Orilotimod

phase 2 Comments Off

Aug 182015

ChemSpider 2D Image | Orilotimod | C16H19N3O5

Orilotimod

(2R)-2-amino-5-{[(1R)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-5-oxopentanoic acid

186087-26-3

Apo805,UNII-Q66Z43C5XM; Thymodepressin; Orilotimod [USAN]; AC1OIBUF;

C₁₆H₁₉N₃O₅
MW 333.339

Apotex Technologies Inc. INNOVATOR

2D chemical structure of 960155-19-5

Orilotimod potassium,

APO805K1

D-Tryptophan, D-gamma-glutamyl-, potassium salt (1:1), CAS 960155-19-5

The drug, orilotimod, was originally developed and launched by Immunotech Developments; however, ApoPharma (a subsidiary of Apotex) is developing orilotimod, presumably a topical formulation, for the treatment of psoriasis. In August 2015, the ApoPharma’s drug was reported to be in phase 2 clinical development.

Thymodepressin is the free diacid having Chemical Abstracts Service (CAS) Registry Number@ of 186087-26-3. U.S. Pat. No. 5,736,519 discloses H-D-iGlu-D-Trp-OH and a process for its preparation wherein it is purified by ion exchange chromatography. It is an immunosuppressant and selectively inhibits proliferation of hemopoietic precursor cells and stimulates granulocyte and lymphocyte apoptosis (Sapuntsova, S. G., et al. (May 2002), Bulletin of Experimental Biology and Medicine, 133(5), 488-490).

Thymodepressin is currently being sold in Russia as the disodium salt of D-isoglutamyl-D-tryptophan in liquid formulation for injection and intranasal administration for the treatment of psoriasis and atopic dermatitis. The solid form of the disodium salt of D-isoglutamyl-D-tryptophan is an amorphous powder which is hygroscopic and very difficult to handle. The disodium salt of D-isoglutamyl-D-tryptophan has the molecular formula C16H17N3Na2O5 and is reported in Kashirin, D. M., et al. (2000), Pharmaceutical Chemistry Journal, 34(11), 619-622.

PAPENT

BEAWARE EXAMPLE WITH AN ESTER GP

http://www.google.im/patents/WO2012129671A1?cl=en

Preparation of H-D-Glu( -Trp-OH)-0-Et hydrochloride salt (Apo836.HCI)

A. Preparation of Boc-D-Glu(D-Trp-0-Bzl)-0-Et

Proceeding in a similar manner as described under Example 3A, Boc-D- Glu(D-Trp-0-Bzl)-0-Et was prepared in 87% yield.¹H NMR ( DMSO-D_6l 400 MHz) δ ppm: 10.87, (s, 1 H), 8.35 (d, J = 7.2 Hz, 1 H), 7.48 (d, J = 7.8 Hz, 1 H), 7.35 (d, J = 7.9 Hz, 1 H), 7.29-7.33 (m, 3H), 7.23 (d, J = 7.7 Hz, 1H), 7.09-7.22 (m, 3H), 7.08 (t, J = 7.6 Hz, 1H), 6.98 (t, J = 7,7 Hz, 1 H), 4.98 – 5.06 (m, 2H), 4.55 (apparent q, J = 7.3 Hz, 1 H), 4.04 – 4.11 (m, 2H), 3.90 – 3.95 (m, 1 H), 3.04 – 3.19 (m, 2H), 2.18 – 2.23 (m, 2H), 1.84 – 1.89 (m, 1 H), 1.70 – 1.77 (m, 1 H), 1.38 (s, 9H), 1.16 (t, J = 7.1 Hz, 3H); MS-ESI (m/z): 552 [ +1]⁺.

B. Preparation of Boc-D-Glu(D-Trp-OH)-0-Et

Proceeding in a similar manner as described under Example 3B, Boc-D-

Glu(D-Trp-OH)-0-Et was prepared in quantitative yield. ¹H NMR ( DMSO-D₆, 400 MHz) δ ppm: 12.62 (br. 1H), 10.82, (s, 1 H), 8.10 (d, J = 7.7 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1 H), 7.33 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1 H), 7.12 (s, 1 H), 7.06 (t, J = 7.3 Hz, 1 H), 6.98 (t, J = 7.5 Hz, 1 H)„ 4.45 (apparent q, J = 7.7 Hz, 1 H), 4.03 – 4.11 (m, 2H), 3.87 – 3.92 (m, 1 H), 3.13 – 3.18 (m, 1H), 2.96 – 3.03 (m,

1 H), 2.13 – 2.20 (m, 2H), 1.82 – 1.88 (m, 1H), 1.69-1.75 (m, 1 H), 1.38 (s, 9H>, 1.17 (t, J = 7.1 Hz, 3H); MS-ESI (m/z): 462 [M+1]⁺.

C. Preparation of H-D-Glu(D-Trp-OH)-0-Et.HCI (Apo836 HCI)

To an ice-cooled solution of Boc-D-Glu(D-Trp-OH)-0-Et (4.55 g, 9.8 mmol) obtained in Section B above in dichloromethane (100 mL) was bubbled HCI gas for 15 min. The reaction mixture was concentrated under vacuum by rotary evaporation to give H-D-Glu(D-Trp-OH)-0-Et hydrochloride (Apo836.HCI, 4.0 g) as a foamy solid. ¹ H NMR ( DMSO-D₆, 400 MHz) δ ppm: 12.68 (br. s, 1 H), 10.90, (s, 1H), 8.66 (br, s, 3H), 8.33 (d, J = 7.8 Hz, 1 H), 7.52 (d, J = 7.8 Hz, 1 H), 7.33 (d, J = 8.0 Hz, 1 H), 7.12 (d, J = 1.5 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1 H), 6.98 (t, J = 7.2 Hz, 1 H), 4.47 (apparent q, J = 4.8 Hz, 1 H), 4.13 – 4.19 (m, 2H), 3.90 (br, 1 H), 3.16 – 3.20 (m, 1H), 2.98 – 3.04 (m, 1 H), 2.29 – 2.33 (m, 2H), 1.94 – 1.98

(m, 2H), 1.20 (t, J = 7.1 Hz, 3H); MS-ESI (m/z): 362 [M+1]⁺ (free base).

……………………..

US 20150225341

file:///H:/ORILOTIMODUS20150225341A1.pdf

Novel crystalline and amorphous salts of thymodepressin (orilotimod), particularly potassium salt, useful for treating psoriasis and atopic dermatitis. Also claims salt exchange method for preparing thymodepressin salts.

hymodepressin is the free diacid having Chemical Abstracts Service (CAS) Registry Number@ of 186087-26-3. U.S. Pat. No. 5,736,519 discloses H-D-iGlu-D-Trp-OH and a process for its preparation wherein it is purified by ion exchange chromatography. It is an immunosuppressant and selectively inhibits proliferation of hemopoietic precursor cells and stimulates granulocyte and lymphocyte apoptosis (Sapuntsova, S. G., et al. (May 2002), Bulletin of Experimental Biology and Medicine, 133(5), 488-490).

Thymodepressin is currently being sold in Russia as the disodium salt of D-isoglutamyl-D-tryptophan in liquid formulation for injection and intranasal administration for the treatment of psoriasis and atopic dermatitis. The solid form of the disodium salt of D-isoglutamyl-D-tryptophan is an amorphous powder which is hygroscopic and very difficult to handle. The disodium salt of D-isoglutamyl-D-tryptophan has the molecular formula C16H17N3Na2O5 and which is reported in Kashirin, D. M., et al. (2000), Pharmaceutical Chemistry Journal, 34(11), 619-622.

Through investigations in our laboratory, we have determined that the freeze-dried disodium salt of D-isoglutamyl-D-tryptophan is extremely hygroscopic turning into a gel in a matter of minutes in air and cannot easily be handled.

A powdery or amorphous form of a compound, intended for pharmaceutical use may give rise to manufacturing problems due to bulk density issues, hygroscopicity and variable water content that cannot be corrected by vacuum drying. D-isoglutamyl-D-tryptophan is a dipeptide and the drying of an amorphous form at elevated temperature, for example, 80-100° C. under vacuum is not recommended. Thus, there are serious difficulties experienced during the purification of the disodium salt of D-isoglutamyl-D-tryptophan and obtaining the pure disodium salt on a manufacturing scale. Further, there is no published procedure for its preparation.

The monosodium salt of D-isoglutamyl-D-tryptophan is identified by the CAS Registry System and is listed in the CAS REGISTRYSM File with a CAS Registry Number@ of 863988-88-9. However, there are no references citing the substance and thus no publication of its identity, its physical and/or chemical properties, its characterization or a procedure for its preparation. Freeze-dried powders of mono sodium and disodium salts of peptide drugs may not have controllable powder bulk density ranges for formulation. They may require significant investment in freeze-dried dispersion technology.

EXAMPLES

Example 1

Preparation of potassium salt of D-isoglutamyl-D-tryptophan (1:1) from D-isoglutamyl-D-tryptophan and potassium hydroxide

In a 100-mL round bottom flask equipped with a magnetic stir bar was placed 5 mL of potassium hydroxide solution (0.5 N). The solution was cooled to 0° C. in an ice-water bath, and solid H-D-iGlu-D-Trp-OH (1.00 g, 3 mmol) was added. The mixture was stirred while the pH of the solution was adjusted to ca. 6.0 by adding a few drops of potassium hydroxide solution (0.5 N). The solution was filtered to remove any solid particulates. The filtrate was evaporated to dryness at a bath temperature of about 30° C. to afford a solid. After drying under vacuum at room temperature for overnight, the salt was obtained in quantitative yield, with a HPLC purity (peak area percent) of 98.3%. HPLC method; Column: XTerra MS C18; 5 μm, 4.6×250 mm; Mobile phase: A=the aqueous phase: 4 mM Tris, 2 mM EDTA, pH 7.4; B=the organic phase: CH3CN; gradient: B %: 0 min. 5%, 15 min. 55%, 30 min. 55%, 32 min. 5%, 35 min. 5%; Flow rate: 1 mL/min; injection volume: 5 μL; λ: 222, 254, 282, 450 nm; retention time of the product: 6.41 min. The XRPD pattern of this crystalline material is shown in FIG. 1A; the water content by Karl-Fischer test is 0.7%; UV (water, c=23.8 ρM, λmax nm): 221 (ε 33270), 280 (ε 5417); MS (m/z): 372.0 [M]+, 334.2 [C16H20N3O5]+, 187.9 (100%). The FT-IR (KBr) spectrum is shown in FIG. 1B.

Example 2

A. Preparation of mono potassium salt of D-isoglutamyl-D-tryptophan (1:1) from the mono ammonium salt of D-isoglutamyl-D-tryptophan (1:1)

A solution of H-D-iGlu-D-Trp-OH, mono ammonium salt (1:1), (1.66 g, 4.05 mmol) and potassium hydroxide (253 mg, 4.50 mmol) in water (20 mL) was stirred at room temperature for 15 min. The pH of the solution was about 9. The reaction mixture was evaporated under reduced pressure to a volume of about 1 mL. After cooling to room temperature, isopropanol was added until a solid precipitated out. The resulting suspension was stirred at room temperature for 15 min, then filtered. The solid was washed with isopropanol (2×20 mL) and ethyl acetate (20 mL), then dried under vacuum in an oven at 42° C. overnight. An off white solid was obtained (1.49 g, 99% yield). The water content by Karl-Fischer test is 2.5%. Analytical data (XRPD pattern, FT-IR and MS spectra) are similar to those described in Example 1.

B. Preparation of amorphous form of potassium salt of D-isoglutamyl-D-tryptophan (1:1) from the mono ammonium salt of D-isoglutamyl-D-tryptophan (1:1)

A solution of H-D-iGlu-D-Trp-OH, mono ammonium salt (1:1), (517 mg, 1.40 mmol) and potassium hydroxide (82 mg, 1.46 mmol) in water (10 mL) was stirred at room temperature for 30 minutes. The resulting mixture was freeze-dried overnight. An off white solid was obtained in quantitative yield. The XRPD pattern spectrum confirmed that this material is amorphous.

1H NMR (D2O) δ: 7.69 (d, J=7.9 Hz, 1H), 7.48 (d, J=8.2 Hz, 1H), 7.23 (t, J=7.6 Hz, 1H), 7.22 (s, 1H), 7.16 (t, J=7.4 Hz, 1H), 4.59 (dd, J=8.7, 4.8 Hz, 1H), 3.51 (dd, J=6.8, 5.8 Hz, 1H), 3.38 (dd, J=14.8, 4.8 Hz, 1H), 3.11 (dd, J=14.8, 8.8 Hz, 1H), 2.20-2.49 (m, 2H) and 1.85-1.94 (m, 2H);

13C NMR (D2O) δ: 181.4, 177.0, 176.6, 138.8, 129.9, 126.9, 124.5, 121.9, 121.4, 114.5, 113.2, 58.6, 57.0, 34.6 (CH2), 30.2 (CH2) and 29.3 (CH2);

the water content by Karl-Fischer test is 5.4%;

the FT-IR (KBr) spectrum is shown in FIG. 1C;

MS (m/z): 371.7 [M]+, 334.2 [C16H20N3O5]+, 187.9 (100%);

HPLC purity (peak area percent): 99.8%, Retention time: 5.04 min; HPLC conditions: Column Waters Symmetry C18, 3.9×150 mm, 5 μm; Mobile phase: 0.035% HClO4, pH 2/CH3CN, 85/15, isocratic, Flow rate: 1 mL/min; λ: 220, 254, 280 nm.

Patent	Submitted	Granted
GAMMA-GLUTAMYL AND BETA-ASPARTYL CONTAINING IMMUNOMODULATOR COMPOUNDS AND METHODS THEREWITH [EP1042286]	2000-10-11	2010-08-25
CRYSTALLINE D-ISOGLUTAMYL-D-TRYPTOPHAN AND THE MONO AMMONIUM SALT OF D-ISOGLUTAMYL-D-TRYPTOPHAN [US8119606]	2010-01-21	2012-02-21
Pharmaceutically Acceptable Salts of Thymodepressin and Processes for their Manufacture [US8138221]	2010-03-04	2012-03-20
CRYSTALLINE FORMS OF THE MONO-SODIUM SALT OF D-ISOGLUTAMYL-D-TRYPTOPHAN [US8207217]	2010-02-04	2012-06-26

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Read all about Organic Spectroscopy on ORGANIC SPECTROSCOPY INTERNATIONAL

////////Orilotimod, PHASE 2, thymodepressin, APO 805K1

C1=CC=C2C(=C1)C(=CN2)CC(C(=O)O)NC(=O)CCC(C(=O)O)N

Fispemifene for hypogonadism

phase 2, Uncategorized Comments Off

Jul 142015

Fispemifene, HM 101

Fispemifene; UNII-3VZ2833V08;

cas 341524-89-8

Molecular Formula:	C₂₆H₂₇ClO₃
Molecular Weight:	422.94378 g/mol

2-[2-[4-[(Z)-4-chloro-1,2-diphenylbut-1-enyl]phenoxy]ethoxy]ethanol

Treatment of Hypogonadism

Androgen Decline in the Aging Male (Andropause) in phase 2

Fispemifene is the Z-isomer of the compound of formula (I)

WO 01/36360 describes a group of SERMs, which are tissue-specific estrogens and which can be used in women in the treatment of climacteric symptoms, osteoporosis, Alzheimer’s disease and/or cardiovascular diseases without the carcinogenic risk. Certain compounds can be given to men to protect them against osteoporosis, cardiovascular diseases and Alzheimer’s disease without estrogenic adverse events (gynecomastia, decreased libido etc.). Of the compounds described in said patent publication, the compound (Z)-2-{2-[4-(4-chloro-1,2-diphenylbut-1-enyl)phenoxy]ethoxy}ethanol (also known under the generic name fispemifene) has shown a very interesting hormonal profile suggesting that it will be especially valuable for treating disorders in men. WO 2004/108645 and WO 2006/024689 suggest the use of fispemifene for treatment or prevention of age-related symptoms in men, such as lower urinary tract symptoms and diseases or disorders related to androgen deficiency in men.

Quatrx had been conducting phase II clinical development for the treatment of androgen decline in the aging male. Unlike testosterone replacement therapies that are typically topical or injection therapies, fispemifene is an oral treatment and is not a formulation of testosterone. Fispemifene utilizes the body’s normal feedback mechanism to increase testosterone levels. Originally developed at Hormos, QuatRx gained rights to the drug candidate following a merger of the companies pursuant to which Hormos became a wholly-owned subsidiary of QuatRx.

Known methods for the syntheses of compounds like ospemifene and fispemifene include rather many steps. WO 02/090305 describes a method for the preparation of fispemifene, where, in a first step, a triphenylbutane compound with a dihydroxysubstituted butane chain is obtained. This compound is in a second step converted to a triphenylbutene where the chain is 4-chlorosubstituted. Then the desired Z-isomer is crystallized. Finally, the protecting group is removed to release the ethanol-ethoxy chain of the molecule.

Fispemifene is a selective estrogen receptor modulator (SERM) studied in phase II clinical trials at Forendo Pharma for the treatment low testosterone in men. The compound is also in phase II clinical studies at Apricus for the treatment of men with secondary hypogonadism.

In 2013, Forendo Pharma acquired the drug from Hormos Medical for the treatment of male low testosterone.

In 2014, Apricus Biosciences acquired U.S. rights for development and commercialization

PATENT

https://www.google.com/patents/US7504530

EXAMPLE 2 2-{2-[4-(4-Chloro-1,2-diphenyl-but-1-enyl)-phenoxy]-ethoxy}-ethanol (Compound I)

{2-[4-(4-Chloro-1,2-diphenyl-but-1-enyl)-phenoxy]-ethoxy}-acetic acid ethyl ester was dissolved in tetrahydrofuran at room temperature under nitrogen atmosphere. Lithium aluminium hydride was added to the solution in small portions until the reduction reaction was complete. The reaction was quenched with saturated aqueous ammonium chloride solution. The product was extracted into toluene, which was dried and evaporated in vacuo. The residue was purified with flash chromatography with toluene/triethyl amine (9.5:0.5) as eluent. Yield 68%.

¹H NMR (200 MHz, CDCl₃):

2.92 (t, 2H, ═CH ₂CH₂Cl),

3.42 (t, 2H, ═CH₂ CH₂ Cl),

3.59-3.64 (m, 2H, OCH₂CH₂O CH₂CH ₂OH),

3.69-3.80 (m, 4H, OCH₂ CH ₂OCH ₂CH₂OH),

3.97-4.02 (m, 2H, OCH₂CH₂OCH₂CH₂OH),

6.57 (d, 2H, aromatic proton ortho to oxygen),

6.78 (d, 2H, aromatic proton meta to oxygen),

7.1-7.43 (m, 10H, aromatic protons).

………….

PATENT

WO 2001036360

https://www.google.com/patents/WO2001036360A1?cl=en

……………

PATENT

WO 2002090305

http://www.google.co.in/patents/WO2002090305A1?cl=en

EXAMPLE

a) [2-(2-chloroethoxy)ethoxymethyl]benzene

is prepared from benzyl bromide and 2-(2-chloroethoxy)ethanol by the method described in literature (Bessodes, 1996).

b) {4-[2-(2-Benzyloxyethoxy)ethoxy]phenyl}phenylmethanone

The mixture of 4-hydroxybenzophenone (16.7 g, 84.7 mmol) and 48 % aqueous sodium hydroxide solution (170 ml) is heated to 80 °C. Tetrabutylammonium bromide (TBABr) (1.6 g, 5.1 mmol) is added and the mixture is heated to 90 °C. [2-(2-Chloroethoxy)ethoxymethyl]benzene (18. g, 84.7 mmol) is added to the mixture during 15 min and the stirring is continued for additional 3.5 h at 115-120 °C. Then the mixture is cooled to 70 °C and 170 ml of water and 170 ml of toluene are added to the reaction mixture and stirring is continued for 5 min. The layers are separated and the aqueous phase is extracted twice with 50 ml of toluene. The organic phases are combined and washed with water, dried with sodium sulphate and evaporated to dryness. Yield 31.2 g.

Another method to prepare {4-[2-(2-benzyloxyethoxy)ethoxy]phenyl}phenyl- methanone is the reaction of 2-(2-benzyloxyethoxy)ethyl mesylate with 4- hydroxybenzophenone in PTC-conditions.

Η NMR (CDCI3): 3.64-3.69 (m, 2H), 3.74-3.79 (m, 2H), 3.90 (dist.t, 2H), 4.22 (dist.t, 2H), 4.58 (s, 2H), 6.98 (d, 2H), 7.28-7.62 (m, 8H), 7.75 (td, 2H), 7.81 (d, 2H).

c) 1- {4-[2-(2-Benzyloxyethoxy)ethoxy]phenyl} – 1 ,2-diphenyl -butane- 1 ,4-diol

Figure imgf000013_0002 R = BENZYL

Lithium aluminum hydride (1.08 g, 28.6 mmol) is added into dry tetrahydrofuran (60 ml) under nitrogen atmosphere. Cinnamaldehyde (6.65 g, 50 mmol) in dry tetrahydrofuran (16 ml) is added at 24-28 °C. The reaction mixture is stirred at ambient temperature for 1 h. {4-[2-(2- Benzyloxyethoxy)ethoxy]phenyl}-phenyl-methanone (14.0 g, 37 mmol) in dry tetrahydrofuran (16 ml) is added at 50-55 °C. The reaction mixture is stirred at 60 °C for 3 h. Most of tetrahydrofuran is evaporated. Toluene (70 ml) and 2 M aqueous hydrogen chloride (50 ml) are added. The mixture is stirred for 5 min and the aqueous layer is separated and extracted with toluene (30 ml). The toluene layers are combined and washed with 2M HC1 and water, dried and evaporated. The product is crystallized from isopropanol as a mixture of stereoisomers (8.8 g, 50 %).

Η NMR (CDCI3 ): 1.75-2.10 (m, 2H), 3.20-4.16 (m, 1 OH), 4.52 and 4.55 (2s, together 2H), 6.61 and 6.88 (2d, together 2H), 6.95-7.39 (m, 15H), 7.49 and 7.57 (2d, together 2H).

d) Z- 1 – {4-[2-(2-Benzyloxyethoxy)ethoxy]phenyl} -4-chloro- 1 ,2-diphenyl-but- 1-ene

Figure imgf000013_0003 R = BENZYL

1 – {4- [2-(2-Benzyloxy-ethoxy)ethoxy]phenyl} – 1 ,2-diphenyl -butane- 1 ,4-diol (10.0 g, 19.5 mmol) is dissolved in toluene (50 ml). Triethylamine (2.17 g, 21.4 mmol) is added to the solution and the mixture is cooled to -10 °C. Thionyl chloride (6.9 g, 58.5 mmol) is added to the mixture at -10 – ±0 °C. The mixture is stirred for 1 hour at 0-5 °C, warmed up to 70 °C and stirred at this temperature for 4 hours. Solvent is evaporated, the residue is dissolved to toluene, washed three times with 1M HC1 solution and twice with water. The Z-isomer of the product is crystallized from isopropanol-ethyl acetate. Yield 3.0 g. The filtrate is purified by flash chromatography to give E-isomer.

Z-isomer: Η NMR (CDCI3): 2.91 (t, 2H), 3.41 (t, 2H), 3.55-3.85 (m, 6H), 3.99 (dist.t, 2H), 4.54 (s, 2H), 6.40 (s, 1H), 6.56 (d, 2H), 6.77 (d, 2H), 7.10- 7.50 (m, 15H)

E-isomer: 1H NMR (CDCI3): 2.97 (t, 2H), 3.43 (t, 2H), 3.65-3.82 (m, 4H), 3.88 (dist.t, 2H), 4.15 (dist.t, 2H), 4.58 (s, 2H), 6.86 -7.45 (m, 19H)

FINAL STEP

e) 2- {2-[4-(4-Chloro- 1 ,2-diphenyl-but- 1 -enyl)phenoxy]ethoxy } ethanol:

Z- 1 – {4-[2-(2-Benzyloxy-ethoxy)ethoxy]phenyl} -4-chloro- 1 ,2-diphenyl -but- 1-ene (3.8 g, 7.4 mmol) is dissolved in ethyl acetate under nitrogen atmosphere , Zn powder (0.12 g, 1.85 mmol) and acetyl chloride (1.27 g, 16.3 mmol) are added and the mixture is stirred at 50 °C for 3 h (Bhar, 1995). The reaction mixture is cooled to room temperature, water (10 ml) is added and stirring is continued for additional 10 min. The aqueous layer is separated and the organic phase is washed with 1 M aqueous hydrogen chloride solution and with water. Ethyl acetate is evaporated and the residue is dissolved in methanol (16 ml) and water (4 ml). The acetate ester of the product is hydrolysed by making the mixture alkaline with sodium hydroxide (1 g) and stirring the mixture at room temperature for 1 h. Methanol is evaporated, water is added and the residue is extracted in ethyl acetate and washed with 1 M hydrogen chloride solution and with water. Ethyl acetate is evaporated and the residue is dissolved in toluene (25 ml), silica gel (0.25 g) is added and mixture is stirred for 15 min. Toluene is filtered and evaporated to dryness. The residue is crystallised from heptane-ethyl acetate (2:1). The yield is 71 %.

Z-isomer: 1H NMR (CDCI3): 2.92 (t, 2H), 3.41 (t, 2H), 3.58-3.63 (m, 2H), 3.69-3.80 (m, 4H), 3.96-4.01 (m, 2H), 6.56 (d, 2H), 6.78 (d, 2H), 7.10-7.40 (m, 10H).

Z ISOMER IE FISPEMIFENE

E-2- {2- [4-(4-Chloro- 1 ,2-diphenyl-but- 1 -enyl)phenoxy]ethoxy} ethanol is prepared analogously starting from E-l-{4-[2-(2-benzyloxy- ethoxy)ethoxy]phenyl} -4-chloro- 1,2-diphenyl-but-l-ene. The product is purified by flash chromatography with toluene-methanol (10:0.5) as eluent.

E-isomer: 1H NMR (CDCI3): 2.97 (t, 2H), 3.43 (t, 2H), 3.65-3.79 (m, 4H), 3.85-3.90 (m, 2H), 4.13-4.17 (m, 2H), 6.85-7.25 (m, 2H).

Debenzylation of 1 – {4-[2-(2-benzyloxy-ethoxy)ethoxy]phenyl} -4-chloro- 1 ,2- diphenyl-but- 1-ene is also carried out by hydrogenation with Pd on carbon as a catalyst in ethyl acetate-ethanol solution at room temperature.

………….

PATENT

http://www.google.com/patents/US5491173

Patent	Submitted	Granted
Method for the preparation of 2-{2-[4-(4-chloro-1,2-diphenylbut-1-enyl)phenoxy]ethoxy}ethanol and its isomers [US6891070]	2004-06-17	2005-05-10
Formulations of fispemifene [US2007104743]	2007-05-10
METHODS FOR THE PREPARATION OF FISPEMIFENE FROM OSPEMIFENE [US7504530]	2008-09-04	2009-03-17
METHOD FOR THE PREPARATION OF THERAPEUTICALLY VALUABLE TRIPHENYLBUTENE DERIVATIVES [US2011015448]	2011-01-20
METHOD FOR THE PREPARATION OF THERAPEUTICALLY VALUABLE TRIPHENYLBUTENE DERIVATIVES [US7812197]	2008-08-28	2010-10-12

WO2001036360A1	1 Nov 2000	25 May 2001	Pirkko Haerkoenen	Triphenylalkene derivatives and their use as selective estrogen receptor modulators
EP0095875A2	20 May 1983	7 Dec 1983	Farmos Group Ltd.	Novel tri-phenyl alkane and alkene derivatives and their preparation and use

Citing Patent	Filing date	Publication date	Applicant	Title
WO2008099059A1 *	13 Feb 2008	21 Aug 2008	Hormos Medical Ltd	Method for the preparation of therapeutically valuable triphenylbutene derivatives
WO2008099060A2 *	13 Feb 2008	21 Aug 2008	Hormos Medical Ltd	Methods for the preparation of fispemifene from ospemifene
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MELOGLIPTIN

diabetes, phase 2, Uncategorized Comments Off

Jul 032015

GRC 8200; 868771-57-7, EMD-675992

4-fluoro-1-[2-[[(1R,3S)-3-(1,2,4-triazol-1-ylmethyl)cyclopentyl]amino]acetyl]pyrrolidine-2-carbonitrile

4(S)-Fluoro-1-[2-[(1R,3S)-3-(1H-1,2,4-triazol-1-ylmethyl)cyclopentylamino]acetyl]pyrrolidine-2(S)-carbonitrile

GRC-8200, a dipeptidyl peptidase IV inhibitor (DPP-IV), is currently undergoing phase II clinical trials at Glenmark Pharmaceuticals and Merck KGaA for the treatment of type 2 diabetes. In 2006, the compound was licensed by Glenmark Pharmaceuticals to Merck KGaA in Europe, Japan and N. America for the treatment of type 2 diabetes, however, these rights were reaquired by Glenmark in 2008.

ALTERNATE……….

see gliptins at………….http://drugsynthesisint.blogspot.in/p/gliptin-series.html

http://organicsynthesisinternational.blogspot.in/p/gliptin-series-22.html

GOSOGLIPTIN

diabetes, Uncategorized 1 Response »

Jul 032015

ChemSpider 2D Image | gosogliptin | C17H24F2N6O

GOSOGLIPTIN

CAS 869490-23-3 FREE BASE

DIHYDROCHLORIDE..869490-47-1

GOSOGLIPTIN; UNII-GI718UO477; PF-00734200; PF-734200;

(3,3-difluoropyrrolidin-1-yl)-[(2S,4S)-4-(4-pyrimidin-2-ylpiperazin-1-yl)pyrrolidin-2-yl]methanone

Molecular Formula:	C17H24F2N6O
Molecular Weight:	366.408866 g/mol

Company	Pfizer Inc.
Description	Dipeptidyl peptidase-4 (DPP-4) inhibitor
Molecular Target	Dipeptidyl peptidase-4 (DPP-4) (CD26)
Mechanism of Action	Dipeptidyl peptidase-4 (DPP-4) inhibitor

Latest Stage of Development	Phase II
Standard Indication	Diabetes
Indication Details	Treat Type II diabetes

Type 2 diabetes mellitus is a chronic disorder characterized by hyperglycemia coupled with a gradual decline in insulin sensitivity and insulin secretion. The incretin hormone glucagon-like peptide-1 (GLP-1), which is released post-prandially from the L-cells of the intestine, stimulates the release of insulin from pancreatic β-cells. However, GLP-1 is rapidly degraded in vivo by peptidases, including dipeptidyl peptidase IV (DPP-4), which is a widely distributed serine protease that specifically cleaves N-terminal dipeptides from polypeptides with proline or alanine at the penultimate position.

In vivo administration of DPP-4 inhibitors to human subjects results in higher circulating concentrations of endogenous GLP-1 and subsequent decrease in plasma glucose. Long term treatment with a DPP-4 inhibitor leads to a reduction in circulating HbA1c (glycosylated hemoglobin). DPP-4 inhibition also offers the potential to improve the insulin producing function of the pancreas through either β-cell preservation or regeneration. Therefore, DPP-4 inhibition has emerged as a promising new treatment of Type 2 diabetes

PF-734200 is a potent, selective, orally active dipeptidyl peptidase IV inhibitor. It had been in phase II clinical development at Pfizer for the treatment of type 2 diabetes; however, in 2010 the company discontinued these trials. In 2012, the product was licensed to SatRx, a spin-off of the ChemRar High Tech Center, by Pfizer on an exclusive worldwide basis (with the exception of China) for the development and commercialization as monotherapy or in combination with other therapies for the treatment of type 2 diabetes. SatRx is conducting phase II clinical trials for the treatment of type 2 diabetes.

……………………….

PAPER

New synthetic route to a dipeptidyl peptidase-4 inhibitor
Org Process Res Dev 2012, 16(3): 409

http://pubs.acs.org/doi/abs/10.1021/op200309z

A new synthetic route to a dipeptidyl peptidase-4 (DPP4) inhibitor was developed and demonstrated on a multigram scale. This approach takes advantage of the cheap and readily available Boc-trans-4-hydroxy-l-proline methyl ester as starting material which was derivatized through an SN2 reaction. Several leaving groups were studied, and the nosylate group showed superiority over other derivatives. Formation of an amide using the most costly starting material, 3,3-difluoropyrrolidine, was performed late in the synthesis to minimize its economical impact on the overall cost of the API.

(3,3-Difluoropyrrolidin-1-yl)-(2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)methanone.FREE BASE

Mp 149 °C (decomp).

[α]d = −31.1 (T = 24 °C, c = 1, CHCl3). Specific rotation of product 4 prepared using the initial route: [α]d = −31.5 (T = 24 °C, c = 1, CHCl3).

1H NMR (400 MHz; CDCl3) δ 8.30 (d, J = 4 Hz, 2H), 6.48 (t, J = 4 Hz, 1H), 3.95–3.6 (m, 9H), 3.25–2.85 (m, 4H), 2.6–2.25 (m, 7H), 1.75–1.6 (m, 1H).

13C NMR (100 MHz; CDCl3) δ 172.28; 161.55; 157.70; 127.22 (t, 1J C–F = 248 Hz), 126.22 (t, 1J C–F = 246 Hz), 109.95; 66.54; 58.87; 57.99; 52.71 (t, 2 J C–F = 32 Hz); 52.00; 50.41; 43.03; 34.46, 34.37, 34.25; 19F NMR (377 MHz, CDCl3) δ −102.1 (m, 2F).

IR (neat): 2951w, 2864w, 2799w, 2759w, 1630s, 1585vs, 1547m, 1449m, 1172m, 1254m, 1129m, 982w, 923m, 796m, 638w.

HRMS (ES, N2) Calcd for C17H24F2N6O: 367.20524, found: 367.20592.

……………………….

PAPER

(3,3-difluoro-pyrrolidin-1-yl)-((2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl)-methanone: A potent, selective, orally active dipeptidyl peptidase IV inhibitor
Bioorg Med Chem Lett 2009, 19(7): 1991

http://www.sciencedirect.com/science/article/pii/S0960894X09001966?np=y

Pfizer Global Research & Development, Groton/New London Laboratories, Pfizer Inc, Groton, CT 06340, United States

doi:10.1016/j.bmcl.2009.02.041

A series of 4-substituted proline amides was evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC₅₀= 13 nM) and selective compound, with high oral bioavailability in preclinical species.

SEE………….https://docs.google.com/viewer?url=http%3A%2F%2Fwww.sciencedirect.com%2Fscience%2FMiamiMultiMediaURL%2F1-s2.0-S0960894X09001966%2F1-s2.0-S0960894X09001966-mmc1.doc%2F271398%2Fhtml%2FS0960894X09001966%2Fce1f70bd989d6d4b79b40c26570693d2%2Fmmc1.doc

………………….

PATENT

WO 2005116014

http://www.google.co.in/patents/WO2005116014A1?cl=en

Example 113 (3.3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)-methanone

Step 1 – (S)-2-(3.3-Difluoro-pyrrolidine-1-carbonyl)-4-oxo-pyrrolidine-1 -carboxylic acid tert-butyl ester

3,3-Difluoro- pyrrolidine hydrochloride (4.13 kg, 1.0 equivalent) was charged to the above mixture over 10 minutes at – 10°C to 0°C. Triethylamine (4.0 liters, 1.0 equiv) was introduced slowly over 70 minutes at -10°C to 0°C. Upon completion of triethylamine addition, the mixture was stirred for 1h at 0 to 5°C. The reaction was complete by HPLC assay (-1% starting material). The reaction was quenched with water (10 volumes) at 0°C to 5 °C. The mixture was heated to 20°C to 25 °C. The layers were separated, and the organic layer was washed with 0.5 M HCI (5 volumes). The organic layer was again washed with combined 5% NaHC0₃ (2 volumes) and half saturated brine solution (1.64 M, 3 volumes). The organic solution was concentrated atmospherically to a low stirrable volume (approximately 20 liters). Ethyl acetate (12.6 volumes, 82.8 liters) was added, the solution was concentrated atmospherically to -6 volumes. The mixture was held at 60°C to 65 °C for 2 hours and cooled to room temperature over 3 hours. The mixture was held at 20°C to 25 °C for 8 hours. Heptane (8 volumes) was added, and the mixture was granulated for a minimum of 2 hours. The solid was filtered, rinsed with 2:1 heptane/ethyl acetate (1 volume), and dried in a tray dryer at 25°C to 35°C for a minimum of 12 h. Yield: 7.26 kg, 79%. HPLC purity: 99.7%. The mother liquor (86 liters) was concentrated to 12 liters under partial vacuum at 65°C to 70°C. The mixture was cooled to 60°C to 65 °C. Ethyl acetate (4.0 liters) was added slowly over 15 minutes. The mixture was cooled to 20°C to 25 °C over 2 hours and was held at that temperature for at least 2 hours. The solid was filtered and rinsed with heptane/ethyl acetate (3:1 v/v, 1.7 liters). Drying in a tray dryer for 12 hours at 35°C to 45 °C yielded 435 grams of product. HPLC purity: 96.4%.

Step 2 – (2S.4S)-2-(3.3-Dif luoro-pyrrolidine-1 -carbonyl)-4-(4-pyrimidin-2-yl-piperazin-1 -yl)-pyrrolidine-1 – carboxylic acid tert-butyl ester A reactor was charged with THF (20 volumes), 2-piperazin-1-yl-pyrimidine (2.17 kg, 1.05 equivalents) and the product from Step 1 (4.00 kg, 1.0 equivalent). The mixture was held at 20°C to 25°C until all material was dissolved over 30 minutes. Acetic acid (0.792 kg, 1.05 equivalents) as added. The mixture was stirred for 1 hour during which the reaction mixture turned to cloudy. The reaction mixture was refluxed for 30 minutes and then concentrated at 60°C to 70°C until a steady temperature of 66.9°C was observed in the overheads indicating complete removal of water from the system. More THF was added as necessary. At the end, THF was added to bring the total volume in the reactor to 15 volumes of the limit reagent. The reaction mixture was cooled to -3°C to 7°C and sampled for complete formation of imine by HPLC (using sodium triacetoxyborohydride to reduce imine). Sodium triacetoxyborohydride (5.33 kg, 2.0 equivalents) was added portion-wise to the suspension at -5°C to 15°C. The reaction mixture was heated to 20°C to 25°C and held for 12 hours. HPLC results confirmed the reaction was complete by 99.8%. Sodium bicarbonate aqueous solution (10% w/w, 10 volumes) was added. The slurry was concentrated to remove 10 volumes of THF under partial vacuum at 30°C to 60°C. Ethyl acetate (10 volumes) was added to the suspension after it cooled to 20°C to 25^CC. The organic phase was separated and the aqueous phase was checked by HPLC. It contained less than 2% of the product. The organic phase was washed with water (5 volumes), saturated brine solution (5 volumes) and concentrated to a small volume (2 volumes) under partial vacuum at 45°C to 50°C. To the slurry was added heptane (10 volumes) at 45°C to 50°C over 30 minutes. The mixture was cooled to 20°C to 25°C and granulated for 2 hours. Solid was collected by filtration, rinsed with heptane (2 volumes). Drying in a tray dryer for 12 hours at 35°C to 45°C yield 5.35 kg (91.3%) of the product. Step 3 – (3.3-Dif luoro-pyrrolidin-1 -yl)-f(2S.4S)-4-(4-pyrimidin-2-yl-piperazin-1 -yl)-pyrrolidin-2-yll- methanone Water (19 liters, 2 volumes) was charged to a reactor followed by the product from Step 2 (9.57 kg,

1.0 equivalent). To the slurry was added concentrated HCI (37 wt% in water, 19.1 liters, 2 volumes) slowly at 20°C to 30°C over 4 hours. The slurry went into solution after 12 liters of HCI was added. After the addition completion, the reaction was complete by HPLC assay. The reaction mixture was cooled to 5°C to 15°C. To the mixture was added 50% NaOH aqueous solution slowly with agitation to pH 10 to pH 11. The pH was monitored with a pH meter closely during the neutralization. The total volume of 50% NaOH added was 12.45 liters. The mixture was warmed to 20°C to 25°C and extracted with ethyl acetate twice (115 liters, 12 volumes and 57 liters, 6 volumes, respectively). The sample from aqueous layer after second extraction was analyzed by HPLC and showed only 1% of the product in that aqueous solution.

The organic layers were combined and treated with magnesium sulfate (5 kg) for 1 hour. The mixture was filtered. The filter cake was rinsed with ethyl acetate (10 liters). The filtrate was charged back to the reactor via a 0.2 micron in-line filter for speck free operation. (The following operations were performed under speck free conditions.) The solution was concentrated to 20 liters (2 volumes) under partial vacuum at 50°C to 60°C. The mixture was cooled to 20°C to 25°C over 30 minutes. Upon cooling to room temperature, crystallization occurred. The mixture was held for 30 minutes. Hexanes (20 liters, 2 volumes) was added slowly over 1 hour. The mixture was granulated for 2 hours. The solid product was collected by filtration and rinsed with hexanes/ethyl acetate (10 liters, 1 :1 v/v). The filter was blown dry with nitrogen for a minimum of 2 hours. The product was dried in a tray dryer at 44°C for 12 hours.

Yield: 5.7 kg, 75.9%.

m.p. 156°C. MS m/z 367 (MH⁺).

FREE BASE

¹H NMR (400 MHz, D₂0): δ 8.15 (d, 2H, J = 5.0 Hz, CH of pyrimidine), 6.55 (t, 1 H, J = 4.8 Hz, CH of pyrimidine), 3.87-3.81 (dd, 1 H, H_2b of proline, rotomeric), 3.78-3.50 (m, 4H, N-CH₂ of pyrrolidide), 3.55-3.40 (m, 4H, N-CH₂ of piperazine), 2.97 (dd, 1 H, J = 10.2, 6.6 Hz, H_5a of proline), 2.85-2.75 (m, 1 H, H_4b of proline), 2.69 (dd, 1 H, J = 10.0, 9.1 Hz, H_5b of proline), 2.55-2.20 (m, 7H, overlapping N-CH₂ of piperazine, CH₂ of pyrrolidide and H_3b of proline), 1.47-1.38 (m, 1 H, H_3a of proline).

Alternatively, the dihydrochloride salt of the titled compound was prepared according to the method of Example 1.

………………

US 2005/0256310

http://www.google.com/patents/US20050256310

This approach begins with N–t-Boc-4-oxo-l-proline (1) that undergoes a mixed anhydride activation with pivaloyl chloride at 0 °C, followed by amidation with 3,3-difluoropyrrolidine to yield the intermediate 2. Reductive amination with 1-(2-pyrimidyl)piperazine using sodium triacetoxyborohydride in THF/AcOH provided the desired stereoisomer 3 in high yield and selectivity, the undesired diastereomer being completely removed by crystallization. Deprotection of 3 with 6 N HCl, followed by neutralization with 50% NaOH and extraction provided PF-734200 (4) in good yield.

EXAMPLE 113 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrolidin-2-yl)-methanone

Step 1—(S)-2-(3,3-Difluoro-pyrrolidine-1-carbonyl)-4-oxo-pyrrolidine-1-carboxylic acid tert-butyl

(S)-4-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (6.6 kg, 1.0 equivalent) was charged to a reactor, followed by addition of dichloromethane (15 volumes). The reaction mixture was cooled to 0° C. Triethylamine (4.82 liters, 1.2 equiv) was added over 30 minutes. The mixture turned from suspension to a clear solution at the end of triethylamine addition. The mixture was held at 0° C. to 5° C. for 10 minutes. Pivaloyl chloride (3.65 kg, 1.05 equivalents) was added slowly while keeping the reaction temperature at 0° C. to 5° C. The reaction mixture turned back to a slurry. The reaction mixture was sampled for completion by HPLC (using diethylamine to derivatize) after held for 1 hour at 0° C. to 5° C. 3,3-Difluoro-pyrrolidine hydrochloride (4.13 kg, 1.0 equivalent) was charged to the above mixture over 10 minutes at −10° C. to 0° C. Triethylamine (4.0 liters, 1.0 equiv) was introduced slowly over 70 minutes at −10° C. to 0° C. Upon completion of triethylamine addition, the mixture was stirred for 1 h at 0 to 5° C. The reaction was complete by HPLC assay (˜1% starting material). The reaction was quenched with water (10 volumes) at 0° C. to 5 ° C. The mixture was heated to 20° C. to 25 ° C. The layers were separated, organic layer was washed with 0.5 M HCl (5 volumes). The organic layer was again washed with combined 5% NaHCO₃(2 volumes) and half saturated brine solution (1.64 M, 3 volumes). The organic solution was concentrated atmospherically to a low stirrable volume (approximately 20 liters). Ethyl acetate (12.6 volumes, 82.8 liters) was added, the solution was concentrated atmospherically to ˜6 volumes. The mixture was held at 60° C. to 65° C. for 2 hours and cooled to room temperature over 3 hours. The mixture was held at 20° C. to 25 ° C. for 8 hours. Heptane (8 volumes) was added, and the mixture was granulated for a minimum of 2 hours. The solid was filtered, rinsed with 2:1 heptane/ethyl acetate (1 volume), and dried in a tray dryer at 25° C. to 35° C. for a minimum of 12 h. Yield: 7.26 kg, 79%. HPLC purity: 99.7%. The mother liquor (86 liters) was concentrated to 12 liters under partial vacuum at 65° C. to 70° C. The mixture was cooled to 60° C. to 65° C. Ethyl acetate (4.0 liters) was added slowly over 15 minutes. The mixture was cooled to 20° C. to 25° C. over 2 hours and was held at that temperature for at least 2 hours. The solid was filtered and rinsed with heptane/ethyl acetate (3:1 v/v, 1.7 liters). Drying in a tray dryer for 12 hours at 35° C. to 45° C. yielded 435 grams of product. HPLC purity: 96.4%.

Step 2—(2S,4S)-2-(3,3-Difluoro-pyrrolidine-1-carbonyl)-4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidine-1-carboxylic acid tert-butyl ester

A reactor was charged with THF (20 volumes), 2-piperazin-1-yl-pyrimidine (2.17 kg, 1.05 equivalents) and the product from Step 1 (4.00 kg, 1.0 equivalent). The mixture was held at 20° C. to 25° C. until all material was dissolved over 30 minutes. Acetic acid (0.792 kg, 1.05 equivalents) as added. The mixture was stirred for 1 hour during which the reaction mixture turned to cloudy. The reaction mixture was refluxed for 30 minutes and then concentrated at 60° C. to 70° C. until a steady temperature of 66.9° C. was observed in the overheads indicating complete removal of water from the system. More THF was added as necessary. At the end, THF was added to bring the total volume in the reactor to 15 volumes of the limit reagent. The reaction mixture was cooled to −3° C. to 7° C. and sampled for complete formation of imine by HPLC (using sodium triacetoxyborohydride to reduce imine). Sodium triacetoxyborohydride (5.33 kg, 2.0 equivalents) was added portion-wise to the suspension at −5° C. to 15° C. The reaction mixture was heated to 20° C. to 25° C. and held for 12 hours. HPLC results confirmed the reaction was complete by 99.8%. Sodium bicarbonate aqueous solution (10% w/w, 10 volumes) was added. The slurry was concentrated to remove 10 volumes of THF under partial vacuum at 30° C. to 60° C. Ethyl acetate (10 volumes) was added to the suspension after it cooled to 20° C. to 25° C. The organic phase was separated and the aqueous phase was checked by HPLC. It contained less than 2% of the product. The organic phase was washed with water (5 volumes), saturated brine solution (5 volumes) and concentrated to a small volume (2 volumes) under partial vacuum at 45° C. to 50° C. To the slurry was added heptane (10 volumes) at 45° C. to 50° C. over 30 minutes. The mixture was cooled to 20° C. to 25° C. and granulated for 2 hours. Solid was collected by filtration, rinsed with heptane (2 volumes). Drying in a tray dryer for 12 hours at 35° C. to 45° C. yield 5.35 kg (91.3%) of the product.

Step 3—(3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone

Water (19 liters, 2 volumes) was charged to a reactor followed by the product from Step 2 (9.57 kg, 1.0 equivalent). To the slurry was added concentrated HCl (37 wt % in water, 19.1 liters, 2 volumes) slowly at 20° C. to 30° C. over 4 hours. The slurry went into solution after 12 liters of HCl was added. After the addition completion, the reaction was complete by HPLC assay. The reaction mixture was cooled to 5° C. to 15° C. To the mixture was added 50% NaOH aqueous solution slowly with agitation to pH 10 to pH 11. The pH was monitored with a pH meter closely during the neutralization. The total volume of 50% NaOH added was 12.45 liters. The mixture was warmed to 20° C. to 25° C. and extracted with ethyl acetate twice (115 liters, 12 volumes and 57 liters, 6 volumes, respectively). The sample from aqueous layer after second extraction was analyzed by HPLC and showed only 1% of the product in that aqueous solution. The organic layers were combined and treated with magnesium sulfate (5 kg) for 1 hour. The mixture was filtered. The filter cake was rinsed with ethyl acetate (10 liters). The filtrate was charged back to the reactor via a 0.2 micron in-line filter for speck free operation. (The following operations were performed under speck free conditions.) The solution was concentrated to 20 liters (2 volumes) under partial vacuum at 50° C. to 60° C. The mixture was cooled to 20° C. to 25° C. over 30 minutes. Upon cooling to room temperature, crystallization occurred. The mixture was held for 30 minutes. Hexanes (20 liters, 2 volumes) was added slowly over 1 hour. The mixture was granulated for 2 hours. The solid product was collected by filtration and rinsed with hexanes/ethyl acetate (10 liters, 1:1 v/v). The filter was blown dry with nitrogen for a minimum of 2 hours. The product was dried in a tray dryer at 44° C. for 12 hours.

Yield: 5.7 kg, 75.9%. m.p. 156° C. MS m/z 367 (MH⁺).

¹H NMR (400 MHz, D₂O): δ 8.15 (d, 2H, J=5.0 Hz, CH of pyrimidine), 6.55 (t, 1H, J=4.8 Hz, CH of pyrimidine), 3.87-3.81 (dd, 1H, H_2bof proline, rotomeric), 3.78-3.50 (m, 4H, N—CH₂of pyrrolidide), 3.55-3.40 (m, 4H, N—CH₂of piperazine), 2.97 (dd, 1H, J=10.2, 6.6 Hz, H_5aof proline), 2.85-2.75 (m, 1H, H_4bof proline), 2.69 (dd, 1H, J=10.0, 9.1 Hz, H_5bof proline), 2.55-2.20 (m, 7H, overlapping N—CH₂of piperazine, CH₂of pyrrolidide and H_3bof proline), 1.47-1.38 (m, 1H, H_3aof proline).

Alternatively, the dihydrochloride salt of the titled compound was prepared according to the method of Example 1.

……………..

PAPER

Scheme 1.

Reagents and conditions: (a) 3,3-difluoropyrrolidine hydrochloride, EDC, HOBt, TEA, DCM, rt; (b) NaBH₄, MeOH, (c) (1) trifluoromethane-sulphonyl chloride, DIPEA, DCM; (2) 2-(1-piperazinyl)pyrimidine, DCM, −10 °C; (d) 4 N HCl in dioxane, rt; (e) 2-(1-piperazinyl)pyrimidine, NaBH(OAc)₃, AcOH, DCE; (f) R₁R₂NH hydrochloride, EDC, HOBt TEA, DCM, 0–rt; (g) N-heterocyclic piperazine, NaBH(OAc)_3, AcOH, DCE.

……………………….

Patent	Submitted	Granted
Therapeutic compounds [US7291618]	2005-11-17	2007-11-06
(2S,4S)-4-(piperazin-1-yl)pyrrolidine-2-methanone derivatives [US7465732]	2007-05-03	2008-12-16
THERAPEUTIC COMPOUNDS [US2007161664]	2007-07-12
Therapeutic compounds [US2006079498]	2006-04-13

see gliptins at………….http://drugsynthesisint.blogspot.in/p/gliptin-series.html

http://organicsynthesisinternational.blogspot.in/p/gliptin-series-22.html

see gliptins at…………http://drugsynthesisint.blogspot.in/p/gliptin-series.html

Burixafor 布利沙福

phase 2 Comments Off

Mar 102015

Burixafor is a potent and selective chemokine CXCR4 antagonist developed by TaiGen Biotechnology (www.taigenbiotech.com.tw).

The SDF1/CXCR4 pathway plays key roles in homing and mobilization of hematopoietic stem cells and endothelial progenitor cells. In a mouse model, burixafor efficiently mobilizes stem cells (CD34+) and endothelial progenitor cells (CD133+) from bone marrow into peripheral circulation. It can be used in hematopoietic stem cell transplantation, chemotherapy sensitization and other ischemic diseases.

Because TaiGen has filed an IND (CXHL1200371) for burixafor as a chemotherapy sensitizer in October 2012, the new application (CXHL1400844) may supplement a new indication. Phase II clinical trials (NCT02104427) are currently underway in the US, with Phase IIa (NCT01018979, NCT01458288) already completed.

TaiGen plans to initiate clinical trials of burixafor as a chemotherapy sensitizer in China shortly. Burixafor’s annual sales are estimated at $1.1 billion by consultancy company JSB. This compound is protected by patent WO2009131598.

SEE……….http://newdrugapprovals.org/2014/06/09/scinopharm-to-provide-active-pharmaceutical-ingredient-%E8%8B%B1%E6%96%87%E5%90%8D%E7%A7%B0-burixafor-to-ftaigen-for-novel-stem-cell-drug/

英文名称Burixafor

TG-0054

(2-{4-[6-amino-2-({[(1r,4r)-4-({[3-(cyclohexylamino)propyl]amino}methyl)cyclohexyl]methyl}amino)pyrimidin-4-yl]piperazin-1-yl}ethyl)phosphonic acid

[2-[4-[6-Amino-2-[[[trans-4-[[[3-(cyclohexylamino)propyl]amino]methyl]cyclohexyl]methyl]amino]pyrimidin-4-yl]piperazin-1-yl]ethyl]phosphonic acid

1191448-17-5

C27H51N8O3P, 566.7194

chemokine CXCR 4 receptor antagonist;

Taigen Biotechnology Co., Ltd.

ScinoPharm to Provide Active Pharmaceutical Ingredient to F*TaiGen for Novel Stem Cell Drug
MarketWatch
The drug has received a Clinical Trial Application from China’s FDA for the initiation of … In addition, six products have entered Phase III clinical trials.

read at

http://www.marketwatch.com/story/scinopharm-to-provide-active-pharmaceutical-ingredient-to-ftaigen-for-novel-stem-cell-drug-2014-06-08

2D chemical structure of 1191448-17-5

TAINAN, June 8, 2014 — ScinoPharm Taiwan, Ltd. (twse:1789) specializing in the development and manufacture of active pharmaceutical ingredients, and TaiGen Biotechnology (4157.TW; F*TaiGen) jointly announced today the signing of a manufacturing contract for the clinical supply of the API of Burixafor, a new chemical entity discovered and developed by TaiGen. The API will be manufactured in ScinoPharm’s plant in Changshu, China. This cooperation not only demonstrates Taiwan’s international competitive strength in new drug development, but also sees the beginning of a domestic pharmaceutical specialization and cooperation mechanisms, thus establishing a groundbreaking milestone for Taiwan’s pharmaceutical industry.

Dr. Jo Shen, President and CEO of ScinoPharm said, “This cooperation with TaiGen is of representative significance in the domestic pharmaceutical companies’ upstream and downstream cooperation and self-development of new drugs, and indicates the Taiwanese pharmaceutical industry’s cumulative research and development momentum is paving the way forward.” Dr. Jo Shen emphasized, “ScinoPharm’s Changshu Plant provides high-quality API R&D and manufacturing services through its fast, flexible, reliable competitive advantages, effectively assisting clients of new drugs in gaining entry into China, Europe, the United States, and other international markets.”

ScinoPharm President, CEO and Co-Founder Dr. Jo Shen

According to Dr. Ming-Chu Hsu, Chairman and CEO of TaiGen, “R&D is the foundation of the pharmaceutical industry. Once a drug is successfully developed, players at all levels of the value chain could reap the benefit. Burixafor is a 100% in-house developed product that can be used in the treatment of various intractable diseases. The cooperation between TaiGen and ScinoPharm will not only be a win-win for both sides, but will also provide high-quality novel dug for patients from around the world.”

Burixafor is a novel stem cell mobilizer that can efficiently mobilize bone marrow stem cells and tissue precursor cells to the peripheral blood. It can be used in hematopoietic stem cell transplantation, chemotherapy sensitization and other ischemic diseases. The results of the ongoing Phase II clinical trial in the United States are very impressive. The drug has received a Clinical Trial Application from China’s FDA for the initiation of a Phase II clinical trial in chemotherapy sensitization under the 1.1 category. According to the pharmaceutical consultancy company JSB, with only stem cell transplant and chemotherapy sensitizer as the indicator, Burixafor’s annual sales are estimated at USD1.1 billion.

ScinoPharm currently has accepted over 80 new drug API process research and development plans, of which five new drugs have been launched in the market. In addition, six products have entered Phase III clinical trials. Through the Changshu Plant’s operation in line with the latest international cGMP plant equipment and quality management standards, the company provides customers with one stop shopping services in professional R&D, manufacturing, and outsourcing, thereby shortening the customer development cycle of customers’ products and accelerating the launch of new products to the market.

TaiGen’s focus is on the research and development of novel drugs. Besides Burixafor, the products also include anti-infective, Taigexyn®, and an anti-hepatitis C drug, TG-2349. Taigexyn® is the first in-house developed novel drug that received new drug application approval from Taiwan’s FDA. TG-2349 is intended for the 160 million global patients with hepatitis C with huge market potential. TaiGen hopes to file one IND with the US FDA every 3-4 years to expand TaiGen’s product line.

About ScinoPharm

ScinoPharm Taiwan, Ltd. is a leading process R&D and API manufacturing service provider to the global pharmaceutical industry. With research and manufacturing facilities in both Taiwan and China, ScinoPharm offers a wide portfolio of services ranging from custom synthesis for early phase pharmaceutical activities to contract services for brand companies as well as APIs for the generic industry. For more information, please visit the Company’s website at http://www.scinopharm.com

About TaiGen Biotechnology

TaiGen Biotechnology is a leading research-based and product-driven biotechnology company in Taiwan with a wholly-owned subsidiary in Beijing, China. The company’s first product, Taigexyn®, have already received NDA approval from Taiwan’s FDA. In addition to Taigexyn®, TaiGen has two other in-house discovered NCEs in clinical development under IND with US FDA: TG-0054, a chemokine receptor antagonist for stem cell transplantation and chemosensitization, in Phase 2 and TG-2349, a HCV protease inhibitor for treatment of chronic hepatitis infection, in Phase 2. Both TG-0054 and TG-2349 are currently in clinical trials in patients in the US.

SOURCE ScinoPharm Taiwan Ltd.

TG-0054 is a potent and selective chemokine CXCR4 (SDF-1) antagonist in phase II clinical studies at TaiGen Biotechnology for use in stem cell transplantation in cancer patients. Specifically, the compound is being developed for the treatment of stem cell transplantation in multiple myeloma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma and myocardial ischemia.

Preclinical studies had also been undertaken for the treatment of diabetic retinopathy, critical limb ischemia (CLI) and age-related macular degeneration. In a mouse model, TG-0054 efficiently mobilizes stem cells (CD34+) and endothelial progenitor cells (CD133+) from bone marrow into peripheral circulation.

BACKGROUND

Chemokines are a family of cytokines that regulate the adhesion and transendothelial migration of leukocytes during an immune or inflammatory reaction (Mackay C.R., Nat. Immunol, 2001, 2:95; Olson et al, Am. J. Physiol. Regul. Integr. Comp. Physiol, 2002, 283 :R7). Chemokines also regulate T cells and B cells trafficking and homing, and contribute to the development of lymphopoietic and hematopoietic systems (Ajuebor et al, Biochem. Pharmacol, 2002, 63:1191). Approximately 50 chemokines have been identified in humans. They can be classified into 4 subfamilies, i.e., CXC, CX3C, CC, and C chemokines, based on the positions of the conserved cysteine residues at the N-terminal (Onuffer et al, Trends Pharmacol ScI, 2002, 23:459). The biological functions of chemokines are mediated by their binding and activation of G protein-coupled receptors (GPCRs) on the cell surface.

Stromal-derived factor- 1 (SDF-I) is a member of CXC chemokines. It is originally cloned from bone marrow stromal cell lines and found to act as a growth factor for progenitor B cells (Nishikawa et al, Eur. J. Immunol, 1988, 18:1767). SDF-I plays key roles in homing and mobilization of hematopoietic stem cells and endothelial progenitor cells (Bleul et al, J. Exp. Med., 1996, 184:1101; and Gazzit et al, Stem Cells, 2004, 22:65-73). The physiological function of SDF-I is mediated by CXCR4 receptor. Mice lacking SDF-I or CXCR4 receptor show lethal abnormality in bone marrow myelopoiesis, B cell lymphopoiesis, and cerebellar development (Nagasawa et al, Nature, 1996, 382:635; Ma et al, Proc. Natl. Acad. ScI, 1998, 95:9448; Zou et al, Nature, 1998, 393:595; Lu et al, Proc. Natl. Acad. ScI, 2002, 99:7090). CXCR4 receptor is expressed broadly in a variety of tissues, particularly in immune and central nervous systems, and has been described as the major co-receptor for HIV- 1/2 on T lymphocytes. Although initial interest in CXCR4 antagonism focused on its potential application to AIDS treatment (Bleul et al, Nature, 1996, 382:829), it is now becoming clear that CXCR4 receptor and SDF-I are also involved in other pathological conditions such as rheumatoid arthritis, asthma, and tumor metastases (Buckley et al., J. Immunol., 2000, 165:3423). Recently, it has been reported that a CXCR4 antagonist and an anticancer drug act synergistically in inhibiting cancer such as acute promuelocutic leukemia (Liesveld et al., Leukemia

Research 2007, 31 : 1553). Further, the CXCR4/SDF-1 pathway has been shown to be critically involved in the regeneration of several tissue injury models. Specifically, it has been found that the SDF-I level is elevated at an injured site and CXCR4-positive cells actively participate in the tissue regenerating process.

………………………………………………………………………..

http://www.google.com/patents/WO2009131598A1?cl=en

Compound 52

Example 1 : Preparation of Compounds 1

1-1 1-Ii 1-m

^ ^–\\ Λ xCUNN H ‘ ‘22.. P rdu/’C^ ^. , Λ>\V>v

Et3N, TFAA , H_, r [ Y I RRaanneeyy–NNiicckkeell u H f [ Y | NH2

CH2CI2, -10 0C Boc^ ‘NNA/ 11,,44–ddιιooxxaannee B Boocer”1^”–^^ LiOH, H2O, 50 0C

1-IV 1-V

Water (10.0 L) and (BoC)2O (3.33 kgg, 15.3 mol) were added to a solution of trans-4-aminomethyl-cyclohexanecarboxylic acid (compound 1-1, 2.0 kg, 12.7 mol) and sodium bicarbonate (2.67 kg, 31.8 mol). The reaction mixture was stirred at ambient temperature for 18 hours. The aqueous layer was acidified with concentrated hydrochloric acid (2.95 L, pH = 2) and then filtered. The resultant solid was collected, washed three times with water (15 L), and dried in a hot box (60 0C) to give trα/?5-4-(tert-butoxycarbonylamino-methyl)-cyclo-hexanecarboxylic acid (Compound l-II, 3.17 kg, 97%) as a white solid. Rf = 0.58 (EtOAc). LC-MS m/e 280 (M+Na+). 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, IH), 2.98 (t, J= 6.3 Hz, 2H), 2.25 (td, J = 12, 3.3 Hz, IH), 2.04 (d, J= 11.1 Hz, 2H), 1.83 (d, J= 11.1 Hz, 2H), 1.44 (s, 9H), 1.35-1.50 (m, 3H), 0.89-1.03 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 181.31, 156.08, 79.12, 46.41, 42.99, 37.57, 29.47, 28.29, 27.96. M.p. 134.8-135.0 0C. A suspension of compound l-II (1.0 kg, 3.89 mol) in THF (5 L) was cooled at

-10 0C and triethyl amine (1.076 L, 7.78 mol) and ethyl chloroformate (0.441 L, 4.47 mol) were added below -10 0C. The reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was then cooled at -100C again and NH4OH (3.6 L, 23.34 mol) was added below -10 0C. The reaction mixture was stirred at ambient temperature for 18 hours and filtered. The solid was collected and washed three times with water (10 L) and dried in a hot box (6O0C) to give trans-4- (tert-butoxycarbonyl-amino-methyl)-cyclohexanecarboxylic acid amide (Compound l-III, 0.8 kg, 80%) as a white solid. Rf= 0.23 (EtOAc). LC-MS m/e 279, M+Na+. 1H NMR (300 MHz, CD3OD) δ 6.63 (brs, IH), 2.89 (t, J= 6.3 Hz, 2H), 2.16 (td, J = 12.2, 3.3 Hz, IH), 1.80-1.89 (m, 4H), 1.43 (s, 9H), 1.37-1.51 (m, 3H), 0.90-1.05 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 182.26, 158.85, 79.97, 47.65, 46.02, 39.28, 31.11, 30.41, 28.93. M.p. 221.6-222.0 0C.

A suspension of compound l-III (1.2 kg, 4.68 mol) in CH2Cl2 (8 L) was cooled at -1O0C and triethyl amine (1.3 L, 9.36 mol) and trifluoroacetic anhydride (0.717 L, 5.16 mol) were added below -10 0C. The reaction mixture was stirred for 3 hours. After water (2.0 L) was added, the organic layer was separated and washed with water (3.0 L) twice. The organic layer was then passed through silica gel and concentrated. The resultant oil was crystallized by methylene chloride. The crystals were washed with hexane to give £rαns-(4-cyano-cyclohexylmethyl)-carbamic acid tert-butyl ester (Compound 1-IV, 0.95 kg, 85%) as a white crystal. Rf = 0.78 (EtOAc). LC-MS m/e 261, M+Na+. 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, IH), 2.96 (t, J = 6.3 Hz, 2H), 2.36 (td, J= 12, 3.3 Hz, IH), 2.12 (dd, J= 13.3, 3.3 Hz, 2H), 1.83 (dd, J = 13.8, 2.7 Hz, 2H), 1.42 (s, 9H), 1.47-1.63 (m, 3H), 0.88-1.02 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 155.96, 122.41, 79.09, 45.89, 36.92, 29.06, 28.80, 28.25, 28.00. M.p. 100.4~100.6°C.

Compound 1-IV (1.0 kg, 4.196 mol) was dissolved in a mixture of 1 ,4-dioxane (8.0 L) and water (2.0 L). To the reaction mixture were added lithium hydroxide monohydrate (0.314 kg, 4.191), Raney-nickel (0.4 kg, 2.334 mol), and 10% palladium on carbon (0.46 kg, 0.216 mol) as a 50% suspension in water. The reaction mixture was stirred under hydrogen atmosphere at 5O0C for 20 hours. After the catalysts were removed by filtration and the solvents were removed in vacuum, a mixture of water (1.0 L) and CH2Cl2 (0.3 L) was added. After phase separation, the organic phase was washed with water (1.0 L) and concentrated to give £rα/?s-(4-aminomethyl- cyclohexylmethyl)-carbamic acid tert- butyl ester (compound 1-V, 0.97 kg, 95%) as pale yellow thick oil. Rf = 0.20 (MeOH/EtOAc = 9/1). LC-MS m/e 243, M+H+. 1H NMR (300 MHz, CDCl3) δ 4.67 (brs, IH), 2.93 (t, J= 6.3 Hz, 2H), 2.48 (d, J= 6.3 Hz, 2H), 1.73-1.78 (m, 4H), 1.40 (s, 9H), 1.35 (brs, 3H), 1.19-1.21 (m, IH), 0.77-0.97 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 155.85, 78.33, 48.27, 46.38, 40.80, 38.19, 29.87, 29.76, 28.07. A solution of compound 1-V (806 g) and Et3N (1010 g, 3 eq) in 1-pentanol

(2.7 L) was treated with compound 1-VI, 540 g, 1 eq) at 900C for 15 hours. TLC showed that the reaction was completed. Ethyl acetate (1.5 L) was added to the reaction mixture at 25°C. The solution was stirred for 1 hour. The Et3NHCl salt was filtered. The filtrate was then concentrated to 1.5 L (1/6 of original volume) by vacuum at 500C. Then, diethyl ether (2.5 L) was added to the concentrated solution to afford the desired product 1-VII (841 g, 68% yield) after filtration at 250C .

A solution of intermediate 1-VII (841 g) was treated with 4 N HCl/dioxane (2.7 L) in MeOH (8.1 L) and stirred at 25°C for 15 hours. TLC showed that the reaction was completed. The mixture was concentrated to 1.5 L (1/7 of original volume) by vacuum at 500C. Then, diethyl ether (5 L) was added to the solution slowly, and HCl salt of 1-VIII (774 g) was formed, filtered, and dried under vacuum (<10 torr). For neutralization, K2CO3 (2.5 kg, 8 eq) was added to the solution of HCl salt of 1-VIII in MeOH (17 L) at 25°C. The mixture was stirred at the same temperature for 3 hours (pH > 12) and filtered (estimated amount of 1-VIII in the filtrate is 504 g). Aldehyde 1-IX (581 g, 1.0 eq based on mole of 1-VII) was added to the filtrate of 1-VIII at 0-100C. The reaction was stirred at 0-100C for 3 hours. TLC showed that the reaction was completed. Then, NaBH4 (81 g, 1.0 eq based on mole of 1-VII) was added at less than 100C and the solution was stirred at 10-150C for Ih. The solution was concentrated to get a residue, which then treated with CH2Cl2 (15 L). The mixture was washed with saturated aq. NH4Cl solution (300 mL) diluted with H2O (1.2 L). The CH2Cl2 layer was concentrated and the residue was purified by chromatography on silica gel (short column, EtOAc as mobile phase for removing other components; MeOH/28% NH4OH = 97/3 as mobile phase for collecting 1-X) afforded crude 1-X (841 g). Then Et3N (167 g, leq) and BoC2O (360 g, leq) were added to the solution of

1-X (841 g) in CH2Cl2 (8.4 L) at 25°C. The mixture was stirred at 25°C for 15 hours. After the reaction was completed as evidenced by TLC, the solution was concentrated and EtOAc (5 L) was added to the resultant residue. The solution was concentrated to 3L (1/2 of the original volume) under low pressure at 500C. Then, n-hexane (3 L) was added to the concentrated solution. The solid product formed at 500C by seeding to afford the desired crude product 1-XI (600 g, 60% yield) after filtration and evaporation. To compound 1-XI (120.0 g) and piperazine (1-XII, 50.0 g, 3 eq) in 1- pentanol (360 niL) was added Et3N (60.0 g, 3.0 eq) at 25°C. The mixture was stirred at 1200C for 8 hours. Ethyl acetate (480 mL) was added to the reaction mixture at 25°C. The solution was stirred for Ih. The Et3NHCl salt was filtered and the solution was concentrated and purified by silica gel (EtOAc/MeOH = 2:8) to afforded 1-XIII (96 g) in a 74% yield.

A solution of intermediate 1-XIII (100 mg) was treated with 4 N HCl/dioxane (2 mL) in CH2Cl2 (1 mL) and stirred at 25°C for 15 hours. The mixture was concentrated to give hydrochloride salt of compound 1 (51 mg). CI-MS (M+ + 1): 459.4

Example 2: Preparation of Compound 2

Compound 2 Intermediate 1-XIII was prepared as described in Example 1.

To a solution of 1-XIII (120 g) in MeOH (2.4 L) were added diethyl vinyl phosphonate (2-1, 45 g, 1.5 eq) at 25°C. The mixture was stirred under 65°C for 24 hours. TLC and HPLC showed that the reaction was completed. The solution was concentrated and purified by silica gel (MeOH/CH2Cl2 = 8/92) to get 87 g of 2-11 (53% yield, purity > 98%, each single impurity <1%) after analyzing the purity of the product by HPLC.

A solution of 20% TFA/CH2C12 (36 mL) was added to a solution of intermediate 2-11 (1.8 g) in CH2Cl2 (5 mL). The reaction mixture was stirred for 15 hours at room temperature and concentrated by removing the solvent to afford trifluoracetic acid salt of compound 2 (1.3 g). CI-MS (M+ + 1): 623.1

Example 3 : Preparation of Compound 3

TMSBr H H

s U

Intermediate 2-11 was prepared as described in Example 2. To a solution of 2-11 (300 g) in CH2Cl2 (1800 mL) was added TMSBr (450 g, 8 eq) at 10-150C for 1 hour. The mixture was stirred at 25°C for 15 hours. The solution was concentrated to remove TMSBr and solvent under vacuum at 400C.

CH2Cl2 was added to the mixture to dissolve the residue. TMSBr and solvent were removed under vacuum again to obtain 36O g crude solid after drying under vacuum (<1 torr) for 3 hours. Then, the crude solid was washed with 7.5 L IPA/MeOH (9/1) to afford compound 3 (280 g) after filtration and drying at 25°C under vacuum (<1 torr) for 3 hours. Crystallization by EtOH gave hydrobromide salt of compound 3 (19Og). CI-MS (M+ + 1): 567.0.

The hydrobromide salt of compound 3 (5.27 g) was dissolved in 20 mL water and treated with concentrated aqueous ammonia (pH=9-10), and the mixture was evaporated in vacuo. The residue in water (30 mL) was applied onto a column (100 mL, 4.5×8 cm) of Dowex 50WX8 (H+ form, 100-200 mesh) and eluted (elution rate, 6 mL/min). Elution was performed with water (2000 mL) and then with 0.2 M aqueous ammonia. The UV-absorbing ammonia eluate was evaporated to dryness to afford ammonia salt of compound 3 (2.41 g). CI-MS (M+ + 1): 567.3.

The ammonia salt of compound 3 (1.5 g) was dissolved in water (8 mL) and alkalified with concentrated aqueous ammonia (pH=l 1), and the mixture solution was applied onto a column (75 mL, 3×14 cm) of Dowex 1X2 (acetate form, 100-200 mesh) and eluted (elution rate, 3 mL/min). Elution was performed with water (900 mL) and then with 0.1 M acetic acid. The UV-absorbing acetic acid eluate was evaporated, and the residue was codistilled with water (5×50 mL) to afford compound 3 (1.44 g). CI-MS (M+ + 1): 567.4. Example 4: Preparation of Compound 4

Compound 4

Intermediate 1-XIII was obtained during the preparation of compound 1. To a solution of diethyl vinyl phosphonate (4-1, 4 g) in CH2Cl2 (120 mL) was added oxalyl chloride (15.5 g, 5 eq) and the mixture was stirred at 300C for 36 hours. The mixture were concentrated under vacuum on a rotatory evaporated to give quantitatively the corresponding phosphochloridate, which was added to a mixture of cyclohexyl amine (4-II, 5.3 g, 2.2 eq), CH2Cl2 (40 mL), and Et3N (6.2 g, 2.5 eq). The mixture was stirred at 35°C for 36 hours, and then was washed with water. The organic layer was dried (MgSO4), filtered, and evaporated to afford 4-III (4.7 g, 85% yield) as brown oil.

Compound 4-III (505 mg) was added to a solution of intermediate 1-XIII (500 mg) in MeOH (4 mL). The solution was stirred at 45°C for 24 hours. The solution was concentrated and the residue was purified by column chromatography on silica gel (EtOAc/ MeOH = 4: 1) to afford intermediate 4-IV (420 mg) in a 63% yield.

A solution of HCl in ether (5 mL) was added to a solution of intermediate 4- IV (420 mg) in CH2Cl2 (1.0 mL). The reaction mixture was stirred for 12 hours at room temperature and concentrated by removing the solvent. The resultant residue was washed with ether to afford hydrochloride salt of compound 4 (214 mg). CI-MS (M+ + 1): 595.1

Preparation of compound 51

TMSBr

Intermediate l-II was prepared as described in Example 1. To a suspension of the intermediate l-II (31.9 g) in toluene (150 mL) were added phosphorazidic acid diphenyl ester (51-1, 32.4 g) and Et3N (11.9 g) at 25°C for 1 hour. The reaction mixture was stirred at 800C for 3 hours and then cooled to 25°C. After benzyl alcohol (51-11, 20 g) was added, the reaction mixture was stirred at 800C for additional 3 hours and then warmed to 1200C overnight. It was then concentrated and dissolved again in EtOAc and H2O. The organic layer was collected. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with 2.5 N HCl, saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (EtOAc/Hexane = 1 :2) to give Intermediate 51-111 (35 g) in a 79% yield. A solution of intermediate 51-111 (35 g) treated with 4 N HCl/dioxane (210 rnL) in MeOH (350 mL) was stirred at room temperature overnight. After ether (700 mL) was added, the solution was filtered. The solid was dried under vacuum. K2CO3 was added to a suspension of this solid in CH3CN and ώo-propanol at room temperature for 10 minutes. After water was added, the reaction mixture was stirred at room temperature for 2 hours, filtered, dried over anhydrous MgSO4, and concentrated. The resultant residue was purified by column chromatography on silica gel (using CH2Cl2 and MeOH as an eluant) to give intermediate 51-IV (19 g) in a 76% yield. Intermediate 1-IX (21 g) was added to a solution of intermediate 51-IV (19 g) in CH2Cl2 (570 mL). The mixture was stirred at 25°C for 2 hours. NaBH(OAc)3 (23 g) was then added at 25°C overnight. After the solution was concentrated, a saturated aqueous NaHCO3solution was added to the resultant residue. The mixture was then extracted with CH2Cl2. The solution was concentrated and the residue was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 51-V (23.9 g) in a 66% yield.

A solution of intermediate 51-V (23.9 g) and BoC2O (11.4 g) in CH2Cl2 (200 mL) was added to Et3N (5.8 mL) at 25°C for overnight. The solution was then concentrated and the resultant residue was purified by column chromatography on silica gel (using EtOAc and Hexane as an eluant) to give intermediate 51-VI (22 g) in a 77% yield.

10% Pd/C (2.2 g) was added to a suspension of intermediate 51-VI (22 g) in MeOH (44 mL). The mixture was stirred at ambient temperature under hydrogen atmosphere overnight, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 51-VII (16.5 g) in a 97% yield.

Intermediate 51-VII (16.5 g) and Et3N (4.4 mL) in 1-pentanol (75 mL) was allowed to react with 2,4-dichloro-6-aminopyrimidine (1-VI, 21 g) at 1200C overnight. The solvent was then removed and the residue was purified by column chromatography on silica gel (using EtOAc and hexane as an eluant) to afford intermediate 51-VIII (16.2 g) in a 77% yield.

A solution of intermediate 51-VIII (16.2 g) and piperazine (1-XII, 11.7 g) in 1-pentanol (32 mL) was added to Et3N (3.3 mL) at 1200C overnight. After the solution was concentrated, the residue was treated with water and extracted with CH2Cl2. The organic layer was collected and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc/ MeOH to 28% NH40H/Me0H as an eluant) to afford Intermediate 51-IX (13.2 g) in a 75% yield. Diethyl vinyl phosphonate (2-1) was treated with 51-IX as described in

Example 3 to afford hydrobromide salt of compound 51. CI-MS (M+ + 1): 553.3

………………………………….

Preparation of Compound 1

Water (10.0 L) and (Boc)2O (3.33 kgg, 15.3 mol) were added to a solution of trans-4-aminomethyl-cyclohexanecarboxylic acid (compound 1-I, 2.0 kg, 12.7 mol) and sodium bicarbonate (2.67 kg, 31.8 mol). The reaction mixture was stirred at ambient temperature for 18 hours. The aqueous layer was acidified with concentrated hydrochloric acid (2.95 L, pH=2) and then filtered. The resultant solid was collected, washed three times with water (15 L), and dried in a hot box (60° C.) to give trans-4-(tert-butoxycarbonylamino-methyl)-cyclo-hexanecarboxylic acid (Compound 1-II, 3.17 kg, 97%) as a white solid. Rf=0.58 (EtOAc). LC-MS m/e 280 (M+Na+). 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, 1H), 2.98 (t, J=6.3 Hz, 2H), 2.25 (td, J=12, 3.3 Hz, 1H), 2.04 (d, J=11.1 Hz, 2H), 1.83 (d, J=11.1 Hz, 2H), 1.44 (s, 9H), 1.35˜1.50 (m, 3H), 0.89˜1.03 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 181.31, 156.08, 79.12, 46.41, 42.99, 37.57, 29.47, 28.29, 27.96. M.p. 134.8˜135.0° C.

A suspension of compound 1-II (1.0 kg, 3.89 mol) in THF (5 L) was cooled at 10° C. and triethyl amine (1.076 L, 7.78 mol) and ethyl chloroformate (0.441 L, 4.47 mol) were added below 10° C. The reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was then cooled at 10° C. again and NH4OH (3.6 L, 23.34 mol) was added below 10° C. The reaction mixture was stirred at ambient temperature for 18 hours and filtered. The solid was collected and washed three times with water (10 L) and dried in a hot box (60° C.) to give trans-4-(tert-butoxycarbonyl-amino-methyl)-cyclohexanecarboxylic acid amide (Compound 1-III, 0.8 kg, 80%) as a white solid. Rf=0.23 (EtOAc). LC-MS m/e 279, M+Na+. 1H NMR (300 MHz, CD3OD) δ 6.63 (brs, 1H), 2.89 (t, J=6.3 Hz, 2H), 2.16 (td, J=12.2, 3.3 Hz, 1H), 1.80˜1.89 (m, 4H), 1.43 (s, 9H), 1.37˜1.51 (m, 3H), 0.90˜1.05 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 182.26, 158.85, 79.97, 47.65, 46.02, 39.28, 31.11, 30.41, 28.93. M.p. 221.6˜222.0° C.

A suspension of compound 1-III (1.2 kg, 4.68 mol) in CH2Cl2 (8 L) was cooled at 10° C. and triethyl amine (1.3 L, 9.36 mol) and trifluoroacetic anhydride (0.717 L, 5.16 mol) were added below 10° C. The reaction mixture was stirred for 3 hours. After water (2.0 L) was added, the organic layer was separated and washed with water (3.0 L) twice. The organic layer was then passed through silica gel and concentrated. The resultant oil was crystallized by methylene chloride. The crystals were washed with hexane to give trans-(4-cyano-cyclohexylmethyl)-carbamic acid tent-butyl ester (Compound 1-IV, 0.95 kg, 85%) as a white crystal. Rf=0.78 (EtOAc). LC-MS m/e 261, M+Na+. 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, 1H), 2.96 (t, J=6.3 Hz, 2H), 2.36 (td, J=12, 3.3 Hz, 1H), 2.12 (dd, J=13.3, 3.3 Hz, 2H), 1.83 (dd, J=13.8, 2.7 Hz, 2H), 1.42 (s, 9H), 1.47˜1.63 (m, 3H), 0.88˜1.02 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 155.96, 122.41, 79.09, 45.89, 36.92, 29.06, 28.80, 28.25, 28.00. M.p. 100.4˜100.6° C.

Compound 1-IV (1.0 kg, 4.196 mol) was dissolved in a mixture of 1,4-dioxane (8.0 L) and water (2.0 L). To the reaction mixture were added lithium hydroxide monohydrate (0.314 kg, 4.191), Raney-nickel (0.4 kg, 2.334 mol), and 10% palladium on carbon (0.46 kg, 0.216 mol) as a 50% suspension in water. The reaction mixture was stirred under hydrogen atmosphere at 50° C. for 20 hours. After the catalysts were removed by filtration and the solvents were removed in vacuum, a mixture of water (1.0 L) and CH2Cl2 (0.3 L) was added. After phase separation, the organic phase was washed with water (1.0 L) and concentrated to give trans-(4-aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester (compound 1-V, 0.97 kg, 95%) as pale yellow thick oil. Rf=0.20 (MeOH/EtOAc=9/1). LC-MS m/e 243, M+H+. 1H NMR (300 MHz, CDCl3) δ 4.67 (brs, 1H), 2.93 (t, J=6.3 Hz, 2H), 2.48 (d, J=6.3 Hz, 2H), 1.73˜1.78 (m, 4H), 1.40 (s, 9H), 1.35 (brs, 3H), 1.19˜1.21 (m, 1H), 0.77˜0.97 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 155.85, 78.33, 48.27, 46.38, 40.80, 38.19, 29.87, 29.76, 28.07.

A solution of compound 1-V (806 g) and Et3N (1010 g, 3 eq) in 1-pentanol (2.7 L) was treated with compound 1-VI, 540 g, 1 eq) at 90° C. for 15 hours. TLC showed that the reaction was completed.

Ethyl acetate (1.5 L) was added to the reaction mixture at 25° C. The solution was stirred for 1 hour. The Et3NHCl salt was filtered. The filtrate was then concentrated to 1.5 L (1/6 of original volume) by vacuum at 50° C. Then, diethyl ether (2.5 L) was added to the concentrated solution to afford the desired product 1-VII (841 g, 68% yield) after filtration at 25° C.

A solution of intermediate 1-VII (841 g) was treated with 4 N HCl/dioxane (2.7 L) in MeOH (8.1 L) and stirred at 25° C. for 15 hours. TLC showed that the reaction was completed. The mixture was concentrated to 1.5 L (1/7 of original volume) by vacuum at 50° C. Then, diethyl ether (5 L) was added to the solution slowly, and HCl salt of 1-VIII (774 g) was formed, filtered, and dried under vacuum (<10 ton). For neutralization, K2CO3 (2.5 kg, 8 eq) was added to the solution of HCl salt of 1-VIII in MeOH (17 L) at 25° C. The mixture was stirred at the same temperature for 3 hours (pH>12) and filtered (estimated amount of 1-VIII in the filtrate is 504 g).

Aldehyde 1-IX (581 g, 1.0 eq based on mole of 1-VII) was added to the filtrate of 1-VIII at 0-10° C. The reaction was stirred at 0-10° C. for 3 hours. TLC showed that the reaction was completed. Then, NaBH4 (81 g, 1.0 eq based on mole of 1-VII) was added at less than 10° C. and the solution was stirred at 10-15° C. for 1 h. The solution was concentrated to get a residue, which then treated with CH2Cl2 (15 L). The mixture was washed with saturated aq. NH4Cl solution (300 mL) diluted with H2O (1.2 L). The CH2Cl2 layer was concentrated and the residue was purified by chromatography on silica gel (short column, EtOAc as mobile phase for removing other components; MeOH/28% NH4OH=97/3 as mobile phase for collecting 1-X) afforded crude 1-X (841 g).

Then Et3N (167 g, 1 eq) and Boc2O (360 g, 1 eq) were added to the solution of 1-X (841 g) in CH2Cl2 (8.4 L) at 25° C. The mixture was stirred at 25° C. for 15 hours. After the reaction was completed as evidenced by TLC, the solution was concentrated and EtOAc (5 L) was added to the resultant residue. The solution was concentrated to 3 L (1/2 of the original volume) under low pressure at 50° C. Then, n-hexane (3 L) was added to the concentrated solution. The solid product formed at 50° C. by seeding to afford the desired crude product 1-XI (600 g, 60% yield) after filtration and evaporation.

To compound 1-XI (120.0 g) and piperazine (1-XII, 50.0 g, 3 eq) in 1-pentanol (360 mL) was added Et3N (60.0 g, 3.0 eq) at 25° C. The mixture was stirred at 120° C. for 8 hours. Ethyl acetate (480 mL) was added to the reaction mixture at 25° C. The solution was stirred for 1 h. The Et3NHCl salt was filtered and the solution was concentrated and purified by silica gel (EtOAc/MeOH=2:8) to afforded 1-XIII (96 g) in a 74% yield.

To a solution of 1-XIII (120 g) in MeOH (2.4 L) were added diethyl vinyl phosphonate (1-XIV, 45 g, 1.5 eq) at 25° C. The mixture was stirred under 65° C. for 24 hours. TLC and HPLC showed that the reaction was completed. The solution was concentrated and purified by silica gel (MeOH/CH2Cl2=8/92) to get 87 g of 1-XV (53% yield, purity>98%, each single impurity<1%) after analyzing the purity of the product by HPLC.

A solution of 20% TFA/CH2Cl2 (36 mL) was added to a solution of intermediate 1-XV (1.8 g) in CH2Cl2 (5 mL). The reaction mixture was stirred for 15 hours at room temperature and concentrated by removing the solvent to afford trifluoracetic acid salt of compound 1 (1.3 g).

CI-MS (M++1): 623.1.

(2) Preparation of Compound 2

Intermediate 1-XV was prepared as described in Example 1.

To a solution of 1-XV (300 g) in CH2Cl2 (1800 mL) was added TMSBr (450 g, 8 eq) at 10-15° C. for 1 hour. The mixture was stirred at 25° C. for 15 hours. The solution was concentrated to remove TMSBr and solvent under vacuum at 40° C. CH2Cl2 was added to the mixture to dissolve the residue. TMSBr and solvent were removed under vacuum again to obtain 360 g crude solid after drying under vacuum (<1 torr) for 3 hours. Then, the crude solid was washed with 7.5 L IPA/MeOH (9/1) to afford compound 2 (280 g) after filtration and drying at 25° C. under vacuum (<1 ton) for 3 hours. Crystallization by EtOH gave hydrobromide salt of compound 2 (190 g). CI-MS (M++1): 567.0.

The hydrobromide salt of compound 2 (5.27 g) was dissolved in 20 mL water and treated with concentrated aqueous ammonia (pH=9-10), and the mixture was evaporated in vacuo. The residue in water (30 mL) was applied onto a column (100 mL, 4.5×8 cm) of Dowex 50WX8 (H+ form, 100-200 mesh) and eluted (elution rate, 6 mL/min). Elution was performed with water (2000 mL) and then with 0.2 M aqueous ammonia. The UV-absorbing ammonia eluate was evaporated to dryness to afford ammonia salt of compound 2 (2.41 g). CI-MS (M++1): 567.3.

The ammonia salt of compound 2 (1.5 g) was dissolved in water (8 mL) and alkalified with concentrated aqueous ammonia (pH=11), and the mixture solution was applied onto a column (75 mL, 3×14 cm) of Dowex 1×2 (acetate form, 100-200 mesh) and eluted (elution rate, 3 mL/min). Elution was performed with water (900 mL) and then with 0.1 M acetic acid. The UV-absorbing acetic acid eluate was evaporated, and the residue was codistilled with water (5×50 mL) to afford compound 2 (1.44 g). CI-MS (M++1): 567.4.

(3) Preparation of Compound 3

Intermediate 1-XIII was obtained during the preparation of compound 1.

To a solution of diethyl vinyl phosphonate (3-I, 4 g) in CH2Cl2 (120 mL) was added oxalyl chloride (15.5 g, 5 eq) and the mixture was stirred at 30° C. for 36 hours. The mixture were concentrated under vacuum on a rotatory evaporated to give quantitatively the corresponding phosphochloridate, which was added to a mixture of cyclohexyl amine (3-II, 5.3 g, 2.2 eq), CH2Cl2 (40 mL), and Et3N (6.2 g, 2.5 eq). The mixture was stirred at 35° C. for 36 hours, and then was washed with water. The organic layer was dried (MgSO4), filtered, and evaporated to afford 3-III (4.7 g, 85% yield) as brown oil.

Compound 3-III (505 mg) was added to a solution of intermediate 1-XIII (500 mg) in MeOH (4 mL). The solution was stirred at 45° C. for 24 hours. The solution was concentrated and the residue was purified by column chromatography on silica gel (EtOAc/MeOH=4:1) to afford intermediate 3-IV (420 mg) in a 63% yield.

A solution of HCl in ether (5 mL) was added to a solution of intermediate 3-IV (420 mg) in CH2Cl2 (1.0 mL). The reaction mixture was stirred for 12 hours at room temperature and concentrated by removing the solvent. The resultant residue was washed with ether to afford hydrochloride salt of compound 3 (214 mg).

CI-MS (M++1): 595.1.

(4) Preparation of Compound 4

Compound 4 was prepared in the same manner as that described in Example 2 except that sodium 2-bromoethanesulfonate in the presence of Et3N in DMF at 45° C. was used instead of diethyl vinyl phosphonate. Deportations of amino-protecting group by hydrochloride to afford hydrochloride salt of compound 4.

CI-MS (M++1): 567.3

(5) Preparation of Compound 5

Compound 5 was prepared in the same manner as that described in Example 2 except that diethyl-1-bromopropylphosphonate in the presence of K2CO3 in CH3CN was used instead of diethyl vinyl phosphonate.

CI-MS (M++1): 581.4

(6) Preparation of Compound 6

Compound 6 was prepared in the same manner as that described in Example 5 except that 1,4-diaza-spiro[5.5]undecane dihydrochloride was used instead of piperazine.

CI-MS (M++1): 649.5

(7) Preparation of Compound 7

Intermediate 1-II was prepared as described in Example 1.

To a suspension of the intermediate 1-II (31.9 g) in toluene (150 mL) were added phosphorazidic acid diphenyl ester (7-I, 32.4 g) and Et3N (11.9 g) at 25° C. for 1 hour. The reaction mixture was stirred at 80° C. for 3 hours and then cooled to 25° C. After benzyl alcohol (7-II, 20 g) was added, the reaction mixture was stirred at 80° C. for additional 3 hours and then warmed to 120° C. overnight. It was then concentrated and dissolved again in EtOAc and H2O. The organic layer was collected. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with 2.5 N HCl, saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (EtOAc/Hexane=1:2) to give Intermediate 7-III (35 g) in a 79% yield.

A solution of intermediate 7-III (35 g) treated with 4 N HCl/dioxane (210 mL) in MeOH (350 mL) was stirred at room temperature overnight. After ether (700 mL) was added, the solution was filtered. The solid was dried under vacuum. K2CO3 was added to a suspension of this solid in CH3CN and iso-propanol at room temperature for 10 minutes. After water was added, the reaction mixture was stirred at room temperature for 2 hours, filtered, dried over anhydrous MgSO4, and concentrated. The resultant residue was purified by column chromatography on silica gel (using CH2Cl2 and MeOH as an eluant) to give intermediate 7-IV (19 g) in a 76% yield.

Intermediate 1-IX (21 g) was added to a solution of intermediate 7-IV (19 g) in CH2Cl2 (570 mL). The mixture was stirred at 25° C. for 2 hours. NaBH(OAc)3(23 g) was then added at 25° C. overnight. After the solution was concentrated, a saturated aqueous NaHCO3 solution was added to the resultant residue. The mixture was then extracted with CH2Cl2. The solution was concentrated and the residue was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 7-V (23.9 g) in a 66% yield.

A solution of intermediate 7-V (23.9 g) and Boc2O (11.4 g) in CH2Cl2 (200 mL) was added to Et3N (5.8 mL) at 25° C. for overnight. The solution was then concentrated and the resultant residue was purified by column chromatography on silica gel (using EtOAc and Hexane as an eluant) to give intermediate 7-VI (22 g) in a 77% yield. 10% Pd/C (2.2 g) was added to a suspension of intermediate 7-VI (22 g) in MeOH (44 mL). The mixture was stirred at ambient temperature under hydrogen atmosphere overnight, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 7-VII (16.5 g) in a 97% yield.

Intermediate 7-VII (16.5 g) and Et3N (4.4 mL) in 1-pentanol (75 mL) was allowed to react with 2,4-dichloro-6-aminopyrimidine (1-VI, 21 g) at 120° C. overnight. The solvent was then removed and the residue was purified by column chromatography on silica gel (using EtOAc and hexane as an eluant) to afford intermediate 7-VIII (16.2 g) in a 77% yield.

A solution of intermediate 7-VIII (16.2 g) and piperazine (1-XII, 11.7 g) in 1-pentanol (32 mL) was added to Et3N (3.3 mL) at 120° C. overnight. After the solution was concentrated, the residue was treated with water and extracted with CH2Cl2. The organic layer was collected and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc/MeOH to 28% NH4OH/MeOH as an eluant) to afford Intermediate 7-IX (13.2 g) in a 75% yield.

Diethyl vinyl phosphonate (2-I) was treated with 7-IX as described in Example 3 to afford hydrobromide salt of compound 7.

CI-MS (M++1): 553.3

(8) Preparation of Compound 8

Cis-1,4-cyclohexanedicarboxylic acid (8-I, 10 g) in THF (100 ml) was added oxalyl chloride (8-II, 15.5 g) at 0° C. and then DMF (few drops). The mixture was stirred at room temperature for 15 hours. The solution was concentrated and the residue was dissolved in THF (100 ml). The mixture solution was added to ammonium hydroxide (80 ml) and stirred for 1 hour. The solution was concentrated and filtration to afford crude product 8-III (7.7 g).

Compound 8-III (7.7 g) in THF (200 ml) was slowly added to LiAlH4 (8.6 g) in THF (200 ml) solution at 0° C. The mixture solution was stirred at 65° C. for 15 hours. NaSO4.10H2O was added at room temperature and stirred for 1 hours. The resultant mixture was filtered to get filtrate and concentrated. The residue was dissolved in CH2Cl2 (100 ml). Et3N (27 g) and (Boc)2O (10 g) were added at room temperature. The solution was stirred for 15 h, and then concentrated to get resultant residue. Ether was added to the resultant residue. Filtration and drying under vacuum afforded solid crude product 8-IV (8.8 g).

A solution of compound 8-IV (1.1 g) and Et3N (1.7 g) in 1-pentanol (10 ml) was reacted with 2,4-dichloro-6-aminopyrimidine (1-VI, 910 mg) at 90° C. for 15 hours. TLC showed that the reaction was completed. Ethyl acetate (10 mL) was added to the reaction mixture at 25° C. The solution was stirred for 1 hour. The Et3NHCl salt was removed. The filtrate was concentrated and purified by silica gel (EtOAc/Hex=1:2) to afford the desired product 8-V (1.1 g, 65% yield).

A solution of intermediate 8-V (1.1 g) was treated with 4 N HCl/dioxane (10 ml) in MeOH (10 ml) and stirred at 25° C. for 15 hours. TLC showed that the reaction was completed. The mixture was concentrated, filtered, and dried under vacuum (<10 ton). For neutralization, K2CO3 (3.2 g) was added to the solution of HCl salt in MeOH (20 ml) at 25° C. The mixture was stirred at the same temperature for 3 hours (pH>12) and filtered. Aldehyde 1-IX (759 mg) was added to the filtrate at 0-10° C. The reaction was stirred at 0-10° C. for 3 hours. TLC showed that the reaction was completed. Then, NaBH4 (112 mg) was added at less than 10° C. and the solution was stirred at 10-15° C. for 1 hour. The solution was concentrated to get a residue, which was then treated with CH2Cl2 (10 mL). The mixture was washed with saturated NH4Cl (aq) solution. The CH2Cl2 layer was concentrated and the residue was purified by chromatography on silica gel (MeOH/28% NH4OH=97/3) to afford intermediate 8-VI (1.0 g, 66% yield).

Et3N (600 mg) and Boc2O (428 mg) were added to the solution of 8-VI (1.0 g) in CH2Cl2 (10 ml) at 25° C. The mixture was stirred at 25° C. for 15 hours. TLC showed that the reaction was completed. The solution was concentrated and purified by chromatography on silica gel (EtOAc/Hex=1:1) to afford intermediate 8-VII (720 mg, 60% yield).

To a solution compound 8-VII (720 mg) and piperazine (1-XII, 1.22 g) in 1-pentanol (10 mL) was added Et3N (1.43 g) at 25° C. The mixture was stirred at 120° C. for 24 hours. TLC showed that the reaction was completed. Ethyl acetate (20 mL) was added at 25° C. The solution was stirred for 1 hour. The Et3NHCl salt was removed and the solution was concentrated and purified by silica gel (EtOAc/MeOH=2:8) to afford 8-VIII (537 mg) in 69% yield.

To a solution of 8-VIII (537 mg) in MeOH (11 ml) was added diethyl vinyl phosphonate (2-I, 201 mg) at 25° C. The mixture was stirred under 65° C. for 24 hours. TLC and HPLC showed that the reaction was completed. The solution was concentrated and purified by silica gel (MeOH/CH2Cl2=1:9) to get 8-IX (380 mg) in a 57% yield.

To a solution of 8-IX (210 mg) in CH2Cl2 (5 ml) was added TMSBr (312 mg) at 10-15° C. for 1 hour. The mixture was stirred at 25° C. for 15 hours. The solution was concentrated to remove TMSBr and solvent under vacuum at 40° C., then, CH2Cl2 was added to dissolve the residue. Then TMSBr and solvent were further removed under vacuum and CH2Cl2 was added for four times repeatedly. The solution was concentrated to get hydrobromide salt of compound 8 (190 mg).

CI-MS (M++1): 566.9

To do a job well is one thing, but to consistently deliver a product that is nearly flawless is quite a different challenge. For its new molecule burixafor, the Taiwanese drug discovery firm TaiGen Biotechnology instructed its contract manufacturing partners to achieve 99.8% purity in the production of the active pharmaceutical ingredient (API).

Discovered in TaiGen’s labs in 2006, burixafor is in Phase II clinical trials in both the U.S. and China for use in stem cell transplants and cancer chemotherapy. Avecia, a unit of Japan’s Nitto Denko, manufactures the drug substance in the U.S., where burixafor was tested for the first time on human patients. When TaiGen later initiated clinical trials in China, it chose the Taiwanese firm ScinoPharm to produce the drug at its plant in Changshu, near Shanghai. Under Chinese law, only drugs made domestically can be tested in China.

NITTO DENKO Avecia Inc.

It is rare for a drug discovery firm to select two companies to scale up the production of a new molecule. TaiGen went one step further by paying both contract manufacturers to reach an extremely high level of purity.

“We are trying to avoid any unwanted side effects during the trials,” says C. Richard King, TaiGen’s senior vice president of research. Drug regulators in the U.S. and China “need very tight specifications these days for new drugs,” he adds.

TaiGen registered burixafor with the U.S. Food & Drug Administration in 2007. When it contracted Girindus America (bought by Avecia in 2013) to manufacture it that year, TaiGen specified purification by column chromatography, a cumbersome and relatively expensive procedure when carried out on a large scale. “Our process development efforts were racing against the clinical trials launch schedule,” King recalls. Column chromatography, he points out, is a “tedious approach, but it works.”

By the time ScinoPharm was hired last year, TaiGen’s process development team had come up with a simpler and more elegant process. But its purity demands hadn’t changed.

“Usually, clients are satisfied with a purity level of 98% to 99%,” says Koksuan Tang, head of operations at ScinoPharm’s Changshu plant. “To go from 99% to 99.8% is very different.” The manufacturing of burixafor, he adds, involves five chemical steps and two purification steps. Upstream of the API, ScinoPharm also produces burixafor’s starting material.

Purity level aside, burixafor is not a particularly difficult compound to make, Tang says. Nonetheless, the process supplied by TaiGen had to be adjusted for larger-scale production. “If you heat up 10 g in the lab, it takes two minutes, but in a plant, it could take as long as two hours,” he says.

Although, while hydrogen chloride gas can be controlled effectively when making minute quantities of a compound in the lab, it’s another challenge to handle large volumes of the toxic substance at the plant level. To safely execute one reaction step, ScinoPharm dissolved HCl in a special solvent that does not affect the purity profile of burixafor.

TaiGen selected ScinoPharm as its China contractor after a careful process that involved two visits to Changshu by TaiGen’s senior managers, Tang recalls. ScinoPharm’s track record of meeting regulatory requirements in different countries, including China, was a plus, Tang believes. Its ability to produce both for clinical trials and in larger quantities after commercial launch was also decisive.

Operational since 2012, ScinoPharm’s Changshu site can deliver products under Good Manufacturing Practices in quantities ranging from grams to kilograms. It employs 220 people.

“Moving from the single-kilogram quantities we make now to hundreds of kilograms will require some adjustment to the process, but we believe we can deliver,” says Tang’s colleague Sing Ping Lee, senior director of product technical support in Changshu. One thing to keep in mind, he notes, is that Chinese regulatory standards for drug production are actually more restrictive than those in the U.S. or Europe, going so far as specifying what equipment manufacturers need to use.

Other than complying with Chinese regulators, one reason TaiGen needed to carefully select its China contractor is that the two companies could well be long-term partners, since TaiGen believes it has the ability to market the drug on its own in China, Taiwan, and Southeast Asia. In the event of approvals elsewhere, TaiGen plans to license the compound to a large drug company, which may or may not stick with ScinoPharm or Avecia.

Relatively unknown outside Taiwan, TaiGen was formed in 2001 by Ming-Chu Hsu, the founder of the Division of Biotechnology & Pharmaceutical Research at Taiwan’s National Health Research Institutes. The holder of a Ph.D. in biochemistry from the University of Illinois, Urbana-Champaign, she headed oncology and virology research at Roche for more than 10 years before returning to Taiwan in 1998.

Ming-Chu Hsu, Chairman & CEO, TaiGen Biotechnology, Taiwan

TaiGen employs about 80 people, three-quarters of whom are in R&D. The company develops its own drugs in-house and also in-licenses molecules that are in early stages of development. The company licenses out the molecules for the European Union and U.S. markets but seeks to retain Asian marketing rights. Burixafor was discovered in TaiGen’s own labs in Taipei. To come up with it, researchers used a high-throughput screening approach that involved 130,000 compounds, including the design and synthesis of 1,500 new compounds. “It went back and forth between chemistry and biology many times,” recalls King, TaiGen’s research head.

A so-called CXCR4 chemokine receptor antagonist, burixafor mobilizes hematopoietic stem cells and endothelial progenitor cells in human bone marrow and channels them into the peripheral blood within three hours of ingestion, according to results of Phase I and Phase II trials.

In the U.S., burixafor is undergoing clinical trials for use during stem cell transplantation in patients with multiple myeloma, non-Hodgkin’s lymphoma, or Hodgkin’s disease. In China, TaiGen is testing it as a chemotherapy sensitizer in relapsed or refractory adult acute myeloid leukemia.

Owing to its activity on CXCR4 chemokine receptors, the drug could also fight age-related macular degeneration and diabetic retinopathy diseases, as well as find use in tissue repair, King says. For clinical trials in the U.S., TaiGen has partnered with Michael W. Schuster, a medical doctor who conducts research at Stony Brook University Hospital in New York.

Dr. Michael Schuster is Gift of Life’s Medical Director, as well as the Director of the Hematopoietic Stem Cell Transplantation Program and Hematologic Malignancy Program of Stony Brook University Hospital in New York

Typical structure of a chemokine receptor

TaiGen sees particular potential for burixafor in stem cell applications. For example, patients undergoing hematopoietic stem cell transplantation often must take a granulocyte colony-stimulating factor plus a Sanofi drug called Mozobil to stimulate stem cell production. TaiGen says burixafor could accomplish this goal on its own in multiple myeloma patients. It cites one consulting firm forecast that puts eventual sales at more than $1 billion per year.

Sanofi drug called Mozobil to stimulate stem cell production

With that kind of potential, the company is counting on significant interest among licensors, any one of which might want to engage its own contract producer of burixafor. If that happens, a third manufacturer will have to learn to reach 99.8% purity.

TaiGen Biotechnology Co., Ltd.

7F,138 Shin Ming Rd. Neihu Dist., Taipei, Taiwan 114 R.O.C

Tel: 886-2-81777072　|　886-2-27901861

Fax: 886-2-27963606

Taipei Railway Station front

Taipei Songshan Airport

Scinopharm

ScinoPharm (Changshu) Pharmaceuticals, Ltd.

ScinoPharm is currently expanding its manufacturing and process development capabilities by adding significant production and technical capacity in Mainland China at its new Changshu site.

ScinoPharm Changshu is located in the Changshu Economic Development Zone (CEDZ), near Suzhou City, Jingsu Province, China on a 6.6-hectare site.

The facilities will include a R&D centre and production plants fully compliant with U.S. and international GMP standards. The Changshu plant, slated to be fully completed by 2012, will be used for the production of GMP grade pharmaceutical intermediates initially, and later be equipped to handle API production. China’s market for better quality APIs has grown considerably, and local formulation companies are encouraged to utilize APIs from companies having DMFs filed in advanced countries. ScinoPharm had closed its site in Kunshan and relocated the production and R&D groups to Changshu in the 4th quarter of 2011. These groups will continue to be expanded to meet growing demand for ScinoPharm products by both multinational and local formulation companies.

The small and medium-sized production units had been operational in the 4th quarter of 2011. The large production Bays plus a peptide purification unit, a high potency unit and a physical property processing facility will be operational by the end of 2012. Using advanced engineering designs, this site will also have the capability to process high potency, injectable grade products.

ScinoPharm Changshu will adopt the same quality systems as ScinoPharm Taiwan, and will therefore comply with ICH guidelines and FDA 21 CFR Parts 210 & 211.

TAIPEI

Clockwise from top: Taipei skyline, Grand Hotel, Far Eastern Plaza, National Palace Museum,Chiang Kai-shek Memorial Hall, Jiantan Station

Old street in Taipei. 2013

Flag

Seal

Nickname(s): The City of Azaleas

Satellite image of Taipei City

Coordinates:

25°02′N 121°38′E

Karyopharm Announces Initiation of Phase 2 Study of Selinexor (KPT-330) an orphan drug

orphan status, phase 2 1 Response »

Jun 102014

2D chemical structure of 1393477-72-9

Selinexor (KPT-330)

1393477-72-9

Karyopharm Therapeutics, Inc.

WO2011109799A1

WO2013019548A1

C17-H11-F6-N7-O

443.3099

Synonyms

(Z)-3-(3-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N’-(pyrazin-2-yl)acrylohydrazide
2-Propenoic acid, 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-, 2-(2-pyrazinyl)hydrazide, (2Z)-
3-[3-[3,5-Bis(trifluoromethyl)phenyl]-1H-1,2,4-triazol-1-yl]-N’-(pyrazin-2-yl)acrylohydrazide

KPT-330
Selinexor

Karyopharm Announces Initiation of Phase 2 Study of Selinexor (KPT-330) in Patients with …

MarketWatch

“These patients were treated in our Phase 1 clinical trial of Selinexor in … Additional Phase 1 and Phase 2 studies are ongoing or currently planned and … the discovery and development of novel first-in-class drugs directed against …

Selinexor, a Exportin-1 (CRM1/XPO1) agonist, is in phase II clinical trials at Karyopharm for the treatment of advanced or metastatic gynecological malignancies (cervical, ovarian and uterine carcinomas) and recurrent glioblastomas. The company is also evaluating the compound in early clinical trials for the treatment of advanced solid tumors, hematological cancer (non-Hodgkin’s lymphoma, multiple myeloma and Waldenstrom’s macroglobulinemia), soft tissue or bone sarcoma, relapsed or refractory acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL).

In 2014, orphan drug designation was assigned in U.S. for the treatment of acute myeloid leukemia and diffuse large B-cell lymphoma

Cells from most major human solid and hematologic malignancies exhibit abnormal cellular localization of a variety of oncogenic proteins, tumor suppressor proteins, and cell cycle regulators (Cronshaw et al. 2004, Falini et al 2006). For example, certain p53 mutations lead to localization in the cytoplasm rather than in the nucleus. This results in the loss of normal growth regulation, despite intact tumor suppressor function. In other tumors, wild-type p53 is sequestered in the cytoplasm or rapidly degraded, again leading to loss of its suppressor function. Restoration of appropriate nuclear localization of functional p53 protein can normalize some properties of neoplastic cells (Cai et al. 2008; Hoshino et al. 2008; Lain et al. 1999a; Lain et al. 1999b; Smart et al. 1999), can restore sensitivity of cancer cells to DNA damaging agents (Cai et al. 2008), and can lead to regression of established tumors (Sharpless & DePinho 2007, Xue et al. 2007). Similar data have been obtained for other tumor suppressor proteins such as forkhead (Turner and Sullivan 2008) and c-Abl (Vignari and Wang 2001). In addition, abnormal localization of several tumor suppressor and growth regulatory proteins may be involved in the pathogenesis of autoimmune diseases (Davis 2007, Nakahara 2009). CRMl inhibition may provide particularly interesting utility in familial cancer syndromes (e.g. , Li-Fraumeni Syndrome due to loss of one p53 allele,

BRCA1 or 2 cancer syndromes), where specific tumor suppressor proteins (TSP) are deleted or dysfunctional and where increasing TSP levels by systemic (or local) administration of CRMl inhibitors could help restore normal tumor suppressor function. Specific proteins and R As are carried into and out of the nucleus by specialized transport molecules, which are classified as importins if they transport molecules into the nucleus, and exportins if they transport molecules out of the nucleus (Terry et al. 2007;

Sorokin et al. 2007). Proteins that are transported into or out of the nucleus contain nuclear import/localization (NLS) or export (NES) sequences that allow them to interact with the relevant transporters. Chromosomal Region Maintenance 1 (Crml or CRM1), which is also called exportin-1 or Xpol, is a major exportin.

Overexpression of Crml has been reported in several tumors, including human ovarian cancer (Noske et al. 2008), cervical cancer (van der Watt et al. 2009), pancreatic cancer (Huang et al. 2009), hepatocellular carcinoma (Pascale et al. 2005) and osteosarcoma (Yao et al. 2009) and is independently correlated with poor clinical outcomes in these tumor types.

Inhibition of Crml blocks the exodus of tumor suppressor proteins and/or growth regulators such as p53, c-Abl, p21, p27, pRB, BRCA1, IkB, ICp27, E2F4, KLF5, YAP1, ZAP, KLF5, HDAC4, HDAC5 or forkhead proteins (e.g., FOX03a) from the nucleus that are associated with gene expression, cell proliferation, angiogenesis and epigenetics. Crml inhibitors have been shown to induce apoptosis in cancer cells even in the presence of activating oncogenic or growth stimulating signals, while sparing normal (untransformed) cells. Most studies of Crml inhibition have utilized the natural product Crml inhibitor Leptomycin B (LMB). LMB itself is highly toxic to neoplastic cells, but poorly tolerated with marked gastrointestinal toxicity in animals (Roberts et al. 1986) and humans (Newlands et al. 1996). Derivatization of LMB to improve drug-like properties leads to compounds that retain antitumor activity and are better tolerated in animal tumor models (Yang et al. 2007, Yang et al. 2008, Mutka et al. 2009). Therefore, nuclear export inhibitors could have beneficial effects in neoplastic and other proliferative disorders.

In addition to tumor suppressor proteins, Crml also exports several key proteins that are involved in many inflammatory processes. These include IkB, NF-kB, Cox-2, RXRa, Commdl, HIFl, HMGBl, FOXO, FOXP and others. The nuclear factor kappa B (NF-kB/rel) family of transcriptional activators, named for the discovery that it drives immunoglobulin kappa gene expression, regulate the mRNA expression of variety of genes involved in inflammation, proliferation, immunity and cell survival. Under basal conditions, a protein inhibitor of NF-kB, called IkB, binds to NF-kB in the nucleus and the complex IkB-NF-kB renders the NF-kB transcriptional function inactive. In response to inflammatory stimuli, IkB dissociates from the IkB-NF-kB complex, which releases NF-kB and unmasks its potent transcriptional activity. Many signals that activate NF-kB do so by targeting IkB for proteolysis (phosphorylation of IkB renders it “marked” for ubiquitination and then proteolysis). The nuclear IkBa-NF-kB complex can be exported to the cytoplasm by Crml where it dissociates and NF-kB can be reactivated. Ubiquitinated IkB may also dissociate from the NF-kB complex, restoring NF-kB transcriptional activity. Inhibition of Crml induced export in human neutrophils and macrophage like cells (U937) by LMB not only results in accumulation of transcriptionally inactive, nuclear IkBa-NF-kB complex but also prevents the initial activation of NF-kB even upon cell stimulation (Ghosh 2008, Huang 2000). In a different study, treatment with LMB inhibited IL-Ιβ induced NF-kB DNA binding (the first step in NF-kB transcriptional activation), IL-8 expression and intercellular adhesion molecule expression in pulmonary microvascular endothelial cells (Walsh 2008). COMMDl is another nuclear inhibitor of both NF-kB and hypoxia-inducible factor 1 (HIFl) transcriptional activity. Blocking the nuclear export of COMMDl by inhibiting Crml results in increased inhibition of NF-kB and HIFl transcriptional activity (Muller 2009).

Crml also mediates retinoid X receptor a (RXRa) transport. RXRa is highly expressed in the liver and plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. During liver inflammation, nuclear RXRa levels are significantly reduced, mainly due to inflammation-mediated nuclear export of RXRa by Crml . LMB is able to prevent IL-Ιβ induced cytoplasmic increase in RXRa levels in human liver derived cells (Zimmerman 2006).

The role of Crml -mediated nuclear export in NF-kB, HIF-1 and RXRa signalling suggests that blocking nuclear export can be potentially beneficial in many inflammatory processes across multiple tissues and organs including the vasculature (vasculitis, arteritis, polymyalgia rheumatic, atherosclerosis), dermatologic (see below), rheumatologic

(rheumatoid and related arthritis, psoriatic arthritis, spondyloarthropathies, crystal arthropathies, systemic lupus erythematosus, mixed connective tissue disease, myositis syndromes, dermatomyositis, inclusion body myositis, undifferentiated connective tissue disease, Sjogren’s syndrome, scleroderma and overlap syndromes, etc.).

CRM1 inhibition affects gene expression by inhibiting/activating a series of transcription factors like ICp27, E2F4, KLF5, YAP1, and ZAP.

Crml inhibition has potential therapeutic effects across many dermatologic syndromes including inflammatory dermatoses (atopy, allergic dermatitis, chemical dermatitis, psoriasis), sun-damage (ultraviolet (UV) damage), and infections. CRMl inhibition, best studied with LMB, showed minimal effects on normal keratinocytes, and exerted anti-inflammatory activity on keratinocytes subjected to UV, TNFa, or other inflammatory stimuli (Kobayashi & Shinkai 2005, Kannan & Jaiswal 2006). Crml inhibition also upregulates NRF2 (nuclear factor erythroid-related factor 2) activity, which protects keratinocytes (Schafer et al. 2010, Kannan & Jaiswal 2006) and other cell types (Wang et al. 2009) from oxidative damage. LMB induces apoptosis in keratinocytes infected with oncogenic human papillomavirus (HPV) strains such as HPV 16, but not in uninfected keratinocytes (Jolly et al. 2009).

Crml also mediates the transport of key neuroprotectant proteins that may be useful in neurodegenerative diseases including Parkinson’s disease (PD), Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). For example, by (1) forcing nuclear retention of key neuroprotective regulators such as NRF2 (Wang 2009), FOXA2 (Kittappa et al. 2007), parking in neuronal cells, and/or (2) inhibiting NFKB transcriptional activity by sequestering IKB to the nucleus in glial cells, Crml inhibition could slow or prevent neuronal cell death found in these disorders. There is also evidence linking abnormal glial cell proliferation to abnormalities in CRMl levels or CRMl function (Shen 2008).

Intact nuclear export, primarily mediated through CRMl, is also required for the intact maturation of many viruses. Viruses where nuclear export, and/or CRMl itself, has been implicated in their lifecycle include human immunodeficiency virus (HIV), adenovirus, simian retrovirus type 1, Borna disease virus, influenza (usual strains as well as H1N1 and avian H5N1 strains), hepatitis B (HBV) and C (HCV) viruses, human papillomavirus (HPV), respiratory syncytial virus (RSV), Dungee, Severe Acute Respiratory Syndrome coronavirus, yellow fever virus, West Nile virus, herpes simplex virus (HSV), cytomegalovirus (CMV), and Merkel cell polyomavirus (MCV). (Bhuvanakantham 2010, Cohen 2010, Whittaker 1998). It is anticipated that additional viral infections reliant on intact nuclear export will be uncovered in the future.

The HIV-1 Rev protein, which traffics through nucleolus and shuttles between the nucleus and cytoplasm, facilitates export of unspliced and singly spliced HIV transcripts containing Rev Response Elements (RRE) RNA by the CRMl export pathway. Inhibition of Rev-mediated RNA transport using CRMl inhibitors such as LMBor PKF050-638 can arrest the HIV-1 transcriptional process, inhibit the production of new HIV-1 virions, and thereby reduce HIV-1 levels (Pollard 1998, Daelemans 2002). Dengue virus (DENV) is the causative agent of the common arthropod-borne viral disease, Dengue fever (DF), and its more severe and potentially deadly Dengue hemorrhagic fever (DHF). DHF appears to be the result of an over exuberant inflammatory response to DENV. NS5 is the largest and most conserved protein of DENV. CRMl regulates the transport of NS5 from the nucleus to the cytoplasm, where most of the NS5 functions are mediated. Inhibition of CRMl -mediated export of NS5 results in altered kinetics of virus production and reduces induction of the inflammatory chemokine interleukin-8 (IL-8), presenting a new avenue for the treatment of diseases caused by DENV and other medically important flaviviruses including hepatitis C virus (Rawlinson 2009).

Other virus-encoded RNA-binding proteins that use CRMl to exit the nucleus include the HSV type 1 tegument protein (VP 13/14, or hUL47), human CMV protein pp65, the SARS Coronavirus ORF 3b Protein, and the RSV matrix (M) protein (Williams 2008, Sanchez 2007, Freundt 2009, Ghildyal 2009).

Interestingly, many of these viruses are associated with specific types of human cancer including hepatocellular carcinoma (HCC) due to chronic HBV or HCV infection, cervical cancer due to HPV, and Merkel cell carcinoma associated with MCV. CRMl inhibitors could therefore have beneficial effects on both the viral infectious process as well as on the process of neoplastic transformation due to these viruses.

CRMl controls the nuclear localization and therefore activity of multiple DNA metabolizing enzymes including histone deacetylases (HDAC), histone acetyltransferases (HAT), and histone methyltransferases (HMT). Suppression of cardiomyocyte hypertrophy with irreversible CRMl inhibitors has been demonstrated and is believed to be linked to nuclear retention (and activation) of HDAC 5, an enzyme known to suppress a hypertrophic genetic program (Monovich et al. 2009). Thus, CRMl inhibition may have beneficial effects in hypertrophic syndromes, including certain forms of congestive heart failure and hypertrophic cardiomyopathies.

CRMl has also been linked to other disorders. Leber’s disorder, a hereditary disorder characterized by degeneration of retinal ganglion cells and visual loss, is associated with inaction of the CRMl switch (Gupta N 2008). There is also evidence linking

neurodegenerative disorders to abnormalities in nuclear transport.

…………………………………………

PATENT

http://www.google.com/patents/WO2013019548A1?cl=en

To date, however, small-molecule, drug-like Crml inhibitors for use in vitro and in vivo are uncommon. SUMMARY OF THE INVENTION

The present invention relates to compounds, or pharmaceutically acceptable salts thereof, useful as nuclear transport modulators. The invention also provides

pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compounds and compositions in the treatment of various disorders, such as those associated with abnormal cellular responses triggered by improper nuclear transport..

In one embodiment of the invention, the compounds are represented by formula I:

Figure imgf000013_0001

http://www.google.com/patents/WO2013019548A1?cl=en

HERE IT REFERS AS 1-16 READER PLEASE CHECKABOVE AND BELOW FOR ERROR

Figure imgf000101_0001

HERE IT REFERS AS 1-18 READER PLEASE CHECK

http://www.google.com/patents/WO2013019548A1?cl=en

Example 1 : Synthesis of Intermediate (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4- triazol-l-yl)acrylic acid.

Synthesis of 3,5-bis(trifluoromethyl)benzothioamid

A 2-L, 3-necked, round-bottomed flask was charged with a solution of 3,5- bis(trifluoromethyl)benzonitrile (200 g) in DMF (1 L). The solution was then treated with NaSH (123.7 g, 2.0 eq.) and MgCl₂ (186.7 g, 1.0 eq.) and the reaction mixture was stirred at RT for 3 hours. The mixture was poured into an ice-water slurry (10 L) and the compound was extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated brine (3 x 100 mL), dried over anhydrous Na₂S0₄, filtered, and concentrated under reduced pressure to afford 205 g of desired crude 3,5- bis(trifluoromethyl)benzothioamide (yield: 90 %), which wasused without purification in the following step.

Synthesi -(3,5-bis(trifluoromethyl)phenyl)-lH-l 2,4-triazole:

A 5-L, 3-necked, round-bottomed flask was charged with a solution of 3,5- bis(trifluoromethyl)benzothioamide (205.65 g) in DMF (1.03 L). Hydrazine hydrate (73.2 mL, 2.0 eq.) was added dropwise and the reaction mixture was stirred at RT for 1 h. HCOOH (1.03 L) was added dropwise and the reaction mixture was refluxed at 90 °C for 3 hours. After being allowed to cool to RT, the reaction mixture was poured into saturated aqueous sodium bicarbonate solution (7 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated brine (3 x 500 mL), dried over anhydrous Na₂S0₄, filtered, and concentrated under reduced pressure (35 °C, 20 mmHg) to afford 180 g of crude compound. This crude material was stirred with petroleum ether (3 x 500 mL) , filtered and dried to obtain 160 g. of 3-(3,5-bis(trifluoromethyl)phenyl)-lH- 1,2,4-triazole obtained as a pale yellow solid (yield: 75%).

Synthesis of (Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l- yl)acrylate:

A 2-L, 3-necked, round-bottomed flask was charged with a solution of 3-(3,5- bis(trifluoromethyl)phenyl)-lH-l ,2,4-triazole (160 g) in DMF (960 mL). The solution was treated with DABCO (127.74 g, 2 eq.) and stirred for 30 min before adding (Z)-isopropyl 3- iodoacrylate (150.32 g, 1.1 eq.) dropwise. After ca. 1 hour, the reaction mixture was poured into an ice-water slurry (5 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated brine (3 x 100 mL), dried over anhydrous Na₂S0₄, filtered, and concentrated under reduced pressure (35 °C, 20 mmHg) to afford 250 g of crude compound that was purified by column chromatography (60/120 silica gel) using a ethyl acetate/n-hexane gradient (the column was packed in hexane and the desired compound started eluting from 2% EtOAC/n-hexane). Fractions containing the desired compounds were combined to afford 138 g the pure desired compound (yield: 61%).

Synthesis of (Z)-3 -(3 -(3 ,5-bis(trifluoromethyl)phenyl)- 1 H- 1 ,2,4-triazol- 1 -yl)acrylic acid:

In a 5-L, 3-necked, round-bottomed flask, (Z)-isopropyl 3-(3-(3,5- bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l-yl)acrylate (130 g, 1.0 eq.) was dissolved in THF (1.3 L). A solution of LiOH (69.3 g, 5.0 eq.) in water (1.3 L) was added dropwise to the solution and the reaction mixture was stirred at room temperature for 4 h before being quenched with 400 mL ice-water slurry and made acidic (pH = 2-3) with dilute aqueous HC1. The mixture was extracted with EtOAc (3 x 1 L) and the combined organic layers were washed with brine, dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to afford 110 g of desired carboxylic acid (yield: 94 %) (cis content = 90.0%, trans content = 8.2% by LCMS).

Example 17: Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l-yl)- ‘-(pyrazin-2-yl)acrylohydrazide

Synthesis of 3,5-bis(trifluoromethyl)benzothioamide:

A 2-L, 3 -necked, round-bottomed flask, charged with a solution of 3,5- bis(trifluoromethyl)benzonitrile (200 g) in DMF (1 L), was treated with NaSH (123.7 g, 2.0 eq.) and MgCl₂ (186.7 g, 1 eq.). The reaction mixture was stirred at RT for 3 h before being poured into an ice-water slurry (10 L) and was extracted with EtOAc (3 x 1 L). The combined organic extracts were washed with brine (3 x 100 niL), dried over anhydrous Na₂S0₄, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to afford 205 g of crude compound (yield: 90 %), which was used in the following step without further purification.

Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazole:

A 5-L, 3-necked, round-bottomed flask, charged with a solution of 3,5- bis(trifluoromethyl)benzothioamide (205.65 g) in DMF (1.03 L) was treated with hydrazine hydrate (73.16 mL, 2.0 eq.) added dropwise. The reaction mixture was stirred at room temperature for 1 h before being treated with HCOOH (1.028 L) added dropwise. The reaction mixture was refluxed at 90°C for 3 h then cooled to room temperature and poured into saturated aqueous NaHC0₃ solution (7 L) and extracted with EtOAc (3 x 1L). The combined organic layers were washed with brine (3 x 500 mL), dried over anhydrous Na₂S0₄, filtered, and concentrated under reduced pressure (35°C, 20 mmHg) to afford 180 g of a solid. The solid was suspended in petroleum ether and the suspension was stirred, filtered and dried to afford the desired triazole as a pale yellow solid (160 g, yield: 75%).

Synthesis of (Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l- yl)acrylate and (E)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l- yl)acrylate:

A 2-L, 3-necked, round-bottomed flask, charged with a solution of 3-(3,5- bis(trifluoromethyl)phenyl)-lH-l,2,4-triazole (160 g,) in DMF (0.96 L, 6V), was treated with DAB CO (127.74 g, 2 eq.) and stirred for 30 min. (Z)-isopropyl 3-iodoacrylate (150.32 g, 1.1 eq.) was added dropwise to the above reaction mixture and stirred for 1 h before being poured into an ice-water slurry (5 L) and extracted with EtOAc (3 x 1 L). The combined organic extracts were washed with brine (3 x 100 mL), dried over anhydrous Na₂S0₄, filtered, and concentrated under reduced pressure (35°C, 20 mmHg) to afford 250 g of crude compound. Purification by column chromatography (Si02, 60/120 mesh, elution with EtOAc:hexanes gradient; the desired compounds started eluting in 2-2.5 % EtOAc in hexanes) afforded pure cis ester (138 g, yield: 61.6%) and pure trans ester (11.6 g, yield: 5.2%). Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l ,2,4-triazol-l-yl);

acid:

A 500-mL, 3 -necked, round-bottomed flask was charged with a solution of (E)- isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l ,2,4-triazol-l-yl)acrylate (5.0 g) in THF (50 mL). The solution was treated with a solution of LiOH (2.66 g, 5.0 eq.) in water (50 mL) and the reaction mixture was stirred at room temperature for 4 h. before being diluted with 40 mL water, acidified (pH = 2-3) with dilute aqueous HC1 and extracted with EtOAc (3 x 100 mL). The organic extract was washed with brine, dried over anhydrous Na₂S0₄, filtered and concentrated under reduced pressure to afford 2.75 g of the desired unsaturated carboxylic acid (yield: 61.6 %, purity: 99.0 % by LCMS).

Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l-yl)-N’- (pyrazin-2-yl)acrylohydrazide :

To a solution of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l- yl)acrylic acid (0.75 g,) in EtOAc (25 mL) and THF (12.5 mL) was added a solution of 2- hydrazinopyrazine (0.23 g) in 12 mL THF at room temperature. T3P (50% in ethyl acetate, 1.52 mL) and DIPEA (1.46 mL) were added dropwise and simultaneously and the reaction mixture was stirred for 30 min at room temperature before being quenched with ice-cold water and extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂S0₄ and concentrated under reduced pressure (35°C, 20 mmHg), affording 0.698 g of a crude solid. Trituration first with petroleum ether then with Et₂0 afforded 275 mg (yield: 29%) (E)-3-(3-(3,5-bis(trifiuoromethyl) phenyl)- 1H- 1,2,4- triazol-l-yl)-N’-(pyrazin-2-yl)acrylohydrazide. 1H NMR (400 MHz, DMSO-d6) δ ,10.3 (s, 1H), 9.15 (s, 2H), 8.59 (s, 2H), 8.30-8.26 (d, J= 14.8 Hz, 1H), 8.13 (s, 1H), 8.06-8.07 (m, 1H), 6.98-6.95 (d, J= 13.4 Hz, 1H); LCMS for Ci₇H₁₂F₆N₇0 [M+H]⁺ 443.31 ; found 444.19 (RT 2.625 min, purity: 99.06%).

MY SUGESTION TO U

http://www.google.com/patents/WO2013019548A1?cl=en

(Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-lH-l,2,4-triazol-l- yl)acrylate IS THE INTERMEDIATE

any discussion mail amcrasto@gmail.com

NOTE IF U USE Z OR CIS STARTING INTERMEDIATE U WILL GET Z ISOMER

…………………………………………………………

int 75 in

http://www.google.com/patents/WO2011109799A1?cl=en

Figure imgf000307_0001

Exam le 75

Molecular Weight: 239.12 Molecular Weight: 273.2 Molecular Weight: 281 .2

Molecular Weight: 393.3

[00715] Synthesis of Intermediate 1)

Molecular Weight: 239.12 Molecular Weight: 273.2

[00716] In a 100-mL, 3N round-bottomed flask equipped with nitrogen inlet, and a rubber septum, 3,5-bis(trifluoromethyl)benzonitrile (5.0 g,1.0 eq) dissolved in DMF (50 mL,10V),Added NaSH(3.09 g,2.0eq) and MgC12 (4.24 g,l eq).Reaction mixture was stirred at RT for 2-3h. The progress of reaction was followed by TLC analysis on silica gel with 40%EtOAc- hexane as mobile phase. SM Rf=0.5 and Product Rf=0.3. Reaction mixture was poured in to ice water (250mL) and extracted with EtOAc ( 3x 100 mL). The combined organic layers were washed with brine solution (3xl00mL), dried over MgS0₄, filtered, and concentrated by rotary evaporation (25°C, 20mmHg) to afford 5.0g of Crude compound which was used for next step without any purification, Yield (87.5%). Mass [M+l]⁺: 273.8

[00717] Synthesis of Intermediate-2

Molecular Weight: 273.20 Molecular Weight: 281 .16

[00718] In a 250-mL, 3N round-bottomed flask equipped with nitrogen inlet, and a rubber septum, Intermediate- 1(5.0 g, 1.0 eq.) was dissolved in DMF (50 mL,10V),added NH₂NH₂.H₂0 (25.0 mL,5V). The reaction mixture was stirred at RT for 1 h. To this reaction mixture HCOOH (25.0 mL, 5V) was added and reaction mixture was refluxed at 90 ⁰ for 2-3 h. The progress of reaction was followed by TLC analysis on silica gel with 50% Ethyl acetate-n-Hexane as mobile phase. SM Rf=0.50 and Product Rf=0.3. Reaction mixture was poured into ice water (500 mL) and neutralized with saturated sodium bicarbonate solution. The reaction mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine solution,(3xl00mL), dried over MgS0₄, filtered, and concentrated by rotary evaporation (25°C, 20mmHg) to afford 4.6g of crude compound, yield (89.49%). Mass: 279.6(-ve mode).

Molecular Weight: 281.2 Molecular Weight: 393.3

[00719] In a 100-mL, 3N round-bottomed flask equipped with nitrogen inlet, and a rubber septum, Intermediate-2(4.5 g, 1.0 eq.) was dissolved in DCM(45 mL,10V),added TEA (2.10 g, 1.3 eq) and isopropyl propiolate (2.33 g, 1.3 eq). The Reaction mixture was stirred at RT for 30 min. The progress of reaction was followed by TLC analysis on silica gel with 50% Ethyl acetate-Hexane as mobile phase, SM f=0.30 and Product Rf=0.5. Reaction mixture was concentrated by rotary evaporation (25°C, 20mmHg) to afford 5.8 g of Crude compound. The crude reaction mixture was purified by column chromatography using silica 60/120 using Ethyl acetate: Hexane as mobile phase. The column (5x10cm) was packed in Hexane and started eluting in Ethyl acetate in gradient manner starting with fraction collection(50-mL fractions) from 5 % to 20 % Ethyl acetate in hexane. Compound started eluting with 20% Ethyl acetate in Hexane. Fraction containing such TLC profile was collected together to obtain pure compound (1.4 g), Yield (22.26%).1H NMR: CDC1₃, 400 MHz) δ 9.74(s,lH),5 8.63(s,2H),5 7.95(s,lH),5 7.28-7.3 l(d,J: 12.0 Hz,lH),55.75-5.78(d,J: 11.2 Ηζ,ΙΗ) δ 5.14-5.17 (m,lH),5 1.27-1.35(m,6H). LCMS of Ci₆Hi₃F₆N₃0₂(M+l)⁺:393.28 found 393.77 at 4.707 min (LCMS 99.25%).

[00720] General method for Example 76, Example 77, Example 78, Example 79, Example 83: A mixture of 5-(3-Chlorophenyl)-l,2,4-triazole (0.50 g, 3.4 mmol), respective propiolate (0.52 ml, 5.1 mmol) and some drops of triethylamine in acetonitrile under nitrogen was stirred at room temperature for 12-16 h. Acetonitrile was removed under reduced pressure to give a residual oil, which was purified by flash chromatography (3-5%> EtOAc/hexanes) to afford the both cis and trans isomers. Cis isomer was isolated 10-30%) and trans was isolated in 30-50%) with overall yield of 50-80%.

WO2011109799A1 *	Mar 5, 2011	Sep 9, 2011	Karyopharm Therapeutics, Inc.	Nuclear transport modulatiors and uses thereof
US20110275607	Mar 5, 2011	Nov 10, 2011	Karyopharm Therapeutics, Inc.	Nuclear transport modulators and uses thereof

Cerecor’s selective NMDA receptor subunit 2B antagonist CERC-301 (MK-0657) for depression in phase 2

phase 2, Uncategorized 1 Response »

Apr 172014

CERC-301 (MK-0657) MK-657, c-6161, AGN-PC-00887R

structure source….http://www.google.com/patents/WO2013156614A1?cl=en my id is amcrasto@gmail.com

Treat depression; Treat major depressive disorder (MDD); Treat suicidality

808732-98-1 free form, C₁₉ H₂₃ F N₄ O₂

(-) (3S,4R) – 1-Piperidinecarboxylic acid, 3-fluoro-4-[(2-pyrimidinylamino)methyl]-, (4-methylphenyl)methyl ester,

AND

1-Piperidinecarboxylic acid, 3-fluoro-4-[(2-pyrimidinylamino)methyl]-, (4-methylphenyl)methyl ester, (3S,4R)-

(-)-(3S,4R)-4-Methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

(3S,4R)-4-methylbenzyl 3-fluor-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

cas no of hydrochloride 808733-06-4

Company	Merck & Co. Inc.
Description	Small molecule NMDA receptor NR2B subtype (GRIN2B; NR2B) antagonist
Molecular Target	NMDA receptor NR2B subtype (GRIN2B) (NR2B)
Mechanism of Action	NMDA receptor antagonist

PLEASE NOTE THE + FORM

(+)-(3R,4S)-4-Methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate HAS CAS NO…..808732-99-2 AND ITS HYDROCHLORIDE 808733-07-5

also NOTE

1-Piperidinecarboxylic acid, 3-fluoro-4-[(2-pyrimidinylamino)methyl]-, (4-methylphenyl)methyl ester, (3R,4S)-rel-;

cis-4-Methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

HAS CAS NO 808733-05-3 AND DELETED CAS 1221592-28-4

MY email ID IS amcrasto@gmail.com

AGN-PC-00887R, (4-methylphenyl)methyl (3S,4R)-3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

Molecular Formula: C₁₉H₂₃FN₄O₂ Molecular Weight: 358.409923

https://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=69053724

Cerecor is developing the selective NMDA receptor subunit 2B antagonist CERC-301 (MK-0657) for depression.

CERC-301 (formerly MK-0657) is an oral, selective NMDA receptor subunit 2B (NR2B) antagonist in phase II clinical trials as adjunctive treatment for major depressive disorder (MDD) at Cerecor.

The compound had been in early trials at the National Institute of Mental Health (NIMH) for the treatment of major depression and at Merck & Co. for the treatment of Parkinson’s disease; however, no recent development has been reported in either case.

In 2013, the product was acquired by Cerecor from Merck & Co. on a worldwide basis for development and commercialization.

A phase II trial began in November 2013 and later that month, the FDA granted fast track designation for major depressive disorder.

………………………………………………

wo 2004108705 or http://www.google.co.in/patents/EP1648882B1?cl=en

METHODS OF SYNTHESIS

EXAMPLES 1 AND 2EXAMPLE 1

(35,4R)-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylateEXAMPLE 2

(3R,4S)-4-methylbenzyl 3-fluor-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

Step 1

Preparation of 4-Methylbenzyl 4-oxopiperidine-1-carboxylate:

4-Methylbenzyl alcohol (37.6 g, 308 mmol) was added to a solution of 1,1′-carbonyldiimidazole (50.0 g, 308 mmol) in DMF at RT and stirred for 1 h. 4-Piperidone hydrate hydrochloride (commercially available from Sigma-Aldrich, 47.0 g, 308 mmol) was added, resulting in a reaction mixture that was then heated to 50°C and stirred for 15 h. The reaction mixture was diluted with EtOAc and washed with 0.1 M HCl, H₂O (four times), and brine, dried over Na₂SO₄, filtered and concentrated. Purification by silica gel chromatography (step gradient elution: 10%, 25%, 50% EtOAc in hexanes) produced the title compound (42.4 g, 85% yield) as a clear oil.
¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, 2 H), 7.15 (d, 2 H), 5.08 (s, 2 H), 3.79 (t, 4 H), 2.45 (br s, 4 H) 2.31 (s, 3 H) ppm;
HRMS (ES) m/z 248.1281 [(M+H)⁺; calcd for C₁₄H₁₈NO₃: 248.1287];
Anal. C₁₄H₁₇NO₃: C, 68.03; H, 7.05; N, 5.59. Found: C, 68.00; H, 6.93; N, 5.66.

Step 2Preparation of (±)-4-methylbenzyl 3-fluoro-4-oxopiperidine-1-carboxylate:

A solution of 4-methylbenzyl 4-oxopiperidine-1-carboxylate (21.2 g, 85.7 mmol) and diisopropylethylamine (71.3 mL, 428 mmol) in dichloromethane (425 mL) was cooled to 0 °C and stirred. TBSOTf (29.5 mL, 129 mmol) was added slowly, maintaining the internal temperature below 5 °C. Aqueous NaHCO₃ (20 mL) was added and the layers were separated. The organic layer was washed with NaHCO₃, H₂O (two times), and brine, dried over Na₂SO₄, filtered and concentrated to give the crude TBS enol ether.
The crude TBS enol ether was dissolved in DMF (125 mL) at RT. Selectfluor® reagent (commercially available from Air Products and Chemicals, Inc., 30.4 g, 85.7 mmol) was added and the reaction mixture was stirred for 10 min. The reaction mixture was partitioned between EtOAc and H₂O and the organic layer was washed with H₂O (three times). The combined aqueous layers were extracted with EtOAc (two times) and the combined organics were dried over Na₂SO₄, filtered and concentrated. The entire reaction above was repeated and the resulting reaction products were combined to give the title compound (40 g), which was used in the next step without purification. NMR and mass spectral data suggest the ketone functionality in the product exists as a hydrate.
¹H NMR (400 MHz, CDCl₃) δ 7.24 (m, 2 H), 7.19 (m, 2 H), 5.18 (s, 2 H), 4.81 (br d, 1 H), 4.50(br d, 1 H), 4.23 (d, 1 H), 3.90 (m, 1 H), 3.60 (m, 1 H), 3.35 (t, 1 H), 2.58 (m, 2 H), 2.35 (s, 3 H) ppm;
HRMS (ES) m/z 284.1292 [(M+H)⁺; calcd for C₁₄H₁₈FNO₄: 284.1293];
Anal. C₁₄H₁₈FNO₄•1.2H₂O: C, 58.61; H, 6.46; N, 4.88. Found: C, 58.28; H, 6.06; N, 4.72.

Step 3Preparation of:

(±)-4-methylbenzyl (E)-4-(2-ethoxy-2-oxoethylidene)-3-fluoropiperidine-1-carboxylate

and

(±)-4-methylbenzyl (Z)-4-(2-ethoxy-2-oxoethylidene)-3-fluoropiperidine-1-carboxylate

To a solution of (±)-4-methylbenzyl 3-fluoro-4-oxopiperidine-1-carboxylate (40 g, 150 mmol) in toluene (200 mL) at RT was added (carbethoxymethylene)triphenylphosphorane (63.0 g, 181 mmol) and the reaction mixture stirred for 1 h. The reaction mixture was concentrated and purified by silica gel chromatography (gradient elution: 10% to 20% EtOAc in hexanes) to give the olefins (±)-4-methylbenzyl (E)-4-(2-ethoxy-2-oxoethylidene)-3-fluoropiperidine-1-carboxylate and (±)-4-methylbenzyl (Z)-4-(2-ethoxy-2-oxoethylidene)-3-fluoropiperidine-1-carboxylate (41.0 g, 78% yield, 3 steps) as a 3:1 E:Z mixture. This mixture was utilized directly in the next step. A small sample of the mixture was separated by silica gel chromatography for characterization purposes.
(±)-4-methylbenzyl (E)-4-(2-ethoxy-2-oxoethylidene)-3-fluoropiperidine-1-carboxylate: white solid, ¹H NMR (400 MHz, CDCl₃) δ 7.26 (d, 2 H), 7.17 (d, 2 H), 5.98 (s, 1 H), 5.11 (s, 2 H), 4.85 (m, 1 H), 4.18 (q, 2 H), 4.08 (br d, 1 H), 3.70 (m, 1 H), 3.55 (m, 1 H) 3.41 (m, 1 H), 3.33, (m, 1 H), 2.63 (br d, 1 H), 2.35 (s, 3 H), 1.29 (t, 3 H) ppm;
HRMS (ES) m/z 358.1420 [(M+Na)⁺; calcd for C₁₈H₂₂FNO₄Na: 358.1425];
Anal. C₁₈H₂₂FNO₄: C, 64.21; H, 6.58; N, 4.27. Found: C, 64.46; H, 6.61; N, 4.18.
(±)-4-methylbenzyl (Z)-4-(2-ethoxy-2-oxoethylidene)-3-fluoropiperidine-1-carboxylate: white solid, ¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, 2 H), 7.15 (d, 2 H), 6.41(m, 1 H), 5.82 (s, 1 H), 5.11 (d, 2 H), 4.61 (m, 1H), 4.38 (br d, 1 H), 4.16 (q, 2 H), 3.05-2.95 (m, 1 H), 2.9-2.75 (m, 2 H), 2.33 (s, 3 H), 2.13 (m, 1 H), 1.27 (t, 3 H) ppm;
HRMS (ES) m/z 358.1422 [(M+Na)⁺; calcd for C₁₈H₂₂FNO₄Na: 358.1425].

Step 4:Preparation of:

(±)-cis 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate

and

(±)-trans 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate

[0081]

To a solution of the olefin mixture from Step 3 (10.0 g, 29.8 mmol) in toluene (160 mL) and CH₂Cl₂ (120 mL) was added diphenylsilane (5.53 mL, 29.8 mmol) and (R)-BINAP (1.86 g, 2.98 mmol). Sodium t-butoxide (0.29 g, 2.98 mmol) and CuCl (0.30 g, 2.98 mmol) were then added, the reaction mixture was protected from light and stirred for 15 h. Additional portions of diphenylsilane (2.76 mL), NaOtBu (0.29 g) and CuCl (0.30 g) were added and the reaction mixture was stirred at RT for 24h. The mixture was then filtered through celite and concentrated. Purification on silica gel (step gradient elution: 5%, 10%, 15%, 25%, 30% EtOAc in hexanes) gave recovered starting materials (3.5 g, 35% yield), (±)-cis 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate (5.0 g, 50% yield) and (±)-trans 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate (1.2 g, 12% yield).
(±)-cis 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate: clear oil, ¹H NMR (400 MHz, CDCl₃) δ 7.25 (d, 2 H), 7.15 (d, 2 H), 5.10 (s, 2 H), 4.80-4.20 (m, 3 H), 4.15 (q, 2 H), 3.10-2.73 (m, 2 H), 2.52 (dd, 1 H), 2.35 (s, 3 H), 2.30 (dd, 1 H), 2.10 (m, 1 H), 1.72-1.48 (m, 2 H), 1.29 (t, 3 H) ppm;
HRMS (ES) m/z 338.1689 [(M+H)⁺; calcd for C₁₈H₂₅FNO₄: 338.1762].
(±)-trans 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate: clear oil, ¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, 2 H), 7.15 (d, 2 H), 5.08 (s, 2 H), 4.50-3.95 (m, 3 H), 4.15 (q, 2 H), 2.81 (br t, 2 H), 2.70 (br d, 1 H), 2.35 (s, 3 H), 2.17 (m, 2 H), 1.89 (br d, 1 H), 1.25 (m, 1 H), 1.22 (t, 3 H) ppm;
HRMS (ES) m/z 338.1699 [(M+H)⁺; calcd for C₁₈H₂₅FNO₄: 338.1762].

Step 5Preparation of (±)-((cis)-3-fluoro-1-{[(4-methylbenzyl)oxy]carbonyl}piperidin-4-yl)acetic acid:

To a solution of (±)-cis 4-methylbenzyl 4-(2-ethoxy-2-oxoethyl)-3-fluoropiperidine-1-carboxylate (10.0 g, 29.6 mmol) in THF (50 mL) was added aqueous NaOH (1M, 50 mL). The reaction mixture was stirred at RT for 5 h and then diluted with EtOAc and 1M HCl. The layers were separated and the aqueous extracted with EtOAc twice. The combined organics were washed with brine, dried over Na₂SO₄, filtered and concentrated to give the title compound (9.1 g) as a white solid which was used in the next step without further purification.
¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, 2 H), 7.15 (d, 2 H), 5.08 (s, 2 H), 4.79-4.16 (m, 3 H), 3.05-2.75 (m, 2 H), 2.59 (dd, 1 H), 2.36 (dd, 1 H), 2.31 (s, 3 H), 2.20-2.02 (m, 1 H), 1.60 (m, 2 H) ppm;
HRMS (ES) m/z 310.1457 [(M+H)⁺; calcd for C₁₆H₂₁FNO₄: 310.1449].
Anal. C₁₆H₂₀FNO₄•0.15 H₂O: C, 62.13; H, 6.52; N, 4.53. Found: C, 61.55; H, 6.37; N, 4.41.

Step 6Preparation of (±)-cis-4-methylbenzyl 4-(aminomethyl)-3-fluoropiperidine-1-carboxylate:

To a suspension of crude acid (±)-((cis)-3-fluoro-1-{[(4-methylbenzyl)oxy]carbonyl}piperidin-4-yl)acetic acid (9.1 g, 29.4 mmol) in toluene (80 mL) was added triethylamine (10.2 mL, 73.5 mmol) and diphenylphosphoryl azide (9.52 mL, 44.1 mmol). The reaction mixture was heated to 70 °C and stirred for 20 min. A mixture of dioxane (80 mL) and 1 M NaOH (80 mL) was added and the reaction mixture was cooled to RT. The reaction mixture was concentrated to remove the dioxane and extracted with EtOAc three times, dried over Na₂SO₄, filtered and concentrated. The residue was suspended in CH₂Cl₂, stirred for 30 min, and the white preciptate filtered off. The filtrate was concentrated to give crude product (7.5 g) as a yellow oil, used directly in the next step. An analytical sample was purified by silica gel chromatography (gradient elution: CH₂Cl₂ to 80:20:2 CH₂Cl₂ : MeOH : NH₄OH) for characterization:
¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, 2 H), 7.15 (d, 2 H), 5.08 (s, 2 H), 4.90-4.18 (m, 3 H), 2.95-2.75 (m, 2 H), 2.79 (dd, 1 H), 2.70 (dd, 1 H), 2.35 (s, 3 H), 1.59 (m, 3 H) ppm;
HRMS (ES) m/z 281.1658 [(M+H)⁺; calcd for C₁₅H₂₂FN₂O₂: 281.1660].

Step 7

Preparation of:

(3S,4R)-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

and

(3R,4S)-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

Two sealed tubes were each charged with a mixture of crude (±)-cis-4-methylbenzyl 4-(aminomethyl)-3-fluoropiperidine-1-carboxylate (Step 6, 3.7 g, 13.2 mmol) and 2-chloropyrimidine (1.51 g, 13.2 mmol) in n-butanol/diisopropyl-ethylamine (1:1, 13 mL). The tubes were sealed and the mixtures heated to 140 °C and stirred for 2 h. After cooling to RT, the reaction mixtures were combined and diluted with EtOAc and sat NaHCO₃. The layers were separated and the organic was washed with H₂O and brine, dried over Na₂SO₄, filtered and concentrated. Purification by silica gel chromatography (gradient elution: 1:1 hexanes:EtOAc to EtOAc) gave racemic cis-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate (6.9 g, 65% yield, 3 steps) as a white solid.
The enantiomers were separated by preparative HPLC on a ChiralPak AD column (5 cm x 50 cm, 20µM) with MeOH:MeCN (15:85, 150 mL/min) as eluant. The HCl salt of Example 1 was prepared by dissolving (3S,4R)-cis-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate (6.9 g, 19.3 mmol) in iPrOH (100 mL) at 65 °C. A solution of HCl in iPrOH (1.608 M, 12.6 mL, 20.2 mmol) was added and the solution was slowly cooled to RT over 15 h. Et₂O (25 mL) was added, the mixture stirred for 3h, cooled to 0 °C, stirred for 1h and filtered to give (3S,4R)-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate hydrochloride as a white solid (7.0 g, 92% recovery).
The hydrochloride salt of (3R,4S)-4-methylbenzyl-3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate was prepared using a similar procedure.

(3S,4R)-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate•HCl:

[α]_D -36.4° (c 0.17, MeOH);
Melting Point 149-150 °C;
¹H NMR (400 MHz, CD₃OD) δ 8.58 (br s, 2 H), 7.21 (d, 2 H), 7.17 (d, 2 H), 6.99 (t, 1 H), 5.06 (s, 2 H), 4.79 (m, 1 H), 4.42 (t, 1 H), 4.21 (d, 1 H), 3.60 (dd, 1 H), 3.50 (dd, 1 H), 3.15-2.80 (m, 2 H), 2.30 (s, 3 H), 2.10 (m, 1 H), 1.61 (m, 2 H) ppm;
HRMS (ES) m/z 359.1879 [(M+H)⁺; calcd for C₁₉H₂₄FN₄O₂: 359.1878];
Anal. C₁₉H₂₃FN₄O₂•HCl•0.2 H₂O: C, 57.27; H, 6.17; N, 14.06. Found: C, 57.22; H, 6.37; N, 14.16.

(3R,4S)-4-methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate •HCl:

[α]_D +34.9° (c 0.18, MeOH);
Melting Point 149-150 °C;
¹H NMR (400 MHz, CD₃OD) δ 8.58 (br s, 2 H), 7.21 (d, 2 H), 7.17 (d, 2 H), 6.99 (t, 1 H), 5.06 (s, 2 H), 4.79 (m, 1 H), 4.42 (t, 1 H), 4.21 (d, 1 H), 3.60 (dd, 1 H), 3.50 (dd, 1 H), 3.15-2.80 (m, 2 H), 2.30 (s, 3 H), 2.10 (m, 1 H), 1.61 (m, 2 H) ppm;
HRMS (ES) m/z 359.1870 [(M+H)⁺; calcd for C₁₉H₂₄FN₄O₂: 359.1878].
Anal. C₁₉H₂₃FN₄O₂•HCl•0.5H₂O: C, 56.50; H, 6.24; N, 13.87. Found: C, 56.68; H, 6.27; N, 13.80.

……………….

WO 2006069287

http://www.google.com/patents/WO2006069287A1?cl=en

Scheme 1:

4-MeBnOH CDI

Scheme 2:

R¹ X- R1

X” Rhodium metal precursor/

H I iiR² chiral phosphine ligand |_] p — R^:

14 13

Representative Examples include:

EXAMPLE 1

Step A:

¹¹ -‘ .OH

A 5 L round bottom flask was charged with THF (1.87 L, KF< 50 ppm) and cooling to -75 ^°C was begun. When the temperature of THF had reached < – 20 ^°C, n-BuLi (11 M in hex, 123 mL) was added over 15 minutes in order to keep the solution temperature below -10 ^“C. When the solution reached -35 ^°C, controlled addition of diisopropylamine (197 mL, KF < 50 ppm) over 15 minutes was carried out so the exotherm did not cause the solution temperature to exceed -16 ^°C. The solution was then allowed to continue to cool until it reached -75 ^“C. 3-Fluoropyridine (compound 1 from Scheme 1) (125 g, KF < 150 ppm) was then added neat to this solution via addition funnel while maintaining the batch temperature below -70 ^°C.

Neat DMF (168 mL, KF < 50 ppm) was then added to the batch over 1 hour maintaining the temperature < -70 ^°C. After confirming complete formation of the aldehyde, the reaction was warmed to 0 ^“C, and H₂O (230 mL, 10 eq.) was added. NaBH₄ (48.4 g) was then added in two portions over 5 minutes at 0 ^°C. Addition of concentrated HCl (6 M, 1.17 L) was completed in 1 hour at temperatures between 0- 25^°C. The rection batch was then heated to 40 ^°C and kept at this temperature for 1 hour.

The reaction was then allowed to cool to room temperature. Then, to the aqueous layer 6 M NaOH (747 mL) was slowly added at 0-15 ^°C to adjust the pH to 12. Approximately 700 mL of H₂O was added to dissolve any precipitate in the aqueous layer. The aqueous layer was then extracted with IPAc (1 x 1.275 L, 2 x 800 mL). The organic layer was treated with 20 wt. % Darco-G60 carbon (based on product assay) and the solution was heated to 40 ^°C for 1 hour followed by filtration over solka floe. After filtration the organic layer was solvent switched from IPAc to IPAc:heptane (15-20% v/v IPAc:heptane). The product crystallized as a white solid. This solution was then cooled to 0 ^°C for 30 minutes and filtered. An additional 250 mL of heptane was cooled to 0 ^°C and used to wash the wet cake. Typical Yield = 79% (128.5 g).

Step B:

To a 2 L flask under N₂ atmosphere were charged compound 2 from Scheme 1 (50.0Ig), acetone (524 mL), and BnBr (50.0 mL). This homogenous solution was heated to reflux for ~ 12 h. The reaction mixture was cooled to room temperature and diluted with heptane (550 mL). The pyridinium salt (compound 3 from Scheme 1) was collected by filtration. The wet cake was then slurry washed at ambient temperature with 25% acetone/heptane (200 mL) and filtered. The wet cake was then dried under vacuum at ambient temperature exposed to the atmosphere, affording a slight-pinkish solid ca. 98% pure by 1 H NMR

Typical Yield – 93% (109.5 g)

Step C:

To a 2 L round bottom flask were charged compound 3 from Scheme 1 (100.30 g, 1.00 eq.) and methanol (960 mL). The homogenous solution was then cooled to 10⁰C. The NaBH₄ (19.10 g, 1.50 Eq) was added portion wise (using a solid addition funnel) while keeping the temperature < 0 ⁰C. The batch was diluted with IPAc (1.0 L), followed by addition of 1 L 11.25 wt% brine. The resulting mixture was aged 15 min, then allowed to separate into two clear layers. The lower brine layer was removed. The organic stream was then washed with 500 mL 15wt% brine, then allowed to separate into two clear layers. The lower brine layer was removed. The batch was adjusted to roughly 1:1 MeOHrIPAc (c = 100 g/L) and then treated with 25 wt% Ecosorb C-941 at 50 ⁰C in for ~ 2 h. This was then filtered through a plug of celite, while rinsing with 1 : 1 MeOH:IPAc (rinse was roughly 25% of total batch volume). The batch was then concentrated to a residue.

The batch was then dissolved in 5% MeOH in IPAc at ~ 100 g/L (~ 636 mL). The batch was warmed to 50 ⁰C, followed by addition of a solution of 4M HCl in dioxane (1.10 eq)) slowly over ~ 1 h. At this point, the batch was seeded with a small spatula tip full of seed. After complete addition of the HCl solution, the batch was allowed to cool to room temperature slowly overnight. The solids were isolated by filtration. A slurry cake wash was then performed with 5% MeOH/IPAc (200 mL), followed by a displacement wash of 5% MeOH/IPAc (200 mL). The batch was then dried under vacuum at ambient temperature exposed to the atmosphere to afford compound 4 as a white solid (77% yield).

This material, 66.1O g of crude 4, was dissolved in 450 mL MeOH to which was added 450 mL IPAc. This mixture was treated with 25wt% Ecosorb C-941 (16.53 g) and heated to 50 ⁰C for 2 h. The mixture was then filtered through a pad of celite, washing the Ecosorb C-941 with ~ 500 ml 25% MeOH in IPAc. The mixture was then solvent switched on a rotovap to roughly 10% MeOH in IPAc. During the solvent switch, after concentrating to roughly 60% of its original volume, a small spatula tip full of seed was introduced, causing instant crystal growth. This mixture was concentrated until the final volume was ~ 350 mL. The slurry was then isolated, using a slurry wash of- 200 mL 5% MeOH/IPAc. The solids were dried over night under vacuum, exposed to the atmosphere, affording 60.23 g of 4 (70% yield).

Typical Yield = 70% (60.2 g).

Step D:

In a N₂ atmosphere glovebox, (R,R)-Walphos (SL-W003-1) (60.1 mg, commercially available from Solvias, Inc., Fort Lee, New Jersey 07024) and [(COD)RhCl]₂ (20.3 mg) were dissolved in dichloromethane (3 mL, anhydrous, N₂ degassed) and aged for 45 min at room temperature. Compound 4 from Scheme 1 (15.0 g) was charged to a 6 oz. glass pressure vessel (Andrews Glass Co., Vineland, NJ) containing a magnetic stir bar. MeOH (69 mL, anhydrous, N₂ degassed) was added, followed by the catalyst solution and a dichloromethane (3 mL) rinse.

The reactor was degassed with H₂ (40 psig) and immersed in a preheated 50 ⁰C oil bath. After a few minutes, the vessel was further pressurized with H₂ to 85 psig and allowed to age for 18.75 h. After this time, the vessel was vented and cooled to room temperature. HPLC analysis indicated >99% conversion of the vinyl fluoride. HPLC analysis indicated 99.3% ee.

The reaction mixture from above was concentrated in vacuo to a dark brown oil, which was then diluted with 50 mL EtOAc, to which was added 50 mL saturated NaHCO₃ (aq). This biphasic mixture was stirred at room temperature for 30 min. This mixture was separated, the aqueous layer was extracted 3 x 10 mL EtOAc, then the combined organic layers were dried over Na₂SO₄ and concentrated in vacuo to a residue, which was purified by column chromatography (1 : 1 EtOAc:hexanes) to afford 9.45 g of free base compound 5 (74.4% isolated yield) as a pale yellow oil.

Typical Yield = 74% (9.5 g).

HC1 HN^>”^F

To a 100 mL round bottom flask was charged the free base compound 5 from Example Scheme 1 , (1.00 eq), the Pd(OH)₂/C (1.29g), MeOH (23 mL), and 6M HCl (3.89 mL, 1.00 eq.). This mixture was degassed three times, finally filling the vessel with H₂ (1 atm, balloon pressure). The reaction was stirred at room temperature for 18 h. The mixture was filtered through a plug of Celite 521, rinsed with 50 mL MeOH, then concentrated to a residue. The residue was redissolved in ~ 150 mL 1 : 1 MeOH:IPAc, then refiltered through a sintered glass funnel to remove inorganics. Theis resulting solution was then solvent switched to roughly 10% MeOH in IPAc, during which spontaneous crystallization of compound 6 from Scheme 1 was observed. The solids were isolated by vacuum, washed twice with ~ 10 mL 10% MeOH in IPAc, then dried under vacuum over night, affording a pale white, crystalline solid.

Typical Yield = 81% (3.2 g).

JV,iV -Carbonyldiimidazole, 2.39 g (1.00 eq) was charged to a 50 mL round bottom flask, to which was added the DMF (19.7 ml). Then, the 4- methylbenzyl alcohol (1.80 g 1.00 eq) was added as a solid. This mixture was stirred for 15 min. at room temperature, during which an exotherm was noted (ΔT = +6.1 ⁰C, 18.5 ⁰C to 24.6 ⁰C). The fluoroalcohol HCl salt 6, 2.50 g (1.00 eq) was then added as a solid to this mixture. This was heated to 50 ⁰C for 1O h, and then allowed to cool to room temperature over night. The resulting mixture was diluted with 40 mL EtOAc. This mixture was washed 2 x 25 mL 3M HCl and separated, then 1 x 25 mL 15wt% brine and separated. This was extracted with 1 x 15 mL EtOAc and combined with the previous organic stream. The organic stream was concentrated to a residue and subjected to column chromatography eluting with a gradient (0% to 50% EtOAc in hexanes, TLCs developed in 50% EtOAc:hexanes, visualizing with UV and KMnO₄), to afford 3.35 g of a clear colorless oil.

Typical Yield = 81 % (3.4 g).

Step G:

A solution of fluoro alcohol compound 7 from Scheme 1 (1.22 g) in CH₃CN was cooled to -20 ^°C and Hunig’s base (2.2 equiv., 1.66 mL) was added. To this, Tf₂O – (1.1 equiv., 0.81 mL) was slowly added while maintaining the internal temperature < -10^°C. Aqueous NH₄OH (15 equiv., 2.7 mL) was then added to the reaction mixture at low temperature (-20^°C) and then warmed up to room temperature and aged for Ih. After completion, toluene (15 mL) and 10% NaOH (10 mL) were added and the layers separated. After extraction, the organic layer was washed with H₂O (IO mL).

The toluene stream of the amine was dried (-400 μg/mL) and concentrated to 100 g/L. Methanol was then added to obtain an overall solvent composition of toluene/MeOH (95:5), followed by the slow addition of HCl (1.05 equiv, 1.12 ml) at 50 ^°C. The amine hydrochloride 8 from Scheme 1 crystallized immediately, and the reaction was aged 20 min. The light yellow salt was then filtered and washed with cold toluene (15 mL) to offer amine hydrochloride 8 in 82% as a white crystalline solid.

Into a 100-L round bottom flask were charged 1.67 kg amine HCl salt 8 from Scheme 1, 912.4 g chloropyrimidine, 4.6 L of diisopropylethyl amine and 25.78 L ethylene glycol. The resulting slurry gradually became a solution, which was degassed and stirred under a nitrogen atmosphere. The contents were heated to 100 ° C for 12 h. The heat was turned off and the reaction solution slowly cooled to room temperature, which resulted in the formation of a slurry. To the slurry was added 77.3 L water over 1 h period and the slurry was aged at room temperature for 3 h. The mixture was filtered and the cake was washed with additional 80 L. The wet cake was left under nitrogen to dry overnight. After drying, 1.90 kg of an off white solid was collected.

1.77 kg of the above solid was dissolved into 71 L EtOAc and treated with 531 g Darco G-60 carbon at room temperature for 3 h. Filtration through Solka Floe was followed by washing with 2 x 20 L EtOAc. A solvent switch to MeOH under reduced pressure resulted in a slurry, and the final MeOH volume was adjusted to 19 L. The slurry in MeOH was heated to ca. 60 °C. Gradually cooling to room temperature resulted in a slurry, to which 57 L GMP water was added over 1 h with cooling (exothermic mixing, temperature controlled below 30 “C). The mixture was aged at room temperature for 3 h and filtered to collect solid, the cake was washed with 30 L GMP water and left to dry under nitrogen. 1.55 kg dried product was collected. (89% yield).

Typical Yield = 89% (1.55 kg).

………….

European Journal of Medicinal Chemistry (2012), 53, 408-415

http://www.sciencedirect.com/science/article/pii/S0223523412002310

Two diastereoisomeric NR2B NMDA antagonists were radiolabelled with fluorine-18. ► The radiolabelling of 3-[¹⁸F]fluoro-1,4-substituted-piperidine pattern was achieved. ► In vitro study showed high specific and selective binding for NR2B NMDAR receptors. ► B_max/K_d ratios and logD_7.4 demonstrated appropriate properties for in vivo imaging.

Full-size image (30 K)

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Organic & Biomolecular Chemistry (2012), 10(42), 8493-8500

http://pubs.rsc.org/en/content/articlelanding/2012/ob/c2ob26378e#!divAbstract

In order to develop a novel and useful building block for the development of radiotracers forpositron emission tomography (PET), we studied the radiolabelling of 1,4-disubstituted 3-[¹⁸F]fluoropiperidines. Indeed, 3-fluoropiperidine became a useful building block in medicinal chemistry for the pharmacomodulation of piperidine-containing compounds. The radiofluorination was studied on substituted piperidines with electron-donating and electron-withdrawing N-substituents. In the instance of electron-donating N-substituents such as benzylor butyl, configuration retention and satisfactory fluoride-18 incorporation yields up to 80% were observed. In the case of electron-withdrawing N-substituents leading to carbamate or amidefunctions, the incorporation yields depend on the 4-susbtitutent (2 to 63%). The radiolabelling of this building block was applied to the automated radiosynthesis of NR2B NMDA receptor antagonists and effected by a commercially available radiochemistry module. The in vivoevaluation of three radiotracers demonstrated minimal brain uptakes incompatible with the imaging of NR2B NMDA receptors in the living brain. Nevertheless, moderate radiometabolism was observed and, in particular, no radiodefluorination was observed which demonstrates the stability of the 3-position of the fluorine-18 atom. In conclusion, the 1,4-disubstituted 3-[¹⁸F]fluoropiperidine moiety could be of value in the development of other radiotracers for PET even if the evaluation of the NR2B NMDA receptor antagonists failed to demonstrate satisfactory properties for PET imaging of this receptor.

Graphical abstract: Radiolabelling of 1,4-disubstituted 3-[18F]fluoropiperidines and its application to new radiotracers for NR2B NMDA receptor visualization

http://www.rsc.org/suppdata/ob/c2/c2ob26378e/c2ob26378e.pdf

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WO 2013156614

The chemical structure of MK-0657 is as follows

http://www.google.com/patents/WO2013156614A1?cl=en

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

Phase 3 drug Comments Off

Feb 202014

Sonidegib/Erismodegib

CODE DESIGNATION ..LDE225, NVP-LDE-225

Treatment of medulloblastoma PHASE3 2014 FDA FILING

Treatment of advanced basal cell carcinoma PHASE3 2014 FDA FILING

Treatment of SOLID TUMORS..PHASE1 2017 FDA FILING

READMalignant Solid Tumors of Childhood

THERAPEUTIC CLAIM Oncology, Antineoplastics & Adjunctive Therapies

CHEMICAL NAMES

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

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

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

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

MOLECULAR FORMULA C26H26F3N3O3

MOLECULAR WEIGHT 485.5

SPONSOR Novartis Pharma AG

CAS REGISTRY NUMBER 956697-53-3 free form

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

sonidegib – European Medicines Agency READ THIS..

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

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

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

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

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

About the Study

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

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

Date: February 19, 2013

Source: Novartis

MORE ABOUT SONIDEGIB

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

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

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

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

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

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

NVP-LDE225 Diphosphate salt (Erismodegib, Sonidegib)

Formula Image

Synonym:Erismodegib, Sonidegib

CAS Number:1218778-77-8

Mol. Formula:C26H26F3N3O3 ∙ 2H3PO4

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

Brief Description:

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

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

About LDE225

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

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

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

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

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

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

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

About Novartis

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

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

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

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

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

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

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

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

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

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

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

PATENTS

1 WO 2007131201

2 WO 2008154259

3 WO 2010033481

4 WO 2011009852

5 WO 2011062939

………………………………………

SYNTHESIS

WO2011009852A2

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

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

Step 1:

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

Step 2:

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

Step 3:

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

Step 4:

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

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

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

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PAPER

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

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

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

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

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Links

SYNTHESIS

US20120196849, ENTRY…..95

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

USE SIMILAR METHODOLOGY

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

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

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

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

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

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

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

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

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

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

USE THIS COMPD IN ABOPVE AND YOU WILL GET SONIDEGIB

4-(Trifluoromethoxy)phenylboronic acid

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

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

……………………………………………….

PAPER

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

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

ENTRY 5m

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

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

Reference

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

sonidegib

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

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

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

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

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

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

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

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

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

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

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

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

SONIDEGIB

Links

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

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Dasantafil for Treatment of Erectile Dysfunction

phase 2, Uncategorized 2 Responses »

Jan 292014

Dasantafil

569351-91-3 CAS NO

405214-79-1 (racemate)

UNII-48P711MI2G, SCH 446132, D03657,

Molecular Formula: C₂₂H₂₈BrN₅O₅

Molecular Weight: 522.39222

Merck & Co. (Originator) IN PHASE 2

THERAPEUTIC CLAIM treatment of erectile dysfunction (phosphodiesterase (PDE) 5 isoenzyme inhibitor)

CHEMICAL NAMES

1H-purine-2,6-dione, 7-[(3-bromo-4-methoxyphenyl)methyl]-1-ethyl-3,7-dihydro-8-[[(1R,2R)-2-hydroxycyclopentyl]amino]-3-(2-hydroxyethyl)
7-(3-bromo-4-methoxybenzyl)-1-ethyl-8-[[(1R,2R)-2-hydroxycyclopentyl]amino]-3-(2-hydroxyethyl)-3,7-dihydro-1H-purine-2,6-dione

7-[(3-bromo-4-methoxyphenyl)methyl]-l-ethyl-8-[[(lR,2R)-2- hydroxycyclopentyl]amino]-3-(2-hydroxyethyl)purine-2,6-dione

Treatment of Erectile Dysfunction , Phosphodiesterase PDE5A Inhibitors

Dasantafil (SCH-446132) is a phosphodiesterase type 5 (PDE5) inhibitor which had been in early clinical development at Merck & Co. for the treatment of erectile dysfunction (ED); however, no recent development has been reported for this research. Phosphodiesterases regulate the tissue concentration of cyclic guanosine monophosphate (cGMP), which in turn triggers smooth muscle relaxation, allowing blood to flow into the penis and resulting in erection. PDE5 is the most abundant phosphodiesterase in the human corpus cavernosum, and as such its inhibition by dasantafil enhances erectile function by increasing the concentration of cGMP.

DASANTAFIL

PDE V inhibitor compounds and their use in treating a variety of physiological conditions are described in a number of patents {e.g., U.S. Pat. Nos. 5,409,934, 5,470,579, 5,939,419 and 5,393,755) and foreign publications (e.g., WO 93/23401 , WO 92/05176, WO 92/05175, and WO 99/24433).

Specific PDE V inhibitors have been found useful for specific indications. For example, the use of PDE V inhibitors for treating impotence has met with commercial success with the introduction of sildenafil citrate, vardenafil, and tadalafil (i.e., Viagra®, Levitra®, and Cialis®, respectively). The chemistry and use of Viagra®, including its mechanism of action in treating erectile dysfunction, are taught in EP 0 702 555 B1. Accordingly, it is an object of this invention to provide a method of using a PDE V inhibitor to treat a patient who has, or is at risk of, congestive heart failure, and/or other cardiovascular conditions.

Processes for preparing PDE V inhibitor compounds can be found in US

6,207,829, US 6,066,735, US 5,955,611 , US 5,939,419, US 5,393,755, US 5,409,934, US 5,470,579, US 5,250,534, WO 02/24698, WO 99/24433, WO 93/23401 , WO 92/05176, WO 92/05175, EP 740,668 and EP 702,555. One type of PDE V inhibitor compound contains a xanthine functionality in its structure. Xanthines can be prepared as described by Peter K. Bridson and Xiaodong Wang in 1 -Substituted Xanthines, Synthesis, 855 (July, 1995), which is incorporated herein by reference in its entirety. WO 02/24698, which is incorporated herein by reference in its entirety, teaches a class of xanthine PDE V inhibitor compounds useful for the treatment of impotence. A general process disclosed therein for preparing xanthine PDE V inhibitor compounds having the formula (I) follows:

(III) (I) (i) reacting a compound having the formula (III) with an alkyl halide in the presence of a base (introduction of R¹¹ or a protected form of R¹¹); (ii) (a) debenzylating and then (b) alkylating the compound resulting from step (i) with an alkyl halide, XCH₂R¹“; (iii) (a) deprotonating and then (b) halogenating the compound resulting from step (ii);

(iv) reacting the compound resulting from step (iii) with an amine having the formula R^lvNH₂; and (v) removing a protecting portion of Rⁿ, if present, on the compound resulting from step (iv) to form the compound having the formula (I). R¹, R”, R^m and R^lv correspond to R¹, R², R³ and R⁴, respectively, in WO02/24698, and are defined therein. WO 02/24698 (pages 44 and 68-73) also teaches a synthesis for the following xanthine compound (identified therein as Compound 13 or Compound 114 of Table II): 1-ethyl-3,7-dihydro-8-[(1 R,2R)- (hydroxycyclopentyl) amino]-3-(2-hydroxyethyl)-7-[(3-bromo-4- methoxyphenyl)methyl]-1 H-purine-2,6-dione:

Compound 13. It would be beneficial to provide an improved process for preparing polycyclic xanthine PDE V inhibitor compounds

………………….

Patent description

Links

WO2006055573A2

entry 129 is dasantafil

Figure imgf000050_0001

…………………

SYNTHESIS

Links

WO2002024698A1

Figure imgf000069_0001

14X^‘ _CHs ^{‘ B}” tX is Experimental Procedure: Compound 114 in Table II (13)

1 (20.0 g, 74.0 mmol) was dissolved in dimethylformamide (370 mL) under nitrogen and (2-bromoethoxy)-terf-butyldimethylsilane (31.8 mL, 148 mmol) was added dropwise. The reaction was stirred at room temperature for 115 hrs., then diluted with ethyl acetate and washed with water several times.

The organic mixture was dried over potassium carbonate, filtered and concentrated under vacuum. Purification via flash chromatography (30/70 ethyl acetate/hexanes) yielded 2 (28.1 g, 88%).

¹H NMR (400 MHz, CDCI₃): δ 7.52 (s, 1 H), 7.29-7.39 (m, 5H), 5.49 (s,

2H), 4.25 (t, 2H, J = 6.0 Hz), 4.07 (q, 2H, J = 7.2 Hz), 3.93 (t, 2H, J =

6.0 Hz), 1.24 (t, 3H, J = 7.2 Hz), 0.75 (s, 9H), 0.08 (s, 6H). HRMS: Calcd for C₂₂H₃₂N₄0₃Si (M+H): 429.2322. Found: 429.2329.

To a solution of 2 (2.10 g, 4.89 mmol) in methanol (375 mL) was added ammonium formate (4.64g, 73.6 mmol) and 20% palladium hydroxide on carbon (980 mg). The reaction was heated to reflux for 1.5 hrs., then cooled to room temperature, filtered and concentrated under vacuum. Purification via flash chromatography (50/50 ethyl acetate/hexanes) yielded 3 (1.26 g, 94%).

¹H NMR (400 MHz, CDCI₃): δ 7.82 (s, 1 H), 4.33 (t, 2H, J = 6.0 Hz), 4.16

(q, 2H, J = 7.2 Hz), 3.99 (t, 2H, J = 6.0 Hz), 1.29 (t, 3H, J = 7.2 Hz),

0.78 (s, 9H), 0.06 (s, 6H). HRMS: Calcd for Cι₅H₂₆N₄O₃Si (M+H): 339.1852. Found: 339.1864. To 3 (970 mg, 2.86 mmol) was added dimethylformamide (25 mL), 3- bromo-4-methoxybenzyl bromide 15 (1.62 g, 5.79 mmol), and potassium carbonate (800 mg, 5.79 mmol) under nitrogen. The reaction mixture was stirred at room temperature for 21 hrs., then diluted with ethyl acetate and washed with water several times. The organic mixture was dried over potassium carbonate, filtered and concentrated under vacuum. Purification by flash chromatography (30/70 ethyl acetate/hexanes) yielded 10 (1.55 g, 100%).

¹H NMR (400 MHz, CDCI₃): δ 7.52 (s, 1 H), 7.51 (d, 1 H, J = 2.4 Hz),

7.30 (dd 1 H, J = 2.0 Hz, J = 8.4 Hz), 6.87 (d, 1 H, J = 8.8 Hz), 5.40 (s,

2H), 4.25 (t, 2H, J = 6.0 Hz), 4.07 (q, 2H, J = 7.0 Hz), 3.93 (t, 2H, J =

6.0 Hz), 3.88 (s, 3H), 1.25 (t, 3H, J = 7.0 Hz), 0.75 (s, 9H), 0.08 (s, 6H).

HRMS: Calcd for C₂₃H₃₃BrN₄O₄Si (M+H): 537.1533. Found: 537.1540.

To solution of 10 (1.50 g, 2.80 mmol) in tetrahydrofuran (24 mL) under nitrogen at -78 °C (dry ice/acetone bath) was added lithium diisopropylamide (2M in THF/heptane, 2.2 mL, 4.33 mmol). After stirring for thirty minutes, 1 ,2- dibromotetrafluoroethane (0.69 mL, 5.77 mmol) was added dropwise over five minutes. The reaction was stirred for 1.25 hrs. at -78 °C then quenched with saturated aqueous sodium bicarbonate and warmed to room temperature.

The mixture was extracted with dichloromethane, dried over potassium carbonate, filtered and concentrated under vacuum. Purification via flash chromatography (30/70 ethyl acetate/hexanes) yielded 11 (600 mg, 34%). ¹H NMR (400 MHz, CDCI₃): δ 7.60 (d, 1 H, J = 2.4 Hz), 7.35 (dd, 1 H, J =

2.0 Hz, J = 8.4 Hz), 6.84 (d, 1 H, J = 8.4 Hz), 5.45 (s, 2H), 4.21 (t, 2H, J = 5.6 Hz), 4.07 (q, 2H, J = 6.8 Hz), 3.90 (t, 2H, J = 5.6 Hz), 3.87 (s, 3H), 1.24 (t, 3H, J = 6.8 Hz), 0.73 (s, 9H), 0.08 (s, 6H). HRMS: Calcd for C₂₃H₃₂Br₂N₄O₄Si (M+H): 615.0638. Found: 615.0633.

To 11 (1.89 g, 3.07 mmol) was added the amino alcohol hydrochloride salt (1.31 g, 12.27 mmol), diisopropylethylamine (15.4 mL), and 1-methyl-2- pyrrolidinone (15.4 mL). The reaction mixture was heated to 160 °C in a sealed tube for 13 hrs., then cooled to room temperature. Water was added, then the mixture was extracted with ethyl acetate and washed with water several times. The organic mixture was dried over potassium carbonate, filtered and concentrated under vacuum. Purification via flash chromatography (3/97 methanol/dichloromethane) yielded 12 (1.77 g, 90%).

¹H NMR (400 MHz, CDCI₃): δ 7.45 (d, 1 H, J = 2.0 Hz), 7.17 (dd, 1 H, J =

2.4 Hz, J = 8.6 Hz), 6.86 (d, 1 H, J = 8.4 Hz), 5.18-4.34 (m, 3H), 4.00- 4.23 (m, 5H), 3.86-3.98 (m, 6H), 3.69-3.79 (m, 1 H), 2.10-2.21 (m, 1 H), 1.99-2.10 (m, 1 H), 1.60-1.84 (m, 3H), 1.32-1.43 (m, 1 H), 1.24 (t, 3H, J = 7.2 Hz), 0.75 (s, 9H), 0.07 (d, 6H, J = 4.0 Hz). HRMS: Calcd for C₂₈H₄₃BrN₅θ₅Si (M+H): 636.2217. Found: 636.2207.

12 (1.77 g, 2.78 mmol) was dissolved in tetrahydrofuran (28 mL) under nitrogen and tetrabutylammonium fluoride (1M in THF, 28 mL) was added dropwise. The reaction was stirred at room temperature for 15 hrs., then diluted with dichloromethane and washed with water several times. The organic mixture was dried over potassium carbonate, filtered and concentrated under vacuum. Purification via flash chromatography (3/97 methanol/dichloromethane) yielded 13 (compound no. 114 in Table II) (760 mg, 52%).

DASANTAFIL

¹H NMR (400 MHz, CDCI₃):

δ 7.47 (d, 1 H, J = 2.0 Hz), 7.19 (dd, 1 H, J =2.0 Hz, J = 8.4 Hz), 6.88 (d, 1 H, J = 8.4 Hz), 5.25 (s, 2H), 5.09 (s, 1H), 4.21-4.27 (m, 3H), 4.06 (q, 2H, J = 7.0 Hz), 3.90-3.97 (m, 3H), 3.89 (s, 1 H), 3.74-3.82 (m, 1 H), 3.08 (s, 1 H), 2.12-2.22 (m, 1 H), 1.98-2.08 (m, 1 H), 1.60-1.86 (m, 3H), 1.33-1.43 (m, 1 H), 1.25 (t, 3H, J = 7.0 Hz),1.06-1.22 (m, 3H). HRMS: Calcd for C₂₂H₂₈BrN₅O₅ (M+H): 522.1352. Found: 522.1346.

2-Bromo-4-methyl anisole 14 (2.2 mL, 14.9 mmol) was dissolved in dichlomethane (30 mL) and N-bromosuccinimide (3.75 g, 16.4 mmol) was added followed by AIBN (26.0 mg). The reaction was heated to reflux for 19 hrs., then cooled to room temperature and the precipitate was filtered off. The filtrate was diluted with dichloromethane and washed with 0.5 M aqueous sodium bicarbonate, followed by water. The organic mixture was dried over sodium sulfate, filtered and concentrated under vacuum to yield 15 (4.16 g,

100%). The benzyl bromide was used as the crude material without further purification.

¹H NMR (400 MHz, CDCI₃): δ 7.59 (d, 1 H, J = 2.0 Hz), 7.30 (dd, 1 H, J =

2.4 Hz, J = 8.4 Hz), 6.85 (d, 1 H, J = 8.4 Hz), 4.37 (s, 2H), 3.90 (s, 3H).

General Synthesis of Compound No. 114 in Table II (13) a) Reacting 1 with an alkyl halide and base to form 2; b) Debenzylation of 2 to form 3; c) Alkylation of 3 with a benzyl halide to form 10; d) Deprotonation of 10 followed. by addition of a brominating agent to form 11 ; e) Displacement of bromo 11 with an amine to form 12; and f) Silyl ether cleavage of 12 to form compound no. 114 in Table II (13).

114 IN TABLE II./(13)

Figure imgf000045_0001

……………

WO2003101992A1

GENERAL SCHEME

Figure imgf000018_0001

SYNTHESIOS

Figure imgf000027_0001

9A 13A DASANTAFIL

SYNTHESIS

Compound 1A:

glycine-A/-r(4-methoxyphenyl)methyl1 ethyl ester

To a mixture of glycine ethyl ester hydrochloride (about 1.4 equiv) and potassium carbonate (about 1.0 equiv) was added anhydrous ethanol. The mixture

was stirred at about 40-45 °C for about 3 hours. Then, p-anisaldehyde (about 1.0

equiv.) was added, and the reaction mixture was stirred for a minimum of about 3 hours to provide an imine (not shown). Upon reaction completion (about <5.0 % p- anisaldehyde remaining by GC analysis), the reaction mixture was cooled to about 0-

10 °C. Then, an aqueous solution of sodium borohydride (about 0.50 equiv) was

added to the reaction mixture at a temperature of between about 0 °C and about 20

°C, and stirred for about 1 hour to provide Compound 1 A. Upon completion of the

reduction reaction, the reaction mixture was quenched with the slow addition of an aqueous solution of aqueous glacial acetic acid. After quenching, the reaction mixture was warmed to room temperature and filtered to remove solids. The filtrate was then concentrated under vacuum, followed by the addition of toluene and water to facilitate layer separation. Aqueous potassium carbonate solution was added to adjust the pH of the mixture to about 8-9. The organic layer was separated and the aqueous layer was extracted with toluene. The combined toluene extracts were concentrated to provide the product in about a 80-85% yield (based on GC and HPLC in solution assay). ¹H NMR 400 MHz (CDCI₃): δ 7.23 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H),

4.17 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 3.73 (s, 2H), 3.38 (s, 2H), 1.88 (s, br, 1 H), 1.26

(t, J = 7.1 Hz, 3H); ¹³C NMR 100 MHz (CDCI₃): δ 172.8, 159.2, 132.0, 129.9, 114.2,

61.1, 55.6, 53.1 , 50.4, 14.6.

Compound 2:

/V-cvanomethanimidic acid ethyl ester

To cyanamide (about 1.2 mole) was added triethylorthoformate (about 1.33 mole), and the reaction mixture was heated to about 85-95 °C for approximately 2 hours to form Compound 2. Estimated in-solution yield was about 95-100%. The product was optionally purified by vacuum distillation.

¹H NMR 400 MHz (CDCI₃): δ 8.38 (s, 1H), 4.28 (t, J = 6.7 Hz, 2H), 1.29 (t, J =

6.8 Hz, 3H); ¹³C NMR 100 MHz (CDCI₃): δ 171.5, 113.4, 65.5, 13.1.

Compound 3A:

Figure imgf000051_0001

cis- and frans-glvcine Λ/-r(cvanoimino,methyl1-Λ/-r(4- methoxyphenvDmethvπ ethyl ester

A solution of Compound 1A (about 1.0 mole) in toluene was concentrated under vacuum to distill off toluene. Anhydrous tetrahydrofuran (“THF”) was added to the concentrate, then Compound 2 (about 1.2 moles, obtained above) was added to that, and the solution was heated at reflux for about 1 hour. At this stage, the formation of Compound 3A was complete. Estimated in-solution yield was about

95% (about 2:1 mixture of cis and trans isomers). Compound 4A: 1H-imidazole-5-carboxylic acid, 4-amino-1-[(4- methoxyphenvDmethvn ethyl ester

Compound 3A (obtained above) was concentrated by distilling off THF. Then, anhydrous ethanol was added to afford a reaction mixture solution. Separately, potassium t-butoxide (about 0.15 mole) was dissolved in anhydrous ethanol to afford a solution. The potassium t-butoxide solution was added to the reaction mixture solution and heated to about 75-85 °C for about 1 hour. The overall in-solution yield of Compound 4A was about 85-90%.

Figure imgf000051_0002

¹H NMR 400 MHz (CDCI₃): δ 7.16 (s, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.82 (d, J

=8.7 Hz, 2H), 5.23 (s, 2H), 4.93 (s, br, 2H), 4.23 (q, J = 7.1 , 2H), 3.76 (s, 3H), 1.26 (t,

J = 7.1 Hz, 3H); ¹³C NMR 400 MHz (CDCI₃): δ 160.9, 159.2, 139.0, 128.6, 128.5,

114.0, 101.8, 59.5, 55.2, 50.1 , 14.4.

Compound 5AK:

Figure imgf000052_0001

4A 5AK

1 -ethyl-3,7-dihydro-7-F(4-methoxyphenyl)methvπ-1 H-Purine-2.6- dione potassium salt

The reaction mixture containing Compound 4A in ethanol (obtained above) was added to diglyme and distilled under vacuum to remove the ethanol. After being cooled to room temperature, Λ/-ethylurethane (about 1.2 equiv.) was added and the

reaction mixture was heated to about 110-120 °C. A solution of potassium t-butoxide

(2.2 equiv.) in diglyme was added to the hot solution. The reaction mixture was cooled to room temperature. THF was added to precipitate additional product, which was filtered and washed to provide Compound Salt 5AK in 55-65% overall yield. The wet cake can be used as such for conversion to Compound 6A.

¹H NMR (DMSO-de, 400 MHz): δ 7.73 (s, 1H) 7.31 (d, J = 8.6 Hz , 2H) 6.86 (d,

J = 8.6 Hz, 2H) 5.24 (s, 1 H) 3.88 (q, J = 6.8 Hz, 2H) 3.71 (s, 3H) 1.07 (t, J = 6.8 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 161.1 , 159.0, 158.4, 157.2, 141.4, 131.0,

129.5, 114.1 , 105.6, 55.4, 48.2, 34.4, 14.3.

Optional Neutralization of Compound Salt 5AK to Compound 5A: Compound 5A: 1-ethyl-3,7-dihvdro-7-r(4-methoχyphenyl,methvπ-1 H-Purine-2,6- dione

The wet cake filtered solid of Compound Salt 5AK (obtained above) was suspended in water and then acidified to a pH of about 5 using glacial acetic acid. The resulting slurry was filtered to obtain the neutralized product, which was then washed with water and dried. The overall isolated yield of neutralized Compound 5A from Compound 1 A was about 45-55%. Spectroscopic data for neutralized Compound 5A was identical to that of Compound Salt 5AK.

Compound 6A:

3-r2-(acetyloxy,ethvn-1-ethyl-3,7-dihvdro-7-r(4- methoxyphenyl,methvπ-1H-purine-2,6-dione

To the wet cake filtered solid of Compound Salt 5AK (obtained above) were added tetrabutylammonium bromide (about 0.05 mole) and 2-bromoethyl acetate

(about 1.2 moles) in THF. After being heated to reflux for about 2 hours, part of the THF was distilled off, and isopropyl alcohol was added to the reaction mixture. The reaction mixture was then concentrated under reduced pressure and cooled to around room temperature. Water was added to precipitate the product. After being cooled to about 0-5 °C for about a few hours, the product was isolated by filtration. The wet cake was washed with aqueous isopropyl alcohol (about 30% in water), and dried under vacuum to afford Compound 6A as a pale yellow solid in about a 45- 55% overall yield (based on Compound 1A). The crude product may be purified further by decolorizing with Darco in methanol, followed by filtration and concentration to afford crystalline Compound 6A.

¹H NMR (CDCI3 , 400 MHz): δ 7.54 (s, 1 H) 7.32 (d, J = 8.6 Hz, 2H) 6.90 (d, J =

8.6 Hz, 2H) 5.43 (s, 2H) 4.41 (m, 2H) 4.38 (m, 2H) 4.10 (q, J = 7.2 Hz, 2H) 3.79 (s,

3H) 1.96 (s, 3H) 1.25 (t, J = 7.2 Hz, 3H); ¹³C NMR (CDCI₃ , 100 MHz): δ 171.1 ,

160.2, 155.3, 151.4, 148.9, 140.9, 130.1 , 127.7, 114.8, 107.5, 61.7, 55.6, 50.2, 42.4, 36.9, 21.2, 13.6.

After Optional Neutralization of Compound Salt 5AK to Compound 5A:

Compound 6A:

3-r2-(acetyloxy.ethvπ-1-ethyl-3,7-dihvdro-7-r.4- methoxyphenyl)methyn-1H-purine-2,6-dione

Acetonitrile was added to a mixture of Compound 5A (about 1.0 mole), anhydrous potassium carbonate (about 1.5 moles) and tetrabutylammonium hydrogen sulfate (about 0.05 mole). 2-bromoethyl acetate (about 1.5 moles) was added in three separate portions (0.72 mole in the beginning, another 0.45 mole after about 2 hours of reaction, and then the remaining 0.33 mole after about another

1 hour of reaction) during the course of the reaction at about 80-85 °C. The total reaction time was about 7 hours. The reaction mixture was cooled to about room temperature and filtered. The filtrate was concentrated. Aqueous isopropanol was added to crystallize the product. The product was filtered, washed with aqueous isopropanol, and dried to provide Compound 6A in about a 75-80% yield. Compound 7A: 8-bromo-1 -ethyl-3-r2-(acetyloxy)ethvπ-3,7-dihvdro-7-r(3-bromo-4- methoxyphenyl)methvπ-1 – -Purine-2,6-dione

Compound 6A (about 1 mole) and NBS (about 2.8 moles) were dissolved in

dry acetonitrile and agitated at about 15-20 °C. To this reaction mixture, a solution of

sulfuric acid (about 0.03 mol) in acetonitrile was added, while maintaining the

reaction temperature below about 25 °C. The reaction mixture was agitated at about

20-25 °C for about 12-15 hours until complete consumption of the starting material

was indicated. The reaction mixture was cooled to about 0-5 °C and a cold (about 5-

10 °C) aqueous solution of sodium sulfite was added, keeping the temperature below

about 10 °C. The reaction was agitated for about 2 hours at about 0-10 °C, and then

filtered. The isolated cake was washed with water, followed by methanol, then dried under a vacuum to obtain Compound 7A in about an 85% yield.

Figure imgf000053_0001

¹H NMR (CDCIs, 400 MHz): D 7.60 (d, J=2.0 Hz, 1H), 7.35 (dd, J=8.4 Hz, 2.0 Hz, 1 H), 6.83 (d, J=8.4 Hz, 1 H), 5.43 (s, 2H), 4.35 (m, 4H), 4.05 (q, J=7.0 Hz, 2H), 3.85 (s, 3H), 1.96 (s, 3H), 1.23 (t, J=7.0 Hz, 3H); ¹³C NMR (CDCI₃, 100 MHz): D 171.0, 156.2, 154.2, 150.8, 148.2, 138.3, 128.9, 128.7, 127.5, 112.1 , 112.0, 109.1 , 61.5, 56.5, 49.3, 42.5, 37.0, 21.0, 13.3. MS (ES) m/e 545.2 (M+H)⁺.

Compound 13A:

1-ethyl-3.7-dihvdro-8-r(1f?,2 )-(hvdroxycvclopentyl)amino1-3-(2- hvdroxyethvπ-7-r(3-bromo-4-methoxyphenvhmethvπ-1/–purine-2.6-dione

Compound 7A (about 1 mole) was combined with (R,R)-2-amino-1- cyclopentanol hydrochloride (Compound 8A, about 1.2 moles) and sodium bicarbonate (about 3 moles). To this reaction mixture was added N,N- dimethylacetamide (“DMA”), and the reaction mixture was agitated at about 135-140 °C for about 15-17 hours until complete consumption of the starting material was

indicated.

Figure imgf000053_0002

Compound 9A is an intermediate that is formed, but not isolated, from the

reaction mixture. The reaction mixture was then cooled to about 45-50 °C, and

tetrabutylammonium hydroxide (about 0.05 moles of about a 40% solution in water) was charged therein, followed by methanol. The reaction mixture was refluxed at

about 80-85 °C for about 8-9 hours until complete deprotection of the acetate group

was indicated. The reaction mixture was cooled to about 40-45 °C and concentrated

under vacuum. The pH of the reaction mixture was adjusted to about 5-6 with dilute

acetic acid, and the reaction mixture was heated to about 55-65 °C, and seeded with

a small amount of Compound 13A. The reaction mixture was then cooled to about

30-35 °C over a period of about 2 hours, and water was added over a period of

about 1 hour. The reaction mixture was further cooled to about 0-5 °C over a period

of about 1 hour, and agitated at that temperature for about 4 hours. The Compound 13A product was isolated by filtration, washed with water and dried to provide about an 85-90% yield.

Figure imgf000054_0002

9A 13A DASANTAFIL

¹H NMR (CDCI₃, 400 MHz): D 7.47 (d, J=2.1 Hz, 1 H), 7.18 (dd, J=8.4 Hz, 2.0 Hz, 1 H), 6.87 (d, J=8.4 Hz, 1H), 5.23 (s, 2H), 5.01 (s, 1 H), 4.22 (m, 2H), 4.15 (m, 1H), 4.05 (q, J=7.0 Hz, 2H), 3.93 (m, 3H), 3.88 (s, 3H), 3.77 (m, 1H), 2.95 (m, 1H), 2.15 (m, 1H), 2.05 (m, 1 H), 1.60-1.80 (m, 4H), 1.35 (m, 1 H), 1.23 (t, J=7.0 Hz, 3H); ¹³C NMR (CDCI₃, 100 MHz): D 156.2, 154.0, 153.5, 151.8, 148.3, 132.6, 129.1 , 127.9, 112.5, 103.2, 79.5, 77.8, 63.2, 61.3, 56.7, 46.5, 45.9, 36.8, 32.9, 31.5, 21.4, 13.8. MS (ES) m/e 523.4 (M+H)⁺. Micronization

INTERPRETATION

¹H NMR (CDCI₃, 400 MHz): DELTA

7.47 (d, J=2.1 Hz, 1 H), SANDWICHED AROM H BETWEEN BROMO AND -CH2-PY RING

7.18 (dd, J=8.4 Hz, 2.0 Hz, 1 H), AROM H ORTHO TO -CH2-PH RING AND PARA TO BROMO

6.87 (d, J=8.4 Hz, 1H), AROM H ORTHO TO O ATOM OF PH RING

5.23 (s, 2H), CH2 OF N-CH2-PH RING

5.01 (s, 1 H), OH OR NH 1H OUT OF 3 NOS

4.22 (m, 2H), OH OR NH 2H OUT OF 3 NOS

4.15 (m, 1H), –NCH2CH2OH 1H OUT OF 4 NOS

4.05 (q, J=7.0 Hz, 2H), CH2 OF NCH2 CH3

3.93 (m, 3H), —NCH2CH2OH 3H OUT OF 4 NOS

3.88 (s, 3H), -OCH3

3.77 (m, 1H), OH-CH OF CYCLOPENTANE RING

2.95 (m, 1H),NH-CH OF CYCLOPENTANE RING

2.15 (m, 1H),

2.05 (m, 1 H), 1H ON CYCLOPENTANE RING

1.60-1.80 (m, 4H), 4H ON CYCLOPENTANE RING

1.35 (m, 1 H), 1 H PARA TO SUBS IN CYCLOPENTANE RING

1.23 (t, J=7.0 Hz, 3H) –NCH2 CH3

………………………..

shark

DASANTAFIL

Links

REFERENCES

1 WANG Y ET AL: “DESIGN AND SYNTHESIS OF XANTHINE ANALOGUES AS POTENT AND SELECTIVE PDE5 INHIBITORS” BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 12, no. 21, 2002, pages 3149-3152, XP009014973 ISSN: 0960-894X

2. Peter K. Bridson and Xiaodong Wang in 1 -Substituted Xanthines, Synthesis, 855 (July, 1995)

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US2011142956	6-17-2011	USE OF A PDE 5 INHIBITOR FOR TREATING AND PREVENTING HYPOPIGMENTARY DISORDERS
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US2008051408	2-29-2008	Use of a Pde 5 Inhibitor for Treating and Preventing Hypopigmentary Disorders
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US7268141	9-12-2007	Xanthine phosphodiesterase V inhibitors
US2007122355	5-32-2007	RAPIDLY ABSORBING ORAL FORMULATIONS OF PDE 5 INHIBITORS


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US2007031349	2-9-2007	Rapidly absorbing oral formulations of PDE 5 inhibitors
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BELINOSTAT, FAST TRACK, ORPHAN DRUG, A hydroxamate-type inhibitor of histone deacetylase.

phase 2, Uncategorized Comments Off

Jan 232014

Belinostat (PXD101)

PHASE 2, FAST TRACK FDA , ORPHAN STATUS

PDX101
PX 105684
PXD-101
PXD101
UNII-F4H96P17NZ

Belinostat (PXD101) is a novel HDAC inhibitor with IC50 of 27 nM, with activity demonstrated in cisplatin-resistant tumors.

CLINICAL TRIALS…http://clinicaltrials.gov/search/intervention=Belinostat+OR+PXD101

Belinostat inhibits the growth of tumor cells (A2780, HCT116, HT29, WIL, CALU-3, MCF7, PC3 and HS852) with IC50 from 0.2-0.66 μM. PD101 shows low activity in A2780/cp70 and 2780AD cells. Belinostat inhibits bladder cancer cell growth, especially in 5637 cells, which shows accumulation of G0-G1 phase, decrease in S phase, and increase in G2-M phase. Belinostat also shows enhanced tubulin acetylation in ovarian cancer cell lines. A recent study shows that Belinostat activates protein kinase A in a TGF-β signaling-dependent mechanism and decreases survivin mRNA.

MW 318.07
MF	C15H14N2O4S

414864-00-9 cas no

866323-14-0

(2E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]acrylamide

A novel HDAC inhibitor

…………………………

BELINOSTAT

Belinostat (PXD101) is experimental drug candidate under development byTopoTarget for the treatment of hematological malignancies and solid tumors. It is a histone deacetylase inhibitor.[1]

A hydroxamate-type inhibitor of histone deacetylase.

NCI: A novel hydroxamic acid-type histone deacetylase (HDAC) inhibitor with antineoplastic activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cellular differentiation, and inhibiting angiogenesis. This agent may sensitize drug-resistant tumor cells to other antineoplastic agents, possibly through a mechanism involving the down-regulation of thymidylate synthase

In 2007 preliminary results were released from the Phase II clinical trial of intravenous belinostat in combination with carboplatin and paclitaxel for relapsedovarian cancer.[2] Final results in late 2009 of a phase II trial for T cell lymphomawere encouraging.[3] Belinostat has been granted orphan drug and fast trackdesignation by the FDA.[4]

The study of inhibitors of histone deacetylases indicates that these enzymes play an important role in cell proliferation and differentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al., 1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida and Beppu, 1988), reverts the transformed phenotype of different cell lines, and induces differentiation of Friend leukaemia cells and others (Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibit cell growth, induce terminal differentiation, and prevent the formation of tumours in mice (Finnin et al., 1999).

Trichostatin A (TSA)

Suberoylanilide Hydroxamic Acid (SAHA)

Cell cycle arrest by TSA correlates with an increased expression of gelsolin (Hoshikawa et al., 1994), an actin regulatory protein that is down regulated in malignant breast cancer (Mielnicki et al., 1999). Similar effects on cell cycle and differentiation have been observed with a number of deacetylase inhibitors (Kim et al., 1999). Trichostatin A has also been reported to be useful in the treatment of fibrosis, e.g., liver fibrosis and liver cirrhosis. See, e.g., Geerts et al., 1998.

Recently, certain compounds that induce differentiation have been reported to inhibit histone deacetylases. Several experimental antitumour compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been reported to act, at least in part, by inhibiting histone deacetylase (see, e.g., Yoshida et al., 1990; Richon et al., 1998; Kijima et al., 1993). Additionally, diallyl sulfide and related molecules (see, e.g., Lea et al., 1999), oxamflatin (see, e.g., Kim et al., 1999), MS-27-275, a synthetic benzamide derivative (see, e.g., Saito et al., 1999; Suzuki et al., 1999; note that MS-27-275 was later re-named as MS-275), butyrate derivatives (see, e.g., Lea and Tulsyan, 1995), FR901228 (see, e.g., Nokajima et al., 1998), depudecin (see, e.g., Kwon et al., 1998), and m-carboxycinnamic acid bishydroxamide (see, e.g., Richon et al., 1998) have been reported to inhibit histone deacetylases. In vitro, some of these compounds are reported to inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases, and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines (see, e.g., Richon et al, 1996; Kim et al., 1999; Yoshida et al., 1995; Yoshida & Beppu, 1988). In vivo, phenybutyrate is reported to be effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid (see, e.g., Warrell et al., 1998). SAHA is reported to be effective in preventing the formation of mammary tumours in rats, and lung tumours in mice (see, e.g., Desai et al., 1999).

The clear involvement of HDACs in the control of cell proliferation and differentiation suggest that aberrant HDAC activity may play a role in cancer. The most direct demonstration that deacetylases contribute to cancer development comes from the analysis of different acute promyelocytic leukaemias (APL). In most APL patients, a translocation of chromosomes 15 and 17 (t(15;17)) results in the expression of a fusion protein containing the N-terminal portion of PML gene product linked to most of RARσ (retinoic acid receptor). In some cases, a different translocation (t(11 ;17)) causes the fusion between the zinc finger protein PLZF and RARα. In the absence of ligand, the wild type RARα represses target genes by tethering HDAC repressor complexes to the promoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARα and displaces the repressor complex, allowing expression of genes implicated in myeloid differentiation. The RARα fusion proteins occurring in APL patients are no longer responsive to physiological levels of RA and they interfere with the expression of the RA- inducible genes that promote myeloid differentiation. This results in a clonal expansion of promyelocytic cells and development of leukaemia. In vitro experiments have shown that TSA is capable of restoring RA-responsiveness to the fusion RARα proteins and of allowing myeloid differentiation. These results establish a link between HDACs and oncogenesis and suggest that HDACs are potential targets for pharmaceutical intervention in APL patients. (See, for example, Kitamura et al., 2000; David et al., 1998; Lin et al., 1998).

BELINOSTAT

Furthermore, different lines of evidence suggest that HDACs may be important therapeutic targets in other types of cancer. Cell lines derived from many different cancers (prostate, coloreetal, breast, neuronal, hepatic) are induced to differentiate by HDAC inhibitors (Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have been studied in animal models of cancer. They reduce tumour growth and prolong the lifespan of mice bearing different types of transplanted tumours, including melanoma, leukaemia, colon, lung and gastric carcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).

Psoriasis is a common chronic disfiguring skin disease which is characterised by well-demarcated, red, hardened scaly plaques: these may be limited or widespread. The prevalence rate of psoriasis is approximately 2%, i.e., 12.5 million sufferers in the triad countries (US/Europe/Japan). While the disease is rarely fatal, it clearly has serious detrimental effects upon the quality of life of the patient: this is further compounded by the lack of effective therapies. Present treatments are either ineffective, cosmetically unacceptable, or possess undesired side effects. There is therefore a large unmet clinical need for effective and safe drugs for this condition. Psoriasis is a disease of complex etiology. Whilst there is clearly a genetic component, with a number of gene loci being involved, there are also undefined environmental triggers. Whatever the ultimate cause of psoriasis, at the cellular level, it is characterised by local T-cell mediated inflammation, by keratinocyte hyperproliferation, and by localised angiogenesis. These are all processes in which histone deacetylases have been implicated (see, e.g., Saunders et al., 1999; Bernhard et al, 1999; Takahashi et al, 1996; Kim et al , 2001 ). Therefore HDAC inhibitors may be of use in therapy for psoriasis. Candidate drugs may be screened, for example, using proliferation assays with T-cells and/or keratinocytes.

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PXD101/Belinostat®

(E)-N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide, also known as PXD101 and Belinostat®, shown below, is a well known histone deacetylate (HDAC) inhibitor. It is being developed for treatment of a range of disorders mediated by HDAC, including proliferative conditions (such as cancer and psoriasis), malaria, etc.

PXD101 was first described in WO 02/30879 A2. That document describes a multi-step method of synthesis which may conveniently be illustrated by the following scheme.

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GENERAL SYNTHESIS

WO2002030879A2

IGNORE 10

ENTRY 45 IS BELINOSTAT

Scheme 1

By using amines instead of aniline, the corresponding products may be obtained. The use of aniline, 4-methoxyaniline, 4-methylaniline, 4-bromoaniline, 4-chloroaniline, 4-benzylamine, and 4-phenethyamine, among others, is described in the Examples below.

In another method, a suitable amino acid (e.g., ω-amino acid) having a protected carboxylic acid (e.g., as an ester) and an unprotected amino group is reacted with a sulfonyl chloride compound (e.g., RSO₂CI) to give the corresponding sulfonamide having a protected carboxylic acid. The protected carboxylic acid is then deprotected using base to give the free carboxylic acid, which is then reacted with, for example, hydroxylamine 2-chlorotrityl resin followed by acid (e.g., trifluoroacetic acid), to give the desired carbamic acid.

One example of this approach is illustrated below, in Scheme 2, wherein the reaction conditions are as follows: (i) RSO₂CI, pyridine, DCM, room temperature, 12 hours; (ii) 1 M LiOH or 1 M NaOH, dioxane, room temperature, 3-48 hours; (iii) hydroxylamine 2-chlorotrityl resin, HOAt, HATU, DIPEA, DCM, room temperature, 16 hours; and (iv) TFA/DCM (5:95, v/v), room temperature, 1.5 hours.

Scheme 2

Additional methods for the synthesis of compounds of the present invention are illustrated below and are exemplified in the examples below.

Scheme 3A

Scheme 3B

Scheme 4

Scheme 8

Scheme 9

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SYNTHESIS

WO2002030879A2

Example 1

3-Formylbenzenesulfonic acid, sodium salt (1)

Oleum (5 ml) was placed in a reaction vessel and benzaldehyde (2.00 g, 18.84 mmol) was slowly added not exceeding the temperature of the reaction mixture more than 30°C. The obtained solution was stirred at 40°C for ten hours and at ambient temperature overnight. The reaction mixture was poured into ice and extracted with ethyl acetate. The aqueous phase was treated with CaC0₃ until the evolution of C0₂ ceased (pH~6-7), then the precipitated CaSO₄was filtered off and washed with water. The filtrate was treated with Na₂CO₃ until the pH of the reaction medium increased to pH 8, obtained CaCO3 was filtered off and water solution was evaporated in vacuum. The residue was washed with methanol, the washings were evaporated and the residue was dried in desiccator over P₂Oβ affording the title compound (2.00 g, 51%). ¹H NMR (D₂0), δ: 7.56-8.40 (4H, m); 10.04 ppm (1 H, s).

Example 2 3-(3-Sulfophenyl)acrylic acid methyl ester, sodium salt (2)

Sodium salt of 3-formylbenzenesulfonic acid (1) (1.00 g, 4.80 mmol), potassium carbonate (1.32 g, 9.56 mmol), trimethyl phosphonoacetate (1.05 g, 5.77 mmol) and water (2 ml) were stirred at ambient temperature for 30 min., precipitated solid was filtered and washed with methanol. The filtrate was evaporated and the title compound (2) was obtained as a white solid (0.70 g, 55%). ¹H NMR (DMSO- d_βl HMDSO), δ: 3.68 (3H, s); 6.51 (1 H, d, J=16.0 Hz); 7.30-7.88 (5H, m).

Example 3 3-(3-Chlorosulfonylphenyl)acrylic acid methyl ester (3)

To the sodium salt of 3-(3-sulfophenyl)acrylic acid methyl ester (2) (0.670 g, 2.53 mmol) benzene (2 ml), thionyl chloride (1.508 g, 0.9 ml, 12.67 mmol) and 3 drops of dimethylformamide were added and the resultant suspension was stirred at reflux for one hour. The reaction mixture was evaporated, the residue was dissolved in benzene (3 ml), filtered and the filtrate was evaporated to give the title compound (0.6’40 g, 97%).

Example 4 3-(3-Phenylsulfamoylphenyl)acrylic acid methyl ester (4a)

A solution of 3-(3-chlorosulfonylphenyl)acrylic acid methyl ester (3) (0.640 g, 2.45 mmol) in dichloromethane (2 ml) was added to a mixture of aniline (0.465 g, 4.99 mmol) and pyridine (1 ml), and the resultant solution was stirred at 50°C for one hour. The reaction mixture was evaporated and the residue was partitioned between ethyl acetate and 10% HCI. The organic layer was washed successively with water, saturated NaCl, and dried (Na₂S0 ). The solvent was removed and the residue was chromatographed on silica gel with chloroform-ethyl acetate (7:1 , v/v) as eluent. The obtained product was washed with diethyl ether to give the title compound (0.226 g, 29%). ¹H NMR (CDCI₃, HMDSO), δ: 3.72 (3H, s); 6.34 (1H, d, J=16.0 Hz); 6.68 (1 H, br s); 6.92-7.89 (10H, m).

Example 5 3-(3-Phenylsulfamoylphenyl)acrylic acid (5a)

3-(3-Phenylsulfamoylphenyl)acrylic acid methyl ester (4a) (0.220 g, 0.69 mmol) was dissolved in methanol (3 ml), 1N NaOH (2.08 ml, 2.08 mmol) was added and the resultant solution was stirred at ambient temperature overnight. The reaction mixture was partitioned between ethyl acetate and water. The aqueous layer was acidified with 10% HCI and stirred for 30 min. The precipitated solid was filtered, washed with water and dried in desiccator over P₂Os to give the title compound as a white solid (0.173 g, 82%). Example 6 3-(3-Phenylsulfamoylphenyl)acryloyl chloride (6a)

To a suspension of 3-(3-phenylsulfamoylphenyl)acrylic acid (5a) (0.173 g, 0.57 mmol) in dichloromethane (2.3 ml) oxalyl chloride (0.17 ml, 1.95 mmol) and one drop of dimethylformamide were added. The reaction mixture was stirred at 40°C for one hour and concentrated under reduced pressure to give crude title compound (0.185 g).

Example 7

N-Hydroxy-3-(3-phenylsulfamoylphenyl)acrylamide (7a) (PX105684) BELINOSTAT

To a suspension of hydroxylamine hydrochloride (0.200 g, 2.87 mmol) in tetrahydrofuran (3.5 ml) a saturated NaHCOβ solution (2.5 ml) was added and the resultant mixture was stirred at ambient temperature for 10 min. To the reaction mixture a 3-(3-phenylsulfamoylphenyl)acryloyl chloride (6a) (0.185 g) solution in tetrahydrofuran (2.3 ml) was added and stirred at ambient temperature for one hour. The reaction mixture was partitioned between ethyl acetate and 2N HCI. The organic layer was washed successively with water and saturated NaCl, the solvent was removed and the residue was washed with acetonitrile and diethyl ether.

The title compound was obtained as a white solid (0.066 g, 36%), m.p. 172°C. BELINOSTAT

¹H NMR (DMSO-d6, HMDSO), δ: 6.49 (1 H, d, J=16.0 Hz); 7.18-8.05 (10H, m); 9.16 (1 H, br s); 10.34 (1 H, s); 10.85 ppm (1 H, br s).

HPLC analysis on Symmetry C₁₈column: impurities 4% (column size 3.9×150 mm; mobile phase acetonitrile – 0.1 M phosphate buffer (pH 2.5), 40:60; sample concentration 1 mg/ml; flow rate 0.8 ml/ min; detector UV 220 nm).

Anal. Calcd for C₁₅Hι₄N₂0₄S, %: C 56.59, H 4.43, N 8.80. Found, %: C 56.28, H 4.44, N 8.56.

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SYNTHESIS

US20100286279

Figure US20100286279A1-20101111-C00034

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SYNTHESIS AND SPECTRAL DATA

Journal of Medicinal Chemistry, 2011 , vol. 54, 13 pg. 4694 – 4720

(E)-N-Hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (28, belinostat, PXD101).

http://pubs.acs.org/doi/full/10.1021/jm2003552

http://pubs.acs.org/doi/suppl/10.1021/jm2003552/suppl_file/jm2003552_si_001.pdf

The methyl ester (27) (8.0 g) was prepared according to reported synthetic route,

(Watkins, C. J.; Romero-Martin, M.-R.; Moore, K. G.; Ritchie, J.; Finn, P. W.; Kalvinsh, I.;
Loza, E.; Dikvoska, K.; Gailite, V.; Vorona, M.; Piskunova, I.; Starchenkov, I.; Harris, C. J.;
Duffy, J. E. S. Carbamic acid compounds comprising a sulfonamide linkage as HDAC
inhibitors. PCT Int. Appl. WO200230879A2, April 18, 2002.)
but using procedure D (Experimental Section) or method described for 26 to convert the methyl ester to crude
hydroxamic acid which was further purified by chromatography (silica, MeOH/DCM = 1:10) to
afford 28 (PXD101) as off-white or pale yellow powder (2.5 g, 31%).

LC–MS m/z 319.0 ([M +H]+).

1H NMR (DMSO-d6)  12–9 (very broad, 2H), 7.90 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.70 (d, J

= 7.8 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 15.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 2H), 7.08 (d,
J = 7.8 Hz, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.50 (d, J = 15.8 Hz, 1H);

13C NMR (DMSO-d6)  162.1,
140.6, 138.0, 136.5, 135.9, 131.8, 130.0, 129.2, 127.1, 124.8, 124.1, 121.3, 120.4.

Anal.
(C15H14N2O4S) C, H, N

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SYNTHESIS

WO2009040517A2

PXDIOI / Belinostat®

PXD101 was first described in WO 02/30879 A2. That document describes a multi-step method of synthesis which may conveniently be illustrated by the following scheme.

Scheme 1

Not isolated

ed on (A)

on (D)

d on (H)

There is a need for alternative methods for the synthesis of PXD101 and related compounds for example, methods which are simpler and/or employ fewer steps and/or permit higher yields and/or higher purity product.

Scheme 5

DMAP, toluene

Synthesis 1 3-Bromo-N-phenyl-benzenesulfonamide (3)

To a 30 gallon (-136 L) reactor was charged aniline (2) (4.01 kg; 93.13 g/mol; 43 mol), toluene (25 L), and 4-(dimethylamino)pyridine (DMAP) (12 g), and the mixture was heated to 50-60⁰C. 3-Bromobenzenesulfonyl chloride (1) (5 kg; 255.52 g/mol; 19.6 mol) was charged into the reactor over 30 minutes at 50-60⁰C and progress of the reaction was monitored by HPLC. After 19 hours, toluene (5 L) was added due to losses overnight through the vent line and the reaction was deemed to be complete with no compound (1) being detected by HPLC. The reaction mixture was diluted with toluene (10 L) and then quenched with 2 M aqueous hydrochloric acid (20 L). The organic and aqueous layers were separated, the aqueous layer was discarded, and the organic layer was washed with water (20 L), and then 5% (w/w) sodium bicarbonate solution (20 L), while maintaining the batch temperature at 45-55°C. The batch was then used in the next synthesis.

Synthesis 2 (E)-3-(3-Phenylsulfamoyl-phenyl)-acrylic acid ethyl ester (5)

To the batch containing 3-bromo-N-phenyl-benzenesulfonamide (3) (the treated organic layer obtained in the previous synthesis) was added triethylamine (2.97 kg; 101.19 g/mol; 29.4 mol), tri(o-tolyl)phosphine (119 g; 304.37 g/mol; 0.4 mol), and palladium (II) acetate (44 g; 224.51 g/mol; 0.2 mol), and the resulting mixture was degassed four times with a vacuum/nitrogen purge at 45-55°C. Catalytic palladium (0) was formed in situ. The batch was then heated to 80-90⁰C and ethyl acrylate (4) (2.16 kg; 100.12 g/mol; 21.6 mol) was slowly added over 2.75 hours. The batch was sampled after a further 2 hours and was deemed to be complete with no compound (3) being detected by HPLC. The batch was cooled to 45-55°C and for convenience was left at this temperature overnight.

The batch was then reduced in volume under vacuum to 20-25 L, at a batch temperature of 45-55°C, and ethyl acetate (20 L) was added. The batch was filtered and the residue washed with ethyl acetate (3.5 L). The residue was discarded and the filtrates were sent to a 100 gallon (-454 L) reactor, which had been pre-heated to 60⁰C. The 30 gallon (-136 L) reactor was then cleaned to remove any residual Pd, while the batch in the 100 gallon (-454 L) reactor was washed with 2 M aqueous hydrochloric acid and water at 45-55°C. Once the washes were complete and the 30 gallon (-136 L) reactor was clean, the batch was transferred from the 100 gallon (-454 L) reactor back to the 30 gallon (-136 L) reactor and the solvent was swapped under vacuum from ethyl acetate/toluene to toluene while maintaining a batch temperature of 45-55°C (the volume was reduced to 20-25 L). At this point, the batch had precipitated and heptanes (10 L) were added to re-dissolve it. The batch was then cooled to 0-10⁰C and held at this temperature over the weekend in order to precipitate the product. The batch was filtered and the residue was washed with heptanes (5 L). A sample of the wet-cake was taken for Pd analysis. The Pd content of the crude product (5) was determined to be 12.9 ppm.

The wet-cake was then charged back into the 30 gallon (-136 L) reactor along with ethyl acetate (50 L) and heated to 40-50⁰C in order to obtain a solution. A sparkler filter loaded with 12 impregnated Darco G60® carbon pads was then connected to the reactor and the solution was pumped around in a loop through the sparkler filter. After 1 hour, a sample was taken and evaporated to dryness and analysed for Pd content. The amount of Pd was found to be 1.4 ppm. A second sample was taken after 2 hours and evaporated to dryness and analysed for Pd content. The amount of Pd had been reduced to 0.6 ppm. The batch was blown back into the reactor and held at 40-50⁰C overnight before the solvent was swapped under vacuum from ethyl acetate to toluene while maintaining a batch temperature of 45-55°C (the volume was reduced to 20-25 L). At this point, the batch had precipitated and heptanes (10 L) were added to re-dissolve it and the batch was cooled to 0-10⁰C and held at this temperature overnight in order to precipitate the product. The batch was filtered and the residue was washed with heptanes (5 L). The filtrate was discarded and the residue was dried at 45-55°C under vacuum for 25 hours. A first lot of the title compound (5) was obtained as an off-white solid (4.48 kg, 69% overall yield from 3-bromobenzenesulfonyl chloride (1)) with a Pd content of 0.4 ppm and a purity of 99.22% (AUC) by HPLC.

Synthesis 3 (E)-3-(3-Phenylsulfamoyl-phenyl)-acrvlic acid (6)

To the 30 gallon (-136 L) reactor was charged the (E)-3-(3-phenylsulfamoyl-phenyl)- acrylic acid ethyl ester (5) (4.48 kg; 331.39 g/mol; 13.5 mol) along with 2 M aqueous sodium hydroxide (17.76 L; -35 mol). The mixture was heated to 40-50°C and held at this temperature for 2 hours before sampling, at which point the reaction was deemed to be complete with no compound (5) being detected by HPLC. The batch was adjusted to pH 2.2 using 1 M aqueous hydrochloric acid while maintaining the batch temperature between 40-50⁰C. The product had precipitated and the batch was cooled to 20-30⁰C and held at this temperature for 1 hour before filtering and washing the cake with water (8.9 L). The filtrate was discarded. The batch was allowed to condition on the filter overnight before being charged back into the reactor and slurried in water (44.4 L) at 40-50⁰C for 2 hours. The batch was cooled to 15-20°C, held for 1 hour, and then filtered and the residue washed with water (8.9 L). The filtrate was discarded. The crude title compound (6) was transferred to an oven for drying at 45-55°C under vacuum with a slight nitrogen bleed for 5 days (this was done for convenience) to give a white solid (3.93 kg, 97% yield). The moisture content of the crude material was measured using Karl Fischer (KF) titration and found to be <0.1% (w/w). To the 30 gallon (-136 L) reactor was charged the crude compound (6) along with acetonitrile (47.2 L). The batch was heated to reflux (about 80°C) and held at reflux for 2 hours before cooling to 0-10°C and holding at this temperature overnight in order to precipitate the product. The batch was filtered and the residue was washed with cold acetonitrile (7.9 L). The filtrate was discarded and the residue was dried under vacuum at 45-55°C for 21.5 hours. The title compound (6) was obtained as a fluffy white solid (3.37 kg, 84% yield with respect to compound (5)) with a purity of 99.89% (AUC) by HPLC.

Synthesis 4 (E)-N-Hvdroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide (PXD101) BELINOSTAT

To the 30 gallon (-136 L) reactor was charged (E)-3-(3-phenylsulfamoyl-phenyl)-acrylic acid (6) (3.37 kg; 303.34 g/mol; 11.1 mol) and a pre-mixed solution of 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in isopropyl acetate (IPAc) (27 g in 30 L; 152.24 g/mol; 0.18 mol). The slurry was stirred and thionyl chloride (SOCI₂) (960 mL; density ~1.631 g/mL; 118.97 g/mol; -13 mol) was added to the reaction mixture and the batch was stirred at 20-30⁰C overnight. After 18.5 hours, the batch was sampled and deemed to be complete with no compound (6) being detected by HPLC. The resulting solution was transferred to a 100 L Schott reactor for temporary storage while the

30 gallon (-136 L) reactor was rinsed with isopropyl acetate (IPAc) and water. Deionized water (28.9 L) was then added to the 30 gallon (-136 L) reactor followed by 50% (w/w) hydroxylamine (6.57 L; -1.078 g/mL; 33.03 g/mol; -214 mol) and another charge of deionized water (1.66 L) to rinse the lines free of hydroxylamine to make a 10% (w/w) hydroxylamine solution. Tetrahydrofuran (THF) (6.64 L) was then charged to the

30 gallon (-136 L) reactor and the mixture was stirred and cooled to 0-10⁰C. The acid chloride solution (from the 100 L Schott reactor) was then slowly charged into the hydroxylamine solution over 1 hour maintaining a batch temperature of 0-10°C during the addition. The batch was then allowed to warm to 20-30⁰C. The aqueous layer was separated and discarded. The organic layer was then reduced in volume under vacuum while maintaining a batch temperature of less than 30⁰C. The intention was to distill out 10-13 L of solvent, but this level was overshot. A larger volume of isopropyl acetate (IPAc) (16.6 L) was added and about 6 L of solvent was distilled out. The batch had precipitated and heptanes (24.9 L) were added and the batch was held at 20-30°C overnight. The batch was filtered and the residue was washed with heptanes (6.64 L). The filtrate was discarded and the residue was dried at 45-55°C under vacuum with a slight nitrogen bleed over the weekend. The title compound (PXD101) was obtained as a light orange solid (3.11 kg, 89% yield with respect to compound (6)) with a purity of 99.25% (AUC) by HPLC.

The title compound (PXD101) (1.2 kg, 3.77 mol) was dissolved in 8 volumes of 1:1 (EtOH/water) at 60⁰C. Sodium bicarbonate (15.8 g, 5 mol%) was added to the solution. Water (HPLC grade) was then added at a rate of 65 mL/min while keeping the internal temperature >57°C. After water (6.6 L) had been added, crystals started to form and the water addition was stopped. The reaction mixture was then cooled at a rate of 10°C/90 min to a temperature of 0-10^cC and then stirred at ambient temperature overnight. The crystals were then filtered and collected. The filter cake was washed by slurrying in water (2 x 1.2 L) and then dried in an oven at 45°C for 60 hours with a slight nitrogen bleed. 1.048 kg (87% recovery) of a light orange solid was recovered. Microscopy and XRPD data showed a conglomerate of irregularly shaped birefringant crystalline particles. The compound was found to contain 0.02% water.

As discussed above: the yield of compound (5) with respect to compound (1) was 69%. the yield of compound (6) with respect to compound (5) was 84%. the yield of PXD101 with respect to compound (6) was 89%.

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FORMULATION

WO2006120456A1

Formulation Studies

These studies demonstrate a substantial enhancement of HDACi solubility (on the order of a 500-fold increase for PXD-101) using one or more of: cyclodextrin, arginine, and meglumine. The resulting compositions are stable and can be diluted to the desired target concentration without the risk of precipitation. Furthermore, the compositions have a pH that, while higher than ideal, is acceptable for use.

UV Absorbance

The ultraviolet (UV absorbance E\ value for PXD-101 was determined by plotting a calibration curve of PXD-101 concentration in 50:50 methanol/water at the λ_max for the material, 269 nm. Using this method, the E¹i value was determined as 715.7.

Methanol/water was selected as the subsequent diluting medium for solubility studies rather than neat methanol (or other organic solvent) to reduce the risk of precipitation of the cyclodextrin.

Solubility in Demineralised Water

The solubility of PXD-101 was determined to be 0.14 mg/mL for demineralised water. Solubility Enhancement with Cvclodextrins

Saturated samples of PXD-101 were prepared in aqueous solutions of two natural cyclodextrins (α-CD and γ-CD) and hydroxypropyl derivatives of the α, β and Y cyclodextrins (HP-α-CD, HP-β-CD and HP-γ-CD). All experiments were completed with cyclodextrin concentrations of 250 mg/mL, except for α-CD, where the solubility of the cyclodextrin was not sufficient to achieve this concentration. The data are summarised in the following table. HP-β-CD offers the best solubility enhancement for PXD-101.

Phase Solubility Determination of HP-β-CD

The phase solubility diagram for HP-β-CD was prepared for concentrations of cyclodextrin between 50 and 500 mg/mL (5-50% w/v). The calculated saturated solubilities of the complexed HDACi were plotted against the concentration of cyclodextrin. See Figure 1.

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Plumb, Jane A.; Finn, Paul W.; Williams, Robert J.; Bandara, Morwenna J.; Romero, M. Rosario; Watkins, Claire J.; La Thangue, Nicholas B.; Brown, Robert (2003). “Pharmacodynamic Response and Inhibition of Growth of Human Tumor Xenografts by the Novel Histone Deacetylase Inhibitor PXD101”. Molecular Cancer Therapeutics 2 (8): 721–728. PMID 12939461.
“CuraGen Corporation (CRGN) and TopoTarget A/S Announce Presentation of Belinostat Clinical Trial Results at AACR-NCI-EORTC International Conference”. October 2007.
Final Results of a Phase II Trial of Belinostat (PXD101) in Patients with Recurrent or Refractory Peripheral or Cutaneous T-Cell Lymphoma, December 2009
“Spectrum adds to cancer pipeline with $350M deal.”. February 2010.
Helvetica Chimica Acta, 2005 , vol. 88, 7 PG. 1630 – 1657, MP 172
WO2009/40517 A2, ….
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SPECTRUM

Tiny Biotech With Three Cancer Drugs Is More Alluring Takeover Bet Now
Forbes
The drug is one of Spectrum’s two drugs undergoing phase 3 clinical trials. Allergan paid Spectrum $41.5 million and will make additional payments of up to $304 million based on achieving certain milestones. So far, Raj Shrotriya, Spectrum’s chairman, …

http://www.forbes.com/sites/genemarcial/2013/07/14/tiny-biotech-with-three-cancer-drugs-is-more-alluring-takeover-bet-now/

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Copenhagen, December 10, 2013

Topotarget announces the submission of a New Drug Application (NDA) for belinostat for the treatment of relapsed or refractory (R/R) peripheral T-cell lymphoma (PTCL) to the US Food and Drug Administration (FDA). The NDA has been filed for Accelerated Approval with a request for Priority Review. Response from the FDA regarding acceptance to file is expected within 60 days from the FDA receipt date.

read all this here

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