RANBEZOLID FROM RANBAXY

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Apr 022014

Ranbezolid

392659-39-1 hydrochloride

392659-38-0 (free base)

N-{[(5S)-3-(3-Fluoro-4-{4-[(5-nitro-2-furyl)methyl]-1-piperazinyl}phenyl)-2-oxo-1,3-oxazolidin-5-yl]methyl}acetamide

(S)-N-[[3-fluoro-4-[N-1[4-{2-furyl-(5-nitro)methyl}]piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide

AC1LAX1P, RBx7644 (*Hydrochloride*),RBx-7644

Molecular Formula: C₂₁H₂₄FN₅O₆ Molecular Weight: 461.443563

Ranbaxy Lab Ltd ORIGINATOR

Ranbezolid is a novel oxazolidinone antibacterial. It competitively inhibits monoamine oxidase-A (MAO-A).^[1]

Infections due to Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and penicillin-resistant Streptococcus pneumoniae(PRSP) are the leading cause of morbidity and mortality in hospital settings and community today. Oxazolidinones are a new class of totally synthetic antibacterial agents active against Gram-positive infections. Linezolid (Zyvox™, Pharmacia/Pfizer, is a drug in this class, approved in the United States and Europe for treatment of Gram-positive nosocomial and community-acquired pneumoniae and skin infections. Oxazolidinones inhibit the bacterial protein synthesis prior to the chain initiation step, by binding to the 23S rRNA of 50S ribosomal subunit, and interfering with the initiator fMet–tRNA binding to the P-site of the ribosomal peptidyltransferase centre

Ranbezolid hydrochloride, RBx-7644

US2005209248

9-23-2005

Plymorphic forms of phenyl oxazolidinone derivatives

The title compound is prepared by reductive alkylation of the known piperazinyl oxazolidinone derivative (I) with 5-nitro-2-furfural (II) in the presence of NaBH(OAc)3, followed by conversion to the corresponding hydrochloride salt.

EP 1303511; US 2002103186; WO 0206278; WO 0307870; WO 0308389

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synthesis

The antibacterial activity of RBx-7644 is due to the 5(S)-acetamidomethyl configuration at the oxazolidinone ring, and thus, asymmetric synthesis of only the 5(S)-enantiomer was desirable: 3,4-Difluoronitrobenzene (I) is condensed with piperazine in acetonitrile to give 4-(2-fluoro-4-nitrophenyl)-piperazine (II) as a light yellow compound. Compound (II) is dissolved in dichloromethane and triethylamine, followed by the addition of Boc-anhydride, to provide compound (III). 4-(tert-Butoxycarbonyl)-1-(2-fluoro-4-nitrophenyl)piperazine (III), upon hydrogenation with H2 over Pd/C in methanol at 50 psi, yields 4-(tert-butoxycarbonyl)-1-(2-fluoro-4-aminophenyl)piperazine (IV) as a dark solid. Compound (IV) reacts with benzylchloroformate in dry THF in the presence of solid sodium bicarbonate to afford the desired compound (V). 4-(tert-Butoxycarbonyl)-1-[2-fluoro-4-(benzyloxycarbonylamino)phenyl]piperazine (V), upon treatment with n-BuLi and (R)-glycidyl butyrate at -78 癈, gives the desired (R)-(-)-3-[3-fluoro-4-[4-(tert-butoxycarbonyl)piperazin-1-yl]phenyl]-5-(hydroxymethyl)-2-oxazolidinone (VI). The hydroxymethyl compound (VI) is treated with methanesulfonyl chloride in dichloromethane in the presence of triethylamine to give (R)-(-)-3-[3-fluoro-4-[4-(tert-butoxycarbonyl)piperazin-1-yl]phenyl]-5-(methylsulfonyloxymethyl)-2-oxazolidinone (VII). The sulfonyl derivative (VII) is treated with sodium azide in dimethylformamide to provide the azide (VIII) as a white solid. (R)-(-)-3-[3-Fluoro-4-[4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl]-5-(azidomethyl)-2-oxazolidinone (VIII), upon hydrogenation with H2 over Pd/C at 45 psi, gives (S)-(-)-3-[3-fluoro-4-[4-(tert-butoxycarbonyl)-piperazin-1-yl]phenyl]-5-(aminomethyl)-2-oxazolidinone (IX). The aminomethyl compound (IX), upon treatment with acetic anhydride in dichloromethane in the presence of triethylamine, affords the acetamide derivative (X). The acetamidomethyl-oxazolidinone derivative (X), upon treatment with trifluoroacetic acid, gives (S)-(-)-3-[3-fluoro-4-(1-piperazinyl)phenyl]-5-(acetamidomethyl)-2-oxazolidinone, which, without isolation, is treated with 5-nitro-2-furaldehyde in the presence of sodium triacetoxy borohydride to provide compound (XI). Compound (XI), upon treatment with ethanolic HCl, affords RBx-7644 as a light yellow crystalline solid.

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polymorphs

http://www.google.com/patents/US20050209248

(S)-N-[[3-fluoro-4-[N-1[4-{2-furyl-(5-nitro)methyl}]piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamidehydrochloride having the Formula I.

The compound of Formula I, namely, (S)-N-[[3-fluoro-4-[N-1 [4-{2-furyl-(5-nitro)methyl}] piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide hydrochloride is a phenyl oxazolidinone derivative, as disclosed in PCT application WO 02/06278. It is said to be useful as antimicrobial agent, effective against a number of human and veterinary pathogens, including gram-positive aerobic bacteria, such as multiply resistant staphylococci, streptococci and enterococci as well as anaerobic organisms such as Bacterioides spp. andClostridia spp. species, and acid fast organisms such as Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium spp.

The PCT application WO 02/06278 describes the preparation of compounds of Formula I. The products of Formula I obtained by following the cited methods tend to be hygroscopic and difficult to filter. These types of disadvantageous properties have proven to be serious obstacles to the large-scale manufacture of a compound. Further, handling problems are encountered during the preparation of pharmaceutical compositions comprising the hygroscopic compound of Formula I obtained by following the method disclosed in WO 02/06278.

EXAMPLE 1 Preparation of Polymorphic ‘Form A’ of the Compound of Formula I

50 gm of free base of Formula I was dissolved in ethanol (750 ml) by heating at about 60° C. and to this solution was added ethanolic HCl (13.36 ml, 8.9 N) at about 45-50° C. The reaction mixture was cooled to about 10° C., and stirred for about 4 hours. The separated solid was filtered off and dried under vacuum at 60° C. The solid was then digested in ethanol (150 ml) at 70-80° C. for about 4 hours. It was then cooled to about 10° C., the solid was filtered and dried under vacuum at 60-65° C. to give 30 gm of the pure polymorphic ‘Form A’ of compound of Formula I.

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Synthesis and SAR of novel oxazolidinones: Discovery of ranbezolid

Bioorg Med Chem Lett 2005, 15(19): 4261

http://www.sciencedirect.com/science/article/pii/S0960894X05008310

Synthesis and SAR of novel oxazolidinones: Discovery of ranbezolid

Pages 4261-4267
Biswajit Das, Sonali Rudra, Ajay Yadav, Abhijit Ray, A.V.S. Raja Rao, A.S.S.V. Srinivas, Ajay Soni, Suman Saini, Shalini Shukla, Manisha Pandya, Pragya Bhateja, Sunita Malhotra, Tarun Mathur, S.K. Arora, Ashok Rattan, Anita Mehta

Graphical abstract

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the ‘piperazinyl–phenyl–oxazolidinone’ core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible Gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.

Substitution of five-membered heterocycles on to the ‘piperazinyl–phenyl–oxazolidinone’ core structure led to the identification of ranbezolid as a clinical candidate. Further replacement of piperazine ring with other diamino-heterocycles led to compounds with potent antibacterial activity.

Scheme 5.

Reagents and conditions: (a) Method A: TFA, CH₂Cl₂, 0 °C → rt; 5-chloromethyl-2-furaldehyde, potassium carbonate, DMF, rt; or (b) Method B: TFA, CH₂Cl₂, 0 °C → rt; 5-nitrofuran-2-carboxaldehyde, sodiumtriacetoxyborohydride, THF, molecular sieves 3 Å, rt. 7 = ranbezolid

Synthesis of compound 7: (S)-N-[[3-[3-Fluoro-4-(N-4-tert-butoxycarbonyl-piperazin-1-yl)phenyl]-2-oxo-5-oxa-zolidinyl]-methyl]acetamide (28a, 3.65 kg, 8.37 mol) was dissolved in dichloromethane (30.86 L) and cooled to 5 °C. To it trifluoroacetic acid (6.17 L) added dropwise and stirred for 14 h allowing the reaction mixture to warm to rt. The reaction mixture was evaporated in vacuo and the residue dissolved in tetrahydrofuran (58 L) followed by addition of molecular sieves 4 Å (4.2 kg). To the resulting mixture 5-nitro-2-furaldehyde (1.5 kg, 10.77 mol) was added followed by sodium triacetoxyborohydride (5.32 kg, 25.1 mol) and stirred for 14 h. The reaction mixture was filtered over Celite and filtrate evaporated in vacuo. The residue was dissolved in ethylacetate (85.6 L) and washed with satd sodium bicarbonate solution (36 L) and water (36 L). The organic layer was dried over anhyd sodium sulfate (3 kg) and evaporated in vacuo. The crude residue was purified by column chromatography (1–3% methanol in ethylacetate) to obtain (S)-N-[[3-[3-fluoro-4-[N-4-(5-nitro-2-furylmethyl)-piperazin-1-yl]phenyl]-2-oxo-5-oxa-zolidinyl]methyl]acetamide (39, 2.6 kg, yield 67%). Mp: 136 °C. ¹H NMR (CDCl₃): δ 7.42 (dd, 1H, phenyl–H), 7.29 (m, 2H, furyl–H), 7.07 (d, 1H, phenyl–H), 6.92 (t, 1H, phenyl–H), 6.51 (d, 1H, furyl–H), 6.11 (t, 1H, –NHCO–), 4.77 (m, 1H, oxazolidinone ring C₅–H), 4.01 (t, 1H), 3.85–3.45 (m, 5H), 3.09 (m, 4H, piperazine–H), 2.72 (m, 4H, piperazine–H), 2.02 (s, 3H, –COCH₃). MS m/z (rel. int.): 462.1 [(M+H)⁺, 100%], 484 [(M+Na)⁺, 25%], 500.2 [(M+K)⁺, 20%]. HPLC purity: 98%.
Compound 39(3.6 kg, 7.81 mol) was dissolved in abs ethanol (53.8 L) by heating to 60 °C. The resulting solution was cooled to 45 °C and ethanolic hydrochloride (1.48 L, 7.9 N) was added dropwise in 10 min. The mixture was then cooled to 10 °C and stirred for 4 h and the precipitate formed was filtered and washed with ethanol and dried to obtain (S)-N-[[3-[3-fluoro-4-[N-4-(5-nitro-2-furylmethyl)-piperazin-1-yl]phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide hydrochloride, ranbezolid (7, 3.2 kg, yield from 39: 82%, yield from 28a: 55%).
Ranbezolid
Mp: 207–209 °C.
¹H NMR (DMSO, 300 MHz): δ 8.30 (t, 1H, –NHCO–), 7.75 (d, J = 3.3 Hz, 1H, furyl–H), 7.52 (dd, 1H, phenyl–H), 7.3–7.0 (m, 3H, phenyl–H, furyl–H), 4.70 (m, 1H, oxazolidinone ring C₅–H), 4.63 (s, 2H), 4.08 (t, J = 8.8 Hz, 1H, –CH₂–), 3.73 (t, J = 7.5 Hz, 1H), 3.43 (br m, piperazine–H merged with H₂O in DMSO), 1.83 (s, 3H, –COCH₃).
HPLC purity: 98%. Anal. Calcd for C₂₁H₂₅ClN₅O₆·0.5H₂O: C, 50.76; H, 5.48; N, 14.09. Anal. Found: C, 50.83; H, 5.17; N, 13.83.

References

European Journal of Pharmacology. 2006. 545, 167–172
US2005209248, 9-23-2005
Plymorphic forms of phenyl oxazolidinone derivatives

23142085	1-1-2013	Anti-anaerobic potential of ranbezolid: insight into its mechanism of action against Bacteroides fragilis.	International journal of antimicrobial agents
19811911	11-15-2009	Synthesis and biological activity of novel oxazolidinones.	Bioorganic & medicinal chemistry letters
19075051	4-1-2009	Mode of action of Ranbezolid against staphylococci and structural modeling studies of its interaction with ribosomes.	Antimicrobial agents and chemotherapy
18676219	8-1-2008	Effect of oxazolidinone, RBx 7644 (ranbezolid), on inhibition of staphylococcal adherence to plastic surfaces.	Journal of chemotherapy (Florence, Italy)
18622683	4-1-2008	Utilization of Bombyx mori larvae as a surrogate animal model for evaluation of the anti-infective potential of oxazolidinones.	Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy
17624776	9-15-2007	Synthesis and in vitro antibacterial activity of novel methylamino piperidinyl oxazolidinones.	Bioorganic & medicinal chemistry letters
16899241	9-18-2006	Ranbezolid, a novel oxazolidinone antibacterial: in vivo characterisation of monoamine oxidase inhibitory potential in conscious rats.	European journal of pharmacology
16054358	10-1-2005	Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid.	Bioorganic & medicinal chemistry letters
15885988	6-1-2005	Activity of RBx 7644 and RBx 8700, new investigational oxazolidinones, against Mycobacterium tuberculosis infected murine macrophages.	International journal of antimicrobial agents
15380263	10-1-2004	In vitro activity of RBx 7644 (ranbezolid) on biofilm producing bacteria.	International journal of antimicrobial agents

12604558	3-1-2003	Antianaerobe activity of RBX 7644 (ranbezolid), a new oxazolidinone, compared with those of eight other agents.	Antimicrobial agents and chemotherapy
12604559	3-1-2003	Antipneumococcal and antistaphylococcal activities of ranbezolid (RBX 7644), a new oxazolidinone, compared to those of other agents.	Antimicrobial agents and chemotherapy

MK 2048 an HIV integrase inhibitor from Merck

Uncategorized Comments Off

Apr 022014

MK 2048

Molecular Formula: C₂₁H₂₁ClFN₅O₄ Molecular Weight: 461.873943

869901-69-9, 3oyl, 3oyn

Merck & Co., Inc.

(6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-1,9-dioxo-1,2,6,7,8,9-hexahydropyrazino‌[1′,2′:1,5]‌pyrrolo‌[2,3-d]‌pyridazine-4-carboxamide

6(S)-2-(3-Chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-l,9-dioxo-l,2,6,7,8,9- hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide

US7538112

5-27-2009

Hiv Integrase Inhibitors

MK-2048 is a second generation integrase inhibitor, intended to be used against HIV infection. It is superior to the first available integrase inhibitor,raltegravir, in that it inhibits the HIV enzyme integrase 4 times longer. It is being investigated for use as part of pre-exposure prophylaxis (PrEP). ^[1]

It is being developed by Merck & Co.^[2]

MK-2048 is a second generation integrase inhibitor for HIV-1 integrase. MK-2048 inhibits subtype B and subtype C integrase activities. MK-2048 inhibits R263K mutants slightly more effectively than G118R mutants.

MK-2048 inhibits S217H intasome and, by contrast, MK2048 remains fully active against the N224H intasome. MK2048 displays substantially lower dissociation rates compared with raltegravir, another integrase inhibitor.

MK-2048 is active against viruses resistant to RAL and EVG. MK-2048 exposure leads to the selection of G118R as a possible novel resistance mutation after 19 weeks. MK-2048, with continued pressure, subsequently leads to an additional substitution, at position E138K, after 29 weeks, within the IN gene.

Although the G118R mutation alone confers only slight resistance to MK-2048 but not to RAL or EVG, its presence arouses a dramatic reduction in viral replication capacity compared to wild-type NL4-3. E138K both partially restores viral replication capacity and also contributes to increased levels of resistance against MK-2048.

Structure of MK-2048 with important pharmacophore highlighted

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Synthesis

WO2005110415A1

http://www.google.as/patents/WO2005110415A1?cl=en

EXAMPLE 62 6(S)-2-(3-Chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-l,9-dioxo-l,2,6,7,8,9- hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide

Step 1: te rt-Butyl[( 1 S)-2-(ethylamino)- 1 -methylethyl] carbamate To a cold (0 °C) solution of N-(tø/ -butoxycarbonyl)-L-alanine N’-methoxy-N’- methylamide (15.6 g, 67.2 mmol) in anhydrous THF (150 mL) and diethyl ether (400 mL), solid lithium aluminum hydride (5.1 g, 134.3 mmol) was added portionwise over a period of 30 minutes. The mixture was stirred at room temperature for 3 hours and cooled back to 0 °C. The reaction was treated carefully with an aqueous solution of potassium hydrogen sulfate (250 mL, 1M). The resultant mixture was diluted with diethyl ether.

The organic extract was washed successively with dilute hydrochloric acid, and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to provide the corresponding aldehyde as colorless solid. Without further purification, a cold (0 °C), stirred solution of the intermediate aldehyde (10.7 g, 61.8 mmol) and ethylamine hydrogen chloride (10.1 g, 123.5 mmol) in methanol (72 mL) was treated with sodium triacetoxyborohydride (17.2 g, 80.9 mmol) in one portion. The mixture was allowed to warm up to room temperature.

After stirring at room temperature overnight, the solution was concentrated under vacuum. The residue was partitioned between diethyl ether and cold aqueous sodium hydroxide (1.5 M). The ethereal extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to provide the titled compound. lH NMR (400 MHz, CDCI3) δ 4.68 (br s, IH), 3.75 (br t, IH), 2.62 (m, 5 H), 1.13 (d, J = 6.7 Hz, 3H),

1.09 (t, J = 7.0 Hz, 3H). ES MS M+l = 203

Step 2: ført-Butyl { ( 1 S)-2-[(bromoacetyl)ethylamino] – 1 -methylethyl } carbamate To a cold (0 °C) stirred solution of ?ert-butyl[(lS)-2-(ethylamino)-l- methylethyl]carbamate (11.0 g, 54.6 mmol) in a mixture of ethyl acetate (107 mL) and saturated aqueous sodium bicarbonate (65 mL), bromoacetyl bromide (12.1 g, 60.0 mmol) was added portionwise under an atmosphere of nitrogen. The mixture was allowed to warm up to room temperature over a period of 3.5 hours. The organic phase was separated, washed successively with saturated aqueous sodium bicarbonate, and brine. The organic extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was concentrated as a solution in toluene under vacuum to afford the title compound. ES MS M+l = 323, 325.

Step 3: fe7 -Butyl (2S)-4-ethyl-2-methyl-5-oxopiperazine-l-carboxylate To a stirred slurry of sodium hydride (1.7 g, 69.8 mmol) in anhydrous THF (800 mL), a solution of tert-butyl{(lS)-2-[(bromoacetyl)ethylamino]-l-methylethyl}carbamate (17.4 g, 53.7 mmol) in anhydrous THF (100 mL) was added dropwise over a period of 1 hour under an atmosphere of nitrogen. The reaction mixture was stirred at room temperature for two hours, cooled in an ice-water bath, and quenched with dropwise addition of aqueous citric acid (80 mL, 1M). The mixture was concentrated under vacuum. The residue was partitioned between chloroform and saturated aqueous sodium bicarbonate. The organic extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was subjected to column chromatography on silica gel eluting with a gradient of 0-15% acetonitrile in chloroform. Collection and concentration of appropriate fractions provided the title compound. lH NMR (400 MHz, CDCI3) δ 4.46 (br s, IH), 4.24 (d, J = 18.4 Hz, 1 H), 3.78 (d, J = 18.4 Hz, 1 H),

3.64 (dd, J = 12.3, 4.2 Hz, 1 H), 3.54 (heptet, J = 7.1 Hz, 1 H), 3.38 (heptet, J = 7.1 Hz, 1 H), 2.99 (dd, J = 12.3, 1.8 Hz, 1 H), 1.47 (s, 9H), 1.21 (d, J = 6.8 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H). ES MS M+l = 243.

Step 4: (5S)-l-Ethyl-5-methylpiperazin-2-one hydrochloride Anhydrous hydrogen chloride gas was bubbled into a cold (-20 °C) solution of tert-butyl (2S)-4-ethyl-2-methyl-5-oxopiperazine-l-carboxylate (10.5 g, 43.4 mmol) in ethyl acetate (250 mL) under nitrogen. After the solution was saturated with hydrogen chloride, the reaction mixture was stirred in an ice-water bath for 30 minutes. The product mixture was purged with nitrogen, concentrated under vacuum to provide the title hydrogen chloride salt as pale yellow solid. lH NMR (400 MHz, DMSO-d6) δ 10.00 (br d, 2H), 3.72 (d, J = 16.6 Hz, 1 H), 3.62(d, J = 16.6 Hz, 1 H),

3.49-3.35 (m, 5 H), 3.29 (heptet, /= 7.3 Hz, 1 H), 1.31 (d, / = 6.6 Hz, 3H), 1.05 (t, J = 7.1 Hz, 3H).

Step 5: Ethyl (4S)-2-ethyl-8-hydroxy-4-methyl-l-oxo-l,2,3,4-tetrahydropyrrolo[l,2-a]pyrazin-7- carboxy late Anhydrous ammonia gas was bubbled into a cold (0 °C) solution of (5S)-l-Ethyl-5- methylpiperazin-2-one hydrochloride (5.8 g, 32.3 mmol) in chloroform for 30 minutes. The resultant slurry was filtered and concentrated under vacuum. The residual oil was concentrated as a solution in toluene under vacuum, redissolved in toluene (120 mL) and treated with diethyl ethoxymethylenemalonate (7.0 g, 32.3 mmol) and heated in a sealed flask in an oil bath at 100 °C overnight. The resultant solution was concentrated under vacuum. The residual oil was concentrated as a solution in toluene under vacuum to provide the corresponding diethyl { [(2S)-4-ethyl-2-methyl-5- oxopiperazin-l-yl]methylene}malonate. Without further purification, to a solution of the malonate (10.5 g, 33.5 mmol) in anhydrous THF (330 mL) warmed with an external oil bath at 65 °C under an atmosphere of nitrogen, a solution of lithium bis(trimethylsilyl)amide (35.1 mL, 1 M, 35.1 mmol) was added. The solution was heated at the same temperature for one hour and concentrated under vacuum. The residue was partitioned between dichloromethane and hydrochloric acid (1M). The organic extract was washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The residue was triturated with diethyl ether. The solid precipitated was filtered, washed with diethyl ether to provide the title compound as pale brown solid. lH NMR (400 MHz, CDCI3) δ 8.43 (s, IH), 7.11 (s, IH), 4.32 (q, J = 7.1 Hz, 2H), 4.24 (m, IH), 3.65-

3.35 (m, 4H), 1.51 (d, J = 6.4 Hz, 3H), 1.36 (t, J = 7.0 Hz, 3H), 1.19 (t, J = 7.0 Hz, 3H). ES MS M+l = 267

Step 6: Ethyl (4S)-2-ethyl-8-methoxy-4-methyl-l-oxo-l,2,3,4-tetrahydropyrrolo[l,2-a]pyrazin-7- carboxylate A mixture of ethyl (4S)-2-ethyl-8-hydroxy-4-methyl-l -oxo- 1,2,3, 4-tetrahydropyrrolo[ 1,2- a]pyrazin-7-carboxylate (6.6 g, 24.8 mmol), anhydrous potassium carbonate (13.7 g, 99.1 mmol, 325 mesh), and iodomethane (4.2 g, 29.7 mmol) in anhydrous DMF (123 mL) was stirred at room temperature overnight. The mixture was filtered and concentrated under vacuum. The residue was partitioned between chloroform and dilute hydrochloric acid. The organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was subjected to column chromatography on silica gel eluting with a gradient of 0-3% methanol in chloroform. Collection and concentration of appropriate fractions provided the title compound. Residual methanol was removed by concentrating from its solution in toluene under vacuum. lH NMR (400 MHz, CDCI3) δ 7.19 (s, IH), 4.29 (q, J = 7.1 Hz, 2 H), 4.24 (m, IH), 4.03 (s, 3H), 3.70-

3.32 (m, 4 H), 1.52 (d, J = 6.6 Hz, 3H), 1.35 (t, J = 7.0 Hz, 3H), 1.19 (t, J = 7.2 Hz, 3H). ES MS M+l = 281

Step 7: Ethyl (4S)-6-bromo-2-ethyl-8-methoxy-4-methyl-l-oxo-l,2,3,4-tetrahydropyrrolo[l,2- a]pyrazin-7-carboxylate To a mixture of ethyl (4S)-2-ethyl-8-(methoxy)-4-methyl-l-oxo-l,2,3,4- tetrahydropyrrolo[l,2- ]pyrazine-7-carboxylate (6.2 g, 22.1 mmol) and sodium bicarbonate (20.0 g, 238.0 mmol) in dichloromethane (500 mL) at 0 °C, a solution of bromine in dichloromethane (24.2 mmol, 0.5 M) was added over a period of 60 minutes. The reaction mixture was stirred at room temperature for 2 h, filtered, and concentrated under vacuum. The residue was subjected to column chromatography on silica gel eluted with ethyl acetate. Collection and concentration of appropriate fractions provided the corresponding bromide. Residual ethyl acetate was removed by concentrating from its solution in benzene under vacuum. lH NMR (400 MHz, CDCI3) δ 4.58 (br m, IH), 4.34 (m, IH), 3.99 (s, 3H), 3.92 (dd, J = 13.0, 4.0 Hz,

IH), 3.67 (heptet, J = 7.1 Hz, 1 H), 3.49 (heptet, J = 7.1 Hz, 1 H), 3.23 (d, J = 13.0 Hz, IH), 1.40 (d, J = 7.1 Hz, 3H), 1.38 (t, 7 = 7.0 Hz, 3H), 1.20 (t, J = 7.0 Hz, 3H). ES MS M+l = 359, 361.

Step 8: Ethyl (4S)-2-ethyl-8-(methoxy)-6-[methoxy(oxo)acetyl]-4-methyl-l-oxo-l,2,3,4- tetrahydropyrrolo[ 1 ,2- ]pyrazine-7-carboxylate To a cold (-78 °C) solution of ethyl (4S)-6-bromo-2-ethyl-8-methoxy-4-methyl-l-oxo- l,2,3,4-tetrahydropyrrolo[l,2-a]pyrazin-7-carboxylate (8.51 g, 23.7 mmol) in anhydrous THF (800 mL) under an atmosphere of dry nitrogen, a solution of n-BuLi in hexane (10.5 mL, 26.3 mmol, 2.5 M) was added. The resultant mixture was stirred at -78 °C for 20 minutes. A solution of dimethyl oxalate (6.4 g, 53.8 mmol; dried from concentration from benzene under vac) in anhydrous THF (30 mL) was added. The reaction mixture was stirred at -78 °C for 1 hour and cannulated into a mixture of aqueous sulfuric acid (240 mL, 2M) and THF (200 mL) maintained between at -5 to -35 °C. The mixture was extracted with ethyl acetate (3 times). The organic extracts were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was subjected to column chromatography on silica gel eluted with 40 to 100% ethyl acetate- hexane gradient. Collection and concentration of appropriate fractions provided the titled compound. lH NMR (400 MHz, CDCI3) δ 5.07 (m, IH), 4.29 (q, J = 7.2 Hz, 2H), 4.00 (s, 3H), 3.99-3.93 (m, IH), 3.89 (s, 3H), 3.74-3.66 (m, IH), 3.53-3.48 (m, IH), 3.23 (dd, J = 1.3, 13.2 Hz, IH), 1.46 (d, J = 6.6 Hz, 3H), 1.36 (t, J = 7.2 Hz, 3H), 1.22 (t, 7= 7.1 Hz, 3H). ES MS M+l = 367

Step 9: (6S)-8-Ethyl-10-methoxy-6-methyl-l,9-dioxo-l,2,6,7,8,9- hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carbohydrazide A mixture of ethyl (4S)-2-ethyl-8-(methoxy)-6-[methoxy(oxo)acetyl]-4-methyl-l-oxo- l,2,3,4-tetrahydropyrrolo[l,2-α]pyrazine-7-carboxylate (3.3 g, 8.9 mmol) and anhydrous hydrazine (1.7 mL, 53.7 mmol) in methanol (400 mL) was stirred at room temperature for one hour. The reaction mixture was concentrated under vacuum. The residue was concentrated from toluene. The resultant gummy solid was treated with methanol (20 mL). Diethyl ether was added to the resultant slurry which was filtered to provide the title compound as white solid. lH NMR (400 MHz, CDCI3) δ 8.99 (br s, 2H), 5.54 (br m, IH), 4.12 (m, IH), 4.10 (s, 3H), 3.81 (m, IH),

3.39 (m, IH), 3.21 (d, 7 = 12.6 Hz, IH), 1.44 (d, 7 = 6.4 Hz, 3H), 1.23 (t, 7 = 7.3 Hz, 3H). ES MS M+l =

335

Step 10: (6S)-8-Ethyl-10-methoxy-N,6-dimethyl-l,9-dioxo-l,2,6,7,8,9- hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide To a solution of (6S)-8-ethyl-10-methoxy-6-methyl-l,9-dioxo-l,2,6,7,8,9- hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carbohydrazide (0.39 g, 1.2 mmol) and methylamine (5.9 mL, 11.8 mmol; 2 M in THF) in anhydrous dichloromethane (25 mL) in a water bath at room temperature, a solution of iodine (0.60 g, 2.4 mmol) in dichloromethane was added dropwise.

After the addition was completed, an aqueous solution of sodium sulfite was added and the mixture was stirred vigorously for 10 minutes. The organic phase was separated, diluted with chloroform, and washed with brine. The organic extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was triturated with a mixture of ethanol (7 mL) and diethyl ether (25 mL). The white solid precipitated was obtained by filtration and dried from its solution in toluene under vacuum. 1H NMR (400 MHz, CDCI3) δ 11.57 (s, IH), 7.38 (m, IH), 5.95 (br m, IH), 4.17 (s, 3H), 4.03 (dd, 7 =

13.4, 3.8 Hz, 1 H), 3.76 (heptet, 7 = 7.1 Hz, 1 H), 3.50 (heptet, 7 = 7.1 Hz, 1 H), 2.99 (dd, 7 = 12.9, 1.0 Hz, 1 H), 3.03 (d, 7 = 5.0 Hz, 3H), 1.44 (d, 7 = 6.6 Hz, 3H), 1.23 (t, 7 = 7.2 Hz, 3H). ES MS M+l = 334 Step 11: (6S)-2-(3-Chloro-4-fluorobenzyl)-8-ethyl-10-methoxy-N,6-dimethyl-l,9-dioxo- l,2,6,7,8,9-hexahydropyrazino[r,2′: l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide To a cold (0 °C) solution of (6S)-8-ethyl-10-methoxy-N,6-dimethyl-l,9-dioxo- l,2,6,7,8,9-hexahydropyrazino[l’,2′: l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide (1.58 g, 4.73 mmol) in anhydrous DMF (50 mL), a solution of lithium bis(trimethylsilyl)amide (4.97 mL, 4.97 mmol, 1 M in THF) was added. After stirring at the same temperature for 25 minutes, 3-chloro-4-fluorobenzyl bromide (1.27 g, 5.68 mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes and concentrated under vacuum. The residue was partitioned between chloroform and brine. The organic extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was subjected to column chromatography on silica gel eluting with a 1-5% methanol in ethyl acetate gradient. Collection and concentration of appropriate fractions provided the title compound. lH NMR (400 MHz, CDCI3) δ 7.46 (dd, 7 = 6.9, 2.2 Hz, IH), 7.32 (m, IH), 7.09 (t, 7 = 7.6 Hz, IH), 7.03

(br signal, IH), 5.92 (m, IH), 5.32 (d, 7 = 14.1 Hz, IH), 5.26 (d, 7= 14.1 Hz, IH), 4.14 (s, 3H), 3.97 (dd, 7 = 13.2, 3.7 Hz, IH), 3.73 (heptet, 7 = 7.2 Hz, 1 H), 3.51 (heptet, 7 = 7.1 Hz, IH), 3.21 (dd, 7= 13.2, 1.7 Hz, IH), 3.03 (d, 7 = 5.0 Hz, 3H), 1.42 (d, 7 = 6.6 Hz, 3H), 1.23 (t, 7 = 7.1 Hz, 3H). ES MS M+l = 476

Step 12:

(6S)-2-(3-Chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-l,9-dioxo- l,2,6,7,8,9-hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d3pyridazine-4-carboxamide

To a solution of (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-methoxy-N,6-dimethyl-l,9- dioxo-l,2,6,7,8,9-hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide (1.15 g, 2.41 mmol) in anhydrous dichloromethane (800 mL), a solution of boron tribromide in dichloromethane (3.14 mL, 3.14 mmol; 1 M) was added. After stirring at room temperature for 5 minutes, the reaction mixture was treated with anhydrous methanol, stirred for 30 minutes, and concentrated under vacuum. The procedure was repeated twice. The residue was dissolved in a mixture of methanol and acetonitrile and treated with aqueous sodium hydroxide. The mixture was subjected to purification on preparative reverse phase high pressure column chromatography. Collection and lyophilization of appropriate fractions provided the title compound as white amorphous solid.

MK 2048

lH NMR (400 MHz, CDCI3) δ 7.48 (dd, 7 = 7.0, 2.2 Hz, IH), 7.33 (m, IH), 7.09 (t, 7 = 8.7 Hz, IH), 6.01 (m, IH), 5.33 (d, 7= 14.1 Hz, IH), 5.27 (d, 7 = 14.1 Hz, IH), 3.99 (dd, 7= 12.8, 4.0 Hz, 1 H), 3.71(heptet, 7 = 7.1 Hz, 1 H), 3.49 (heptet, 7 = 7.1 Hz, 1 H), 3.24 (dd, 7 = 13.2, 1.5 Hz, 1 H), 3.03 (d, 7 = 5.1 Hz, 3H), 1.42 (d, 7 = 6.6 Hz, 3H), 1.24 (t, 7 = 7.3 Hz, 3H). ES MS M+l = 462

The amorphous product was dissolved in boiling methanol (1.4 g/200 mL). Upon cooling in an ice-water bath, a precipitate formed which was separated by obtained by filtration to afford a white crystalline solid.

MK 2048sodium salt

The corresponding sodium salt was prepared by treatment of a solution of (6S)-2-(3- chloro-4-fluorobenzyl)-8-ethyl- 10-hydroxy-N,6-dimethyl-l ,9-dioxo- 1 ,2,6,7,8,9- hexahydropyrazino[r,2′:l,5]pyrrolo[2,3-d]pyridazine-4-carboxamide (920 mg, 1.99 mmol) in aqueous acetonitrile with aqueous sodium hydroxide (1.03 equivalent), followed by lyophilization of the resultant solution.

ChemSpider 2D Image | (5S)-1-Ethyl-5-methylpiperazin-2-on | C7H14N2O

(5S)-1-ethyl-5-methylpiperazin-2-one

1,5-Cyclooctadiene-iridium(I) chloride dimer, Chloro(1,5-cyclooctadiene)iridium(I) dimer, Di-μ-chlorobis[(1,2,5,6-η)-1,5-cyclooctadiene]diiridium, Iridium(I) chloride 1,5-cyclooctadiene complex dimer, [Ir(1,5-cod)Cl]2, [Ir(1,5-cod)Cl]₂, [Ir(cod)Cl]2

(S)-1-[(R)-2-Di-(4-methoxy-3,5-dimethylphenyl-phosphino)ferrocenyl]-ethyl-dicyclohexylphosphine

SL-J006-2

(5S)-l-Ethyl-5-methylpiperazin-2-one was alternatively prepared as follows:

Step 1: N^rf-Butoxycarbonyl-N^ethylglycinamide Ethylamine (37 g, 0.82 mol) was condensed into a pressure vessel at 0 °C. N-(tert- butoxycarbonyl)glycine methyl ester (50 mL, 0.34 mol) was added. The vessel was sealed and the mixture was stirred at room temperature overnight. The product mixture was concentrated under vacuum and the residue was passed through a pad of silica gel eluting with ethyl acetate. The solution was concentrated under vacuum to provide the title compound as a clear oil. lH NMR (400 MHz, CDCI3) δ 6.11 (br s, IH), 5.18 (br s, IH), 3.77 (d, 7 = 5.7 Hz, 2H), 3.31 (q, 7 = 7.1

Hz, 2H), 1.15 (t, 7 = 7.1 Hz, 3H).

Step 2: l-Ethyl-5-methylpyrazin-2(lH)-one A cold (0 °C) solution of N^tø^butoxycarbonyl-N^ethylglycinamide (68.0 g, 0.33 mol) in anhydrous dichloromethane (500 mL) was saturated with anhydrous hydrogen chloride gas. After stirring at the same temperature for 1.5 hours, the solution was recharged with more hydrogen chloride gas and stirred for additional 15 minutes. The reaction mixture was concentrated under vacuum. The residue was dissolved in methanol, diluted with toluene, and concentrated under vacuum to afford the intermediate N-ethylglycinamide HCI salt.

This was stored under vacuum overnight and used without further purification. A solution of N-ethylglycinamide HCI salt (44.2 g, 0.32 mol), aqueous sodium hydroxide (640 mL, 1M), water (350 mL), pyruvic aldehyde (20.9 mL, 40% solution in water) was heated in an oil bath at 120 °C for one hour. The reaction mixture was cooled and saturated with solid sodium chloride. The mixture was extracted with chloroform (4×250 mL).

The combined organic extract was dried over anhydrous sodium sulfate, filtered, and passed through a plug of silica gel. The silica gel was rinsed successively with ethyl acetate and then 2% methanol in ethyl acetate. The eluted fractions were combined and concentrated under vacuum. The residual solid was recrystallized from diethyl ether to afford the title compound as pale yellow solid. lH NMR (400 MHz, CDCI3) δ 8.11 (s, IH), 6.92 (s, IH), 3.92 (q, 7 = 7.2 Hz, 2H), 2.28 (s, 3H), 1.37 (t, 7 = 7.2 Hz, 3H).

Step 3: (5 S)- 1 -Ethyl-5-methylpiperazin-2-one

A mixture of chloro-l,5-cyclooctadiene iridium (I) dimer (34 mg, 51 μmol) and (S)-l-[(R)-2-di-(3,5-bis(trifluoromethyl)phenyl)phosphino)ferrocenyl]ethyldicyclohexylphosphine (44 mg, 51 μmol; Solvias AG, SL-J006-2) in a mixture of 1:2 toluene and methanol (100 mL; purged with nitrogen for 15 minutes) was sonicated under an atmosphere of nitrogen for 15 minutes. To the resultant mixture, iodine (0.39 g, 1.52 mmol) and l-ethyl-5-methylpyrazin-2(lH)-one (7.0 g, 50.66 mmol) was added. The resultant mixture was heated in an oil bath at 50 °C under an atmosphere of hydrogen gas at 800 psi for 48 hours. The product mixture was filtered through a pad of Celite. The filtrate was concentrated under vacuum. The residue was treated with chloroform saturated with ammonia gas (100 mL). The resultant suspension was filtered through a pad of Celite, which was the rinsed with chloroform saturated with ammonia gas. The combined filtrate was concentrated under vacuum. The residue was concentrated as a solution in toluene for subsequent reaction. lH NMR (400 MHz, CDCI3) δ 3.58 (d, 7 = 17.2 Hz, IH), 3.53(d, 7 = 17.2 Hz, IH), 3.49-3.35 (m, 2H),

1.19 (d, 7 = 5.9 Hz, 3H), 1.14 (t, 7 = 7.2 Hz, 3H).

……………..

US 7538112

http://www.google.com/patents/US7538112

Step 12: (6S)-2-(3-Chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-1,9-dioxo-1,2,6,7,8,9-hexahydropyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyridazine-4-carboxamide

To a solution of (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-methoxy-N,6-dimethyl-1,9-dioxo-1,2,6,7,8,9-hexahydropyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyridazine-4-carboxamide (1.15 g, 2.41 mmol) in anhydrous dichloromethane (800 mL), a solution of boron tribromide in dichloromethane (3.14 mL, 3.14 mmol; 1 M) was added. After stirring at room temperature for 5 minutes, the reaction mixture was treated with anhydrous methanol, stirred for 30 minutes, and concentrated under vacuum. The procedure was repeated twice. The residue was dissolved in a mixture of methanol and acetonitrile and treated with aqueous sodium hydroxide. The mixture was subjected to purification on preparative reverse phase high pressure column chromatography. Collection and lyophilization of appropriate fractions provided the title compound as white amorphous solid.

¹H NMR (400 MHz, CDCl₃) δ 7.48 (dd, J=7.0, 2.2 Hz, 1H), 7.33 (m, 1H), 7.09 (t, J=8.7 Hz, 1H), 6.01 (m, 1H), 5.33 (d, J=14.1 Hz, 1H), 5.27 (d, J=14.1 Hz, 1H), 3.99 (dd, J=12.8, 4.0 Hz, 1 H), 3.71 (heptet, J=7.1 Hz, 1 H), 3.49 (heptet, J=7.1 Hz, 1 H), 3.24 (dd, J=13.2, 1.5 Hz, 1 H), 3.03 (d, J=5.1 Hz, 3H), 1.42 (d, J=6.6 Hz, 3H), 1.24 (t, J=7.3 Hz, 3H). ES MS M+1=462

The corresponding sodium salt was prepared by treatment of a solution of (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-1,9-dioxo-1,2,6,7,8,9-hexahydropyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyridazine-4-carboxamide (920 mg, 1.99 mmol) in aqueous acetonitrile with aqueous sodium hydroxide (1.03 equivalent), followed by lyophilization of the resultant solution.

References

Keith Alcorn. Ralvetgravir shows potential for use as PrEP drug AIDSmap.com. 28 April 2009. Accessed 8 Nov 2009.
Mark Mascolini. Merck Offers Unique Perspective on Second-Generation Integrase Inhibitor. 10th International Workshop on Clinical Pharmacology of HIV Therapy, April 15–17, 2009, Amsterdam. Accessed 8 Nov 2009.

WO2011121105A1	1 Apr 2011	6 Oct 2011	Tibotec Pharmaceuticals	Macrocyclic integrase inhibitors
EP1756114A2 *	3 May 2005	28 Feb 2007	Merck and Co., Inc.	Hiv integrase inhibitors
US7517929	3 Dec 2004	14 Apr 2009	Momentive Performance Materials Inc.	Star-branched silicone polymers as anti-mist additives for coating applications

US5693640 *	6 Jun 1995	2 Dec 1997	Merck, Sharp & Dohme, Ltd.	Pyridazino-indole derivatives
US5756501 *	3 Dec 1996	26 May 1998	American Home Products Corporation	Saturated and unsaturated pyridazino 4,5-B! indolizines useful as antidementia agents

SEROTONIN..makes me feel happy

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Mar 302014

Serotonin /ˌ s ɛr ə ˈ t oʊ n ɨ n / or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter. Biochemically derived from tryptophan, serotonin is primarily found in the gastrointestinal (GI) tract, platelets, and the central nervous system (CNS) of animals, including humans. It is popularly thought to be a contributor to feelings of well-being and happiness.^[6]

In 1935, Italian Vittorio Erspamer showed an extract from enterochromaffin cells made intestines contract. Some believed it contained adrenaline, but two years later, Erspamer was able to show it was a previously unknown amine, which he named “enteramine”. In 1948, Maurice M. Rapport, Arda Green, and Irvine Page of the Cleveland Clinic discovered a vasoconstrictor substance inblood serum, and since it was a serum agent affecting vascular tone, they named it serotonin.

In 1952, enteramine was shown to be the same substance as serotonin, and as the broad range of physiological roles was elucidated, the abbreviation 5-HT of the proper chemical name 5-hydroxytryptamine became the preferred name in the pharmacological field. Synonyms of serotonin include: 5-hydroxytriptamine, thrombotin, enteramin, substance DS, and 3-(β-Aminoethyl)-5-hydroxyindole.In 1953, Betty Twarog and Page discovered serotonin in the central nervous system.

Approximately 90% of the human body’s total serotonin is located in the enterochromaffin cells in the alimentary canal (gut), where it is used to regulate intestinal movements.^[7]^[8] The remainder is synthesized in serotonergic neurons of the CNS, where it has various functions. These include the regulation of mood, appetite, and sleep. Serotonin also has some cognitive functions, including memory and learning. Modulation of serotonin at synapses is thought to be a major action of several classes of pharmacological antidepressants.

Serotonin secreted from the enterochromaffin cells eventually finds its way out of tissues into the blood. There, it is actively taken up by bloodplatelets, which store it. When the platelets bind to a clot, they release serotonin, where it serves as a vasoconstrictor and helps to regulatehemostasis and blood clotting. Serotonin also is a growth factor for some types of cells, which may give it a role in wound healing.

Serotonin is metabolized mainly to 5-HIAA, chiefly by the liver. Metabolism involves first oxidation by monoamine oxidase to the correspondingaldehyde. This is followed by oxidation by aldehyde dehydrogenase to 5-HIAA, the indole acetic acid derivative. The latter is then excreted by the kidneys. One type of tumor, called carcinoid, sometimes secretes large amounts of serotonin into the blood, which causes various forms of thecarcinoid syndrome of flushing (serotonin itself does not cause flushing. Potential causes of flushing in carcinoid syndrome include bradykinins, prostaglandins, tachykinins, substance P, and/or histamine.), diarrhea, and heart problems. Because of serotonin’s growth-promoting effect on cardiac myocytes,^[9] persons with serotonin-secreting carcinoid may suffer a right heart (tricuspid) valve disease syndrome, caused by proliferation of myocytes onto the valve.

In addition to animals, serotonin is found in fungi and plants.^[10] Serotonin’s presence in insect venoms and plant spines serves to cause pain, which is a side-effect of serotonin injection. Serotonin is produced by pathogenic amoebae, and its effect on the gut causes diarrhea. Its widespread presence in many seeds and fruits may serve to stimulate the digestive tract into expelling the seeds.

Serotonin system, contrasted with thedopamine system

Introduction

Serotonin was first recognised as a powerful vasoconstrictor in blood serum. It was isolated in 1948 by Page and was later found to be associated with the central nervous system.

The chemical name for serotonin is 5-hydoxytryptamine which is often abbreviated to 5-HT.

Serotonin is naturally produced in the Pineal gland which lies deep at the centre of the human brain. The average adult human possesses only 5 to 10 mg of serotonin, 90 % of which is in the intestine and the rest in blood platelets and the brain.

One role of this ‘wonder drug’ is as a neurotransmitter, allowing numerous functions in the human body including the control of appetite, sleep, memory and learning, temperature regulation, mood, behaviour, cardiovascular function, muscle contraction, endocrine regulation and depression. Subsequent to his discovery of Serotonin, Page commented that no physiological substance known possesses such diverse actions in the body as does serotonin.

5-HT is also found in wasp stings and scorpion venom where its function is of an irritant, since intravenous injection of serotonin in humans leads to pain, gasping, coughing, a tingling and prickling sensation, nausea, cramps and other unpleasant symptoms.

Serotonin is manufactured in the human brain using the essential amino acid tryptophan which is found in foods such as bananas, pineapples, plums, turkey and milk.

The enzyme tryptophan hydroxylase adds a hydroxyl group to tryptophan’s benzene ring at position 5, creating 5-hydroxytryptophan. Another enzyme, amino acid decarboxylase, then removes a carboxyl group from 5-hydroxytryptophan, forming 5-hydroxytryptamine which is more commonly known as serotonin.

In animals including humans, serotonin is synthesized from the amino acid L–tryptophan by a short metabolic pathway consisting of two enzymes: tryptophan hydroxylase (TPH) and amino acid decarboxylase (DDC). The TPH-mediated reaction is the rate-limiting step in the pathway. TPH has been shown to exist in two forms: TPH1, found in several tissues, and TPH2, which is a neuron-specific isoform.

Serotonin can be synthesized from tryptophan in the lab using Aspergillus niger and Psilocybe coprophila as catalysts. The first phase to 5-hydroxytryptophan would require letting tryptophan sit in ethanol and water for 7 days, then mixing in enough HCl (or other acid) to bring the pH to 3, and then adding NaOH to make a pH of 13 for 1 hour. Asperigillus niger would be the catalyst for this first phase. The second phase to synthesizing tryptophan itself from the 5-hydroxytryptophan intermediate would require adding ethanol and water, and letting sit for 30 days this time. The next two steps would be the same as the first phase: adding HCl to make the pH = 3, and then adding NaOH to make the pH very basic at 13 for 1 hour. This phase uses the Psilocybe coprophila as the catalyst for the reaction.

Serotonin taken orally does not pass into the serotonergic pathways of the central nervous system, because it does not cross theblood–brain barrier. However, tryptophan and its metabolite 5-hydroxytryptophan (5-HTP), from which serotonin is synthesized, can and do cross the blood–brain barrier. These agents are available as dietary supplements, and may be effective serotonergic agents. One product of serotonin breakdown is 5-hydroxyindoleacetic acid (5-HIAA), which is excreted in the urine. Serotonin and 5-HIAA are sometimes produced in excess amounts by certain tumors or cancers, and levels of these substances may be measured in the urine to test for these tumors.

Serotonin is a neurotransmitter involved in the transmission of nerve impulses. Neurotransmitters are chemical messengers within the brain that allow the communication between nerve cells.

Packets of serotonin (vesicles) are released from the end of the presynaptic cell www.thebrain.mcgill.ca/flash/i/i_01/i_01_m/i_01_m_ana/i_01_m_ana.html into the synaptic cleft. The serotonin molecules can then bind to receptor proteins within the postsynaptic cell, which causes a change in the electrical state of the cell. This change in electrical state can either excite the cell, passing along the chemical message, or inhibit it. Excess serotonin molecules are taken back up by the presynaptic cell and reprocessed.

www.thebrain.mcgill.ca/flash/i/i_01/i_01_m/i_01_m_ana/i_01_m_ana.html

The neurons in the brain that release serotonin are found in small dense collections of neurons called Raphe Nuclei. The Raphe Nuclei are found in the medulla, pons and midbrain which are all located at the top of the spinal cord. Serotonergic neurons have axons which project to many different parts of the brain, therefore serotonin affects many different behaviors.

Low serotonin levels are believed to be the cause of many cases of mild to severe depression which can lead to symptoms such as anxiety, apathy, fear, feelings of worthlessness, insomnia and fatigue. The most concrete evidence for the connection between serotonin and depression is the decreased concentrations of serotonin metabolites in the cerebrospinal fluid and brain tissues of depressed people.

http://www.depression.org/

If depression arises as a result of a serotonin deficiency then pharmaceutical agents that increase the amount of serotonin in the brain should be helpful in treating depressed patients. Anti-depressant medications increase serotonin levels at the synapse by blocking the reuptake of serotonin into the presynaptic cell. Anti-depressants are one of the most highly prescribed medications despite the serious side-effects they can cause.

If depression is mild enough it can sometimes be managed without prescribed medications. The most effective way of raising serotonin levels is with vigorous exercise. Studies have shown that serotonin levels are increased with increased activity and the production of serotonin is increased for some days after the activity. This is the safest way of increasing serotonin levels and many other benefits result from regular exercise.

Serotonin levels can also be controlled through the diet. A diet deficient in omega-3 fatty acids may lower brain levels of serotonin and cause depression. Complex carbohydrates raise the level of tryptophan in the brain resulting in a calming effect. Vitamin C is also required for the conversion of tryptophan into serotonin.

Lysergic acid diethylamide, more commonly known as LSD, is a non-toxic, non-addictive molecule which mimics serotonin in the brain. The body ‘mistakes’ LSD for serotonin and shoots it across the synaptic cleft. LSD has a higher affinity for 5-HT receptors than serotonin, thus the presence of LSD prevents

serotonin from sending neural messages in the brain. Once the LSD molecule is bound to the receptor proteins the message is not carried any further. Instead the impulse is redirected to the older parts of the brain, where the bloodstream then takes it to the sense interpretive centres and the motor areas.

There are many similarities between the molecules of serotonin and LSD which allows this process to occur, the most obvious being their close structural similarities, particularly the indole ring shown highlighted in blue.

Serotonin Lysergic acid diethylamide

Another close similarity between LSD and serotonin is the electron density of the highest occupied molecular orbital. The electron density is lowest in the areas around the indole ring in both molecules. This is indicated by the blue areas in the diagrams.

Serotonin LSD

The dipole moment of the two molecules are very close. Serotonin is 2.98 debye and LSD is 3.04 debye, with the dipole moment going towards the NH₂ group in both molecules. The close similarity in dipole moment is key to the ability of LSD to fit into the same receptors as serotonin.

The combination of all of these chemical similarities allows LSD to imitate serotonin and cause psychedelic hallucinations and visions.

serotonin that is ingested will be broken down by the metabolic enzyme MAO. The second point is that even if large quantities of serotonin were to survive the MAO metabolism, it would not directly increase serotonin levels in the brain. The third part of the answer is that simply increasing serotonin levels does not lead to MDMA-like effects.Serotonin, as a molecule, has two particular characteristics that makes it unlikely to be able to be taken orally and reach the brain with a significant concentration to cause a noticeable effect. A key characteristic for this type of molecule is how the nitrogen dangling off the end (see the structure link above and look for the N that is not in the ring structure) behaves with respect to the number of hydrogens that is bound to it and the electrical charge it has (positive or neutral) depending on its environment. The so-called “terminal nitrogen” (on the end) is a base and will change its state (three hydrogens/positive charge vs. two hydrogens/neutral charge) depending on the pH of the solution it is in (acid/base chemistry). At any given time, in most physiological environments (stomach, blood, inside cells, etc.), most serotonin molecules will be positively charged.Now, to get into the brain, molecules have to pass through a layering of cells that surround the brain’s blood vessels commonly referred to as the “blood-brain barrier”. These cells, as with all the cells in your body, have a membrane made up of fat-like, hydrophobic (water-disliking), “non-polar” chains of molecules stacked up on each other (a bi-layer). While the outside and inside of this membrane bi-layer is hydrophilic (water-liking), charged, and/or “polar”, in order to pass into the cell(s) and/or through the membrane(s), a molecule has to pass through this fat-like region. If the molecule itself is charged or has any groups on it that are water-liking/polar, this is unlikely to happen. Think of trying to push a droplet of water to the bottom of a cup of oil with a fork. It can also be thought of as having to get the chemical to dissolve in the fat-layer to get through it: charged / polar chemicals simply will not “dissolve” and pass through this layer.There are ‘active transporters’ or ‘active carriers’ that act as gateways through the blood-brain barrier for molecules of particular shapes. The normal amino acids (building blocks for the neurotransmitters) such as tryptophan, phenylalanine, tyrosine, etc get through the BBB by having special systems for pushing them through. As a side note, there are fewer of these transporters than there are possible molecules and the same transporters work on many different chemicals, so there is competition for transport into the brain between different chemicals, however this does not really impact the question at hand.

Pietra, S.;Farmaco, Edizione Scientifica 1958, Vol. 13, pp. 75–9.
Calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 (©1994–2011 ACD/Labs)
Mazák, K.; Dóczy, V.; Kökösi, J.; Noszál, B. (2009). “Proton Speciation and Microspeciation of Serotonin and 5-Hydroxytryptophan”. Chemistry & Biodiversity 6 (4): 578–90.doi:10.1002/cbdv.200800087. PMID 19353542.
Erspamer, Vittorio (1952). Ricerca Scientifica 22: 694–702.
Tammisto, Tapani (1968). Annales Medicinae Experimentalis et Biologiea Fenniae 46 (3, Pt. 2): 382–4.
Young SN (2007). “How to increase serotonin in the human brain without drugs”. Rev. Psychiatr. Neurosci. 32(6): 394–99. PMC 2077351. PMID 18043762.
King MW. “Serotonin”. The Medical Biochemistry Page. Indiana University School of Medicine. Retrieved 1 December 2009.
Berger M, Gray JA, Roth BL (2009). “The expanded biology of serotonin”. Annu. Rev. Med. 60: 355–66.doi:10.1146/annurev.med.60.042307.110802.PMID 19630576.
Bianchi, P. (2005). “A new hypertrophic mechanism of serotonin in cardiac myocytes: Receptor-independent ROS generation”. The FASEB Journal. doi:10.1096/fj.04-2518fje.
Kang K, Park S, Kim YS, Lee S, Back K (2009). “Biosynthesis and biotechnological production of serotonin derivatives”. Appl. Microbiol. Biotechnol. 83 (1): 27–34.doi:10.1007/s00253-009-1956-1. PMID 19308403.

www.encyclopedia.com/html/s1/serotoni.asp

www.angelfire.com/hi/TheSeer/seratonin.html

www.findthelight.net/Depression/the_chemistry_of_dep.htm

http://www.cmste.uncc.edu/Document%20Hold/Sawsun-%20Serotonin%20FINAL%20PAPER.doc

www.totse.com/en/technology/science_technology/seroton.html

www.macalester.edu/~psych/whathap/ubnrp/mdma/serotonin.html

www.serendipity.li/mcclay/pineal.html#a1.6

www.serendip.brynmawr.edu/bb/neuro/neuro98/202s98-paper3/Frederickson3.html

www.serendip.brynmawr.edu/bb/neuro/neuro99/web1/Byrd.html

BERAPROST….Stable prostacyclin analog.

Uncategorized Comments Off

Mar 242014

BERAPROST

https://www.ama-assn.org/resources/doc/usan/beraprost.pdf

2,3,3a,8b-tetrahydro-2-hydroxy-1-(3-hydroxy-4-methyl-1-octen-6-ynyl)-1H-cyclopenta(b)benzofuran-5-butanoic acid

(±)-(IR*,2R*,3aS*,8bS*)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octene-6-inyl]-1H-cyclopenta[b]benzofuran-5-butyric acid

rac-4-{(1R,2R,3aS,8bS)-2-hydroxy-1-[(1E,3S,4RS)-3-hydroxy-4-methyloct-1-en-6-ynyl]-2,3,3a,8b-tetrahydro-1H-cyclopenta[b][1]benzofuran-5-yl}butanoic acid

88430-50-6 88475-69-8

Beraprost
Beraprostum
Beraprostum [INN-Latin]
MDL 201229
MDL-201229
ML 1229
ML-1229
UNII-35E3NJJ4O6

Beraprostum, Beraprostum [INN-Latin], ML 1229, MDL 201229, 88430-50-6

Molecular Formula: C₂₄H₃₀O₅

Molecular Weight: 398.492

CLINICAL TRIALS..http://clinicaltrials.gov/search/intervention=Beraprost

Beraprost is a synthetic analogue of prostacyclin, under clinical trials for the treatment of pulmonary hypertension. It is also being studied for use in avoiding reperfusion injury.

As an analogue of prostacyclin PGI₂, beraprost effects vasodilation, which in turn lowers the blood pressure. Beraprost also inhibits plateletaggregation, though the role this phenomenon may play in relation to pulmonary hypertension has yet to be determined.

Beraprost …sodium salt

ML 1129; Procyclin; TRK 100 (CAS 88475-69-8)

Beraprost is an analog of prostacyclin in which the unstable enol-ether has been replaced by a benzofuran ether function. This modification increases the plasma half-life from 30 seconds to several hours, and permits the compound to be taken orally. Doses of 20-100 µg in humans, given 1 to 3 times per day, have been demonstrated to improve clinical end points in diseases responsive to prostacyclin. Oral beraprost therapy improved the survival and pulmonary hemodynamics of patients with primary pulmonary hypertension.1 Beraprost inhibits platelet aggregation in healthy subjects and in diabetic patients at similar doses.2,3

Synonyms	ML 1129 Procyclin TRK 100
Formal Name	2,3,3a,8b-tetrahydro-2-hydroxy-1-(3-hydroxy-4-methyl-1-octen-6-ynyl)-1H-cyclopenta[b]benzofuran-5-butanoic acid, monosodium salt
CAS Number	88475-69-8
Molecular Formula	C24H29O5 · Na
Formula Weight	420.5

Beraprost sodium is a prostacyclin analog and an NOS3 expression enhancer that was first launched in 1992 in Japan pursuant to a collaboration between Astellas Pharma and Toray for the oral treatment of peripheral vascular disease (PVD), including Raynaud’s syndrome and Buerger’s disease. In 2000, the drug was commercialized for the treatment of pulmonary hypertension. Development for the oral treatment of intermittent claudication associated with arteriosclerosis obliterans (ASO) was discontinued at Kaken and United Therapeutics after the product failed to demonstrate statistically significant results in a phase III efficacy trial.

In terms of clinical development, beraprost sodium is currently in phase II clinical trials at Kaken for the treatment of lumbar spinal canal stenosis and at Astellas Pharma for the oral treatment of primary chronic renal failure. The company is also conducting phase III trials for the treatment of nephrosclerosis. The drug has also been studied through phase II clinical trials at Kaken for the oral treatment of diabetic neuropathy, but recent progress reports for this indication have not been made available.

Beraprost is an oral form of prostacyclin, a member of the family of lipid molecules known as eicosanoids. Prostacyclin is produced in the endothelial cells from prostaglandin H2 by the action of the enzyme prostacyclin synthase. It has been shown to keep blood vessels dilated and free of platelet aggregation.

Beraprost sodium was originally developed at Toray in Japan, and rights to the drug were subsequently acquired by Astellas Pharma. A 1972 alliance between Toray and Kaken Pharmaceutical to develop and commercialize prostaglandin led to a later collaboration agreement for the development of beraprost. In 1990, Toray granted the right to market the drug to Sanofi (formerly known as sanofi-aventis), a licensing agreement that was later expanded to include Canada, the U.S., South America, Africa, Southeast Asia, South Asia, Korea and China. In September 1996, Bristol-Myers Squibb entered into separate agreements with Sanofi and Toray to acquire all development and marketing rights to beraprost in the U.S. and Canada. In January 1999, United Therapeutics and Toray agreed to cooperatively test the drug in North America, and in July 2000, a new agreement was signed pursuant to which United Therapeutics gained exclusive North American rights to develop and commercialize sustained-release formulations of beraprost for all vascular and cardiovascular diseases. In 1999, orphan drug designation was received in the U.S. for the treatment of pulmonary arterial hypertension associated with any New York Heart Association classification (Class I, II, III, or IV). In 2011, orphan drug designation was assigned in the U.S. for the treatment of pulmonary arterial hypertension.

The compound name of beraprost which is used as an antimetastasis agent of malignant tumors according to the present invention is (±)-(IR*,2R*,3aS*,8bS*)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octene-6-inyl]-1H-cyclopenta[b]benzofuran-5-butyric acid. This compound has the following structure.

Beraprost is described in Japanese Laid-open Patent Application (Kokai) Nos. 58-32277, 57-144276 and 58-124778 and the like as a PGI₂ derivative having a structure in which the exoenol moiety characteristic to beraprost is converted to inter-m-phenylene structure. However, it is not known that beraprost has an activity to inhibit metastasis of malignant tumors.
The beraprost which is an effective ingredient of the agent of the present invention includes not only racemic body, but also d-body and l-body. Beraprost can be produced by, for example, the method described in the above-mentioned Japanese Laid-open Patent Application (Kokai) No. 58-124778. The salts of beraprost include any pharmaceutically acceptable salts including alkaline metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; primary, secondary and tertiary amine salts; and basic amino acid salts.

EP0623346A1

…………………..

US7005527

EXAMPLE 6 Beraprost of the Formula (I)

0.246 g (0.6 mmol) of compound of the general formula (II) obtained in Example 5 is dissolved in 1 ml of methanol and 1 ml of 1 M aqueous sodium hydroxide solution is added dropwise slowly thereto. After stirring for an hour the methanol is distilled off from the reaction mixture in vacuum. The aqueous residue is diluted with 10 ml of water extracted with methyl-tert.butyl-ether and the combined organic phase is washed with saturated NaCl solution, dried on Na₂SO₄and evaporated. The residue of evaporation is crystallized from ethylacetate-hexane mixture and the pure above mentioned title compound is obtained as colourless crystals.

Yield: 0.21 g (87%)

TLC-R_f(toluene-dioxan-acetic acid 20:10:1)=0.41

Melting point: 98–112° C.

¹H NMR (400 MHz, CDCl₃), δH (ppm): 1.00d, 1.03d [3H; J=6.8 Hz; 21-H₃]; 1.79m [1H; 16-H]; 1.80t, 1.81t [3H, J=2.5,2.4 Hz; 20-H₃]; 2.3–1.9m [5H, 3-H₂, 10H_b, 17-H₂]; 2.34t [1H; J=7.4 Hz; 2-H₂]; 2.43m [1H; 12-H]; 2.64m [3H; 10-H_a, 4-H₂]; 3.43t, 3.44t [1H, J=8.7,8.5 Hz; 8-H]; 3.92m [1H; 11-H]; 4.07t, 4.17t [1H, J=7.3,5.6 Hz; 15-H]; 4.3b [2H; OH]; 5.09m [1H, 9-H]; 5.58dd, 5.61dd [1H; J=15.3,6.5 Hz; 14-H]; 5.67dd, 5.68dd [1H; J=15.3,8.0 Hz; 13-H]; 6.77m [1H; 2′-H]; 6.95m [2H; 1′-H,3′-H]¹³C NMR (100 MHz, CDCl₃), δC (ppm): 3.5, 3.6 [C-20]; 14.7, 15.8 [C-21]; 22.3, 22.6 [C-17]; 24.6 [C-2]; 29.1 [C-4]; 33.1 [C-3]; 38.2, 38.3 [C-16]; 41.2 [C-10]; 50.4 [C-8]; 58.8 [C-12]; 75.8, 76.3, 76.4 [C-11, C-15]; 77.2, 77.4 [C-18, C-19]; 84.5, 84.6 [C-9]; 120.6 [C-2′]; 121.9 [C-3′]; 123.2 [C-5]; 129.0 [C-1′]; 129.7 [C-7]; 132.3, 133.0, 133.8, 134.0 [C-13, C-14]; 157.2 [C-6]; 178.3 [C-1].

EXAMPLE 7 Beraprost Sodium Salt (The Sodium Salt of the Compound of Formula (I)

0.199 g of beraprost is dissolved in 2 ml of methanol, 0.5 ml of 1 M aqueous solution of sodium hydroxide is added thereto and after their mixing the solvent is evaporated in vacuum and thus the above title salt is obtained as colourless crystals.

Yield: 0.21 g (100%)

Melting point: >205° C.

¹H NMR (400 MHz, DMSO-d₆), δH (ppm): 0.90d, 0.92d [3H; J=6.7 Hz; 21-H₃]; 1.75–1.55m [7H; 10H_b, 16-H, 3-H₂, 20-H₃]; 1.89t [2H, J=7.6 Hz; 2-H₂]; 1.94m [1H; 17-H_b]; 2.16q [1H, J=8.5 Hz; 12-H]; 2.25m [1H; 17-H_a]; 2.44t [2H; J=7.5 Hz; 4-H₂]; 2.50o [1H; 10-H_a]; 3.39t [1H, J=8.5 Hz; 8-H]; 3.72td [1H; J=8.5,6.1 Hz; 11-H]; 3.84t 3.96t [1H, J=6.5,6.0 Hz; 15-H]; 4.85b [2H, OH]; 5.01dt [1H, J=8.5,6.6 Hz; 9-H]; 5.46dd, 5.47dd [1H; J=15.4,6.5 Hz, J=15.4,6.0 Hz; 14-H]; 5.65dd, 5.66dd [1H; J=15.4,8.5 Hz; 13-H]; 6.71m [1H; 2′-H]; 6.92m [2H; 1′-H, 3′-H] During the above thin layer chromatography (TLC) procedures we used plates MERCK Kieselgel 60 F₂₅₄, thickness of layer is 0.2 mm, length of plates is 5 cm.

…………….

Reaction Scheme A.
The starting material of bromocarboxylic acid, Compound 1, and the process for the preparation thereof are disclosed in Japanese Patent Application No. 29637/81.

Scheme B.

REACTION SCHEME B

https://www.google.com/patents/EP0084856A1

Org Lett 2012, 14(1): 299

http://pubs.acs.org/doi/abs/10.1021/ol203060v

http://pubs.acs.org/doi/suppl/10.1021/ol203060v/suppl_file/ol203060v_si_001.pdf

EP0024943A1	Sep 2, 1980	Mar 11, 1981	Toray Industries, Inc.	5,6,7-Trinor-4,8-inter-m-phenylene PGI2 derivatives and pharmaceutical compositions containing them
EP0084856A1	Jan 19, 1983	Aug 3, 1983	Toray Industries, Inc.	5,6,7-Trinor-4, 8-inter-m-phenylene prostaglandin I2 derivatives
JP3069909B				Title not available

CARBAZITAXEL

Uncategorized Comments Off

Mar 202014

Cabazitaxel

For treatment of patients with hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen.

4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1-hydroxy-7β,10β-dimethoxy-9-oxotax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenyl-propionate

(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-(Acetyloxy)-15-{[(2R,3S)-3-{[(tert-butoxy)carbonyl]amino}-2-hydroxy-3-phenylpropanoyl]oxy}-1-hydroxy-9,12-dimethoxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0^3,10.0^4,7]heptadec-13-ene-2-yl benzoate

183133-96-2

Jevtana, Taxoid XRP6258, Cabazitaxelum, 183133-96-2, Xrp6258, CHEBI:63584, XRP-6258, TXD 258, XRP 6258

Molecular Formula: C₄₅H₅₇NO₁₄ Molecular Weight: 835.93238

EMA:Link, US FDA:link

Cabazitaxel is prepared by semi-synthesis from 10-deacetylbaccatin III (10-DAB) which is extracted from yew tree needles. The chemical name of cabazitaxel is (2α,5β,7β,10β,13α)-4-acetoxy-13-({(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-7,10-dimethoxy-9-oxo-5,20-epoxy-tax-11-en-2-yl benzoate and is marketed as a 1:1 acetone solvate (propan-2-one),

Cabazitaxel is an anti-neoplastic used with the steroid medicine prednisone. Cabazitaxel is used to treat people with prostate cancer that has progressed despite treatment with docetaxel. Cabazitaxel is prepared by semi-synthesis with a precursor extracted from yew needles (10-deacetylbaccatin III). It was approved by the U.S. Food and Drug Administration (FDA) on June 17, 2010.

Cabazitaxel (previously XRP-6258, trade name Jevtana) is a semi-synthetic derivative of a natural taxoid.^[1] It was developed by Sanofi-Aventis and was approved by the U.S. Food and Drug Administration (FDA) for the treatment of hormone-refractory prostate cancer on June 17, 2010. It is a microtubule inhibitor, and the fourth taxane to be approved as a cancer therapy.^[2]

Nagesh Palepu, “CABAZITAXEL FORMULATIONS AND METHODS OF PREPARING THEREOF.” U.S. Patent US20120065255, issued March 15, 2012.

US20120065255 Link out

Cabazitaxel in combination with prednisone is a treatment option for hormone-refractory prostate cancer following docetaxel-based treatment.

Clinical trials

In a phase III trial with 755 men for the treatment of castration-resistant prostate cancer, median survival was 15.1 months for patients receiving cabazitaxel versus 12.7 months for patients receiving mitoxantrone. Cabazitaxel was associated with more grade 3–4 neutropenia (81.7%) than mitoxantrone (58%).^[3]

United States	5438072	2010-06-17	exp 2013-11-22
United States	5698582	2010-06-17	2012-07-03
United States	5847170	2010-06-17	2016-03-26
United States	6331635	2010-06-17	2016-03-26
United States	6372780	2010-06-17	2016-03-26
United States	6387946	2010-06-17	2016-03-26
United States	7241907	2010-06-17	2025-12-10

JEVTANA (cabazitaxel) is an antineoplastic agent belonging to the taxane class. It is prepared by semi-synthesis with a precursor extracted from yew needles.

The chemical name of cabazitaxel is (2α,5β,7β,10β,13α)-4-acetoxy-13-({(2R,3S)-3[(tertbutoxycarbonyl) amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-7,10-dimethoxy-9oxo-5,20-epoxytax-11-en-2-yl benzoate – propan-2-one(1:1).

Cabazitaxel has the following structural formula:

JEVTANA (cabazitaxel) Structural Formula Illustration

Cabazitaxel is a white to almost-white powder with a molecular formula of C₄₅H₅₇NO₁₄^•C₃H₆O and a molecular weight of 894.01 (for the acetone solvate) / 835.93 (for the solvent free). It is lipophilic, practically insoluble in water and soluble in alcohol.

JEVTANA (cabazitaxel) Injection 60 mg/1.5 mL is a sterile, non-pyrogenic, clear yellow to brownish-yellow viscous solution and is available in single-use vials containing 60 mg cabazitaxel (anhydrous and solvent free) and 1.56 g polysorbate 80. Each mL contains 40 mg cabazitaxel (anhydrous) and 1.04 g polysorbate 80.

DILUENT for JEVTANA is a clear, colorless, sterile, and non-pyrogenic solution containing 13% (w/w) ethanol in water for injection, approximately 5.7 mL.

JEVTANA requires two dilutions prior to intravenous infusion. JEVTANA injection should be diluted only with the supplied DILUENT for JEVTANA, followed by dilution in either 0.9% sodium chloride solution or 5% dextrose solution.

The taxane family of terpenes has received much attention in the scientific and medical community, because members of this family have demonstrated broad spectrum of anti-leukemic and tumor-inhibitory activity. A well-known member of this family is paclitaxel (Taxol®).

Paclitaxel (Taxol) Paclitaxel was first isolated from the bark of the pacific yew tree (Taxus brevifolia) in 1971 , and has proved to be a potent natural anti-cancer agent. To date, paclitaxel has been found to have activity against different forms of leukemia and against solid tumors in the breast, ovary, brain, and lung in humans.

As will be appreciated, this beneficial activity has stimulated an intense research effort over recent years with a view to identifying other taxanes having similar or improved properties, and with a view to developing synthetic pathways for making these taxanes, such as paclitaxel.

This research effort led to the discovery of a synthetic analogue of paclitaxel, namely, docetaxel (also known as Taxotere®). As disclosed in U.S. Patent No. 4,814,470, docetaxel has been found to have a very good anti-tumour activity and better bioavailability than paclitaxel. Docetaxel is similar in structure to paclitaxel, having t- butoxycarbonyl instead of benzoyl on the amino group at the 3′ position, and a hydroxy group instead of the acetoxy group at the C-10 position.

As will be appreciated, taxanes are structurally complicated molecules, and the development of commercially viable synthetic methods to make taxanes has been a challenge. A number of semi-synthetic pathways have been developed over the years, which typically begin with the isolation and purification of a naturally occurring starting material, which can be converted to a specific taxane derivative of interest. Cabazitaxel (I) is an anti-tumor drug which belongs to the taxol family. It differs from docetaxel in that it has methoxy groups at positions 7 and 10 of the molecule, as opposed to the hydroxyl groups at equivalent positions in docetaxel. Cabazitaxel is obtained by semi-synthesis from an extract of Chinese yew (Taxus mairei). It is understood that cabazitaxel can be obtained via semi-synthesis from other taxus species including T.candensis, T.baccatta, T.chinensis, T. mairei etc.

Cabazitaxel is a semi-synthetic derivative of the natural taxoid 0-deacetylbaccatin III (10-DAB) with potentially unique antineoplastic activity for a variety of tumors.

Cabazitaxel binds to and stabilizes tubulin, resulting in the inhibition of microtubule depolymerization and cell division, cell cycle arrest in the G2/M phase, and the inhibition of tumor cell proliferation. This drug is a microtubule depolymerization inhibitor, which can penetrate blood brain barrier (BBB).

Cabazitaxel was recently approved by the US Federal Drug Administration (FDA) for the treatment of docetaxel resistant hormone refractory prostate cancer. It has been developed by Sanofi-Aventis under the trade name of Jevtana. The CAS number for the compound is 183133-96-2. A synonym is dimethoxydocetaxel. The compound is also known as RPR-1 16258A; XRP6258; TXD 258; and axoid XRP6258.

The free base form of cabazitaxel has the chemical name

(2aR,4S,4aS,6R,9S, 1 1 S,12S,12aR, 12bS)-12b-acetoxy-9-(((2R,3S)-3-((tert- butoxycarbonyl)amino)-2-hydroxy-3-phenylpropanoyl)oxy)-11-hydroxy-4,6-dimethoxy- 4a,8, 13, 13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9, 10, 11 , 12, 12a, 12b-dodecahydro-1 H- 7, 1 1-methanocyclodeca[3,4]benzo[1 ,2-b]oxet-12-yl benzoate. In a first part of this description, taxel drugs including paclitaxel (taxol), docetaxel (taxotere) and cabazitaxel may be prepared starting from 10-deacetylbaccatin (known as 10-DAB) derived from Taxus plants, via semi-synthesis. Furthermore, the same inventive methodologies can be used to semi-synthesize cabazitaxel starting from 9- dihydro-13-acetylbaccatin III (9-DHB).

Patent numbers CN1213042C, CN152870, CN1179716 and CN1179775 disclose methods to prepare cabazitaxel from 10-DAB (herein compound II).

10-DAB (II)

A typical prior art synthesis route is as follows:

OCOCH₃

OCOC₆H₅

The method above which synthesizes cabazitaxel has many synthetic steps, a very low overall yield and high price.

There is therefore a need in the art to develop new methods to synthesize cabazitaxel and its intermediates to improve the yield of cabazitaxel, simplify the methodology and optimize the synthetic technology.

Cabazitaxel, chemically known as 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1-hydroxy-7β,10β-dimethoxy-9-oxotax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenyl-propionate, is represented by formula (I).

It is a microtubule inhibitor, indicated in combination with prednisone for treatment of patients with hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, under the trade name Jevtana®.

Cabazitaxel is known from U.S. Pat. No. 5,847,170. Process for preparation of Cabazitaxel as described in U.S. Pat. No. 5,847,170 involves column chromatography, which is cumbersome tedious and not commercially viable.

The acetone solvate of 4-acetoxy-2α-benzoyloxy-5β-20-epoxy-1-hydroxy-7β, 10β-dimethoxy-9-oxotan-11-en-13α-yl-(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate (Form A) is formed by crystallization by using acetone and is characterized by XRD in U.S. Pat. No. 7,241,907.

U.S. 20110144362 describes anhydrous crystalline Forms B to Form F, ethanolates Form B, D, E and F and mono and dihydrate Forms of Cabazitaxel. All the anhydrous crystalline forms are prepared either by acetone solvate or ethanol solvate. Mono and dihydrate forms are formed at ambient temperature in an atmosphere containing 10 and 60% relative humidity, respectively.

Cabazitaxel (also called dimethoxy docetaxel) is a dimethyl derivative of docetaxel, which itself is semi-synthetic, and was originally developed by Rhone-Poulenc Rorer and was approved by the U.S. Food and Drug Administration (FDA) for the treatment of hormone-refractory prostate cancer on Jun. 17, 2010. Cabazitaxel is a microtubule inhibitor. The acetone solvate crystalline form of cabazitaxel and a process for its preparation is disclosed in the U.S. Pat. No. 7,241,907.

U.S. Pat. No. 5,847,170 describes cabazitaxel and its preparation methods. One of the methods described in U.S. Pat. No. 5,847,170 includes a step-wise methylation of 10-DAB (the step-wise methylation method is shown in FIG. 1) to provide the key intermediate (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-acetoxy-9,11-dihydroxy-4,6-dimethoxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate, herein referred to as 7,10-di-O-methyl-10-DAB (XVa). The intermediate XVa is coupled with the 3-phenylisoserine side chain derivative VI to provide XVa′, which is followed by removal of the oxazolidine protecting group from the side chain of XVa′ to give cabazitaxel.

Another method described in U.S. Pat. No. 5,847,170 utilizes methylthiomethyl (MTM) ethers as shown in FIG. 2. MTM ethers can be prepared from alcohols using two common methods. One method comprises deprotonation of an alcohol with a strong base to form an alkoxide followed by alkylation of the alkoxide with a methylthiomethyl halide. This approach is only useful when the alcohol is stable to treatment with a strong base. 10-DAB and some of its derivatives in which C7-OH is not protected displays so instability in the presence of strong bases and epimerization of the C7-OH can occur upon contact of 10-DAB and some of its derivatives in which C7-OH is not protected with strong bases. Another method for the synthesis of MTM ethers from alcohols utilizes Ac2O and DMSO. One disadvantage of this method is that it can also lead to the oxidation of alcohols to aldehydes or ketones. For example when the synthesis of the 10-di-O-MTM derivative of 10-DAB without protecting groups at the C13 hydroxyl group is attempted undesired oxidation of the C13-OH to its corresponding ketone occurs.

U.S. Pat. No. 5,962,705 discloses a method for dialkylation of 10-DAB and its derivatives to furnish 7,10-di-O-alkyl derivatives, as shown in FIG. 3. This has been demonstrated as a one-step, one-pot reaction, however, provides the best isolated yield when potassium hydride is used at −30° C. From an industrial point of view, the use of low reaction temperature is less favorable than using ambient temperature. Furthermore the use of a strong base can cause some epimerization of the C7-OH chiral center with an associated loss of yield. Potassium hydride is a very reactive base and must be treated with great caution.

Accordingly, there is a need for an alternative processes for the preparation of cabazitaxel and its key intermediate, 7,10-di-O-methyl-10-DAB (XVa) that is short in number of synthetic steps and avoids the use of low temperatures and strong bases such as metal hydrides in the C7-O methyl ether formation step. Such a process would also be useful for the preparation of analogues of cabazitaxel wherein the C7-O and C10-O functional groups were substituted with other alkyl groups.

FIG. 1 shows the chemistry employed in the examples of U.S. Pat. No. 5,847,170.

FIG. 2 shows the chemistry employed in the examples of U.S. Pat. No. 5,847,170.

FIG. 3 shows the chemistry employed in the examples of U.S. Pat. No. 5,962,705.

FIG. 4 shows key steps of the general synthetic scheme as per Method A/A′ of the present invention for the synthesis of cabazitaxel and cabazitaxel analogues.

FIG. 5 shows key steps of the general synthetic scheme as per Method B/B′ of the present invention for the synthesis of cabazitaxel and cabazitaxel analogues.

FIG. 6 shows the general scheme for the hydrodesulfurization reaction.

FIG. 7 shows the complete synthetic route of Method A that can be used for conversion of 10-DAB to cabazitaxel.

FIG. 8 shows the complete synthetic route of Method B that can be used for conversion of 10-DAB to cabazitaxel.

FIG. 9 shows the complete synthetic route of Method A′ that can be used for conversion of 10-DAB, via XIV′, to cabazitaxel.

FIG. 10 shows the complete synthetic route of Method B′ that can be used for conversion of 10-DAB, via XVI′, to cabazitaxel.

FIG. 11 shows the synthetic relationship between two methods (A and B) used to convert 7,10-di-O-alkyl-10-DAB (XV) to cabazitaxel.

FIG. 12 shows the synthetic scheme for the preparation of XIVa.

FIG. 13 shows the synthetic scheme for the preparation of XIVb from 10-DAB.

FIG. 14 shows the synthetic scheme for the preparation of XIVb from XIVb′.

FIG. 15 shows the synthetic scheme for the preparation of XIVc.

FIG. 16 shows the synthetic scheme for the preparation of XIVa′ from XIVa.

FIG. 17 shows the synthetic scheme for the preparation of XIVa′ from XX.

FIG. 18 shows the synthetic scheme for the preparation of XIVb′.

FIG. 19 shows the synthetic scheme for the preparation of XIVc′.

FIG. 20 shows the synthetic scheme for the preparation of XVa from XIVa.

FIG. 21 shows the synthetic scheme for the preparation of XVa from XIVb.

FIG. 22 shows the synthetic scheme for the preparation of XVa from XIVc.

FIG. 23 shows the synthetic scheme for the preparation of XVa′ from XIVa.

FIG. 24 shows the synthetic scheme for the preparation of XVa′ from XIVa′.

FIG. 25 shows the synthetic scheme for the preparation of XVa′ from XIVb′.

FIG. 26 shows the synthetic scheme for the preparation of XVa′ from XIVc′.

FIG. 27 shows the synthetic scheme for the preparation of XVa′ from XVa.

FIG. 28 shows the synthesis of cabazitaxel.

FIG. 29 shows the synthesis of XVIIa.

FIG. 30 shows the synthesis of XVIIIa.

FIG. 31 shows the synthesis of XIXa.

FIG. 32 shows the synthesis of XVIa.

FIG. 33 shows the synthesis of XVa from XVIa.

see this at http://www.google.com/patents/US20130116444

………..

WO2013057260A1

Detailed description

The invention provides a new method for the preparation of cabazitaxel, one embodiment of which can be summarized as follows, showing the preparation of a protected taxane intermediate and its deprotection to taxane compounds:

OH OCOCH₃

OCOC₆H₅

This reaction is also depicted in Figure 1. The reaction of the invention reduces the number of steps and increases yield of cabazitaxel.

The deprotection methods of the invention can also be used for the preparation of paclitaxel (taxol):

The deprotection methods of the invention can also be applied to the preparation of docetaxel:

10-DAB synthetic routes

Example 12

Dissolve 100 g of 2′-THP-cabazitaxel in 1730 ml of HOAc/H₂0/THF (3:1 :1 ). under N₂ atmosphere, increase temperature to 50 degrees C and stir 4 hrs. Then cool to room temperature. Add 2L of ethyl acetate, 2 L of H₂0, stir, separate layers, wash organic layer with saturated NaHC0₃ (3 L x 2), saturated NaCI (3 L), dry with Na₂S0₄.

Concentrate to obtain white 77.8 g of cabazitaxel (yield 83%).

MS(m/z) :859(M+Na)„ _jHNMR (500MHz) δ 1.21(611, d) , 1.36(911, s) , 1.59(lH, s) , 1.64(lH,s) , 1.79(lH,m) , 1.87 (3H, s) ,2.27 (2H, m) , 2.35(3H,m) ,2.69(lH,m) ,3.30 (3H, s) ,

3.45 (3H, s) , 3.85 (2H, m) , 4.16 (1H, d) , 4.29 (1H, d) , 4.62 (1H, bs) , 4.79 (1H, s) , 5.29 (1H, m),5.42(lH, d),5.62(lH, d),6.21 (1H, t),7.2 ~ 7.4(6H, m) , 7.48 (2H, t),7.59(lH, t) , 8.11 (2H, d) ,

References

http://www.cancer.gov/drugdictionary/?CdrID=534131
“Jevtana (cabazitaxel) Injection Approved by U.S. FDA After Priority Review” (Press release). sanofi-aventis. 2010-06-17. Retrieved June 17, 2010.
“Cabazitaxel Effective for Hormone Refractory Prostate Cancer After Failure of Taxotere”.

Cabazitaxel – Official web site of manufacturer.
Cabazitaxel Prescribing Information – Official prescribing information.
U.S. National Library of Medicine: Drug Information Portal – Cabazitaxel

Patents

Patent :

Patent Number : 5438072

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Approved : 2010-06-17

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Patent :

Patent Number : 5698582

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Expires : 2012-07-03

Patent :

Patent Number : 5847170

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Patent :

Patent Number : 6331635

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Patent Number : 6372780

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Patent Number : 6387946

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US2009069411	3-13-2009	SELF-EMULSIFYING AND SELF-MICROEMULSIFYING FORMULATIONS FOR THE ORAL ADMINISTRATION OF TAXOIDS
US2005070496	3-32-2005	Semi-solid formulations for the oral administration of taxoids
US2005025792	2-4-2005	Self-emulsifying and self-microemulsifying formulations for the oral administration of taxoids
US6403634	6-12-2002	Use of taxoid derivatives

CHEMDRAW PRO 8.0

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Mar 152014

CHEMDRAW PRO 8.0

ChemDraw is a leading chemistry molecules drawing software. ChemDraw is easy to use and best software for drawing a molecule structures and finding stereochemistry of the molecules.

Features

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Screenshot

ChemDraw Pro 8.0

ChemDraw Pro 8.0 provides chemists with a rich set of easy to use tools for creating publication ready, scientifically meaningful drawings of molecules and reactions.

Free download ChemDraw Pro 8.0 full version with product key and serial number from following download link. This download link is test personally and found viruses and spam free.

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Specifications

Operating System: Windows 95/NT/98/2000/XP, Windows 7

Size: 47 MB

Nano drugs..Powder dispersion

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Mar 132014

Powder dispersion

One of the main criteria for the effective drug delivery via lungs is the size of the inhaled particle. Respirable size i.e. the particle is transported to deep lungs (alveoli region) is around 1 – 5 micrometers. The particle size can be easily controlled in the powder production. The powders, however, tend to stick to each other in the collection. Therefore, the dispersion and deagglomeration behavior of the powder should be studied.

The dispersion testing of the fine powders was conducted with the novel deagglomeration apparatus [14]. Powder agglomerates, i.e. the mixture of carrier and fine powders, is fed continuously through a narrow tube with the aid of thin air flow of 1.2 l/min. At the outlet of the needle the agglomerates are subjected to the main flow rate (Q_M) from 15 to 90 l/min intending to disperse the powder i.e. deagglomeration zone, see Figure 8. These Q_M values correspond to the jet Reynolds numbers from 8000 to 48000. The flow forms a highly turbulent space, i.e. the deagglomeration zone, where possible break-up of powder agglomerates takes place. The fine powder particles are isokinetically sampled from homogeneously mixed aerosol into low-pressure impactors. Fine particle fractions (FPF), mass medium aerodynamic diameter (MMAD), and size distribution can be determined at different flow rates.

Figure 9 illustrates a common trend in the change of the particle distribution at different flow rates. As seen, all the dispersed particles, in this case salbutamol 92 w% and L-leucine 8 w%, are within the respirable size range. Also, the increase in the flow rate from 15 to 90 l/min improved the powders dispersion (the increase in particle concentration) and deagglomeration (the size of the dispersed particles decreases).

In dry powder inhaler the fine drug particles are commonly mixed with large lactose particles. These lactose carriers aid the dispersion of fine particles. Figure 10 shows the fine particle fractions of the peptide-coated drug powders that were introduced to the dispersion testing without lactose carriers. The powders with crystalline L-leucine surface exhibited good flowability that made possible to feed these powders as such i.e. without carrier particles. More importantly, they showed excellent deagglomeration performance even at very low flow rate.

Octreotide اکترئتید For treatment of acromegaly and reduction of side effects from cancer chemotherapy

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Mar 122014

Octreotide

(D)-Phe-Cys-Phe-(D)-Trp-Lys-Thr-Cys-Thr-ol.

(4R,7S,10S,13R,16S,19R)-10-(4-aminobutyl)-19-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-16-benzyl-N-[(2R,3R)-1,3-dihydroxybutan-2-yl]-7-(1-hydroxyethyl)-13-(1H-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-
pentazacycloicosane-4-carboxamide

L-cysteinamide, D-phenylalanyl-L-cysteiny-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic (2→7)-disulfide; [R-(R*,R*)].

Octreotide is the acetate salt of a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin.

Canada	1328402	1994-04-12	expiry 2011-04-12
United States	5922338	1997-01-13	2017-01-13
United States	5538739	1993-07-23	2013-07-23

CAS number	83150-76-9 79517-01-4 (acetate) 135467-16-2 (pamoate)

Sandostatin LAR Depot
L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-(2-hydroxy-1-(hydroxymethyl)propyl)-, cyclic(2-7)-disulfide, (R-(R*,R*))-, acetate (salt)
Octreotide Acetate Depot
AC1L1GVR
AC1Q2BPN
CCRIS 8708
Octreotide acetate [USAN:JAN]
UNII-75R0U2568I
83150-76-9 (Parent)
AC-663

Octreotide (brand name Sandostatin,^[1] Novartis Pharmaceuticals) is an octapeptide that mimics natural somatostatin pharmacologically, though it is a more potent inhibitor of growth hormone, glucagon, and insulin than the natural hormone. It was first synthesized in 1979 by the chemist Wilfried Bauer.

Since octreotide resembles somatostatin in physiological activities, it can:

inhibit secretion of many hormones, such as gastrin, cholecystokinin, glucagon, growth hormone, insulin, secretin, pancreatic polypeptide, TSH, and vasoactive intestinal peptide,
reduce secretion of fluids by the intestine and pancreas,
reduce gastrointestinal motility and inhibit contraction of the gallbladder,
inhibit the action of certain hormones from the anterior pituitary,
cause vasoconstriction in the blood vessels, and
reduce portal vessel pressures in bleeding varices.

It has also been shown to produce analgesic effects, most probably acting as a partial agonist at the mu opioid receptor.^[2]^[3]

Acromegaly is a hormonal disorder that results when the pituitary gland produces excess growth hormone (GH). It most commonly affects middle-aged adults and can result in serious illness and premature death. Once recognized, acromegaly is treatable in most patients, but because of its slow and often insidious onset, it frequently is not diagnosed correctly.

Octreotide is one drug used to treat acromegaly. Octreotide exerts pharmacologic actions similar to those of the natural hormone somatostatin. Octreotide decreases GH and IGF-1 levels, as well as glucagons and insulin. Octreotide also suppresses luteinizing hormone (LH) response to gonadotropin releasing hormone (GnRH), decreases splanchnic blood flow, and inhibits the release of serotonin, gastrin, vasoactive intestinal peptide, secretin, motilin, and pancreatic polypeptide. In many patients, GH levels fall within one hour and headaches improve within minutes after the injection of octreotide. Several studies have shown that octreotide is effective for long-term treatment. Octreotide also has been used successfully to treat patients with acromegaly caused by non-pituitary tumors. In some acromegaly patients who already have diabetes, octreotide can reduce the need for insulin and improve blood sugar control.

Octreotide is currently available as Sandostatin LAR® Depot, which is, upon reconstitution, a suspension of microspheres containing octreotide acetate. Sandostatin LAR® Depot is the only medication indicated for the long-term maintenance therapy in acromegalic patients. It is also indicated for the long-term treatment of severe diarrhea and flushing episodes associated with metastatic carcinoid tumors and profuse water diarrhea associated with VIP-secreting tumors. Sandostatin LAR® T Depot is administered via intramuscular injection every four weeks, following a titration period. Octreotide acetate has also been available in an immediate-release formulation, Sandostatin® Injection solution, which was required to be administered by injection three times daily.

Octreotide is an octapeptide with the following amino acid sequence: L-cysteinamide, D-phenylalanyl-L-cysteiny-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic (2→7)-disulfide; [R-(R*,R*)]. The structure of octreotide is shown below.

The chemical formula is C₄₉H₆₆N₁₀O₁₀S₂and its molecular weight is 1019.3 Da. Its therapeutic category is gastric antisecretory agent.

The Food and Drug Administration (FDA) has approved the usage of a salt form of this peptide, octreotide acetate, as an injectable depot formulation for the treatment of growth hormone producing tumors (acromegaly and gigantism), pituitary tumors that secrete thyroid stimulating hormone(thyrotropinoma), diarrhea and flushing episodes associated with carcinoid syndrome, and diarrhea in patients with vasoactive intestinal peptide-secreting tumors (VIPomas).

Octreotide is used in nuclear medicine imaging by labelling with indium-111 (Octreoscan) to noninvasively image neuroendocrine and other tumours expressing somatostatin receptors.^[4] More recently, it has been radiolabelled with carbon-11^[5] as well as gallium-68, enabling imaging with positron emission tomography (PET), which provides higher resolution and sensitivity.

Octreotide can also be labelled with a variety of radionuclides, such as yttrium-90 or lutetium-177, to enable peptide receptor radionuclide therapy(PRRT) for the treatment of unresectable neuroendocrine tumours.

Octreotide is the acetate salt of a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. Octreotide is known chemically as L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1- (hydroxy-methyl) propyl]-, cyclic (2→7)-disulfide; [R-(R*,R*)].

Sandostatin LAR Depot is available in a vial containing the sterile drug product, which when mixed with diluent, becomes a suspension that is given as a monthly intragluteal injection. The octreotide is uniformly distributed within the microspheres which are made of a biodegradable glucose star polymer, D,L-lactic and glycolic acids copolymer. Sterile mannitol is added to the microspheres to improve suspendability.

Sandostatin LAR Depot is available as: sterile 5-mL vials in 3 strengths delivering 10 mg, 20 mg, or 30 mg octreotide-free peptide. Each vial of Sandostatin LAR Depot delivers:

NAME OF INGREDIENT	10 MG	20 MG	30 MG
octreotide acetate	11.2 mg*	22.4 mg*	33.6 mg*
D, L-lactic and glycolic acids copolymer	188.8 mg	377.6 mg	566.4 mg
mannitol	41.0 mg	81.9 mg	122.9 mg
*Equivalent to 10/20/30 mg octreotide base.

Each syringe of diluent contains:

carboxymethylcellulose sodium	12.5 mg
mannitol	15.0 mg
water for injection	2.5 mL

The molecular weight of octreotide is 1019.3 (free peptide, C₄₉H₆₆N₁₀O₁₀S₂) and its amino acid sequence is

Sandostatin LAR® Depot (octreotide acetate) Amino acid sequence - Illustration

Octreotide has also been used off-label for the treatment of severe, refractory diarrhea from other causes. It is used in toxicology for the treatment of prolonged recurrent hypoglycemia after sulfonylurea and possibly meglitinides overdose. It has also been used with varying degrees of success in infants with nesidioblastosis to help decrease insulin hypersecretion.

Octreotide has been used experimentally to treat obesity, particularly obesity caused by lesions in the hunger and satiety centers of thehypothalamus, a region of the brain central to the regulation of food intake and energy expenditure.^[6] The circuit begins with an area of the hypothalamus, the arcuate nucleus, that has outputs to the lateral hypothalamus (LH) and ventromedial hypothalamus (VMH), the brain’s feeding and satiety centers, respectively.^[7]^[8] The VMH is sometimes injured by ongoing treatment for acute lymphoblastic leukemia (ALL) or surgery or radiation to treat posterior cranial fossa tumors.^[6] With the VMH disabled and no longer responding to peripheral energy balance signals,

Octreotide has also been investigated for patients with pain from chronic pancreatitis,^[11] and it may be useful in the treatment of thymic neoplasms.

The drug has been used off-label, injected subcutaneously, in the management of hypertrophic pulmonary osteoarthropathy (HPOA) secondary to non-small cell lung carcinoma. Although its mechanism is not known, it appears to reduce the pain associated with HPOA.^{[citation needed]}

It has been used in the treatment of malignant bowel obstruction.^[12]

Octreotide may be used in conjunction with midodrine to partially reverse peripheral vasodilation in the hepatorenal syndrome. By increasing systemic vascular resistance, these drugs reduce shunting and improve renal perfusion, prolonging survival until definitive treatment with liver transplant.^[13] Similarly, octreotide can be used to treat refractory chronic hypotension.^[14]

While successful treatment has been demonstrated in case reports,^[15]^[16] larger studies have failed to demonstrate efficacy in treating chylothorax.^[17]

Octreotide is often give as an infusion for management of acute haemorrhage from esophageal varices in liver cirrhosis on the basis that it reduces portal venous pressure, though current evidence suggests that this effect is transient and does not improve survival.^[18]

A small study has shown that octreotide may be effective in the treatment of idiopathic intracranial hypertension.^[19]^[20]

Octreotide has not been adequately studied for the treatment of children, pregnant and lactating women. The drug is given to these groups of patients only if a risk-benefit analysis is positive.^[21]^[22]

Acetate

C₅₃H₇₄N₁₀O₁₄S₂ , 1139.34326

The most frequent adverse effects (more than 10% of patients) are headache, hypothyroidism, cardiac conduction changes, gastrointestinal reactions (including cramps, nausea/vomiting and diarrhoea or constipation), gallstones, reduction of insulin release, hyperglycemia^[23] or hypoglycemia, and (usually transient) injection site reactions. Slow heart rate, skin reactions such aspruritus, hyperbilirubinemia, hypothyroidism, dizziness and dyspnoea are also fairly common (more than 1%). Rare side effects include acute anaphylactic reactions, pancreatitis andhepatitis.^[21]^[22] One study reported a possible association with rheumatoid arthritis.^[24]

Some studies reported alopecia in patients who were treated by octreotide.^[25] Rats which were treated by octreotide experienced erectile dysfunction in a 1998 study.^[26]

A prolonged QT interval has been observed in patients, but it is uncertain whether this is a reaction to the drug or part of the patients’ illnesses.^[21]

Octreotide can reduce the intestinal resorption of ciclosporin, possibly making it necessary to increase the dose.^[27] Patients with diabetes mellitusmight need less insulin or oral antidiabetics when treated with octreotide. The bioavailability of bromocriptine is increased;^[22] besides being anantiparkinsonian, bromocriptine is also used for the treatment of acromegaly.

Octreotide is absorbed quickly and completely after subcutaneous application. Maximal plasma concentration is reached after 30 minutes. The elimination half-life is 100 minutes (1.7 hours) on average when applied subcutaneously; after intravenous injection, the substance is eliminated in two phases with half-lives of 10 and 90 minutes, respectively.^[21]^[22]

Conventional synthesis of octreotide may be divided into two main approaches, liquid-phase synthesis and solid-phase synthesis. · Octreotide first disclosed in US4395403, in which Octreotide is prepared by solution phase peptide synthesis. The process comprises; removing protected group from peptide;^‘ linking together by an amide bond to two peptide unit; converting a function group at the N- or C- terminal; oxidizing a straight chain polypeptide by boron tristrifluoroacetate.

Since all the synthesis steps are carried out in liquid phase, US’403 process is a time- consuming, multi-step synthesis and it is difficult to separate octreotide from the reaction mixtures. Another solution phase approach described in US6987167 and WO2007110765A2, in which the cyclization of partially deprotected octreotide is carried out in the solution phase using iodine under specific conditions in presence of alcoholic solvents.

US6346601 B1 , WO2005087794A1 and WO2010089757A2 disclose a process for the preparation of octreotide by hybrid approach i. e synthesis of fragments on solid phase and condensing the obtained fragments in a liquid phase.

US6476186 describes the solid phase synthesis, in which the synthesis of octreotide using Thr(ol)(tBu)-2CI-trityl resin as starting material, followed by the cleavage of the straight chain peptide from the resin using a strong acid and the formation of the intra-molecular disulfide bond on the completely deprotected octreotide by oxidation using charcoal catalyst.

US20040039161A1 provides a solid phase .peptide synthetic method for the preparation of C-terminal alcohols using trichloroacetimidate activated linker, making the required peptide chain on the resin support, cleaving the attached peptide; air oxidation to form said C- terminal amino alcohol containing peptide and a 36.3% yield of octreotide after HPLC purification.

Charcoal oxidation or air oxidation needs longer reaction time and results in low yield. Further, in large scale, the conversion of dithiol to disulfide bond ends in unconverted starting material.

Another solid phase approach describes in Bioconjugate chem. 2009, 20, 1323-1331. This article discloses the process of somatostatin and octreotide analogues using solid phase peptide synthesis with CTC resin.

Journal of Harbin Institute of Technology, 2008, Vol 40 (2), 292-295, discloses the process for the preparation of octreotide using CTC resin. According to this process the obtained octreotide has the purity 70.26% by HPLC. During the process of peptide bond formation which is mediated by a coupling agent, the carboxylic group of amino acid interacts with the coupling agent to form an activated intermediate, which in turn interacts with the amino group of the next amino acid.

Racemization is a side-reaction that occurs during the preparation of a peptide. In large scale production, the formations of small amounts of epimers are possible. Detection and removal of these impurities are very difficult. This constitutes one of the most serious drawbacks for the implementation of peptides in commercial scale production.

WO2005087794A1

Conventional syntheses of OCT may be divided two main approaches, direct solid-phase synthesis and liquid-phase synthesis. Direct solid-phase synthesis comprises attachment of a C-terminal amino acid to a resin, and step-by step elongation of the peptide chain, with pre- activated amino acids.

This route is expensive because it requires large excesses of starting amino acids and additionally is quite labor consuming as the peptide size increases, necessitating complex purification procedures to separate the product from the impurities since they are very similar to the final product. These shortcomings are especially important for large scale industrial production of the product. For example, see Canadian Patent Application 2,309,312 and U.S. Patent No. 6,476,186. With each successive condensation reaction required to add an amino acid, waste of starting materials increases, and purification steps are repeated. Liquid-phase synthesis comprises condensation of amino acids in solution. Several blocks, containing from 2 to 5 amino acids may be synthesized independently, followed by condensation of these synthons to each other in the required sequence.

For example, see WO 03/097668; U.S. Patent No. 4,395,403; and RU 2196144 C1. The advantage of this kind of processes is that it is less expensive than the previous one and the product is easier to purify. This method is also more effective for scale-up. However, liquid phase synthesis of lengthy peptide blocks, for example having more than 3 amino acids, is inefficient. Liquid-phase octreotide synthesis has the drawback is that the method is extremely labor-intensive and time consuming.

U.S. Patent No. 6,346,601 describes a method for octreotide synthesis where a solid-phase method is used to obtain a 7-mer, followed by condensation in solution with the modified amino acid threoninol. However, by using solid- phase synthesis to produce a 7-mer, only one less condensation is required compared to the solid-phase process for forming octreotide itself. Thus, only a marginal efficiency is introduced.

Summary of the invention According to an embodiment of the invention, there is provided a process for obtaining octreotide or a pharmaceutically acceptable salt thereof by hybrid solid-phase – liquid-phase synthesis. The synthesis comprises the steps of condensing two or three peptide blocks using liquid phase condensation to form a condensation product followed by cyclizing the product.

Each peptide block contains two or more amino acid residues, and at least one of the blocks is synthesized by solid-phase synthesis. The condensation product comprises in sequence the amino acids residues of octreotide. In the step of cyclizing, the condensation product is cyclized to form a disulfide bridge between the two cysteine residues, thereby forming octreotide. Further, according to another embodiment of the invention, a process is provided for obtaining an intermediate in octreotide synthesis by hybrid solid- phase – liquid-phase synthesis.

The synthesis of the intermediate comprises the steps of obtaining two or three peptide blocks, each peptide block containing two or more amino acid residues, and at least one of the blocks is synthesized by solid-phase synthesis. Subsequently, the peptide blocks are condensed using liquid phase condensation to form a condensation product, wherein the condensation product comprises in sequence the amino acids residues of octreotide.

This invention provides a more cost-effective and labor-saving method for obtaining OCT and its pharmaceutically acceptable salts by means of hybrid solid-phase – liquid-phase synthesis. The invention involves liquid phase condensation of two peptide blocks, at least one of which is obtained by solid- phase synthesis, the blocks containing more two or more amino acid residue in every block, followed by formation of a disulfide bridge from the two cysteine groups. Optionally, three blocks may be condensed. This hybrid solid phase-liquid phase method involves formation of one or more blocks of the octreotide amino acid sequence by solid-phase synthesis, followed by liquid phase condensation of the block(s) with required supplementary amino acids or other block(s) of amino acids.

This method is a blend of solid-phase and liquid-phase synthesis methods, combining the efficiencies of preparing shorter (6-mer or less) peptides using a solid-phase method with relative cheapness and easiness of purification of the product, characteristic of the liquid-phase method. Generally, the methods of invention comprise synthesizing specific side- chain protected peptide fragment intermediates of OCT on a solid support or in solution, coupling of the protected fragments in solution to form a protected OCT, followed by deprotection of the side chains and oxidation to yield the final OCT. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the OCT

………………

WO2013046233A2

Stage-I: Preparation of protected octreotide anchored to 2-CTC Resin

Method -1:

Octreotide was synthesized manually on 2-chlorotrityl chloride resin (substitution 0.90 mmol/g) by standard Fmoc solid phase synthesis strategy. The resin was soaked in the mixture of DC and DMF for the swelling. Fmoc-Thr(tBu)-OL was treated with the swelled 2- CTC resin in DCM in the presence of DIEA and substitution level was determined by weight gain measurements and also by UV Method. After the coupling of the first amino acid onto the resin, the un-reacted linkers on the resin (polymer) are protected, to avoid the undesired peptide chain formation, with a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to the resin. The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out in DMF. The N- terminal Fmoc group was removed with 20 %( V/V) piperidine in DMF. The couplings were performed by dissolving the Fmoc-Amino acid (2 eq.) and HOBt (2 eq.) in DMF. The solution was cooled on ice and then DIC (2 eq.) was added. The reaction mixture was added to the resin and allowed to react for 2 hrs. The efficiency of the coupling was monitored using the Kaiser Ninhydrin test. The coupling step was repeated if Kaiser test was found positive. The sequence of addition for the synthesis of Octeriotide was Fmoc-Cys(Trt), Fmo-Thr(tBu), Fmoc-Lys(Boc), Fmoc-Trp(Boc), Fmoc-Phe, Fmoc-Cys(Trt), Boc-D-Phe.

Method -2:

Octreotide was synthesized manually on 2-chlorotrityl chloride resin (substitution 0.90 mmol/g) by standard Fmoc solid phase synthesis strategy. The resin was soaked in the mixture of MDC and DMF for the swelling. Fmoc-Thr-OL was treated with the swelled 2-CTC resin in DCM in the presence of DIEA and substitution level was determined by weight gain measurements and also by UV Method. After the coupling of the first amino acid onto the resin, the un-reacted linkers on the resin (polymer) are protected, to avoid the undesired peptide chain formation, with a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to the resin. The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out in DMF. The N- terminal Fmoc group was removed with 20 %( V7V) piperidine in DMF. The couplings were performed by dissolving the Fmoc-Amino acid (2 eq.) and HOBt (2 eq.) in DMF. The solution was cooled on ice and then DIC (2 eq.) was added. The reaction mixture was added to the resin and allowed to react for 2 hrs. The efficiency of the coupling was monitored using the Kaiser Ninhydrin test. The coupling step was repeated if Kaiser test was found positive. The sequence of addition for the synthesis of Octeriotide was Fmoc-Cys(Trt), Fmo-Thr(tBu), Fmoc-Lys(Boc), Fmoc-Trp(Boc), Fmoc-Phe, Fmoc-Cys(Trt), Boc-D-Phe.

Stage-ll: Cleavage of peptide from resin along with global deprotection

The peptide resin (200 g, obtained in stage I) was swelled in DCM (500 mL) for 15 to 20 minutes under nitrogen at 25-30° C. The cocktail mixture (2.0 L – TFA (1.8 L), water (80 mL) DCM (80mL) and TIPS (80 mL)) was charged to the resin at 25-30° C. and the obtained reaction mixture was stirred for 2.5 hours at 25-30°C under nitrogen atmosphere. The reaction mixture was filtered and washed the resin with TFA (250 mL). The obtained filtrate was charged into cold MTBE (4 L, pre-cooled to a temperature of 0 -5° C) under stirring and allowing the temperature to rise more than 5° C. The reaction mixture was stirred for 45-75 minutes at 0-5°C. The obtained suspension was filtered, washed the solid with MTBE (5 L) and dried the solid under nitrogen. The product was stir with 5%ethanol in ethyl acetate at 25-30°C. Filtered the product, wash ith ethyl acetate and dried under vacuum to obtain a desired product

Stage-Ill: Disulphide bridge formation

The free thiol (100 g) obtained above is dissolved in methanol (22.0 L) with small amount of acetic acid and water (4.5 L) and stirred. Iodine solution (20gm iodine in 500 mL methanol) was added to the reaction mass slowly up to yellow color persists. The reaction was maintained for another 2 hrs, and the excess iodine quenched with Indion 830-S Resin (900 g) and filtered the resin. The filtrate was evaporated and precipitated using TBE or directly taken the solution for purification using preparative HPLC.

Stage -IV: Preparative HPLC Purification

Method-1 :

The crude disulphide bridge peptide was purified on a preparative reverse phase HPLC system using Kromasil C-18, 10 micron (50 x 250 mm). and eluting with a solvent system of 0.2% acetic acid in water(A) and 0.2% acetic acid in methanol(B). A linear gradient of 20- 60% B was used at a flow rate of 80mlJmin and detection at 220 nm.

The octreotide was eluted at around 25% methanol. The fractions were collected at regular intervals and assayed by HPLC to determine the purity of fractions. The desired purities fractions were pooled together and evaporated using Rota evaporator. The aqueous layer was lyophilized to isolate octreotide acetate

Method-2:

The crude disulphide bridge peptide was purified on a preparative reverse phase HPLC system using Kromasil C-18, 10 micron (50 x 250 mm) and eluting with a solvent system of 0.4% acetic acid in water(A) and methanol(B). A linear gradient of 25-60% B was used at a flow rate of 80mL/min and detection at 220 nm.

The octreotide was eluted at around 25% methanol. The fractions are collected at regular intervals and are assayed by HPLC to determine the purity and fractions. The desired purities may be pooled together and were evaporated using Rota evaporator. The aqueous layer was lyophilized to isolate octreotide acetate >

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WO2010089757A2

Octreotide is a highly potent and pharmacologically selective analog of somatostatin. It inhibits growth hormone for long duration and is thereof indicated for acromegaly to control and reduce the plasma level of growth hormone. The presence of D-Phe at the N-terminal and an amino alcohol at the C-terminal, along with D-Tryptophan and a cyclic structure makes it very resistant to metabolic degradation.

Octreotide comprises 8 amino acids which has the following structural formula:

(D)Phe-Cys-Phe-{D)Trp-Lys-Thr-Cys-Thr-OL

Formula(l) wherein sulphur atoms of the Cys at the position 2 and of the Cys at the position 7 are mono-cyclic to form an -S-S- bridge.

A considerable number of known, naturally occurring small and medium-sized cyclic peptides as well as some of their artificial derivatives and analogs possessing desirable pharmacological properties have been synthesized. However, wider medical use is often hampered due to complexity of their synthesis and purification. Therefore, improved methods for making these compounds in simple, lesser steps and at lesser cost are desirable and this is the felt need of the industry and the mankind.

Conventional synthesis of octreotide may be divided into two main approaches, direct solid-phase synthesis and liquid-phase synthesis. Solution phase synthesis has been described by Bauer et al., (Sandoz) (Eur. Pat. Appl. 29,579 and U.S. Pat. No. 4,395,403). The process comprises: removing protected group from peptide; linking together by an amide bond two peptide unit; converting a function group at the N- or C-terminal; oxidizing a straight chain polypeptide by boron tristrifluoroacetate. This process involves a time-consuming, multi-step synthesis, and it is difficult to separate octreotide from the reaction mixtures since all the synthesis steps are carried out in liquid phase.Another solution phase approach described by Chaturvedi, et al., (Wockhardt) in U.S. Pat. No. 6,987,167 and EP 1506219 A, claims the cyclization of partially deprotected octreotide in the solution phase using iodine under conditions and for a time sufficient to form the octreotide.

Synthesis in solid phase have been described subsequently (Mergler et al., Alsina et al., Neugebauer). The above prior art for solid phase peptide synthesis cites the octapeptide formation, by starting the synthesis from the threoninol residue which makes it mandatory to protect this residue. Mergler et al., (Peptides: Chemistry and Biology. Proceedings of the 12* American Peptide Symposium. Smith, J.A. And Rivier J.E. Eds ESCOM, Leiden, Poster 292 Presentation, (1991) ) describes a synthetic process, using an aminoethyl resin upon which the Threoninol residue is incorporated with the two alcohol functions protected in acetal form The synthesis is carried out following an Fmoc/tBu protection scheme, forming the disulphide bridge on resin by oxidation of the thiol groups of the previously deprotected cysteine residues and releasing and deprotecting the peptide with a 20% mixture of TFA/DCM.

In early 1997, Alsina J. et al. ( Alsina J., Chiva C, Ortiz M., Rabanal F., Giralt E., and Albericio F., Tetrahedron Letters, 38, 883-886, 1997) described the incorporation, on active carbonate resins, of a Threoninol residue with the amino group protected by the Boc group and the side chain protected by a BzI group. The synthesis was then continued by Boc/Bzl strategy. Formation of the disulfide bridge was carried out directly on resin using iodine and the peptide was cleaved from the resin and its side chain protecting groups were simultaneously removed with HF/anisole 9/1. At the final stage the formyl group was removed with a piperidine/DMF solution.

Neugebauer (Neugebauer W., Lefevre M.R., Laprise R, Escher E., Peptides: Chemistry, Structure and Biology, p 1017, Marshal G.R. And Rivier J.E. Eds. ESCOM.Leiden (1990) described a linear synthesis with a yield of only 7%.

Edwards et al., (Edwards B.W., Fields C.G., Anderson CJ., Pajeau T.S., Welch M.J., Fields G.B., J.Med.Chem. 37, 3749-3757 (1994) carried out another another solid- phase type approximation; they synthesized step-by-step on the resin, the peptide D- Phe-Cys(Acm)-Phe-D-Tφ(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-HMP-Resin. Next they proceeded to form the disulfide on resin and then release the peptide from the resin by means of aminolysis with threoninol, with obtaining a total yield of only 14%.

The solid phase synthesis described by Yao-Tsung Hsieh et. al., in U.S. Pat. No. 6,476,186 involves the synthesis of octreotide by using Thr(ol)(tBu)-2Cl-trityl resin as starting material followed by the cleavage of the straight chain peptide from the resin by using a strong acid and the formation of the intra-molecular disulfide bond on the completely deprotected octreotide by oxidation using charcoal catalyst and a higher yield of >70%.

Another solid phase synthesis described by Berta Ponsati et.al (Lipotec) in U.S. Pat No. 6,346,601 and EP 0953577 B involve the coupling of threoninol on the protected heptapeptide in solution, after a selective acid cleavage from the chlorotrityl resin without affecting the peptide side-chain protecting groups.

A hybrid solid phase-liquid phase method for synthesis of octreotide described by Iarov et al., (Dalton Chemical Laboratories) in WO 2005087794 wherein the method comprises liquid phase condensation of two or three peptide blocks in which at least one peptide block is synthesized by solid-phase method.

EP 1511761 Bl involves cyclization on the semi-protected linear peptide wherein one of the cysteine residue is protected with an orthogonal protecting group. The radioactive isotope labeling of octreotide by the coupling of bifunctional chelating agents like DTPA or DOTA to the peptide was described by Te- Wei Lee et al., in U.S. Pat. No. 5,889,146 (Inst, of Nuclear Energy Research)

The method for cyclization of linear vapreotide by means of intramolecular cysteine formation has been described by Quattrini et. al., (Lonza AG) in WO 2006048144, wherein the process involves the synthesis of linear vapreotide peptide on Sieber-resin (from Novabiochem) by Fmoc standard groups, wherein the side chain protecting groups are D or L-Trp(Boc), Cys(Trt), Lys(Boc), Tyr(tBu). The protected peptide is cleaved off in 5% TFA in dichloromethane and then globally deprotected by acidolysis in a cleavage mix of 300 equivalents of concentrated TFA, 12 equivalents of Dithiothreitol, 12 equivalents of Dichloromethane, 50 equivalents of water forl hour at room temperature. The Boc groups are removed. The product was subjected to charcoal method using trace amounts of activated, powdered charcoal wherein a concentration of the linear cysteinyl peptide of 50 mg/ml (1 eq.) in DMF in the presence of 1 eq. Diisopropyl-ethyl-amine and that additionally air was sparged at low pressure into the liquid under stirring. After 15-20 hrs, 100% conversion was achieved with 84% (w/w) analytical yield of 79% vapreotide.

The formation of intramolecular disulphide formation in a polypeptide by reacting with hydrogen peroxide has been described by Mineo Niwa et al. (Fujisawa Pharmaceutical Co.) in U.S. Pat. No.5, 102,985 wherein the reaction is to be carried out at a pH of about 6 tol 1, wherein the molar ratio of H₂O₂ to polypeptide is within the range of 1:1 to 100:1. The above cited prior art mainly carries out the cyclization of the peptide on the resin or on partially protected or protected peptides. The use of partial or minimal protecting group strategies and improvement in the activation methods have considerable effect on limitations of poor solubility and possible danger of racemization due to the overactivation of carboxyl groups. However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules.

Example 8:

Oxidation of S-H peptide with DMSO-HCl to get S-S peptide:

(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-OL

Formula (1)

S-H peptide ( 9g) was dissolved in 6.5L DMSO and under ice-cooling 6.5L IM HCl was added slowly so that temperature is below 26°C. Stirring was continued for 6 hours. At room temperature after six hours reaction mixture was diluted with 13L of water and filtered through Whatman no. 41 through Celite bed. The filtrate was loaded on C- 18 column for concentration. The compound was eluted with 100% acetonitrile. The eluant was concentrated on rotavap and then the concentrated solution was centri-evaporated to dryness. The RP-HPLC profile of crude octreotide is depicted in Figure 1.

Weight of crude peptide =3.9g.(45%)

Purity: 44.25%

Example 9:

Purification of crude octreotide:

The crude octreotide was loaded on to cation ion exchange column and eluted using a salt gradient using a Akta Purifier (by Amersham, Sweden) low pressure chromatography system. The IEX fractions of purity >70% were further loaded for RP-HPLC purification on Kromacil C-18 column of (250x50mm,100A°.) The peptide was purified by using aqueous TF A(O-0.5%) and methanol/ethanol and/or Acetonitrile in a gradient program on a Shimadzu preparative HPLC System consisting of a controller, 2 LC8A pumps, and UV-Vis detector. The purified peptide was analysed by analytical RP-HPLC (Figure 5). Fractions of > 99% purity were subjected either by RP-HPLC or IEX to salt exchange and concentrated to remove organic solvent either by rota or reverse osmosis and subsequently lyophilized to get final API with purification step yield of 70% or above.The MS spectrum of octreotide is depicted in Figure 6.

References

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US8507432	Jun 11, 2010	Aug 13, 2013	Endo Pharmaceuticals Solutions Inc.	Controlled release formulations of octreotide
US20100247594 *	Jun 11, 2010	Sep 30, 2010	Endo Pharmaceuticals Solutions Inc.	Delivery of dry formulations of octreotide
US20110009338 *	Jun 11, 2010	Jan 13, 2011	Endo Pharmaceuticals Solutions Inc.	Controlled release formulations of octreotide

WO2010089757A2	May 4, 2009	Aug 12, 2010	Usv Limited	An improved process for synthesis of cyclic octapeptide
WO2013046233A2	Sep 28, 2012	Apr 4, 2013	Mylan Laboratories Ltd	Process for the preparation of octreotide acetate
WO2013132505A1	Mar 9, 2012	Sep 12, 2013	Natco Pharma Limited	Improved process for preparation of octreotide by solution phase peptide synthesis
US8377891	May 4, 2009	Feb 19, 2013	Usv, Ltd.	Process for synthesis of cyclic octapeptide

WO2003097668A2 *	Apr 16, 2003	Nov 27, 2003	Suresh Beri	Novel process for production of the somatostatin analog, octreotide
US6346601 *	Jan 29, 1999	Feb 12, 2002	Lipotec S.A.	Procedure for obtaining the somatostatin analog, octreotide
US6476186 *	May 24, 2000	Nov 5, 2002	Institute Of Nuclear Energy Research	Process for preparing octreotide and derivatives thereof

WO2005087794A1	Mar 14, 2005	Sep 22, 2005	Dalton Chemical Lab Inc	Process for octreotide synthesis
WO2007110765A2	Mar 28, 2007	Oct 4, 2007	Deshpande Amol Ashok	Processes for the preparation of octreotide
WO2010089757A2	May 4, 2009	Aug 12, 2010	Usv Limited	An improved process for synthesis of cyclic octapeptide
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US6987167	May 22, 2002	Jan 17, 2006	Wockhardt Limited	Process for production of the somatostatin analog, octreotide
US20040039161	Aug 22, 2002	Feb 26, 2004	Mayer John Philip	Use of trichloroacetimidate linker for peptide synthesis

RUXOLITINIB…FOR THE TREATMENT OF INT OR HIGH-RISK MYELOFIBROSIS

Uncategorized Comments Off

Mar 102014

Ruxolitinib

(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile, cas no 941678-49-5

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile

1H-Pyrazole-1-propanenitrile, beta-cyclopentyl-4-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-,(betaR)-

Formula: C₁₇H₁₈N₆
Molecular Weight: 306.37

JAKAFI® (ruxolitinib) Structural Formula Illustration

Phosphate salt

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate

INCB 018424

INCB 018424
INCB018424
Ruxolitinib
UNII-82S8X8XX8H

CAS No.: 1092939-17-7
M.Wt: 404.36
Formula: C17H21N6O4P
Ruxolitinib phosphate

LAUNCHED 2011, INCYTE FOR MYELOFIBROSIS, NDA202192, 2011-11-16

CLINICAL TRIALS.http://clinicaltrials.gov/search/intervention=INCB018424+OR+ruxolitinib

EMA:Link,

US FDA:link

HPLC, MS, NMR…http://www.medkoo.com/Product-Data/Ruxolitinib/Ruxolitinib-QC-LC20130225.pdf

http://file.selleckchem.com/downloads/nmr/S137803-INCB018424-HNMR-Selleck.pdf

http://file.selleckchem.com/downloads/hplc/S137803-INCB018424-HPLC-Selleck.pdf

Ruxolitinib phosphate is a kinase inhibitor with the chemical name (R)-3-(4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate and a molecular weight of 404.36.

Ruxolitinib is a janus-associated kinase inhibitor indicated to treat bone marrow cancer, specifically intermediate or high-risk myelofibrosis. FDA approved on November 16, 2011.

INCB018424 is the first potent, selective, JAK1/2 inhibitor to enter the clinic with IC50 of 3.3 nM/2.8 nM, >130-fold selectivity for JAK1/2 versus JAK3

Ruxolitinib phosphate has the following structural formula:

JAKAFI® (ruxolitinib) Structural Formula Illustration

Ruxolitinib phosphate is a white to off-white to light pink powder and is soluble in aqueous buffers across a pH range of 1 to 8.

Jakafi (ruxolitinib) Tablets are for oral administration. Each tablet contains ruxolitinib phosphate equivalent to 5 mg, 10 mg, 15 mg, 20 mg and 25 mg of ruxolitinib free base together with microcrystalline cellulose, lactose monohydrate, magnesium stearate, colloidal silicon dioxide, sodium starch glycolate, povidone and hydroxypropyl cellulose

.NCI: /Ruxolitinib phosphate/ The phosphate salt form of ruxolitinib, an orally bioavailable Janus-associated kinase (JAK) inhibitor with potential antineoplastic and immunomodulating activities. Ruxolitinib specifically binds to and inhibits protein tyrosine kinases JAK 1 and 2, which may lead to a reduction in inflammation and an inhibition of cellular proliferation. The JAK-STAT (signal transducer and activator of transcription) pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cellular proliferation, growth, hematopoiesis, and the immune response; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies. (NCI Thesaurus)

patent expiry

US pat 7598257 exp 24/12/27

US pat 8415362 exp 24/12/27

NCE.Nov 16, 2016

Discovered by Incyte, ruxolitinib phosphate is an inhibitor of Janus-associated kinase 2 (JAK2), a protein involved in signal transduction. This orally available compound was approved and launched in the U.S. in 2011 for the treatment of patients with intermediate or high-risk myelofibrosis (MF), including primary MF, post-polycythemia vera MP and post-essential thrombocythemia MF. A regulatory application in the E.U. was filed in 2011 and a positive opinion was assigned in April 2012. Final E.U. approval was obtained in August 2012. In November 2012, the product was launched in the U.K. for the treatment of disease-related splenomegaly or symptoms in primary myelofibrosis or myelofibrosis due to polycythemia vera or essential thrombocythemia. In 2012, the product has been filed for approval in Japan for the treatment of myelofibrosis.

Phase II clinical trials are also ongoing for the treatment of multiple myeloma, leukemia, pancreas cancer, thrombocytopenia and for the treatment of relapsed or refractory diffuse large B-cell or peripheral T-cell non-Hodgkin lymphoma following donor stem cell transplant. In Japan, the product is under development in phase III trials for the treatment of polycythemia vera and in phase II trials for the treatment of myelofibrosis. No recent development has been reported for clinical trials for the treatment of rheumatoid arthritis (RA), for the treatment of psoriasis or for the treatment of metastatic prostate cancer. Columbia University is evaluating the compound in preclinical studies for the treatment of alopecia areata. The University of Pennsylvania is conducting phase II clinical trials for the treatment of breast cancer.

In 2008 and 2009, the compound was assigned orphan drug designation in the U.S. and the E.U., respectively, for the treatment of myelofibrosis. This designation was assigned in Japan for this indication in 2011. Additional orphan drug designation was assigned by the FDA in 2010 for the treatment of polycythemia vera and for the treatment of essential thrombocythemia. In 2013, an orphan drug designation was assigned in U.S. for the treatment of pancreatic cancer. In 2009, fast track designation was assigned to ruxolitinib phosphate in the U.S .for the treatment of myelofibrosis.

Ruxolitinib (trade names Jakafi and Jakavi, by Incyte Pharmaceuticals and Novartis) is a drug for the treatment of intermediate or high-risk myelofibrosis, a type of bone marrow cancer.It is also being investigated for the treatment of other types of cancer (such as lymphomas and pancreatic cancer), for polycythemia vera, and for plaque psoriasis.
The phase III Controlled Myelofibrosis Study with Oral JAK Inhibitor-I (COMFORT-I) and COMFORT-II trials showed significant benefits by reducing spleen size, relieving debilitating symptoms, and improving overall survival.

INCYTE developed in cooperation with companies and NOVARTIS jak2 selective inhibitor Ruxolitinib(INCB-018424) – has been approved by the FDA successfully listed). (Safety and Efficacy of INCB018424, a JAK1 and JAK2 Inhibitor, in Myelofibrosis. Srdan Verstovsek, MD, Ph.D., Hagop Kantarjian, MD, Ruben A. Mesa. MD, et al. N Engl J Med 2010; 363: 1117-1127).

The presently disclosed a series of patent applications JAK inhibitors, including WO2001042246, WO200200066K WO2009054941 and WO2011013785, etc.

Mechanism of action

Ruxolitinib is a Janus kinase inhibitor with selectivity for subtypes 1 and 2 of this enzyme.
Side effects

Immunologic side effects have included herpes zoster (1.9%) and case reports of opportunistic infections.[10] Metabolic side effects have included weight gain (7.1%). Laboratory abnormalities have included alanine transaminase (ALT) abnormalities (25.2%), aspartate transaminase (AST) abnormalities (17.4%), and elevated cholesterol levels (16.8%).
Legal status

In November 2011, ruxolitinib was approved by the USFDA for the treatment of intermediate or high-risk myelofibrosis based on results of the COMFORT-I and COMFORT-II Trials.

Some analysts believe this to be a potential blockbuster drug.[3] As of the end of March 2012, and according to an Incyte spokesman, approximately 1000 physicians had prescribed the drug in the United States, out of a total 6500 hematologists and oncologists nationwide.

The US Food and Drug Administration had approved Incyte’s Jakafi (ruxolitinib) to treat patients with the bone marrow disease myelofibrosis (MF). Jakafi is the first and only drug granted license specifically for the treatment of the rare blood cancer.
Jakafi approved by FDA to treat rare bone marrow disease
Posted By Edward Su On November 17th, 2011

MF is a rare, potentially life-threatening blood cancer with limited treatment methods. Patients with the bone marrow disoder, characterized by bone marrow failure, enlarged spleen (splenomegaly), suffer from the symptoms of fatigue, night sweats and pruritus, poor quality of life, weight loss and shortened survival. The US drug firm Incyte estimates the disease affects about 16,000-18,500 people in the USA. Currently, the disease is treated with chemotherapy or bone marrow transplant.

Incyte’s Jakafi, the first drug to reach market from the Wilmington-based drug company, was approved by the FDA as a twice-a-day pill for the treatment of patients with intermediate or high-risk myelofibrosis (MF), including primary MF, post-polycythemia vera MF and post-essential thrombocythemia MF. The US regulators reviewed Jakafi under its priority review program for important new therapies.

The approval of Jakafi was based on the results from two clinical studies involved 528 patients with the disease. Patients in the Jakafi treatment arm experienced a significant reduction in the size of their spleen as well as a 50 percent decrease in symptoms, including pain, discomfort and night sweats.

Jakafi, generically known as ruxolitinib, works by blocking JAK1 and JAK2 enzymes associated with the disease. The company has co-developed the drug with Novartis as part of their collaboration signed in 2009. The Swiss drug firm has the rights to market Jakafi in other countries.

“The availability of Jakafi is a significant medical advancement for people living with myelofibrosis, a debilitating disease,” said Paul A. Friedman, M.D., President and Chief Executive Officer of Incyte. “This milestone marks a tremendous achievement for Incyte because a scientific discovery from our research laboratories has become the first JAK inhibitor to reach the market and provide a clinical benefit to patients.”

Richard Pazdur, director of the Office of Hematology and Oncology Drug Products in the FDA’s Center for Drug Evaluation and Research, said that Jakafi “represents another example of an increasing trend in oncology where a detailed scientific understanding of the mechanisms of a disease allows a drug to be directed toward specific molecular pathways”.

Incyte says Jakafi will be available next week, and the drug will cost $7,000 per month, or $84,000 for a year’s supply for insured patients. The company plans to provide Jakafi free to uninsured patients and will offer co-pay assistance to patients with financial need.

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NMR free base

http://www.google.com/patents/US8410265

For (R)-13 (free base): ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J=0.42 Hz), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, 1H, J=2.34, 3.51 Hz), 6.98 (dd, 1H, J=1.40, 3.44 Hz), 4.53 (td, 1H, J=19.5, 4.63 Hz), 3.26 (dd, 1H, J=9.77, 17.2 Hz), 3.18 (dd, 1H, J=4.32, 17.3 Hz), 2.40 (m, 1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H); C₁₇H₁₈N₆(MW, 306.37) LCMS (EI) m/e 307 (M⁺+H).

http://www.google.com/patents/US8410265

phosphate

For (R)-14 (phosphate): mp. 197.6° C.; ¹H NMR (DMSO-d₆, 500 MHz) δ ppm 12.10 (s, 1H), 8.78 (s, 1H), 8.68 (s, 1H), 8.36 (s 1H), 7.58 (dd, 1H, J=1.9, 3.5 Hz), 6.97 (d, 1H, J=3.6 Hz), 4.52 (td, 1H, J=3.9, 9.7 Hz), 3.25 (dd, 1H, J=9.8, 17.2 Hz), 3.16 (dd, 1H, J=4.0, 17.0 Hz), 2.41, (m, 1H), 1.79 (m, 1H), 1.59 (m, 1H), 1.51 (m, 2H), 1.42 (m, 1H), 1.29 (m, 2H), 1.18 (m, 1H); ¹³C NMR (DMSO-d₆, 125 MHz) δ ppm 152.1, 150.8, 149.8, 139.2, 131.0, 126.8, 120.4, 118.1, 112.8, 99.8, 62.5, 44.3, 29.1, 29.0, 24.9, 24.3, 22.5; C₁₇H₁₈N₆(MW, 306.37 for free base) LCMS (EI) m/e 307 (M⁺+H, base peak), 329.1 (M⁺+Na).

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SYNTHESIS

http://www.google.com/patents/US8410265

Figure US08410265-20130402-C00204

Figure US08410265-20130402-C00211

Figure US08410265-20130402-C00230

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SYNTHESIS

US20100190981

(R)-Methyl 3-cyclopentyl-3-(4-(7-((2-(trimethylsilypethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoate ((R)-22). A solution of (E)-methyl 3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)acrylate (21, 815 mg) in tetrahydrofuran (THF, 8.0 mL) in a pressure glass tube was treated with the catalyst [Rh(COD)(−)-DuanPhos](BF₄) (4.6 mg) under nitrogen before the reaction mixture was pressurized with hydrogen gas to 50 bar pressure. The reaction mixture was stirred at 35° C. under this hydrogen pressure for 22 h. When HPLC analysis showed that the substrate was almost completely consumed, the reaction mixture was cooled down to room temperature. The enantiomeric excess of the reaction mixture was determined to be 94.7% ee (97.35% of the second peak, (R)-22; 2.65% of the first peak, (S)-22) by chiral HPLC analysis. The reaction mixture was then filtered through a thin silica gel pad and the pad was washed with tetrahydrofuran (THF, 5 mL). The filtrate was then concentrated under reduced pressure to dryness. The resultant foamy solid (778 mg) was analyzed by chiral HPLC analysis and result showed a 94.7% of enantiomeric excess favoring the second peak (97.35% of the second peak, (R)-22; 2.65% of the first peak, (S)-22).

(3R)-3-Cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoic acid ((R)-23). To a stirred solution of (3R)-methyl 3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoate ((R)-22, 2.47 g, 5.26 mmol) in THF (30 mL) at room temperature was added a solution of lithium hydroxide monohydrate (LiOH—H₂O, 265 mg, 6.31 mmol, 1.2 equiv) in water (15 mL). The reaction mixture was stirred at room temperature for 3 h. When LCMS showed the reaction was complete, the reaction mixture was then acidified with 1 N aqueous HCl solution to pH 5 before it was extracted with EtOAc (2×25 mL). The combined organic layers were washed with brine, dried over magnesium sulfate (MgSO₄), filtered and concentrated under reduced pressure to afford (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoic acid ((R)-23, 2.40 g, 2.40 g theoretical, 100% yield) as a colorless oil, which solidified upon standing at room temperature in vacuo. For (R)-23: ¹H NMR (CDCl₃, 300 MHz) δ ppm 8.95 (s, 1H), 8.95 (bs, 1H), 8.36 (s, 1H), 7.57 (d, 1H, J=3.7 Hz), 6.99 (d, 1H, J=3.7 Hz), 5.74 (s, 2H), 4.65 (dt, 1H, J=3.1, 10.3 Hz), 3.58 (t, 2H, J=8.2 Hz), 3.24 (dd, 1H, J=16.5, 10.3 Hz), 3.04 (dd, 1H, J=16.2, 3.1 Hz), 2.59 (m, 1H), 2.00 (m, 1H), 1.77-1.24 (m, 7H), 0.97 (t, 2H, J=8.2 Hz), 0.00 (s, 9H); C₂₃H₃₃N₅O₃Si (MW, 455.63), LCMS (EI) m/e 456.1 (M⁺+H).

(3R)-3-Cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanamide ((R)-24). To a stirred solution of (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoic acid ((R)-23, 20 mg, 0.044 mmol) in DMF (1 mL) at room temperature was added N,N-carbonyldiimidazole (CDI, 21 mg, 0.13 mmol, 3.0 equiv). The reaction mixture was then stirred at room temperature and TLC was used to follow the reaction for formation of acyl imidazole (consumption of acid to a higher Rf spot with 30% EtOAc/hexane). When TLC showed that the acyl imidazole transformation was complete, ammonia gas was then bubbled through the stirred solution for 30 min to afford the amide (followed by LCMS). The excess amount of ammonia gas was evaporated by bubbling nitrogen vigorously through the solution. The crude product, (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanamide ((R)-24), in DMF was used directly to the following reaction to convert amide ((R)-24) into the corresponding nitrile ((R)-20).

(3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-20). Method A. To a stirred solution of (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanamide ((R)-24, 20 mg, 0.044 mmol) in DMF (1 mL) at 0° C. was added methylene chloride (1 mL) and triethylamine (0.12 mL, 0.88 mmol, 20.0 equiv), followed by trichloroacetyl chloride (0.052 ml, 0.462 mmol, 10.5 equiv). The resulting reaction mixture was stirred at 0° C. for 1 h. When LCMS showed the reaction was complete, the reaction mixture was quenched with saturated sodium bicarbonate solution (NaHCO₃, 5 mL) before being extracted with EtOAc (2×10 mL). The combined organic layers were washed with brine, dried over magnesium sulfate (MgSO₄), filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography with 0-75% EtOAc/hexane gradient elution to give (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-20, 10 mg, 19 mg theoretical, 53% yield). For (R)-20: ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 8.83 (s, 1H), 8.75 (s, 1H), 8.39 (s, 1H), 7.77 (d, 1H, J=3.7 Hz), 7.09 (d, 1H, J=3.7 Hz), 5.63 (s, 2H), 4.53 (td, 1H, J=19.4, 4.0 Hz), 3.51 (t, 2H, J=8.1 Hz), 3.23 (dq, 2H, J=9.3, 4.3 Hz), 2.41 (m, 1H), 1.79 (m, 1H), 1.66-1.13 (m, 7H), 0.81 (t, 2H, J=8.2 Hz), 0.124 (s, 9H); C₂₃H₃₂N₆OSi (MW, 436.63), LCMS (EI) m/e 437 (M⁻+H) and 459 (M⁺+Na).

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-13, free base). Method B. To a solution of (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-20, 463 g, 1.06 mol, 98.6% ee) in acetonitrile (4.5 L) was added water (400 mL) followed immediately by lithium tetrafluoroborate (LiBF₄, 987.9 g, 10.5 mol, 10.0 equiv) at room temperature. The reaction temperature was observed to decrease from ambient to 12° C. upon addition of the water and then increase to 33° C. during the addition of lithium tetrafluoroborate (LiBF₄). The resulting reaction mixture was heated to reflux (about 80° C.) for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v). When LCMS and TLC analyses showed both the hydroxyl methyl intermediate ((R)-25) and fully de-protected material ((R)-13, free base) produced but no starting material ((R)-20) left, the reaction mixture was cooled gradually to <5° C. before a 20% aqueous solution of ammonium hydroxide (NH₄OH, 450 mL) was added gradually to adjust the pH of the reaction mixture to 9 (checked with pH strips). The cold bath was removed and the reaction mixture was gradually warmed to room temperature and stirred at room temperature for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v) to confirm complete de-protection. When LCMS and TLC showed the reaction was deemed complete, the reaction mixture was filtered and the solids were washed with acetonitrile (1 L). The combined filtrates were then concentrated under reduce pressure, and the residue was partitioned between ethyl acetate (EtOAc, 6 L) and half-saturated brine (3 L). The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 L). The combined organic layers were washed with half-saturated sodium bicarbonate (NaHCO₃, 3 L) and brine (3 L), dried over sodium sulfate (Na₂SO₄), and concentrated under reduced pressure to give the crude product as an orange oil. The crude material was then purified by flash column chromatography (SiO₂, 40 to 100% ethyl acetate/heptane gradient elution) to afford (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-13, free base, 273 g, 324.9 g theoretical, 84% yield) as a white foam. This material was checked by ¹⁹F NMR to ensure no lithium tetrafluoroborate (LiBF₄) remained and by chiral HPLC (Chiralcel OD, 90:10 hexane/ethanol) to confirm enantiomeric purity and was used without further purification to prepare the corresponding phosphate salt. For (R)-13 (free base): ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J=0.42 Hz), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, 1H, J=2.34, 3.51 Hz), 6.98 (dd, 1H, J=1.40, 3.44 Hz), 4.53 (td, 1H, J=19.5, 4.63 Hz), 3.26 (dd, 1H, J=9.77, 17.2 Hz), 3.18 (dd, 1H, J=4.32, 17.3 Hz), 2.40 (m, 1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H); C₁₇H₁₈N₆(MW, 306.37) LCMS (EI) m/e 307 (M⁺+H).

3-Cyclopentylacrylonitrile (8). A solution of diethyl cyanomethylphosphonate (7, 742.5 g, 4.2 mol, 1.1 equiv) in dry THF (5.75 L) was stirred under nitrogen on an ice-water-methanol bath and a solution of 1 M potassium tert-butoxide in THF (4 L, 4.0 mol, 1.05 equiv) was added at such a rate as to keep the temperature below 0° C. After addition of 1 M potassium tert-butoxide in THF was complete, the stirring was continued on the cold bath for 1 h and a solution of cyclopentanecarbaldehyde (6, 374 g, 3.81 mol) in dry THF (290 mL) was added at such a rate as to maintain the temperature below 0° C. The cold bath was removed, and the reaction mixture was gradually warmed to room temperature and stirred at room temperature for overnight. When the reaction was deemed complete, the reaction mixture was partitioned between methyl tent-butyl ether (MTBE, 14 L), water (10 L) and brine (6 L). The two layers were separated, and the combined organic phase was washed with brine (6 L). The aqueous phase was extracted with MTBE (10 L) and washed with brine (6 L). The combined organic extracts were concentrated under reduced pressure and the residue was distilled (65-78° C./6 torr) to afford 3-cyclopentylacrylonitrile (8, 437.8 g, 461.7 g theoretical, 94.8% yield) as a colorless oil, which was found to be a mixture of E- and Z-isomer. For 8: ¹H NMR (DMSO-d₆, 400 MHz, for Z-isomer) δ ppm 6.58 (t, 1H, J=10.6 Hz), 5.55 (dd, 1H, J=10.8, 0.59 Hz), 2.85 (m, 1H), 1.9-1.46 (m, 6H), 1.34 (m, 2H) and (for E-isomer) δ ppm 6.83 (q, 1H, J=8.3 Hz), 5.66 (dd, 1H, J=16.5, 1.4 Hz), 2.60 (m, 1H), 1.9-1.46 (m, 6H), 1.34 (m, 2H); ¹³C NMR (DMSO-d₆, 100 MHz, for Z-isomer) δ ppm 159.8, 116.6, 97.7, 42.3, 32.3, 25.1 and (for E-isomer) δ ppm 160.4, 118.1, 97.9, 43.2, 31.5, 24.8; C₈H₁₁N (MW, 121.18), GCMS (EI) m/e 120 (M⁺−H).

4-(1H-Pyrazol-4-yl)-7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (5). Method A. To a flask equipped with a reflux condenser, a nitrogen inlet, mechanical stirrer, and a thermowell was added 4-chloro-7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (3a, 817 g, 2.88 mol) and dioxane (8 L). To this solution was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (4, 728 g, 3.75 mol, 1.30 equiv) followed by a solution of potassium carbonate (K₂CO₃, 1196 g, 8.67 mol, 3.0 equiv) in water (4 L). The solution was degassed by passing a stream of nitrogen through the solution for 15 minutes before being treated with tetrakis(triphenylphosphine)palladium(0) (167 g, 0.145 mol, 0.05 equiv) and the resulting reaction mixture was heated at reflux (about 90° C.) for 2 hours. When the reaction was deemed complete by TLC (1:1 heptane/ethyl acetate) and LCMS, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (24 L) and water (4 L). The two layers were separated, and the aqueous layer was extracted with ethyl acetate (4 L). The combined organic layers were washed with water (2×2 L), brine (2 L), dried over sodium sulfate (Na₂SO₄), and concentrated under reduced pressure. The residue was suspended in toluene (4 L) and the solvent was removed under reduced pressure. The residue was finally triturated with methyl tert-butyl ether (MTBE, 3 L) and the solids were collected by filtration and washed with MTBE (1 L) to afford 4-(1H-pyrazol-4-yl)-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (5, 581.4 g, 908.5 g theoretical, 64% yield) as white crystalline solids. For 5: ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 13.41 (bs, 1H), 8.74 (s, 1H), 8.67 (bs, 1H), 8.35 (bs, 1H), 7.72 (d, 1H, J=3.7 Hz), 7.10 (d, 1H, J=3.7 Hz), 5.61 (s, 2H), 3.51 (t, 2H, J=8.2 Hz), 0.81 (t, 2H, J=8.2 Hz), 0.13 (s, 9H); C₁₅H₂₁N₅OSi (MW, 315.45), LCMS (EI) m/e 316 (M⁺+H).

Racemic 3-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile (9, racemic SEM-protected compound). Method A. 3-Cyclopentylacrylonitrile (8, 273.5 g, 2.257 mol, 1.20 equiv) and DBU (28 mL, 0.187 mol, 0.10 equiv) was added to a suspension of 4-(1H-pyrazol-4-yl)-7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (5, 591.8 g, 1.876 mol) in acetonitrile (4.7 L) at room temperature. The resulting reaction mixture was heated to 50-60° C. for 17 hours (a clear solution developed midway through heating) then to 70-80° C. for 8 hours. When LCMS analysis showed the reaction was deemed complete, the reaction mixture was cooled to room temperature. The cooled solution was then concentrated under reduced pressure to give the crude product (9) as a thick amber oil. The crude product was dissolved in dichloromethane (DCM) and absorbed onto silica gel then dry-loaded onto a silica column (3 Kg) packed in 33% EtOAc/heptanes. The column was eluted with 33% EtOAc/heptanes (21 L), 50% EtOAc/heptanes (28 L), 60% EtOAc/heptanes (12 L) and 75% EtOAc/heptanes (8 L). The fractions containing the desired product (9) were combined and concentrated under reduced pressure to generate a yellow oil, which was transferred to a 3 L flask with EtOAc. The solvent was removed under reduced pressure and the residual EtOAc by co-evaporating with heptanes. The residue was further dried under high vacuum for overnight to afford racemic 3-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile (9, racemic SEM-protected compound, 800 g, 819.1 g theoretical, 97.7% yield) as an extremely viscous yellow oil. For 9: ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 8.83 (s, 1H), 8.75 (s, 1H), 8.39 (s, 1H), 7.77 (d, 1H, J=3.7 Hz), 7.09 (d, 1H, J=3.7 Hz), 5.63 (s, 2H), 4.53 (td, 1H, J=19.4, 4.0 Hz), 3.51 (t, 2H, J=8.1 Hz), 3.23 (dq, 2H, J=9.3, 4.3 Hz), 2.41 (m, 1H), 1.79 (m, 1H), 1.66-1.13 (m, 7H), 0.81 (t, 2H, J=8.2 Hz), 0.124 (s, 9H); C₂₃H₃₂N₆OSi (MW, 436.63), LCMS (EI) m/e 437 (M⁺+H) and 459 (M⁺+Na).

(3R)-Cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo [2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-10) and (3S)-Cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((S)-10) A slurry of 1.5 Kg of 20-micron Chiralcel® OD chiral stationary phase (CSP) made by Daicel in 3.0 L of isopropanol (IPA) was packed into a PROCHROM Dynamic Axial Compression Column LC110-1 (11 cm ID×25 cm L; Column Void Vol.: approximate 1.5 L) under 150 bar of packing pressure. The packed column was then installed on a Novasep Hipersep HPLC unit. The column and the Hipersep unit were flushed with methanol (17 L) followed by the mobile phase made of a mixture of isopropanol and hexane (2:8 by volume, 17 L). The feed solution was then prepared by dissolving 3-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile (9, racemic SEM-protected compound, 2795 g, 6.4 mol) in the mobile phase to a concentration of 80 g/L. The feed solution was then sequentially injected into the preparative chiral column for separation. Each injection was 120 ml in volume. The chiral column was eluted with the mobile phase at a flow rate of 570 mL/min at room temperature. The column elution was monitored by UV at a wavelength of 330 nm. Under these conditions a baseline separation of the two enantiomers was achieved. The retention times were 16.4 minutes (Peak 1, the undesired (S)-enantiomer (S)-10) and 21.0 minutes (Peak 2, the desired (R)-enantiomer (R)-10), respectively. The cycle time for each injection was 11 minutes and a total of 317 injections were performed for this separation process. Fractions for Peak 1 (the undesired (S)-enantiomer, (S)-10) and Peak 2 (the desired (R)-enantiomer, (R)-10) were collected separately from each injection. The collected fractions collected were continuously concentrated in the 1-square feet and 2-square feet ROTOTHERM evaporator, respectively, at 40° C. under reduced pressure (40-120 bar). The residue from each evaporator was further dried under high vacuum to constant weight to afford (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-10, 1307 g, 1397.5 g theoretical, 93.5%) from Peak 2 as a light yellow oil and (3S)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((S)-10, 1418 g, 1397.5 g theoretical, 101.5%) from Peak 1 as an yellow oil.

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-12, free base). Method A. To a solution of (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-10, 463 g, 1.06 mol, 98.6% ee) in acetonitrile (4.5 L) was added water (400 mL) followed immediately by lithium tetrafluoroborate (LiBF₄, 987.9 g, 10.5 mol, 10.0 equiv) at room temperature. The reaction temperature was observed to decrease from ambient to 12° C. upon addition of the water and then increase to 33° C. during the addition of lithium tetrafluoroborate (LiBF₄). The resulting reaction mixture was heated to reflux (about 80° C.) for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v). When LCMS and TLC analyses showed both the hydroxyl methyl intermediate ((R)-11) and fully de-protected material ((R)-12, free base) produced but no starting material ((R)-10) left, the reaction mixture was cooled gradually to <5° C. before a 20% aqueous solution of ammonium hydroxide (NH₄OH, 450 mL) was added gradually to adjust the pH of the reaction mixture to 9 (checked with pH strips). The cold bath was removed and the reaction mixture was gradually warmed to room temperature and stirred at room temperature for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v) to confirm complete de-protection. When LCMS and TLC showed the reaction was deemed complete, the reaction mixture was filtered and the solids were washed with acetonitrile (1 L). The combined filtrates were then concentrated under reduce pressure, and the residue was partitioned between ethyl acetate (6 L) and half-saturated brine (3 L). The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 L). The combined organic layers were washed with half-saturated sodium bicarbonate (NaHCO₃, 3 L) and brine (3 L), dried over sodium sulfate (Na₂SO₄), and concentrated under reduced pressure to give the crude product as an orange oil. The crude material was then purified by flash column chromatography (SiO₂, 40 to 100% ethyl acetate/heptane gradient elution) to afford (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-12, free base, 273 g, 324.9 g theoretical, 84% yield) as a white foam. This material was checked by ¹⁹F NMR to ensure no lithium tetrafluoroborate (LiBF₄) remained, and by chiral HPLC (Chiralcel® OD-H, 90:10 hexane/ethanol) to confirm enantiomeric purity (98.7% ee), and was used without further purification to prepare the corresponding phosphate salt. For (R)-12 (free base): ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J=0.42 Hz), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, 1H, J=2.34, 3.51 Hz), 6.98 (dd, 1H, J=1.40, 3.44 Hz), 4.53 (td, 1H, J=19.5, 4.63 Hz), 3.26 (dd, 1H, J=9.77, 17.2 Hz), 3.18 (dd, 1H, J=4.32, 17.3 Hz), 2.40 (m, 1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H); C₁₇H₁₈N₆(MW, 306.37) LCMS (EI) m/e 307 (M⁺+H).

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (R)-10. A solution of (R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile ((R)-10, 75.0 g, 0.172 mol, 98.8% ee) in acetonitrile (600 mL) was cooled to 0-5° C. To the cooled solution was added boron trifluoride diethyl etherate (54.4 mL, 0.429 mol) over 10 minutes while maintaining the internal reaction temperature below 5° C. Following the addition, the cold bath was removed and the reaction mixture was allowed to warm to room temperature. When HPLC analysis indicated that the level of (R)-10 was below 1%, the initial phase of the deprotection reaction was considered complete. The reaction was then cooled to 0-5° C., followed by the slow addition of water (155 mL). Following the water addition, the cold bath was removed and the resulting reaction mixture was allowed to warm to 13-17° C., and stirred for an additional 2-3 hours. The resulting reaction mixture was cooled again to 0-5° C. To the cooled reaction mixture was added slowly a solution of ammonia in water [prepared by mixing aqueous 28% ammonia solution (104.5 mL) and water (210.5 mL)] while maintaining the internal reaction temperature at below 5° C. After the aqueous ammonia solution was added, the cold bath was removed and the reaction was allowed to warm to room temperature. The hydrolysis was deemed complete when the level of the hydroxylmethyl intermediate was below 1% by HPLC analysis.

The resulting reaction mixture was diluted with ethyl acetate (315 mL) and washed with 20% brine (315 mL). The aqueous fraction was back extracted with ethyl acetate (315 mL). The organic fractions were combined and concentrated under vacuum with a bath temperature of 40° C. to a volume of 380 mL. The concentrated residue was diluted with ethyl acetate (600 mL) and washed with 1M NaHCO₃(2×345 mL) and 20% brine (345 mL). The aqueous washes were combined and back extracted with ethyl acetate (345 mL). The organic fractions were combined and polish filtered into a clean 2L round bottom flask. The organic fraction was washed with warm water (50° C., 2×450 mL) and then treated with activated charcoal at 65° C. with stirring for 1.5 hours. The slurry was filtered through a celite bed. The filtrate was concentrated under vacuum with a bath temperature of 40° C. The resulting syrup was placed under high vacuum to provide (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile [(R)-12, 54.2g, 103% yield] as a light yellow foam. This material was checked by ¹⁹F NMR to ensure that the product was not contaminated by any fluorinated impurities. The chemical purity of the isolated free base was 96.3%. The chiral purity of the free base was 98.8% by chiral HPLC (chiralcel OD, 90:10 hexane/ethanol). The free base was used without further purification to prepare the phosphate salt. ¹H NMR (DMSO-d₆, 400 MHz) δ 12.11(bs, 1H), 8.79(d, 1H, J=0.43 Hz), 8.67(s, 1H), 8.37(s, 1H), 7.59(q, 1H, J=2.3 Hz), 6.98(q, 1H, J=1.6 Hz), 4.53(td, 1H, J=19.2, 4.1 Hz), 3.22(dq, 2H, J=9.8, 4.3 Hz), 2.40(m, 1H), 1.79(m, 1H), 1.65-1.13(m, 7H). C₁₇H₁₆N₆(MW, 306.37), LCMS (EI) m/e 307 (M⁺+H).

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile phosphate salt ((R)-13, phosphate).

Method A. To a solution of (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-12, free base, 572 g, 1.87 mol) in isopropanol (IPA, 8 L) at 60-65° C. was added a solution of phosphoric acid (186.2 g, 1.9 mol, 1.10 equiv) in isopropanol (1.6 L). No exotherm was observed while adding a solution of phosphoric acid, and a precipitate was formed almost immediately. The resulting mixture was then heated at 76° C. for 1.5 hours, then cooled gradually to ambient temperature and stirred at room temperature for overnight. The mixture was filtered and the solids were washed with a mixture of heptanes and isopropanol (1/1, v/v, 3 L) before being transferred back to the original flask and stirred in heptanes (8 L) for one hour. The solids were collected by filtration, washed with heptanes (1 L), and dried in a convection oven in vacuum at 40° C. to a constant weight to afford (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile phosphate salt ((R)-13, phosphate, 634.2 g , 755 g theoretical, 84% yield) as white to off-white crystalline solids. For (R)-13, phosphate: mp. 197.6° C.; ¹H NMR (DMSO-d₆, 500 MHz) δ ppm 12.10 (s, 1H), 8.78 (s, 1H), 8.68 (s, 1H), 8.36 (s 1H), 7.58 (dd, 1H, J=1.9, 3.5 Hz), 6.97 (d, 1H, J=3.6 Hz), 4.52 (td, 1H, J=3.9, 9.7 Hz), 3.25 (dd, 1H, J=9.8, 17.2 Hz), 3.16 (dd, 1H, J=4.0, 17.0 Hz), 2.41, (m, 1H), 1.79 (m, 1H), 1.59 (m, 1H), 1.51 (m, 2H), 1.42 (m, 1H), 1.29 (m, 2H), 1.18 (m, 1H); ¹³C NMR (DMSO-d₆, 125 MHz) δ ppm 152.1, 150.8, 149.8, 139.2, 131.0, 126.8, 120.4, 118.1, 112.8, 99.8, 62.5, 44.3, 29.1, 29.0, 24.9, 24.3, 22.5; C₁₇H₁₈N₆(MW, 306.37 for free base) LCMS (EI) m/e 307 (M⁺+H, base peak), 329.1 (M⁺+Na).

Method B. To a solution of (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H -pyrazol-1-yl)-3-cyclopentylpropanenitrile ((R)-12, 54.2 g, 177 mol) in dichloromethane (782 mL) and 2-propanol (104 mL) at reflux was added a solution of phosphoric acid (19.9 g, 0.173 mol, 1.15 equiv) in 2-propanol (34.0 mL) over a period of 47 minutes. Following the acid addition, the resulting mixture was heated to reflux for an additional 1 hour. The mixture was gradually cooled to ambient temperature and stirred for 3 hours. The solids were collected by filtration and washed with dichloromethane (390 mL), followed by n-heptane (390 mL). The solids were partially dried under vacuum at room temperature and then under vacuum at 62° C. to afford (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate (60.1 g, 84% yield) as white to off-white crystalline solids. Analysis by chiral HPLC (chiralcel OD, 90:10 hexane/ethanol) gave the enantiopurity as 99.2% ee.

¹H NMR (DMSO-d₆, 400 MHz) δ 12.11(bs, 1H), 8.79(d, 1H, J=0.59 Hz), 8.67(s, 1H), 8.36(s, 1H), 7.59(q, 1H, J=2.3 Hz), 6.98(q, 1H, J=1.6 Hz), 4.53(td, 1H, J=19.6, 4.4 Hz), 3.22(dq, 2H, J=9.6, 4.3 Hz), 2.40(m, 1H), 1.79(m, 1H), 1.65-1.13(m, 7H). C₁₇H₂₁N₆O₄P (MW, 404.36), LCMS (EI) m/e 307 (M⁺+H) and m/e 329 (M⁺+Na).

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate.

Into a 1L round bottom flask, equipped with stir bar, distillation head, addition funnel and heating mantle, were charged methanol (520 mL) and (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate ((R)-13, phosphate, 40.0 grams, 98.92 mmol). The slurry was heated to 55° C. to generate a slightly pink solution. The solution was cooled to 50° C. and filtered into a 2 L flask equipped with an overhead stirrer, distillation head, addition funnel and heating mantle. The 1 L round bottom flask and the filter funnel were rinsed with additional methanol (104.0 mL). The filtrate solution was heated to reflux to distill methanol (281 mL) over 1 hour under atmospheric pressure. Isopropyl alcohol (IPA) (320 mL) was charged slowly via the addition funnel over 80 minutes while maintaining the internal temperature approximately at 65° C. Precipitation of the phosphate salt was observed during IPA addition. After the addition of IPA was complete, n-heptane (175 mL) was added slowly at the same temperature. Distillation was continued under atmospheric pressure. Additional n-heptane (825 mL) was added at approximately the same rate as the distillation rate while maintaining the internal temperature at approximately 65° C. The distillation was complete when the volume of the distillate reached 742 mL (excluding the volume of 281 mL of methanol from the previous distillation). The distillation took approximately 1 hour. The vapor temperature during the distillation was in the range of 54-64° C. and the internal temperature was 67° C. at the end of the distillation. The mixture was slowly cooled to room temperature and stirred for an additional 3 hours. The solids were collected by filtration. The wet cake was washed with 16.7% (v/v) of isopropyl alcohol in n-heptane (384.0 mL), followed by n-heptane (280.0 mL), and dried under vacuum at 55° C. to provide 36.1 grams of the desired product as white solids in 90% yield. The chemical purity is 99.79% by HPLC analysis. The chiral purity is 99.8% by chiral HPLC analysis.

¹H NMR (499.7 MHz, DMSO-d6) δ (ppm): 12.21 (s, 1H), 10.71 (s, 3H), 8.80 (s, 1H), 8.72 (s, 1H), 8.40 (s, 1H), 7.60 (d, J=3.5 Hz, 1H), 7.00 (d, J=3.5 Hz, 1H), 4.51 (td, J=9.75, 4.0 Hz, 1H), 3.25 (dd, J=17.3, 9.75 Hz, 1H), 3.14 (dd, J=17.0, 4.0 Hz, 1H), 2.43-2.35 (m, 1H), 1.79-1.73 (m, 1H), 1.58-1.42 (m, 3H), 1.41-1.33 (m, 1H), 1.30-1.23 (m, 2H), 1.19-1.12 (m, 1H);

¹³C NMR (125.7 MHz, DMSO-d6) δ (ppm): 152.8, 151.2, 150.3, 140.0, 131.8, 127.7, 120.8, 118.8, 113.5, 100.7, 63.3, 45.0, 29.8, 25.6, 25.0, 23.2;

LCMS m/z: calculated for C₁₇H₁₈N₆(M+H)⁺:=307.2. Found (M+H)⁺: 307.0.

……………………….

US8410265

(JAK1, JAK2) inhibitor, developed by the Incyte Corporation, trade name Jakafi.
Ruxolitinib synthetic route as shown below. 4 – bromo-pyrazole ( 1 ) with ethyl vinyl ether ( 2 ) to protect, and then with a Grignard reagent to a halogen – exchanged with isopropyl magnesiumpinacol ester ( 3 ) quenching to obtain 4 . Compound 5 is obtained consisting of hydrogen is protected 6 , and then with a boronic acid ester 4 Suzuki coupling occurs under acidic conditions after removal of the protecting group pyrazolyl 7 , 7 and α, β-unsaturated aldehyde 8 chiral catalyst 9 of under the catalysis of asymmetric Michael addition to give ( R ) -10 (90% EE). ( R) -10 , after reaction with ammonia to obtain an imine oxidation with iodine nitrile 11 , respectively, with different conditions for the final removal of the protecting group to afford Ruxolitinib.

Ruxolitinib <wbr> 2011 年 11 月 FDA approved drugs treat myelofibrosis

…………………………………

Bioorganic and Medicinal Chemistry Letters, 2013 , vol. 23, # 9 p. 2793 – 2800

http://www.sciencedirect.com/science/article/pii/S0960894X13001868

Figure 1.

Structures of tofacitinib (1a) and ruxolitinib (1b).

………………………….

Organic Letters, 2009 , vol. 11, 9 p. 1999 – 2002

http://pubs.acs.org/doi/abs/10.1021/ol900350k

An enantioselective synthesis of INCB018424 via organocatalytic asymmetric aza-Michael addition of pyrazoles (16 or 20) to (E)-3-cyclopentylacrylaldehyde (23) using diarylprolinol silyl ether as the catalyst was developed. Michael adducts (R)-24 and (R)-27 were isolated in good yield and high ee and were readily converted to INCB018424

http://pubs.acs.org/doi/suppl/10.1021/ol900350k/suppl_file/ol900350k_si_001.pdf

COMPD 1 IS RUXOLITINIB

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol
-1-yl]propionitrile (1, INCB018424).

. Method A. To a solution of (3R)-cyclopentyl-3-
{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-
yl}proprionitrile ((R)-25, INCB032306, 463 g, 1.06 mol, 98.6% ee) in acetonitrile (4.5 L)
was added water (400 mL) followed immediately by lithium tetrafluoroborate (LiBF4,
987.9 g, 10.5 mol, 10.0 equiv) at room temperature. The resulting reaction mixture was
heated to reflux for overnight. An aliquot was quenched into ethyl acetate/water and
checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v). When LCMS and TLC
analyses indicated that both the hydroxyl methyl intermediate (R)-26, INCB021499) and
the fully deprotected product (1, INCB018424) SEE LINKhttp://pubs.acs.org/doi/suppl/10.1021/ol900350k/suppl_file/ol900350k_si_001.pdf

:1H NMR
(DMSO-d6, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J = 0.4 Hz), 8.67 (s, 1H), 8.37
(s, 1H), 7.59 (dd, 1H, J = 2.3, 3.5 Hz), 6.98 (dd, 1H, J = 1.4, 3.4 Hz), 4.53 (td, 1H, J =
19.5, 4.6 Hz), 3.26 (dd, 1H, J = 9.8, 17.2 Hz), 3.18 (dd, 1H, J = 4.3, 17.3 Hz), 2.40 (m, S-12
1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H);

13C NMR (DMSO-d6, 100MHz) δ ppm 152.1,
151.0, 149.9, 139.3, 131.0, 126.8, 120.6, 118.2, 112.8, 99.8, 62.5, 44.3, 29.1, 25.0, 24.3,
22.5; IR (KBr) 3197, 3118, 2956, 2865, 1731, 1581, 1448, 1344, 1251 cm-1;

HRMS (CI)
m/z calculated for C17H19N6 (M + H)+
307.1671, found 307.1665

REFERENCES

Jakafi (ruxolitinib) dosing, indications, interactions, adverse effects, and more”. Medscape Reference. WebMD. Retrieved 16 February 2014.
Mesa, Ruben A.; Yasothan, Uma; Kirkpatrick, Peter (2012). “Ruxolitinib”. Nature Reviews Drug Discovery 11 (2): 103–4.doi:10.1038/nrd3652. PMID 22293561.
Harrison, C; Mesa, R; Ross, D; Mead, A; Keohane, C; Gotlib, J; Verstovsek, S (2013). “Practical management of patients with myelofibrosis receiving ruxolitinib”. Expert Review of Hematology 6 (5): 511–23. doi:10.1586/17474086.2013.827413. PMID 24083419.
Natoli, Cori Anne (May 5, 2012), “Incyte looks to ride on drug’s success”, The News Journal, retrieved May 6, 2012
Harrison, C.; Kiladjian, J. J.; Al-Ali, H. K.; Gisslinger, H.; Waltzman, R.; Stalbovskaya, V.; McQuitty, M.; Hunter, D. S.; Levy, R.; Knoops, L.; Cervantes, F.; Vannucchi, A. M.; Barbui, T.; Barosi, G. (2012). “JAK Inhibition with Ruxolitinib versus Best Available Therapy for Myelofibrosis”. New England Journal of Medicine 366 (9): 787–798.doi:10.1056/NEJMoa1110556. PMID 22375970. edit
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RIDAFOROLIMUS

Uncategorized Comments Off

Mar 072014

Ridaforolimus

572924-54-0

(1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^4,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate

Dimethyl-phosphinic Acid C-43 Rapamycin Ester

42-(dimethylphosphinate) Rapamycin

Deforolimus, MK-8669, AP-23573, S1022_Selleck, AP23573, AP23573, MK-8669, Ridaforolimus, Deforolimus, 572924-54-0, MK 8669

AP 23573
AP23573
Deforolimus
MK 8669
MK-8669
MK8669
Ridaforolimus
Taltorvic
UNII-48Z35KB15K

Molecular Formula: C₅₃H₈₄NO₁₄P Molecular Weight: 990.206122

An mTOR inhibitor for the treatment of cancer.

Ridaforolimus (MK-8669; AP23573; formerly Deforolimus)

Merck, under exclusive worldwide license agreement with Ariad Pharmaceuticals

Method of Action: Oral inhibitor of mammalian target of rapamycin inhibitor (mTOR)

Indications/Phase of Trial: Maintenance therapy for metastatic soft-tissue sarcoma and bone sarcomas after at least four chemotherapy cycles (under review after receiving Complete Response letter from FDA in June; NME)

Ridaforolimus is an investigational small-molecule inhibitor of the protein mTOR, a protein that acts as a central regulator of protein synthesis, cell proliferation, cell cycle progression and cell survival, integrating signals from proteins, such as PI3K, AKT and PTEN, known to be important to malignancy.

TARGET- mTOR

Ridaforolimus (also known as AP23573 and MK-8669; formerly known as Deforolimus^[1]) is an investigational targeted and small-molecule inhibitor of the protein mTOR, a protein that acts as a central regulator of protein synthesis, cell proliferation, cell cycle progression and cell survival, integrating signals from proteins, such as PI3K, AKT and PTEN known to be important to malignancy. Blocking mTOR creates a starvation-like effect in cancer cells by interfering with cell growth, division, metabolism, and angiogenesis.

It has had promising results in a clinical trial for advanced soft tissue and bone sarcoma.

RIDAFOROLIMUS

NMR….http://file.selleckchem.com/downloads/nmr/S102201-Deforolimus-HNMR-Selleck.pdf

HPLC . http://file.selleckchem.com/downloads/hplc/S102201-Deforolimus-HPLC-Selleck.pdf

MSDS..http://www.selleckchem.com/msds/Deforolimus-MSDS.html

Commercial arrangements

Ridaforolimus is being co-developed by Merck and ARIAD Pharmaceuticals. On May 5, 2010, Ariad Pharmaceuticals and Merck & Company announced a clinical development and marketing agreement. With this agreement, Ariad received $125 million in upfront payments from Merck and $53 million in milestone payments. Future payments are triggered upon acceptance of the NDA by the FDA with another payment when the drug receives marketing approval. There are similar milestones for acceptance and approval in both Europe and Japan. Other milestone payments are tied to revenue goals for the drug.^[2] ARIAD has opted to co-promote ridaforolimus in the U.S. Merck plans to submit a New Drug Application (NDA) for ridaforolimus to the U.S. Food and Drug Administration (FDA) and a marketing application in the European Union in 2011.^[3]

Clinical trials

Phase III SUCCEED

On June 6, 2011, Ariad and Merck announced detailed results from the largest randomized study ever in the soft tissue and bone sarcoma population, the Phase III SUCCEED clinical trial. SUCCEED evaluated oral ridaforolimus, in patients with metastatic soft-tissue or bone sarcomas who previously had a favorable response to chemotherapy. In this patient population, ridaforolimus improved progression-free survival (PFS) compared to placebo, the primary endpoint of the study. The complete study results were presented by Sant P. Chawla, M.D., director, Sarcoma Oncology Center, Santa Monica, CA, during the 2011 American Society of Clinical Oncology (ASCO) annual meeting.
The SUCCEED (Sarcoma Multi-Center Clinical Evaluation of the Efficacy of Ridaforolimus) trial was a randomized (1:1), placebo-controlled, double-blind study of oral ridaforolimus administered at 40 mg/day (five of seven days per week) in patients with metastatic soft-tissue or bone sarcomas who previously had a favorable response to chemotherapy. Oral ridaforolimus was granted a Special Protocol Assessment (SPA) by the FDA for the SUCCEED trial.
Based on 552 progression-free survival (PFS) events in 711 patients, (ridaforolimus (N=347), placebo (N=364) determined by an independent radiological review committee, the study achieved its primary endpoint of improvement in PFS, with a statistically significant (p=0.0001) 28 percent reduction in the risk of progression or death observed in those treated with ridaforolimus compared to placebo (hazard ratio=0.72).

Median PFS was 17.7 weeks for those treated with ridaforolimus compared to 14.6 weeks in the placebo group. Furthermore, based on the full analysis of PFS determined by investigator assessment, there was a statistically significant (p<0.0001) 31 percent reduction by ridaforolimus in the risk of progression or death compared to placebo (hazard ratio=0.69). In the investigator assessment analysis, median PFS was 22.4 weeks for those treated with ridaforolimus compared to 14.7 weeks in the placebo group ^[4

EU WITHDRAWAL IN NOV 2012

Merck, known as MSD outside the U.S. and Canada, announced today that it has formally notified the European Medicines Agency (EMA) of Merck’s decision to withdraw the Marketing Authorisation Application (MAA) for ridaforolimus.

The application for Marketing Authorisation for ridaforolimus was accepted by the EMA in August 2011. At the time of the withdrawal it was under review by the Agency’s Committee for Medicinal Products for Human Use (CHMP). In its letter to the EMA, Merck said that the withdrawal of ridaforolimus was based on the provisional view of the CHMP that the data available to date and provided in the Marketing Authorisation Application were not sufficient to permit licensure of ridaforolimus in the European Union for the maintenance treatment of patients with soft tissue sarcoma or primary malignant bone tumor.

Although the application for these uses was withdrawn, Merck is studying ridaforolimus in combination with other drugs in other tumor types. The withdrawal of the European application of ridaforolimus for the maintenance treatment of patients with soft tissue sarcoma or primary malignant bone tumor does not change Merck’s commitment to the ongoing clinical trials with ridaforolimus.

Ridaforolimus

Description

42-(dimethylphosphinate) Rapamycin (Ridaforolimus) represented by the following formula I:

2. Description of RelatedArt

The mammalian target of Rapamycin (mTOR) is known as a mechanistic target of Rapamycin (H), which is found in the studies of Rapamycin. On the other hand, 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I) is a derivative of Rapamycin (II), which is also a kind of mTOR inhibitor. Ridaforolimus (I) can inhibit cell division and possibly lead to tumor cell death. Hence, there are many studies related to solid tumor treatments and blood cancer treatments using Ridaforolimus (I). In addition, in 2011, Merck also applied a certification of this compound against soft tissue and bone cancer.

U.S. Pat. No. 7,091,213 discloses a process for preparing 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I), and the process thereof is shown in the following Scheme I.

In this process, a solution of Rapamycin (II) in dichloromethane (DCM) was respectively added with 2,6-di-tert-butyl-4-methylpyridine or 3,5-lutidine as a base, and followed by the addition of a solution of dimethylphosphinic chloride (DMP-Cl) to perform a phosphorylation reaction at 0° C., under a stream of N_2(g). The crude product was purified by flash chromatography (eluted with MeOH/DCM/EtOAc/hexane=1:10:3:3) to provide 42-(dimethyl- phosphinate) Rapamycin (Ridaforolimus) (I), which is a phosphorylated compound at 42-hydroxyl position of Rapamycin (II). In addition, this patent also disclosed a side product of 31,42-bis(dimethyl phosphinate) Rapamycin (III), which is a phosphorylated compound at both 31- hydroxyl position and 42- hydroxyl position of Rapamycin (II).

…………………..

SYNTHESIS

US7091213

Some additional transformations of potential interest to the practitioner are shown below, including the preparation of reagents for generating the described C-43 phosphorus-containing rapalogs:

Preparation of Diakyl/diaryl Chlorophoshates

Preparation of Alkyl Halide Phosphonates

Illustrative routes for using the foregoing sorts of reagents to prepare certain rapalogs of this invention are shown below.

The synthesis of compounds of this invention often involves preparation of an activated form of the desired moiety “J”, such as a phosphoryl chloride as shown above (e.g. (R)(RO)P—Cl or RR′P(═O)—Cl, etc), and reaction of that reagent with rapamycin (or the appropriate rapalog) under conditions yielding the desired product, which may then be recovered from residual reactants and any undesired side products. Protecting groups may be chosen, added and removed as appropriate using conventional methods and materials.

Purification of Compounds of the Invention

A variety of materials and methods for purifying rapamycin and various rapalogs have been reported in the scientific and patent literatures and may be adapted to purification of the rapalogs disclosed herein. Flash chromatography using a BIOTAGE prepacked cartridge system has been particularly effective. A typical protocol is disclosed in the Examples which follow.

Physicochemical Characterization of Compounds of the Invention

The identity, purity and chemical/physical properties of the rapalogs may be determined or confirmed using known methods and materials, including HPLC, mass spectral analysis, X ray crystallography and NMR spectroscopy. High resolution 1D ¹H and ³¹P NMR spectra acquired using a typical relaxation delay of 3 seconds have proved useful, as has reverse phase HPLC analysis (analytical column, 3 micron particle size, 120 ansgstrom pore size, thermostatted to 50° C. with a mobile phase of 50% acetonitrile, 5% methanol and 45% water (all % s by volume), for example, in an isocratic elution system, with elution of product and impurity peaks followed by UV detection at 280 nanometers). Normal phase HPLC may also be used, especially to evaluate the level of residual rapamycin or rapalog by-products. The presence of residual solvent, heavy metals, moisture and bioburden may be assessed using conventional methods.

Example 9

Dimethyl-phosphinic Acid C-43 Rapamycin Ester

Dimethyl-phosphinic Acid C-43 Rapamycin Ester

To a cooled (0° C.) solution of rapamycin (0.1 g, 0.109 mmol) in 1.8 mL of dichloromethane was added 0.168 g (0.82 mmol) of 2,6-di-t-butyl-4-methyl pyridine, under a stream of N₂, followed immediately by a solution of dimethylphosphinic chloride (0.062 g, 0.547 mmol) in 0.2 mL of dichloromethane. The slightly yellow reaction solution was stirred at 0° C., under an atmosphere of N₂, for 3.5 h (reaction monitored by TLC). The cold (0° C.) reaction solution was diluted with ˜20 mL EtOAc then transferred to a separatory funnel containing EtOAc (150 mL) and saturated NaHCO₃(100 mL). Upon removing the aqueous layer, the organic layer was washed successively with ice cold 1N HCl (1×100 mL), saturated NaHCO₃(1×100 mL), and brine (1×100 mL), then dried over MgSO₄and concentrated. The crude product was purified by silica gel flash chromatography (eluted with 1:10:3:3 MeOH/DCM/EtOAc/hexane) to provide 0.092 g of a white solid:

¹H NMR (300 MHz, CDCl₃) d 4.18 (m, 1H), 4.10 (m, 1H), 3.05 (m, 1H), 1.51 (m, 6H);
³¹P NMR (121 MHz, CDCl₃) d 53.6; 1013 m/z (M+Na).

Example 9

Alternative Synthesis

Rapamycin and dichloromethane are charged into a nitrogen-purged reaction flask. The stirred solution is cooled to approximately 0° C. (an external temperature of −5±5° C. is maintained throughout the reaction). A solution of dimethylphosphinic chloride (2.0 molar equivalents) in dichloromethane is then added over a period of approximately 8–13 minutes.

This is followed immediately by the addition of a solution of 3,5-lutidine (2.2 molar equivalents) in dichloromethane over a period of approximately 15–20 minutes. Throughout both additions, the internal temperature of the reaction sssstays below 0° C. The cooled reaction solution is stirred for 1 hour and then transferred, while still cold, to an extractor containing saturated aqueous NaHCO₃and methyl-t-butyl ether (MTBE), ethyl acetate or diethyl ether. In-process samples are removed at 30 and 60 minute time points.

Samples are prepared in a similar fashion to that described for the reaction workup. Reaction progress is monitored by TLC (1:10:3:3 MeOH/DCM/EtOAc/hexanes) and reverse-phase HPLC analyses. The isolated organic layer is successively washed with ice cold 1N HCl, saturated aqueous NaHCO₃(2×), saturated aqueous NaCl, and dried over sodium sulfate. Upon filtration and solvent removal, the residue undergoes solvent exchange with acetone followed by concentration in vacuo to provide crude product, which may be analyzed for purity by normal- and reversed-phase HPLC.

…………………….

SYNTHESIS

US20140058081

The process of the present invention is shown in the following Scheme II.

EXAMPLE 7

Preparation of 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I)

42-(dimethylphosphinate)-31-triethylsilylether Rapamycin (VI-b) (2.312 g, available from 1.945 mmole of Rapamycin -28-triethylsilylether) and tetrahydrofuran (60 mL) was placed into a flask, and the resulting solution was cooled to 0˜−5° C. Next, a sulthric acid solution (2 N, 6 mL) was slowly added into the resulting solution dropwise. When the 42-(dimethylphosphinate)-31-triethylsilylether Rapamycin (VI-b) was less than 2%, ethyl acetate (1000 mL) was added into the resulting solution. Then, the organic layer was successively washed with a NaCl saturated solution (300 mL), a NaHCO₃saturated solution (200 mL) and a NaCl saturated solution (200 mL), dried over anhydrous sodium sulfate and concentrated to obtain a crude product of 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I) (2.341 g). The crude product was then purified by Licrhoprep RP-18 silica gel chromatography (eluted with acetonitrile: 0.02 M ammonium formate solution=6:4, wherein the pH of the ammonium formate solution was adjusted to 4.0 with formic acid), extracted with ethyl acetate, concentrated and dried to obtain a white foam solid 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I) (1.840 g, purity=99.48%). The yield thereof was 95.55% based on 2.0 g of 31-triethylsilyl ether Rapamycin.

¹H-NMR(400 MHz, CDCl₃)d 4.18(m, 1H), 4.10(m, 1H), 3.05(m, 1H),1.51(m, 6H); ³¹P-NMR(161 MHz, CDCl₃)d 53.33; 1012.6 m/z [M+Na]⁺.

“ARIAD Reports First Quarter 2009 Development Progress and Financial Results- Ridaforolimus New USAN Name to Replace Deforolimus”. ARIAD Pharmaceuticals. 2009. Retrieved 2009-05-07.
“ARIAD – News release”. Phx.corporate-ir.net. Retrieved 2012-10-07.
“ARIAD – News release”. Phx.corporate-ir.net. 2011-03-17. Retrieved 2012-10-07.
“ARIAD – News release”. Phx.corporate-ir.net. 2011-06-06. Retrieved 2012-10-07.

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