93 (d, 1H), 7.01–7.06 (dd, 1H), 7.21–7.24 (dd, 1H), 7.51–7.52 (d, 1H), 7.53–7.56 (dd, 1H), 7.69–7.65 (dd, 1H).

13C NMR (CDCl3): 26.02, 26.40, 30.36, 34.34, 53.36, 54.90, 55.80, 67.16, 97.04, 97.31, 110.20, 111.02, 111.98, 112.23, 117.12, 122.36, 122.46, 123.06, 130.11, 149.00, 152.66, 160.91, 162.60, 163.53, 163.66, 165.09, 198.59.

MS (ESI, m/z): 427.2 [M + H].+

Anal. Calcd (%) for C24H27FN2O4(426.48): C, 67.54; H, 6.33; found (%): C, 67.24; H, 6.18.

HPLC

HPLC analysis developed at Megafine India using a Hypersil BDS C18 column (250 mm × 4.6 mm, particle size 5 μm); mobile phase A comprising a mixture of 5.0 mM ammonium dihydrogen orthophosphate buffer and 0.1% triethylamine; mobile phase B comprising a mixture of acetonitrile/methanol in the ratio 80:20 v/v; gradient elution: time (min)/A (v/v): B (v/v); T0.01/65:35, T8.0/65:35, T25.0/35:65, T35.0/35:65, T37.0/65:35, T45.0/65:35; flow rate 1.0 mL/min; column temperature 30 °C; wavelength 225 nm. The observed retention time of iloperidone under these chromatographic conditions is about 17.0 min.

…….

http://www.asianjournalofchemistry.co.in/User/ViewFreeArticle.aspx?ArticleID=25_10_2

N oxide impurity

m.p. 155-157 ºC;

FT-IR (KBr, νmax, cm-1):
3083, 2958, 2878, 1655, 1606, 1584, 1509, 1467, 1419, 1348,1273, 1223, 1182, 1143, 1121, 1032, 971, 957, 881, 857, 813,
802;

1H NMR (300 MHz, CDCl3)

δ 1.89-1.93 (m, 2H), 2.31-2.40 (m, 2H), 2.55 (s, 3H), 2.60-2.72 (m, 2H), 3.29-3.52 (m,
2H), 3.29-3.52 (m, 2H), 3.29-3.52 (m, 2H), 3.29-3.52 (m, 1H),3.85 (s, 3H), 4.23(t, 2H, J = 6.0 Hz), 7.11 (d, 1H, J = 8.4 Hz),7.30-7.36 (m, 1H), 7.62-7.65 (m, 1H), 7.71-7.74 (dd, J = 9.3and 2.0 Hz, 1H), 8.02-8.07 (dd, J = 8.7 and 5.4 Hz, 1H);

13CNMR (75 MHz, CDCl3)

δ 22.13, 24.70, 26.35, 31.49, 55.54,63.21, 67.07, 67.82, 97.51, 110.35, 111.86, 112.67, 123.11,
123.67, 129.95, 148.63, 152.22, 160.79, 163.10, 163.69,196.40;

MS (ESI, m/z): 443 [M + H]+.

Anal. calcd. (%) forC24H27N2O5F (442.19): C, 65.15; H, 6.15; N, 6.33; found (%):C, 65.11; H, 6.09; N, 6.29.

………………………

INTERMEDIATES

Acetovanillon (4-hydroxy-3-methoxyacetophenone) 6 is also a first-generation fine chemical obtained as a reaction product from the oxidation−hydrolysis of lignosulfonate LS. The compound serves as substrate in synthetic processes leading to several second-generation fine chemicals, such as acetoveratron, veratric acid, and veratric acid chloride. Moreover, recently, a new compound iloperidone REF 20,21 34 [1-(3-(4-acetyl-2-methoxyphenoxy)propyl)-4-(6-fluorobenzisoxazol-3-yl)piperidine] that includes an acetovanillon 6 moiety was reported to be under development for use as an antipsychotic dopamine D2 antagonist and a 5-HT2Aantagonist.

The synthesis of iloperidone 34 is performed by means of an eight-step synthetic process. The acetovanillon 6, which constitutes an integral part of this substance, is condensed with 3-chloropropylbromide 43 in DMF in the presence of potassium carbonate or sodium hydride as base to obtain the key intermediate 44. In the last step of the process 44 is reacted with 42 to afford iloperidone 34. The intermediate 42 is synthesised by reacting piperidine-4-carboxylic acid 35 with formic acid and acetic acid anhydride to obtain 1-formylpiperidine-4-carboxylic acid 36 that upon treatment with thionyl chloride in acetic acid anhydide gives the corresponding acyl chloride 37 (1-formylpiperidine-4-carbonyl chloride). Under Friedel−Craft conditions, the acyl chloride 37 is condensed with 1,3-difluorobenzene 38 to afford 4-(2,4-difluorobenzoyl)piperidine-1-carbaldehyde 39. Treatment of this intermediate with hydroxylamine in refluxing ethanol yields the oxime 40 (4-[(2,4-difluorophenyl)hydroxyiminomethyl]piperidine-1-carbaldehyde). When the oxime 40 is exposed to basic conditions by means of sodium hydride in hot DMF and THF in the following step, a cyclisation proceeds to afford benzo[d]isoxazol 41 (4-(6-fluorobenzo[d]isoxazol-3-yl)piperidine-1-carbaldehyde), which upon treatment with HCl in refluxing ethanol affords the key intermediate 42.

FANAPT is a psychotropic agent belonging to the chemical class of piperidinyl-benzisoxazole derivatives. Its chemical name is 4′-[3-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)piperidino]propoxy]-3′-methoxyacetophenone. Its molecular formula is C24H27FN2O4 and its molecular weight is 426.48. The structural formula is:

FANAPT® (iloperidone) Structural Formula Illustration

Iloperidone is a white to off-white finely crystalline powder. It is practically insoluble in water, very slightly soluble in 0.1 N HCl and freely soluble in chloroform, ethanol, methanol, and acetonitrile.

Title: Iloperidone

CAS Registry Number: 133454-47-4

CAS Name: 1-[4-[3-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]ethanone

Manufacturers’ Codes: HP-873; ILO-522

Trademarks: Zomaril (Novartis)

Molecular Formula: C24H27FN2O4

Molecular Weight: 426.48

Percent Composition: C 67.59%, H 6.38%, F 4.45%, N 6.57%, O 15.01%

Literature References: Combined dopamine (D2) and serotonin (5HT2) receptor antagonist. Prepn: J. T. Strupczewski et al., EP402644; eidem, US 5364866 (1990, 1994 both to Hoechst-Roussel); eidem, J. Med. Chem. 38, 1119 (1995).

Pharmacology: M. R. Szewczak et al., J. Pharmacol. Exp. Ther. 274, 1404 (1995).

Clinical pharmacokinetics: S. M. Sainati et al., J. Clin. Pharmacol.35, 713 (1995).

HPLC determn in plasma: A. E. Mutlib, J. T. Strupczewski, J. Chromatogr. B 669, 237 (1995). Receptor binding study: S. Kongsamut et al., Eur. J. Pharmacol. 317, 417 (1996).

Review of pharmacology and therapeutic potential in schizophrenia: J. M. K. Hesselink, Curr. Opin. Cent. Peripher. Nerv. Syst. Invest. Drugs 2, 71-78 (2000); K. K. Jain, Expert Opin. Invest. Drugs 9, 2935-2943 (2000).

Properties: Crystals from ethanol, mp 118-120°.

Melting point: mp 118-120°

Therap-Cat: Antipsychotic.

Keywords: Antipsychotic; Benzisoxazoles; Serotonin-Dopamine Antagonist.

King, D. R.; Kanavos, P. Croat. Med. J. 2002, 43, 462– 9

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Strupczewski, J. T.; Helsley, G. C.; Chiang, Y.; Bordeau, K. J. EP 0402644A1, 1990.
Ansari, S. A.; Hirpara, H. M.; Yadav, A. K.; Gianchandani, J. P. WO2012164516, 2012.
Azad, M. A. K.; Pandey, G.; Singh, K.; Prasad, M.; Arora, S. K. WO2012/090138 A1, 2012.
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Shiwei, Z.; Feng, J. US 2012/0172699A1, 2012.
Bettoni, P.; Roletto, J.; Paissoni, P. EP 2644608A1, 2013.
Mathad, V. T.; Solanki, P. V.; Pandit, B. S.; Uppelli, S. B. WO2012/123963 A2, 2012.

Strupczewski, J. T.; Allen, R. C.; Gardner, B. A.; Schmid, B. L.; Stache, U.; Glamkowski, E. J.; Jones, M. C.; Ellis, D. B.; Huger, F. P.; Dunn, R. W. J. Med. Chem. 1985, 28, 761–769

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WO2003037337A1 *	Oct 29, 2002	May 8, 2003	Markus Ahlheim	Depot formulations of iloperidone and a star polymer
WO2008027993A2 *	Aug 29, 2007	Mar 6, 2008	Eurand Inc	Drug delivery systems comprising solid solutions of weakly basic drugs
CN101768154A	Sep 19, 2009	Jul 7, 2010	浙江华海药业股份有限公司	New preparation method of iloperidone
EP0402644A1	May 16, 1990	Dec 19, 1990	Hoechst-Roussel Pharmaceuticals Incorporated	N-(aryloxyalkyl)heteroarylpiperidines and -heteroarylpiperazines,a process for their preparation and their use as medicaments
EP0542136A1 *	Nov 5, 1992	May 19, 1993	Hoechst-Roussel Pharmaceuticals Incorporated	Heteroarylpiperidines, pyrrolidines and piperazines and their use as antipsychotics and analgetics
US5364866	Oct 30, 1992	Nov 15, 1994	Hoechst-Roussel Pharmaceuticals, Inc.	Heteroarylpiperidines, pyrrolidines and piperazines and their use as antipsychotics and analetics
US5663449	Jun 6, 1995	Sep 2, 1997	Hoechst Marion Roussel, Inc.	Intermediate compounds in the synthesis of heteroarylpiperidines, pyrrolidines and piperazines
USRE39198	Nov 15, 2000	Jul 18, 2006	Aventis Pharmaceuticals Inc.	Heteroarylpiperidines, pyrrolidines and piperazines and their use as antipsychotics and analgesics

CAPREOMYCIN

GENERIC, Uncategorized Comments Off

Feb 082014

Structure

Capreomycin is a peptide antibiotic, commonly grouped with the aminoglycosides, which is given in combination with other antibiotics for MDR-tuberculosis. Adverse effects include nephrotoxicity and 8th cranial auditory vestibular nerve nerve toxicity.

The drug should not be given with streptomycin or other drugs that may damage the auditory vestibular nerve. Patients on this drug will often require audiology tests.

It is a cyclic peptide. Capreomycin is administered intramuscularly and shows bacteriostatic activity.REF 20

Capreomycin is frequently used to treat Mycobacterium tuberculosis infections. Mycobacterium tuberculosis growth has been found to be inhibited at a concentration of 2.5 μg/mL. REF21

This is the basic structure of capreomycin. The table below identifies the various naturally occuring analogues^{12, 14}.

	R₁	R₂
Capreomycin IA	OH	b-Lys
Capreomycin IB	H	b-Lys
Capreomycin IIA	OH	NH₂
Capreomycin IIB	H	NH₂

Introduction

Capreomycin is a metabolite of Streptomyces capreolus, it is an antimycobacterial agent – and a potent tuberlostatic antibiotic. Capreomycin is effective against a number of Gram-positive and Gram-negative organisms, but is primarily active against mycobacteria. It has been used in the treatment of certain resistant strains of Mycobacterium tuberculosis. The drug was first described in 1960 be Herr, and was subsequently found to contain two components (I and II) and later to be comprised of four (IA, IB, IIA, IIB) as shown on thestructure page.

Tuberculosis

Tuberculosis is a disease of the respiratory system, and is airbourne. The bacilli implant themselves in areas such as the lungs, renal cortex and reticuloendothelial system where there is a high partial pressure of oxygen. This is the Primary infection and does not normally affect the person whilst their immune system is intact as the bacteria lie dormant. When the immune system is depressed, the secondary reactivation occurs, and effects of the disease are seen.

This infectious disease has been known since about 1000B.C., and it stills remains the ‘leading cause of death from a single infectious disease agent’⁷. It is estimated that around eight million people contract TB every year, of which 95% are in developing countries. Deaths from the disease is estimated at 3 million people per year by the World Health Organisation. The occurance of the disease is related directly to the economic state of the country. This is because the spread of the disease is greatly assisted by poor public and personal hygiene and by overcrowding. New drugs were develoed about forty years ago allowing tuberculosis to be regarded as a curable disease. This is no longer the case, as many multidrug-resistant strains of the disease have emerged. This is where capreomycin has it uses.

There are three groups of drugs used to treat TB, which vary in their effectiveness and potential side effects.

First line drugs include: isoniazid, rifampicin and pyrazinamide. These are most effective and have the fewest potential side effects.

Second line drugs include: ethambutol, streptomycin and p-amino salicyclic acid. These are less effective and have more toxic effects.

Third line drugs include: Capreomycin, cycloserine, viomycin, kanamycin and amikacin. These are least effective and have the most toxic effects.

The third line drugs have to be used for infections with tubercle bacilli, likely to be resistant to first and second line drugs or when first and second line drugs have been abandoned because of unwanted reactions. To decrease the possibility of resistant organisms from emerging, ‘Compound Drug Therapy’ is used where a concoction of several drugs is administered.

General Physical Data

Molecular Weight	653.70
Molecular Formula	C₂₅H₄₃N₁₃O₈
CAS Registry number	61394-77-2
Beilstein Registry number	876587
Chemical Name	L-3,6-diamino-hexanoyl->-cyclo-[L-2,3-diamino-propionyl->-L-seryl->-L-alanyl->-2-amino-3-ureido -acryloyl->-(S)-amino-((R)-2-amino-1(3),4,5,6-tetrahydro-pyrimidin-4-yl)-acetyl-(1->N%3&)]
Auto name	3,6-diamino-hexanoic acid [12-hydroxymethyl-3-(2-imino-hexahydro-pyrimidin-4-yl)-9-methyl- 2,5,8,11,14-pentaoxo-6-ureidomethylene-1,4,7,10,13-pentaaza-cyclohexadec-15-yl]-amide

		Cpm IA¹⁰	Cpm IB¹⁰	Cpm IIA¹⁴	Cpm IIB¹⁴
m.p. / ^oC		240-5	250-3	250	252
[a]_D / ^o		-22.0	-42.5	+9.3	-24.9
UV / nm	0.1 M HCl	269 (e 23, 400)	268 (22, 000)
	H₂O	268 (23, 200)	268 (21, 900)
	0.1 M NaOH	288 (15, 800)	290 (13, 100)

According to the literature⁹the following applies to naturally occuring capreomycin:
Ratio of IA to IB = 1.16
Capreomycin II = 1.5%

¹³C NMR Data of Cpm IA

Carbon Number	d / ppm
1	51.92
2	40.28
4	172.76
10	176.29
11	54.15
5, 14	55.66
5, 14	56.23
7	168.0
8	105.90
13	172.00
16	176.6
17	135.79
19	155.32
20	18.86
21	68.33
22	49.20
23	23.53
24	49.83
26	157.0 (b)
1′	172.0
2′	36.93
3′	49.26
4′	23.59
5′	29.77
6′	39.77

The included NMR data is taken from tables in the literature^{8, 14}

The ¹³C NMR data is that of Capreomycin IA only, and the carbons are numbered accordingly in red on the structure shown above.

Below are ¹H NMR tables for the four different naturally occurring forms of capreomycin, the NH protons and CH protons are given in different tables. The NH protons are again numbered on the Cpm IA structure above, but this time in blue. The CH protons are numbered according to their postion in the amino acid residue. These are also numbered in pink on the above diagram.

Chemical Shifts of CH protons in Capreomycin Analogues

Position of Amino Acid Residue		Cpm IA	Cpm IB	Cpm IIA	Cpm IIB
1	a-CH₂	2.63 (dd)	2.5 (dd)
		2.85 (dd)	2.81 (dd)
	b-CH₂	3.8 (m)	3.7 (m)
	g-CH₂	1.8 (m)	1.8 (m)
	d-CH₂	1.8 (m)	1.8 (m)
	e-CH₂	3.10 (m)	3.08 (m)
2	a-CH	4.3-3.5 (m)	4.2-4.5 (m)	4.3-4.6 (m)	4.3-4.6 (m)
	b-CH₂	3.3 (m)	3.3 (m)	3.3 (m)	3.3 (m)
		3.8 (m)	3.8 (m)	4.1 (m)	4.1 (m)
3	a-CH	4.86 (t)	4.67 (q)	4.84 (t)	4.68 (q)
	b-CH₂	3.84 (d)		3.95 (d)
	b-CH₃		1.43 (d)		1.45 (d)
4	a-CH	4.3-4.5(m)	4.2-4.5 (m)	4.3-4.5 (m)	4.3-4.5 (m)
	b-CH₂	3.7-4.2 (m)	3.7-4.2 (m)	3.7-4.2 (m)	3.79 (dd)
					3.8-4.2 (m)
5	b-CH	8.04 (s)	8.03 (s)	8.05 (s)	8.04 (s)
6	a-CH	5.01 (d)	4.96 (d)	5.01 (d)	4.95 (d)
	b-CH	4.5 (m)	4.5 (m)	4.5 (m)	4.5 (m)
	g-CH₂	1.6-2.3 (m)	1.6-2.3 (m)	1.6-2.3 (m)	1.6-2.3 (m)
	d-CH₂	3.3 (m)	3.3 (m)	3.3 (m)	3.3 (m)

Chemical Shifts of NH Protons of Capreomycin Analogues

	Cpm IA	Cpm IB	Cpm IIA	Cpm IIB
1	9.33 (d)	9.72(d)	9.60 (d)	9.50 (d)
2	9.24 (d)	9.24 (d)	9.33 (d)	9.30 (d)
3	8.82 (s)	8.76 (s)	9.10 (s)	9.10 (s)
4	8.64 (d)	8.68(d)	8.73 (d)	8.73 (d)
6	8.22 (t)	8.15 (t)
7	8.10 (t)	8.15 (t)	8.19 (t)	8.08 (t)
8	7.61 (d)	7.62 (d)	7.50 (d)	7.49 (d)
9	7.46 (s)	7.42 (s)	7.44 (s)	7.44 (s)
10	7.46 (s)	7.42 s)	7.31 (s)	7.18 (s)
11	6.48 (s)	6.49 (s)	6.43 (s)	6.34 (s)
12	6.29 (s)	6.34 (s)	6.29 (s)	6.27 (s)

Using the program gNMR I attempted to plot the above data. However, this was not successful as this program can only cope with molecules with up to 23 protons. As this molecule has Capreomycin IA has 43 hydogens, the generated ¹H NMR was lacking many essential peaks, and hence was not included.

IR Spectrum of Capreomycin IA

The same process could have done for any of the other three Capreomycin anlogues. The very broad band around 2000 cm^-1 upwards is due to the presence of so many nitrogen and carbonyl groups and hence hydrogen bonding.

Cyclo[3-[[(3S)-3,6-diamino-1-oxohexyl]amino]-L-alanyl-(2Z)-3-[(aminocarbonyl)amino]-2,3-didehydroalanyl-(2S)-2-[(4R)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]glycyl-(2S)-2-amino-b-alanyl-L-seryl]

capreomycinIA;Cyclo[3-[[(3S)-3,6-diamino-1-oxohexyl]amino]-L-alanyl-(2Z)-3-[(aminocarbonyl)amino]-2,3-didehydroalanyl-(2S)-2-[(4R)-2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl]glycyl-(2S)-2-amino-b-alanyl-L-seryl] (9CI);1,4,7,10,13-Pentaazacyclohexadecane, cyclic peptide deriv.

37280-35-6

Formula:	C25H44 N14 O8
Molecular Weight:	668.83

Properties:Crystals. Mp: 246–248^°C.
Synonyms:capreomycin IA;Cyclo[A2pr*-Ser-N3-[(3S)-3,6-diamino-1-oxohexyl]A2pr-2-[(Z)-aminocarbonylaminomethylene]Gly-2-[(4R)-2-iminohexahydropyrimidine-4-yl]Gly-]SynthesisBelow is the peptide synthesis of capreomycin IA and IB. This was taken directly from the literature¹⁰.

No chemical synthesis of capreomycin could be found in any of the literature references. However, below is a synthesis devised from the peptide synthesis shown above. This is colour coded depending on the various amino residues. Each of the amino groups is added to the molecule in sequence linked by a peptide bond to eventually form the cyclo-structure. This was designed with some help from general references^1,2,3.

This synthesis would be identical for capreomycin IB other than the Serine-Bzl is replaced by Alanine and the synthesis works in exactly the same way.

Capreomycin

The individual components of the capreomycin were colour coded as follows:

Red	DEA / UDA	b, b � diethoxyalanine / b – ureidodehydroalanine
Green	A₂pr	a, b � diaminopropionic acid
Turquoise	Ser	Serine
Blue	Cpd	Capreomycidine
Pink	b–Lys	b–Lysine

The black components of the synthesis were the various protecting groups involved:

Boc	tert � butoxycarbonyl
Z	Benzyloxycarbonyl
ONSu	N-hydroxysuccinimide
Nps	o-Nitrophenylsulphenyl
NO₂	Nitro	NO₂
Bzl	Benzene

Abbreviation	Chemical Name
NMM	N-Methylmorpholine
DCC	N,N�-Dicyclohexylcarbodiimine
HOBt	l-Hydroxybenztriazole
HONSu	N-Hydroxysuccinimide
THF	Tetrahydrofuran

This is the synthesis of capreomycin IA. The IB form is produced in an identical fashion except that Ser � Bzl , is replaced with Ala.

……………………..

US8044186

Capastat Sulfate (capreomycin for injection) is a polypeptide antibiotic isolated from Streptomyces capreolus. It is a complex of 4 microbiologically active components which have been characterized in part; however, complete structural determination of all the components has not been established.

Capreomycin is supplied as the disulfate salt and is soluble in water. In complete solution, it is almost colorless.

Each vial contains the equivalent of 1 g capreomycin activity.

The structural formula is as follows:

Capastat Sulfate Structural Formula Illustration

Biological Action

Capreomycin is part of a group of drugs called aminoglycosides. These act to inhibit bacterial protein synthesis. The oxygen-dependent active transport by a polyamine carrier system affects the penetration of the aminoglycosides through the cell membrane of the bacterium. Minimal action on anaerobic organisms is observed. The effect of the aminoglycosides is bactericidal and is enhanced by agents that interfere with cell wall synthesis.

Very little is known about the mechanism of action of capreomycin specifically, but it is thought to inhibit protein synthesis by binding to the 70s ribosomal unit. Other sources⁶support this theory by suggesting that capreomycin “prevents protein biosynthesis by inhibiting group I intron splicing of RNA as well as blocking translation on the bacterial ribosome via inhibition of ribosomal subunits.” It has been reported¹⁴ that the b-amino group of the A₂pr residue promotes biological potency, and that its location within the molecule is of importance.

Side Effects

This powerful antimycobacterial agent can give rise to several side effects, some of which are listed below:

The following Nephrotoxic effects are reversible once treatment is stopped, but capreomycin is not recommended for people with kidney disorders.

Polyuria (excess urination)
Haematuria (red blood cells in the urine)
Proteinuria (protein in the urine)
Nitrogen metabolism
Electrolyte disturbances
Anorexia
Anaemia
Thirst

Capreomycin is also Ototoxic giving the following side effects. The nerve damage is permanent.

Deafness
Loss of vestibular function
Damage to the cranial nerve 8

References:1. An Introduction to Peptide Chemistry – P.D. Bailey
2. Organic Chemistry – Vollhardt and Schore
3. Peptide Synthesis – M. Bodanszky, Y. Klausner and M. Ondetti
4. Pharmacology – H.P. Rand, M.M. Dale and J.M. Ritter
5. http://www.aidsinfonyc.org/network/access/drugs/capr.html
6. http://rwingo1.chm.colostate.edu/group/duane/duane.html
7. http://www.hucmlrc.howard.edu/Pharmacology/handouts/TBRCLSIS.html
8. J. Org.Chem.,1977, 42, 8 – McGahren, Morton, Kunstmann, Ellestad
9. Bull.W.H.O., 1972, 47(3), 343-56 – Lightbrown et al.
10. Tetrahedron, 1978, 34(7), 912-7 – Nomoto, Teshima, Wakamiya, Shiba
11. Tetrahedron Letters, 1976, 43, 3907-10 – Shiba, Nomoto, Teshima, Wakamiya
12. J.Org.Chem., 1992, 57, 5214-5217 – Gould and Minott
13. Tetrahedron Letters, 1969, 30, 2549-41 – Bycroft, Cameron, Hassanali-Walji and Johnson
14. Bull.Chem.Soc.Jpn, 1979, 52(6), 1709-15 – Nomoto and Shiba
15. Experimentia – 1976, 32(9), 1109-11 – Nomoto and Wakamiya
16. Pharmazie – 1970, 25(8), 471-2 – Voigt and Maa Bared
17. Antimicrobial Agents Chemotherapy, 1964, 522-9 – Black, Griffith and Brickler
18. Antimicrobial Agents Chemotherapy, 1962, 201-12 – Herr
19. www2.chemie.uni-erlangen.de/services/telespec
20 “Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2′-O-methylations in 16S and 23S rRNAs”. Mol. Cell 23 (2): 173–82. July 2006. doi:10.1016/j.molcel.2006.05.044. PMID 16857584
21 http://www.toku-e.com/Assets/MIC/Capreomycin%20sulfate.pdf
CAPREOMYCIN wiki



Systematic (IUPAC) name
(3S)-3,6-diamino-N-[[(2S,5S,8E,11S,15S)-15-amino-11-[(4R)-2-amino-3,4,5,6-tetrahydropyrimidin-4-yl]-8-[(carbamoylamino)methylidene]-2-(hydroxymethyl)-3,6,9,12,16-pentaoxo-1,4,7,10,13-pentazacyclohexadec-5-yl]methyl]hexanamide; (3S)-3,6-diamino-N-[[(2S,5S,8E,11S,15S)-15-amino-11-[(4R)-2-amino-3,4,5,6-tetrahydropyrimidin-4-yl]-8-[(carbamoylamino)methylidene]-2-methyl-3,6,9,12,16-pentaoxo-1,4,7,10,13-pentazacyclohexadec-5-yl]methyl]hexanamide
Clinical data
AHFS/Drugs.com	monograph
MedlinePlus	a682860

Identifiers
CAS number	11003-38-6

Chemical data
Formula	C₂₅H₄₄N₁₄O₈
Mol. mass	668.706 g/mol