ACH-702

7-[3(R)-(2-Aminopropan-2-yl)pyrrolidin-1-yl]-9-cyclopropyl-6-fluoro-8-methoxy-2,3,4,9-tetrahydroisothiazolo[5,4-b]quinoline-3,4-dione

(7^-7-[3-(1-AMrNO-I-METHYLETHYL)PYRROLiDiN-I-YL]-P-CYCLOPROPYL-6-FLUORO-8-METHOXY-PH-ISOTHIAZOLO[5,4-B]QUINOLINE-3,4-DIONE

(R)- 7-[3-(l-amino-l-methyl-ethyl)-pyrrolidin-l-yl]-9-cyclopropyl-6-fluoro-8-methoxy- 9H-isothiazolo[5, 4-b] -quinoline-3, 4-dione

922491-46-1 free base

922491-09-6 (hydrochloride)468.973, C21 H25 F N4 O3 S . Cl H

ACH-0139586
ACH-702

Achillion Pharmaceuticals (USA)

pre clinical

Achillion Pharmaceuticals is working on the discovery of compounds in a new subclass of quinolones, the isothiazoloquinolones. The most advanced compound is ACH-702, which is at the pre-clinical stage of development [1-3].

ACH 702

The utility of isothiazoloquinolines as pharmaceutical agents has been discussed in the literature. For example, Pinol, et al discussed the use of isothiazoloquinolines as medical bactericides in US Patent 5,087,621, including

The Proctor & Gamble Company discussed antimicrobial quinolones including the following compound:

in published application no. US 2003008894.

The use of isothiazoloquinoline compounds as TNF production inhibitors has also been discussed, for example by Sankyo Co., Ltd. in JPl 010149, which includes the following compound

Bayer Aktiengesellschaft has discussed bicycle[3.3.0]oct-7-yl containing compounds useful for treating H. pylori infections in WO 98/26768, including isothiazoloquinolines, having the general structure shown below in which Y may be sulfur joined to the carboxamide group to form a 5-membered ring

Otsuka Pharmaceutical Co., Ltd. has discussed the use of isothiazoloquinolines as antibacterial agents in JP 01193275, including the following carbamate-containing compound

Abbott Laboratories has discussed the use of isothiazoloquinolines as antineoplastic agents in US Patent No. 5,071,848 and has discussed the use of tricyclic quinolones as antibacterial agents in US 4,767,762. The Abbott compounds have hydrogen, halogen, or lower alkyl as substituents at the 6- and 8- positions of the isothiazoloquinoline core.

………………

Synthesis

WO2008021491A2

http://www.google.com/patents/WO2008021491A2?cl=en

EXAMPLE 1. SYNTHESIS OF (7^-7-[3-(1-AMrNO-I-METHYLETHYL)PYRROLiDiN-I-YL]-P-

CYCLOPROPYL-6-FLUORO-8-METHOXY-PH-ISOTHIAZOLO[5,4-B]QUINOLINE-3,4-DIONE (5). Step 1. Ethyl l-cyclopropyl-6, 7-difluoro-2-methanesulfonyl-8-methoxy-4-oxo-l,4-dihydro- quinoline-3-carboxylate (6)

Oxonβ®

MeOHZH₂O

6

Water (180 mL), followed by Oxone^® (Dupont Specialty Chemicals) (170 g, 277 mmol), is added to a suspension of 1 in MeOH (510 mL). The reaction mixture is heated with stirring at 55-60 ⁰C for 3 h. The reaction mixture is cooled to room temperature, diluted with water (40 mL), and stirred at 5 ⁰C (ice bath) for 30 min. The resulting crystals are collected by filtration, washed with water (2 x 100 mL), and dried to afford 6 (13.8 g). This material was used in the next step without further purification, mp 177-178 ⁰C. ¹H NMR (DMF-^₇): J0.62 (m, IH), 1.11 (m, 2H), 1.29 (m, IH), 1.32 (t, J_H-H = 7.0 Hz, 3H), 3.76 (s, 3H), 4.18 (m, IH), 4.21 (d, JH-F = 2.0 Hz, 3H), 4.33 (q, J_H-_H = 7.0 Hz, 2H), 7.64 (dd, J_H-F = 10.0 Hz, 8.5 Hz, IH). Step 2 (R)- 7-[3-(l-amino-l-methyl-ethyl)-pyrrolidin-yl]-l-cyclopropyl-6-fluoro-2- methanesulfonyl-8-methoxy-4-oxo-l , 4-dihydro-quinoline-3-carboxylic acid ethyl ester (7)

10                                                                                  6                                                                                            7

A mixture containing compound (6) (3.88 g, 9.67 mmol), compound 10 (1.64 g, 12.8 mmol), anhydrous DIEA (5.05 g, 39.1 mmol, dried over 4A sieves), and anhydrous DMF (40 mL) is heated at 70 ⁰C under an atmosphere of argon gas. After heating for 4.5 h (LC-MS analysis shows ~7% compound (6) remained), the reaction mixture is cooled to room temperature, diluted with EtOAc (200 mL), and washed with water (100 mL). The aqueous layer is extracted with EtOAc (100 mL), and the combined organic layers are washed with a saturated aqueous solution of sodium bicarbonate (100 mL). The organic layer is diluted with water (100 mL) and treated with an aqueous solution of HCl (4 N) until the aqueous layer is acidic (pH 2—3 after shaking the mixture vigorously). The organic layer is separated, and this process is repeated. The combined aqueous layers are diluted with EtOAc (100 mL) and treated with an aqueous solution of sodium hydroxide (6 N) until the aqueous layer is basic (pH ~8 after shaking the mixture vigorously). The aqueous layer is separated, and this process is repeated. The combined organic layers are dried over magnesium sulfate, filtered, and concentrated under reduced pressure giving an orange solid (3.27 g of an~80:20 mixture of compound (7) and impurity B). This solid is recrystallized from hot EtOAc (~60 mL) furnishing 2.18 g (44% yield) of pure compound 7 as a bright yellow solid. LC-MS mlz calcd for C₂₄H₃₂FN₃O₆S 509 ([M⁺]); found 510 ([M + H]⁺).

This reaction should not be allowed to proceed for more than a few hours (not overnight) as prolonged reaction time can lead to the formation of more side products. The product should be —95% pure (based on HPLC), with only a trace amount of impurity B. Step 3. (R)-7-[3-(l-amino-l-methyl-ethyl)-pyrrolidin-yl]-l-cyclopropyl-6-fluoro-2-mercapto-8- methoxy-4-oxo-l,4-dihydro-quinoline-3-carboxylic acid ethyl ester (8)

7                                                                                                                                                                                          8

Compound 7 (1.04 g, 2.04 mmol) is partially dissolved in DME (40 mL) under an atmosphere of argon. Sodium hydrosulfide hydrate (Aldrich, 72.6% by titration, 465 mg, 6.02 mmol) in water (3.0 mL) is added to this solution. The resulting mixture is sparged slowly with argon for 30 min.

The progress of the reaction is monitored by HPLC-MS, and judged to be complete (<2% of 7 remains) after 11.5 h. Excess sodium hydrosulfide is quenched upon addition of aq HCl (4.5 mL, 4 N).

The resulting orange solution (pH ~2) is sparged with argon (30 min) to remove the generated hydrogen sulfide. Step. 4 (R)- 7-[3-(l-amino-l-methyl-ethyl)-pyrrolidin-l-yl]-9-cyclopropyl-6-fluoro-8-methoxy- 9H-isothiazolo[5, 4-b] -quinoline-3, 4-dione (5)

A solution of potassium carbonate (4.26 g, 30.8 mmol) in water (25 mL) is next added to this solution to give a clear yellow solution (pH 9-10). The clear yellow solution is then sparged with argon for ~5 min. Finally, hydroxylamine-0-sulfonic acid (0.93 g, 8.2 mmol) is added portionwise as a solid, with immediate evolution of gas and formation of the product as a yellow precipitate. After stirring for 16 h, the reaction mixture (pH 10.2) is acidified with aq HCl to pH 8.3 (the approximate isoelectric point of 5) causing additional product to precipitate from solution. The reaction mixture is concentrated under reduced pressure (final volume -40 mL). The yellow precipitate is collected by centrifugation, washed with water (3 x 40 mL, with sonication), and lyophilized to give 0.80 g of 5.

……………………..

WO2007014308A1

http://www.google.com/patents/WO2007014308A1?cl=en

EXAMPLE 5. SYNTHESIS OF I-METHYL-I-PYRROLIDIN-3-YL ETHYLAMINE (5)

1 -Methyl- l-pyrrolidin-3-yl-ethylamine is prepared in accordance with the synthetic scheme below.

N O

5 P

Step 1. Synthesis of (S)-l-benzylpyrrolidin-3-yl methanesulfonate (N).

Methanesulfonyl chloride (15 mL, 0.19 mol) is added to a cooled (0 ⁰C) solution of toluene (300 mL) containing (5)-l-benzylpyrrolidin-3-ol (24.5 g, 0.14 mol) and triethylamine (80 mL, 0.57 mol). The resulting mixture is stirred at 0 °C for 15 min, and allowed to warm to room temperature with stirring for 2h. The mixture is quenched with a 5% aqueous solution of sodium bicarbonate (250 mL). The organic layer is washed with a 5% aqueous solution of sodium bicarbonate (2 x 250 mL), washed with water (I x 250 mL), dried over magnesium sulfate, and concentrated under reduced pressure to give N (35.1 g, 99 %) as an orange oil. ¹H NMR (300 MHz, CDCl₃): £2.07 (m, IH), 2.30 (m, IH), 2.49 (m, IH), 2.75-2.90 (m, 3H), 2.98 (s, 3H), 3.61 (d, J= 13.0 Hz, IH), 3.68 (d, J= 13.0 Hz, IH), 5.18 (m, IH), 7.15-7.30 (m, 5H). LCMS mlz calcd for C₁₂H₁₇NO₃S 255 ([M⁺]); found 256 ([M + H]⁺, 100%), 160 (40%). Steps 2 and 3. Syntheses of(R)-l-benzylpyrrolidine-3~carbonitrile (O) and 2-((R)-I- benzylpyrrolidin-3-yl)propan-2-amine (P).

The syntheses of O and P are described previously by Fedij et al. (Fedij, V.; Lenoir, E. A., Ill; Suto, M. J.; Zeller, J. R.; Wemple, J. Tetrahedron: Asymmetry 1994, J, 1131- 1134). Step 4. Synthesis ofl-((R)-Methyl~l-pyrrolidin-3-yl)-ethylamine (5).

A mixture containing P (7.4 g), 20% palladium hydroxide on carbon (7.5 g), and ethanol (75 niL) is stirred under an atmosphere of hydrogen gas (50 psi) at 45 °C for 24 h. The mixture is filtered and the filtrate is concentrated under reduced pressure to give 5 (4.1 g, 95 %) as a yellow oil. This material is stored under an atmosphere of argon gas. ¹H NMR (300 MHz, CDCl₃): J1.09 (s, 6H), 1.51 (m, IH), 1.64 (br s, 3H), 1.81 (m, IH), 2.06 (apparent pentet, J= 8.5 Hz, IH), 2.69 (dd, J= 11.0 Hz, J= 8.5 Hz, IH), 2.94 (m, 2H), 3.00 (dd, J= 11.0 Hz, J= 8.5 Hz, IH). LCMS mlz calcd for C₇H₁₆N₂ 128 ([M⁺]); found 129 ([M + H]⁺, 60%), 112 (100%).

EXAMPLE 6. GENERAL METHOD FOR THE FINAL AMINE-COUPLING STEP: SYNTHESIS OF 7-((R)-3-

(2-AMINOPROPAN-2-YL)PYRROLIDIN- 1 -YL)-9-CYCLOPROPYL-6-FLUORO-8- METHOXYISOTHIAZOLO[5,4-B]QUINOLINE-3 ,4(2H,9H)-DIONE HYDROCHLORIDE

[0261 ] 7-((R)-3-(2-Aminopropan-2-yl)pyrrolidin- 1 -yl)-9-cyclopropyl-6-fluoro-8- methoxyisothiazolo[5,4-b]quinoline-3,4(2H,9H)-dione hydrochloride is prepared in accordance with the synthetic scheme below.

Synthesis ofJ-ffRJS-^-aminopropan^-ylJpyrrolidin-l-ylJ-P-cyclopropyl-o-fluoro-S- methoxyisothiazolofS, 4-bJguinoline-3,4(2H, 9H)-dione hydrochloride (6).

Under an atmosphere of argon, a reaction vessel is charged with 5 (206.0 mg, 1.6 mmol), 3 (328.6 mg, 1.0 mmol), dimethyl sulfoxide (4.5 mL), and ΛζN-diisopropylethylamine (750 μL, 4.3 mmol). The resulting mixture is irradiated with microwaves (CEM Discover) at 125 ⁰C for 1 h (conventional heating may also be used — 115 °C in an oil bath for 14 h), allowed to cool, and evaporated to dryness under reduced pressure (-70 °C/2-3 mm Hg). The oily residue is triturated with ethyl acetate (15 mL) and the resulting powder is collected by centrifugation. This solid is purified using preparative HPLC to give the desired product. Preparative HPLC is performed using a YMC Pack Pro C18 150 x 30.0 mm 5//m column coupled to a YMC Pack Pro 50 x 20 mm 5/an column with an isocratic elution of 0.37 min at 95:5 H₂OiCHsCN containing 0.1% TFA followed by a 15.94 min linear gradient elution from 95:5 to 25:75, followed by a 0.69 min linear gradient from 25:75 to 5:95 at a flow rate of 30.0 mL/min with UV detection at 254. The crude material is loaded as a solution containing acetic acid (~2 mL), methanol (~1 mL), and water (~1 mL). The purified product is isolated as the TFA salt and is converted to the corresponding hydrochloride salt by addition of a solution of hydrogen chloride (~1.25 M in methanol) followed by evaporation; this process is repeated twice to give a yellow solid. Purity by HPLC: >99%; t_R = 10.08 min. ¹H NMR (300 MHz, TFA-d): δ 1.28 (m, 2H), 1.53 (m, 2H), 1.66 (s, 6H), 2.43 (m, IH), 2.57 (m, IH), 3.35 (m, IH), 3.97 (s, 3H), 4.01-4.38 (m, 5H), 8.17 (d, J= 12.0 Hz, IH, aromatic). ¹⁹F(¹H) (282 MHz, TFA-J): δ-\ 18.0 (s). ¹³C(¹H) (75 MHz, TFA-d): £13.5, 13.9, 25.0, 25.1, 29.1, 39.7, 49.6, 59.4 (br, W_1/2 « 14 Hz), 59.8 (br, W_1/2 « 14 Hz), 60.0, 66.8, 106.0, 112.1 (dJc__F = 23.0 Hz), 137.5 (br m, W_1/2 « 24 Hz), 138.4, 144.8 (br, W_1/2 » 10 Hz), 155.3 (dJc__F = 255.0 Hz), 169.8, 170.1, 171.5 (br, W_1/2 « 9 Hz). LCMS mlz calcd for C₂₁H₂₅FN₄O₃S 432 ([M⁺]); found 433 ([M + H]⁺). Anal. Calcd for C21H25FN4O₃S-l.5HCM.5H2O: C, 49.05; H, 5.78; N, 10.90; Cl, 10.34. Found: C, 49.30; H, 5.60; N, 10.83; Cl, 10.00.

EXAMPLE 3. SYNTHESIS OF 9-CYCLθPRθPYL-6,7-DiFLUθRθ-8-METHθχγ-9H-isoτHiAzθLθ[5,4- 5]QUlNOLlNE-3,4-DIONE (Compound 3).

9-Cyclopropyl-6,7-difluoro-8-methoxy-9H-isothiazolo[5,4-&]quinoline-3,4-dione (3) is prepared in accordance with the synthetic scheme below.

Step 1. Synthesis of 2,4, 5-trifluoro-3-methoxybenzoyl chloride (A)

A mixture of 2,4,5-trifluoro-3-methoxybenzoic acid (154 mg, 0.75 mmol) and thionyl chloride (8 mL) is refluxed for 4 h. Excess thionyl chloride is removed in vacuo, and the remaining residue is used directly in the next synthetic step. Step 2. Synthesis of (Z)-ethyl 3-hydroxy-3-(2,4,5-trifluoro-3-methoxyphenyl)aaγlate (B).

Compound B is prepared using the general method of Wierenga and Skulnick (Wierenga, W.; Skulnick, H. I. J. Org. Chein. (1979) 44: 310-311). H-Butyllithium (1.6 M in hexanes) is added to a cooled (-78 °C) solution of tetrahydrofliran (10 mL) containing ethyl hydrogen malonate (180 juL, 1.50 mmol) and 2,2′-bipyridyl (~1 mg as indicator). The temperature of the reaction mixture is allowed to rise to ca. -5 ⁰C during the addition of n- butyllithium. Sufficient n-butyllithium (2.8 mL, 4.48 mmol) is added until a pink color persists at -5 ⁰C for 5-10 min. A solution of 2,4,5-trifluoro-3-methoxybenzoyl chloride (0.75 mmol, vide supra) in tetrahydrofuran (~3 mL) is added in one portion to the reaction mixture that had been recooled to -78 ⁰C. The resulting mixture is allowed to warm to room temperature, diluted with ethyl acetate (50 mL), and quenched with a 1 M aqueous solution of hydrochloric acid. The organic layer is washed with a 5% aqueous solution of sodium bicarbonate (2 x 30 mL), followed by brine (2 x 50 mL), dried over sodium sulfate, and evaporated under reduced pressure to give the crude product. This material is purified by flash column chromatography (eluting with 20% v/v ethyl acetate in hexanes) to give pure B as a white solid. ¹H NMR (300 MHz, CDCl₃): (enol, predominant tautomer, >90%) δ 1.32 (t, J_H-_H = 7.0 Hz, 3H, CO₂CH₂CH₃), 4.02 (apparent t, J_H-_F = 1.0 Hz, 3H, OCH₃), 4.25 (q, J_H-_H = 7.0 Hz, 2H, CO₂CH₂CH₃), 5.79 (s, IH, CH₃C(OH)=CH- CO₂CH₂CH₃), 7.39 (ddd, J_H__F= 11.0 Hz, 8.5 Hz, 6.5 Hz, IH, aromatic), 12.68 (s, IH, OH). ¹⁹F(¹H) NMR (282 MHz, CDCl₃): <5-146.8 (dd, J_F__F = 21.5 Hz, 10.5 Hz, IF), -140.2 (dd, J_F__F = 21.5 Hz, 13.5 Hz, IF), -131.3 (dd, J_F__F = 13.5 Hz, 10.5 Hz, IF).

Step 3. Synthesis ofζEyethy^-^ZJ-N-cyclopropy^methylthioJcarbonoimidoylJS-hydroxyS- (2, 4, 5-trifluoro-3-methoxyphenyl)acrylate (C)

Sodium hydride (60% in mineral oil, 31 mg, 0.78 mmol) is added portionwise to a cooled (0 °C) solution containing B (200 mg, 0.73 mmol), cyclopropyl isothiocyanate (120 /JL, 1.2 mmol), and dimethylformamide (2 mL). The resulting mixture is allowed to warm to room temperature with stirring overnight (18 h). Methyl iodide (80 juL, 1.2 mmol) is added to the resulting solution and stirred for an additional 4 h (until TLC indicated the complete consumption of B). The reaction mixture is diluted with ethyl acetate (100 mL) and quenched by addition of a saturated aqueous solution of ammonium chloride (30 mL). The organic layer is washed with brine (4 x 30 mL), dried over sodium sulfate, and evaporated under reduced pressure to give the crude product. This material is purified by flash column chromatography (eluting with 40% v/v ethyl acetate in hexanes) to give C as a yellow oil. ¹H NMR (300 MHz, CDCl₃): (50.86 (m, 2H, cyclopropyl CH₂), 0.97 (m, 5H), 2.52 (s, 3H, SCH₃), 3.00 (m, IH, cyclopropyl CH), 3.96 (q, J_H-_H = 7.0 Hz, 2H, CO₂CH₂CH₃), 4.02 (apparent t, J_H-_F = 1.0 Hz, 3H, OCH₃), 6.96 (m, IH, aromatic), 11.71 (s, IH). ¹⁹F(¹H) NMR (282 MHz, CDCl₃): £-149.9 (br, IF), -141.4 (br, IF), -135.7 (br, IF).

Step 4. Synthesis of ethyl l-cyclopropyl-6,7-difluoro-8-methoxy-2-(methylthio)-4-oxo-l,4- dihydroquinoline-3-carboxylate (D)

Sodium hydride (60% in mineral oil, 82 mg, 2.1 mmol) is added portionwise to a solution of C (760 mg, 1.95 mmol) in dimethylformamide (15 mL) at room temperature. The reaction mixture is heated at 80 ⁰C for 3 d (until TLC indicates the complete consumption of B), cooled to room temperature, and quenched by addition of a saturated aqueous solution of ammonium chloride (10 mL). The mixture is extracted with ethyl acetate (3 x 50 mL). The combined organic extracts are washed with brine (4 x 30 mL), dried over sodium sulfate, and evaporated under reduced pressure to give crude D. This material is purified by flash column chromatography (eluting with 30% v/v ethyl acetate in hexanes) to D as a pale yellow oil.¹H NMR (300 MHz, CDCl₃): £0.73 (m, 2H, cyclopropyl CH₂), 1.19 (m, 2H, cyclopropyl CH₂), 1.38 (t, J_H-_H = 7.0 Hz, 3H, CO₂CH₂CH₃), 2.66 (s, 3Η, SCH₃), 3.74 (m, IH, cyclopropyl CH), 4.08 (d, J_H-_F = 2.5 Hz 3H, OCH₃), 4.40 (q, J_H__H = 7.0 Hz, 2H, CO₂CH₂CH₃), 7.76 (dd, J_H__F = 10.5 Hz, 8.5 Hz IH, aromatic). ¹⁹F(¹H) NMR (282 MHz, CDCl₃): £-146.8 (d, J_F__F = 21.0 Hz, IF), – 137.7 (d, J_F-_F = 21.0 Hz, IF). LCMS mlz calcd for C₁₇H₁₇F₂NO₄S 369 ([M⁺]); found 370 ([M + H]⁺).

Step 5. Synthesis of ethyl l-cyclopropyl-6,7-difluoro-8-methoxy-2-(methylsulfinyl)-4-oxo-l,4- dihydroquinoline-3-carboxylate (E)

m-Chloroperoxybenzoic acid (<77%, 34 mg, 0.15 mmol) is added in one portion to a solution of D (50 mg, 0.14 mmol) in methylene chloride (3 mL) at room temperature. The reaction mixture is stirred for 1 h, diluted with ethyl acetate (20 mL), and washed with a 5% aqueous solution of sodium bicarbonate (2 x 10 mL). The organic layer is dried over sodium sulfate and evaporated under reduced pressure to give the crude product. This material is purified by preparative thin-layer chromatography (eluting with 10% v/v hexanes in ethyl acetate) to give pure E as a white solid. ¹H NMR (300 MHz, CDCl₃): £0.62 (m, IH, cyclopropyl CH₂), 1.00 (m, IH, cyclopropyl CH₂), 1.13 (m, IH, cyclopropyl CH₂), 1.29 (m, IH, cyclopropyl CH₂), 1.36 (t, JH__H = 7.5 Hz, 3H, CO₂CH₂CH₃), 3.22 (s, 3Η, S(O)CH₃), 3.85 (m, IH, cyclopropyl CH), 4.09 (d, JH-_F = 2.5 Hz, 3H, OCH₃), 4.37 (q, J_H-_H = 7.5 Hz, 2H, CO₂CH₂CH₃), 7.75 (dd, J_H-_F = 10.0, 8.0 Hz, IH, aromatic). ¹⁹F(¹H) NMR (282 MHz, CDCl₃): £-145.2 (d, J_F__F = 21.0 Hz, IF), -136.2 (d, J_F__F = 21.0 Hz, IF). LCMS mlz calcd for C₁₇H₁₇F₂NO₅S 385 ([M⁺]); found 386 ([M + H]⁺).

Step 6. Synthesis of ethyl l-cyclopropyl-^βJ-difluoro-l-mercaptoS-methoxy-^oxo-lA- dihydroquinoline-3-carboxylate (F).

Anhydrous sodium hydrogen sulfide (Alfa Aesar, 20 mg, 0.36 mmol) is added in one portion to a solution of DMF (6 mL) containing E (93 mg, 0.24 mmol) at room temperature. The resulting solution is heated at 40 ⁰C for 2-3 h (until TLC indicated complete consumption of E) and allowed to cool to room temperature. The reaction mixture is quenched by addition of a 5% aqueous solution of hydrochloric acid (20 mL) and extracted with ethyl acetate (2 x 25 mL). The combined organic extracts are washed with brine (4 x 25 mL), dried over sodium sulfate, and evaporated to dryness under reduced pressure to give crude F in quantitative yield. This material is used directly in the next synthetic step to prevent its oxidative degradation. LCMS mlz calcd for C₁₆H₁₅F₂NO₄S 355 ([M⁺]); found 356 ([M + H]⁺) Step 7. Synthesis of9-cyclopropyl-6,7-difluoro-8-methoxyisothiazolo[5,4-b]quinoline- 3,4(2H,9H)-dione (3).

A solution of sodium bicarbonate (820 mg, 9.8 mmol) in water (14 mL) is added to a solution of F (348 mg, 0.98 mmol) in tetrahydrofuran (10 mL) at room temperature. Hydroxylamine-O-sulfonic acid (465 mg, 4.1 mmol) is added in one portion to this mixture. The reaction mixture is stirred at room temperature for ~3 h and quenched by addition of an aqueous solution of 5% hydrochloric acid (100 mL). The precipitate that formed is collected by filtration, washed with water (3 x 5 mL), and dried in vacuo to give 3 as a white solid. This product is of sufficient purity (>95% by ¹H NMR spectroscopy) to use directly in the final amine-coupling step. ¹HNMR (300 MHz, DMSO-J₆): Jl.12 (m, 4H, cyclopropyl CH₂), 3.85 (m, IH, cyclopropyl CH), 4.01 (d, J_H-_F= 1.5 Hz, 3H, OCH₃), 7.85 (dd, J_H__F = 11.0 Hz, 9.0 Hz, IH, aromatic). ¹⁹F(¹H) NMR (282 MHz, DMSO-J₆): £-146.4 (d, J_F__F = 23.0 Hz, IF), -140.2 (d, J_F_ _F = 23.0 Hz, IF). LCMS mlz calcd for C₁₄H₁₀F₂N₂O₃S 324 ([M*]); found 325 ([M + H]⁺).

REFERENCES

1. Achillion Pharmaceuticals. About ACH-702. Available online: http://www.achillion.com/PL/pdf/04_ach_702_bg.pdf (accessed on 2 May 2013).
2. Pucci, M.J.; Podos, S.D.; Thanassi, J.A.; Leggio, M.J.; Bradbury, B.J.; Deshpande, M. In vitro and in vivoprofiles of ACH-702, an isothiazoloquinolone, against bacterial pathogens. Antimicrob. Agents Chemother. 2011, 55, 2860–2871, doi:10.1128/AAC.01666-10.
3. Achillion Pharmaceuticals, Inc. SEC filling form 10-Q quarterly report filed August 7, 2013. Available online: http://ir.achillion.com/secfiling.cfm?filingID=1193125–13–324297 (accessed on 28 September 2013).
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   2	*	WANG, Q. ET AL.: “Isothiazoloquinolones with Enhanced Antistaphylococcal Activities against Multidrug-Resistant Strains: Effects of Structural Modifications at the 6-, 7-, and 8-Positions” J. MED. CHEM., vol. 50, 2007, pages 199-210, XP002465316

9. WO2005019228A1 *	Aug 4, 2004	Mar 3, 2005	Achillion Pharmaceuticals Inc	Isothiazoloquinolones and related compounds as anti-infective agents
   WO2006118605A2 *	Nov 10, 2005	Nov 9, 2006	Achillion Pharmaceuticals Inc	8a, 9-dihydro-4a-h-isothiazolo[5,4-b] quinoline-3, 4-diones and related compounds as anti-infective agents
   WO2007014308A1 *	Jul 27, 2006	Feb 1, 2007	Achillion Pharmaceuticals Inc	8-methoxy-9h-isothiazolo[5,4-b]quinoline-3,4-diones and related compounds as anti-infective agents

10. Citing Patent	Filing date	Publication date	Applicant	Title
    WO2008021491A2 *	Aug 16, 2007	Feb 21, 2008	Achillion Pharmaceuticals Inc	Method for synthesis of 8-alkoxy-9h-isothiazolo[5,4-b]quinoline-3,4-diones
    WO2011031745A1	Sep 8, 2010	Mar 17, 2011	Achaogen, Inc.	Antibacterial fluoroquinolone analogs
    EP2488532A2 *	Oct 15, 2010	Aug 22, 2012	Rib-X Pharmaceuticals, Inc.	Antimicrobial compounds and methods of making and using the same
    US7902365	Aug 16, 2007	Mar 8, 2011	Achillion Pharmaceuticals, Inc.	Method for synthesis of 8-alkoxy-9H-isothiazolo[5,4-B]quinoline-3,4-diones
    US8138346	Mar 4, 2011	Mar 20, 2012	Achillion Pharmaceuticals, Inc.	Method for synthesis of 8-alkoxy-9H-isothiazolo[5,4-B]quinoline-3,4-diones

Cenicriviroc

TAK-652; TBR-652

1-Benzazocine-5-carboxamide, 8-[4-(2-butoxyethoxy)phenyl]-1,2,3,4-tetrahydro-1-(2-methylpropyl)-N-[4-[[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl]phenyl]-, (5E)-

(-)-(S)-8-[4-(2-Butoxyethoxy)phenyl]-1-isobutyl-N-[4-(1-propyl-1H-imidazol-5-ylmethylsulfinyl)phenyl]-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide

(S)-(−)-8-{4-[2-(Butoxy)ethoxy]phenyl}-1-isobutyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide methanesulfonate

497223-25-3 , Molecular Formula: C41H52N4O4S   Molecular Weight: 696.94098

497223-28-6 (mesylate) ^{C41 H52 N4 O4 S . C H4 O3 S, 793.047}

Cenicriviroc (TAK-652, TBR-652) is an experimental drug candidate for the treatment of HIV infection.^[1] It is being developed by Takeda Pharmaceutical and Tobira Therapeutics.

TBR-652 (formerly TAK-652) is a highly potent and orally active CCR5 antagonist in phase II clinical trials at Takeda for the treatment of HIV infection. Tobira Therapeutics is evaluating the compound in preclinical studies for the treatment of rheumatoid arthritis.

TBR-652 binds CCR5 receptors to interfere with the entry of the HIV-1 virus into macrophages and activated T-cells by inhibiting fusion between viral and cellular membranes. This mechanism of action differs from currently used HIV treatments such as nucleoside reverse transcriptase inhibitors and protease inhibitors.

In 2007, Takeda entered into an agreement with Tobira pursuant to which Tobira obtained exclusive worldwide rights to develop, manufacture and commercialize TBR-652 for the treatment of HIV infection.

Cenicriviroc is an inhibitor of CCR2 and CCR5 receptors,^[2] allowing it to function as an entry inhibitor which prevents the virus from entering into a human cell. Inhibition of CCR2 may have an anti-inflammatory effect.

A double-blind, randomized, placebo-controlled clinical study to assess the antiviral activity, safety, and tolerability of cenicriviroc was conducted in 2010. HIV-infected patients taking cenicriviroc had significant reductions in viral load, with the effect persisting up to two weeks after discontinuation of treatment.^[3] Additional Phase II clinical trials are underway.^[4]

Phase IIb data presented at the 20th Conference on Retroviruses and Opportunistic Infections (CROI) in March 2013 showed similar viral suppression rates of 76% for patients taking 100 mg cenicriviroc, 73% with 200 mg cenicriviroc, and 71% with efavirenz. Non-response rates were higher with cenicriviroc, however, largely due to greater drop-out of patients. A new tablet formulation with lower pill burden may improve adherence. Looking at immune and inflammatory biomarkers, levels of MCP-1 increased and soluble CD14 decreased in the cenicriviroc arms.^[5]

Although HIV has been largely rendered a chronic infection, there remains a need for new drugs because of the virus’s propensity to develop resistance to the drugs used to keep it at bay.

Pfizer’s maraviroc was the first drug that acted on the cells to prevent viral entry by antagonising the CCR5 co-receptor. Several others have been investigated and have failed; another that is undergoing clinical trials is Takeda’s cenicriviroc, which has been licensed to Tobira Therapeutics. Unlike maraviroc, the new agent also acts at the CCR2 co-receptor, which is implicated in cardiovascular and metabolic diseases.

In a Phase I double blind, placebo controlled trial designed to study safety, efficacy and pharmacokinetics, treatment-experienced but CCR5 antagonist-naïve patients with HIV-1 were given doses of 25, 50, 75, 100 or 150mg of the drug, or placebo once a day for 10 days.² The maximum median reductions in HIV-1 RNA values were 0.7, 1.6, 1.8 and 1.7 log10 copies/ml for the respective doses, with a median time to nadir of 10 to 11 days. The effect on CD-4 cell counts was negligible. There was also a significant reduction in levels of monocyte chemotactic protein 1, suggesting that CCR2 was also being blocked. The drug was both generally safe and well tolerated, and no patients withdrew from the trial due to adverse events.

In another Phase I trial, designed to look at pharmacokinetics and pharmacodynamics and carried out in a similar patient population, subjects were given the drug as oral monotherapy for 10 days, again in doses of 25, 50, 75, 100 and 150mg, or placebo.³ The drug was well absorbed into the systemic circulation, and the concentration levels declined slowly, with meant elimination half-lives of one to two days. Potent, dose-dependent reductions in viral load were seen, and again it was generally safe and well tolerated across all levels.

In June 2011, Tobira initiated a multi-centre, double blind, double dummy, 48-week comparative Phase IIb trial in 150 patients with HIV-1 infection. Subjects are being given 100 or 200mg once-daily doses of the drug to evaluate its efficacy, safety and tolerability.

PATENTS

WO  2003014105

WO 2003076411

WO 2005116013

WO 2007144720

WO 2011163389

US 20130079233

WO 2013167743

See also

• Discovery and development of CCR5-receptor antagonists

ancriviroc (formerly known as SCH-C), vicroviroc which has the chemical name (4,6-dimethylprymidine-5-yl){4- [(3S)-4-{(1 R)-2-methoxy-1 -[4-(trifluoromethyl)phenyl]ethyl}-3-methylpiperazin-1 -yl]-4-methylpiperidin-1 – yljmethanone, PRO-140, apliviroc (formerly known as GW-873140, Ono-4128, AK-602), AMD-887, INC- B9471 , CMPD-167 which has the chemical name N-methyl-N-((1R,3S,4S)-3-[4-(3-benzyl-1-ethyl-1H- pyrazol-δ-yOpiperidin-i-ylmethylH-IS-fluorophenyllcyclopent-i-yll-D-valine), methyl1-endo-{8-[(3S)-3- (acetylamino)-3-(3-fluorophenyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-2-methyl-4,5,6,7-tetrahydro-1 H- imidazo[4,5-c]pyridine-5-carboxylate, methyl 3-endo-{8-[(3S)-3-(acetamido)-3-(3-fluorophenyl)propyl]-8- azabicyclo[3.2.1]oct-3-yi}-2-methyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-5-carboxylate, ethyl 1- endo-{8-[(3S)-3-(acetylamino)-3-(3-fiuorophenyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-2-methyl-4,5,6,7- tetrahydro-1 H-imidazo[4,5-c]pyridine-5-carboxylate and N-{(1S)-3-[3-endo-(5-lsobutyryl-2-methyl-4,5,6,7- tetrahydro-1H-imidazo[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-(3-fluorophenyl)propyl}acetamide) and pharmaceutically acceptable salts, solvates or derivatives of the above. The last four compounds are disclosed in WO 03/084954 and WO 05/033107.

http://pubs.acs.org/doi/full/10.1021/jm0509703

Compound (S)-(−)-5b (TAK-652) also inhibited the replication of six macrophage-tropic (CCR5-using or R5) HIV-1 clinical isolates in peripheral blood mononuclear cells (PBMCs) (mean IC₉₀ = 0.25 nM).

(S)-(−)-8-{4-[2-(Butoxy)ethoxy]phenyl}-1-propyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide ((S)-(−)-5a). The 1 N HCl (160 mL) was added to 19³¹ (35.68 g, 53.4 mmol), and the mixture was extracted with EtOAc. To the aqueous layer was added 25% aqueous K₂CO₃ (160 mL), and the mixture was extracted with a mixture of EtOAc and i-PrOH (4:1). The organic layer was washed with brine, dried over MgSO₄, and concentrated in vacuo to give (S)-18. To a solution of 16a (18.0 g, 41.1 mmol) and DMF (0.5 mL) in THF (180 mL) was added thionyl chloride (SOCl₂) (4.50 mL, 61.7 mmol) at room temperature. After being stirred at room temperature for 1.5 h, the reaction mixture was concentrated in vacuo. A solution of the residue in THF (200 mL) was added dropwise to a mixture of (S)-18 and triethylamine (Et₃N) (35.0 mL, 251 mmol) in THF (150 mL) under ice cooling. After being stirred at room temperature for 4 h, water was added to the reaction mixture. The mixture was washed with 10% aqueous AcOH, saturated aqueous NaHCO₃, and brine, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on a NH silica gel (hexane/EtOAc = 1:5 → 1:8 → 1:9) to give 21.14 g (75%) of (S)-(−)-5a as a yellow amorphous powder, [α]_D = −132.5° (C = 0.507%, EtOH). ¹H NMR (300 MHz, CDCl₃) δ 0.87−1.03 (9H, m), 1.34−1.49 (2H, m), 1.50−1.85 (8H, m), 2.55−2.65 (2H, m), 3.15−3.25 (2H, m), 3.52−3.58 (4H, m), 3.75−3.83 (4H. m), 4.02 (1H, d, J = 13.8 Hz), 4.08−4.17 (3H, m), 6.56 (1H, d, J = 1.0 Hz), 6.80 (1H, d, J = 8.8 Hz), 6.96 (2H, d, J = 8.8 Hz), 7.31−7.46 (7H, m), 7.55 (1H, s), 7.76 (2H, d, J = 8.8 Hz), 7.98 (1H, s). Anal. (C₄₀H₅₀N₄O₄S·0.25H₂O) C, H, N.

(S)-(−)-8-{4-[2-(Butoxy)ethoxy]phenyl}-1-isobutyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide methanesulfonate ((S)-(−)-5b). The free base of (S)-(−)-5b was prepared in 80% yield from 16band 19 by a method similar to that described for (S)-(−)-5a. To a solution of the free base of (S)-(−)-5b (64.91 g, 93.1 mmol) in EtOAc (600 mL) was added dropwise a solution of methanesulfonic acid (8.95 g, 93.1 mmol) in EtOAc (160 mL) at room temperature. After being stirred at room temperature for 4 h, the crystals were collected by filtration and washed with EtOAc to give 69.09 g (94%) of (S)-(−)-5b as yellow crystals. The crystals (68.0 g) were purified by recrystallization from 2-butanone to give 58.9 g (85%) of (S)-(−)-5b as yellow crystals, mp 145.5−147.5 °C, [α]_D = −191.2° (c = 0.508%, EtOH). ¹H NMR (300 MHz, DMSO-d₆) δ 0.82−0.97 (12H, m), 1.29−1.39 (2H, m), 1.40−1.55 (4H, m), 1.65−1.85 (2H, m), 2.00−2.25 (1H, m), 2.29 (3H,s), 2.38−2.60 (2H, m), 3.10 (2H, d, J = 7.8 Hz), 3.30−3.60 (4H, m), 3.70 (2H, t, J = 4.8 Hz), 3.98 (2H, t,J = 6.6 Hz), 4.10 (2H, t, J = 4.8 Hz), 4.34 (1H, d, J = 15.0 Hz), 4.68 (1H, d, J = 15.0 Hz), 6.87 (1H, d, J = 8.7 Hz), 6.99 (2H, d, J = 8.7 Hz), 7.16 (1H, s), 7.42−7.60 (8H, m), 7.93 (2H, d, J = 8.7 Hz), 9.05 (1H, s), 10.18 (1H, s). Anal. (C₄₂H₅₆N₄O₇S₂) C, H, N.

…………………

WO 2003014105 OR  US20090030032

http://www.google.st/patents/US20090030032?hl=pt-PT&cl=un

EXAMPLE 7 Preparation of Compounds 9 and 10

8-[4-(2-Butoxyethoxy)phenyl]-1-propyl-N-[4-[[[1-propyl-1H-imidazol-5-yl]methyl]sulfinyl]phenyl]-1,2,3,4-tetrahydro-1-benzazocin-5-carboxamide (317 mg) was resolved by using CHIRAKCEL OJ 50 mm ID×500 mL (hexane/ethanol) to give (−)-8-[4-(2-butoxyethoxy)phenyl]-1-propyl-N-[4-[[[1-propylimidazol-5-yl]methyl]sulfinyl]phenyl]-1,2,3,4-tetrahydro-1-benzoazocine-5-carboxamide (142 mg) (Compound 9) and (+)-8-[4-(2-butoxyethoxy)phenyl]-1-propyl-N-[4-[[[1-propylimidazol-5-yl]methyl]sulfinyl]phenyl]-1,2,3,4-tetrahydro-1-benzoazocine-5-carboxamide (143 mg) (Compound 10).

Compound 9

[α]_D=−127.4° (C=0.533% in ethanol).

Compound 10

[α]_D=+121.0° (C=0.437% in ethanol).

………………………….

WO 2003076411

http://www.google.st/patents/WO2003076411A1?cl=en

http://www.google.st/patents/US20050107606?hl=pt-PT&cl=en

Example 21 (−)-8-[4-(2-Butoxyethoxy)phenyl]-1-isobutyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide

To a solution of 8-[4-(2-butoxyethoxy)phenyl]-1-isobutyl-1,2,3,4-tetrahydro-1-benzazocine-5-carboxylic acid (45 g) in tetrahydrofuran (135 ml) was added N,N-dimethylformamide (230 mg) and added dropwise thionyl chloride (12.45 g) at 10 to 15° C., and the resulting solution was stirred at the same temperature for 40 minutes to prepare an acid chloride.

Separately, to a solution of (−)-4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenylamine in tetrahydrofuran (270 ml) was added pyridine (27.59 g), the resulting mixture was adjusted to 5° C. or lower, and then thereto was added dropwise the acid chloride solution at 5° C. or less, and the resulting mixture was stirred at the same temperature for 2 hours. To the mixture were added water (270 ml) and 20% aqueous citric acid solution (180 ml), tetrahydrofuran was distilled off under reduced pressure and the residue was extracted with ethyl acetate. The extract was sequentially washed with water, saturated sodium bicarbonate solution and water, and then the solvent was distilled off. To the residue was added ethyl acetate (360 ml), added heptane (360 ml) at 40° C. and added seed crystals of (−)-8-[4-(2-butoxyethoxy)phenyl]-1-isobutyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide (10 mg), and the mixture was stirred at 25° C. for 2 hours and stirred at 5° C. for 1 hour. The precipitated crystals were collected by filtration to obtain 63.97 g (yield: 92.1%) of the title compound. Melting point: 120-122° C.

Elemental analysis value: in terms of C₄₁H₅₂N₄O₄S

Calcd. value: C, 70.66; H, 7.52; N, 8.04.

Analytical value: C, 70.42; H, 7.52; N, 8.01

Industrial Applicability

According to the present invention, an optically active sulfoxide derivative having CCR5 antagonism or an intermediate compound thereof can be prepared without causing side reactions such as racemization and Pummerer rearrangement. In particular, Process 7 is industrially advantageous since it is possible to prepare an optically active Compound (II) by asymmetric oxidization in the presence of an optically active acid.

Example 20 (−)-8-[4-(2-Butoxyethoxy)phenyl]-1-propyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide.methanesulfonate

According to the same method as that described in Example 15, the title compound was produced from 8-[4-(2-butoxyethoxy)phenyl]-1-propyl-1,2,3,4-tetrahydro-1-benzazocine-5-carboxylic acid and (−)-4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenylamine.

¹H-NMR (CDCl₃, δ, 300 MHz) 0.88-1.01 (9H, m), 1.37-1.42 (2H, m), 1.57-1.80 (8H, m), 2.63 (2H, br), 2.77 (3H, s), 3.27 (2H, br), 3.51-3.57 (4H, m), 3.77-3.86 (4H, m), 3.90-4.05 (1H, m), 4.14 (2H, t, J=4.6 Hz), 4.25 (1H, d, J=14.6 Hz), 6.73 (1H, s), 6.84 (1H, d, J=8.7 Hz), 6.93 (2H, d, J=8.8 Hz), 7.21 (2H, d, J=8.7 Hz), 7.40-7.48 (4H, m), 7.61 (1H, s), 7.89 (2H, d, J=8.7 Hz), 8.65 (1H, s), 9.27 (1H, br)

Elemental analysis value: in terms of C₄₁H₅₄N₄O₇S₂

Calcd. value: C, 63.21; H, 6.99; N, 7.19; S, 8.23.

Analytical value: C, 63.00; H, 7.09; N, 7.41; S, 8.25

Example 15 (−)-8-[4-(2-Butoxyethoxy)phenyl]-1-isobutyl-N-(4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenyl)-1,2,3,4-tetrahydro-1-benzazocine-5-carboxamide.methanesulfonate

8-[4-(2-Butoxyethoxy)phenyl]-1-isobutyl-1,2,3,4-tetrahydro-1-benzazocine-5-carboxylic acid (986 mg) was dissolved in tetrahydrofuran (3 ml) and thereto was added N,N-dimethylformamide (one drop). Subsequently, to the resulting solution was added dropwise oxalyl chloride (0.2 ml, 2.29 mmol) under ice-cooling and the mixture was stirred for 80 minutes under ice-cooling to prepare an acid chloride.

Separately, (−)-4-{[(1-propyl-1H-imidazol-5-yl)methyl]sulfinyl}phenylamine (689 mg) was added to tetrahydrofuran (7 ml) and the resulting solution was cooled to 5° C. To the solution was added dropwise pyridine (0.62 ml) and added dropwise the acid chloride solution at 3 to 5° C., and the mixture was stirred for 2 hours under ice-cooling. To the mixture was added water (20 ml) at 10° C. or lower and the mixture was extracted with ethyl acetate. The organic layer was sequentially washed with water, saturated sodium bicarbonate solution and water, and concentrated under reduced pressure. Thereto was added toluene and the mixture was concentrated under reduced pressure. Thereto was added acetonitrile and the mixture was concentrated under reduced pressure. The residue was dissolved in acetonitrile (7 ml) and acetone (7 ml), thereto was added dropwise methanesulfonic acid (209 mg), and added seed crystals and the mixture was stirred at room temperature for 100 minutes. Subsequently, to the mixture was added acetone-acetonitrile (1:1, 5 ml). After stirring at room temperature overnight, the mixture was stirred for 2.5 hours under ice-cooling. The precipitated crystals were collected by filtration and washed with the ice-cooled acetone (9 ml). The crystals were dried at 40° C. under reduced pressure to obtain 1.51 g (yield: 87%) of the title compound as yellow crystals.

¹H-NMR (300 MHz, DMSO-d₆, δ): 0.78-0.96 (12H, m), 1.25-1.40 (2H, m), 1.41-1.51 (4H, m), 1.65-1.85 (2H, m), 2.05-2.15 (1H, m), 2.30 (3H, s), 2.35-2.50 (2H, m), 3.05-3.15 (2H, m), 3.30-3.55 (4H, m), 3.65-3.70 (2H, m), 3.90-4.05 (2H, m), 4.05-4.10 (2H, m), 4.30 (1H, d, J=14.73 Hz), 4.65 (1H, d, J=14.73 Hz), 6.85 (1H, d, J=8.97 Hz), 6.97 (1H, d, J=8.79 Hz), 7.17 (1H, s), 7.35-7.75 (6H, m), 7.92 (2H, d, J=8.79 Hz), 9.08 (1H, s), 10.15 (1H, s).

Elemental analysis value: in terms of C₄₁H₅₂N₄O₄S.CH₄SO₃

Calcd. value: C, 63.61; H, 7.12; N, 7.06; S, 8.09.

Found value: C, 63.65; H, 7.23; N, 7.05; S, 8.08.

………………………….

References

……………….

Chemical structures of selected small molecule CCR5 inhibitors. A. Maraviroc (MVC, Selzentry), B. Vicriviroc (VCV), C. Cenicriviroc (TBR-652), D. PF-232798.

http://www.intechopen.com/books/immunodeficiency/chemokine-receptors-as-therapeutic-targets-in-hiv-infection

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Glenmark’s novel molecule ‘GRC 27864’ for chronic inflammatory diseases including pain entering human trials 

• GRC 27864 is a potent, selective, orally bioavailable inhibitor of mPGES-1
• The molecule has successfully completed pre-clinical and Phase 1 enabling studies. Regulatory submission has been filed for Phase 1 trial (first-in-human)with MHRA, UK

• mPGES-1 inhibitors selectively block the production of PGE₂ while sparing other prostanoids of physiological importance
• With this announcement, Glenmark has reaffirmed its position globally in the development of novel pain therapies

Mumbai, India: April 3, 2014: Glenmark Pharmaceuticals today announced that its Novel Chemical Entity (NCE) ‘GRC 27864′ is entering human trials. This NCE program targets Microsomal Prostaglandin E synthase-1 (mPGES-1) as a novel therapeutic target in pain management. Selective mPGES-1 inhibitors are expected to inhibit increased prostaglandin E2 (PGE2) production in the disease state without affecting other prostanoid metabolites and, consequently, may be devoid of the GI(gastrointestinal) and cardiovascular side effects seen with NSAIDs and COX-2 inhibitors, respectively.

Glenmark has completed preclinical studies and Phase 1 enabling GLP studies for its selected lead molecule, GRC 27864 and has filed a Phase 1 application forfirst-in-human trial with the MHRA, UK. The Phase 1 studies are to be initiated soon and are likely to get completed by January 2015. Following this, Glenmark will also be initiating a proof of concept study in patients with acute pain.

Ophiocordyceps sinensis (left) growing out of the head of a dead caterpillar

Ophiocordyceps sinensis is a fungus that parasitizes larvae of ghost moths and produces a fruiting body valued as an herbal remedy. The fungus germinates in the living larva, kills and mummifies it, and then the stalk-like fruiting body emerges from the corpse. It is known in English colloquially as caterpillar fungus, or by its more prominent foreign names (see below): yartsa gunbu or yatsa gunbu (Tibetan), or Dōng chóng xià cǎo (Chinese: 冬虫夏草; literally “winter worm, summer grass”). Of the various entomopathogenic fungi, Ophiocordyceps sinensis is one that has been used for at least 2000 years[2] to treat many diseases related to lungs, kidney, and also used as a natural Viagra. This fungus is not yet cultivated commercially,[3] despite the fact that several fermentable strains of Ophiocordyceps sinensis are isolated by Chinese Scientists.[4] Overharvesting and over exploitation have led to the classification of O. sinensis as an endangered species in China.[5] Additional research needs to be carried out in order to understand its morphology and growth habit for conservation and optimum utilization.

The moths in which O. sinensis grows are ambiguously referred to as “ghost moth”, which identifies either a single species or the genus Thitarodes, and the species parasitized by O. sinensis may be one of several Thitarodes that live on the Tibetan Plateau (Tibet, Qinghai, West-Sichuan, SW-Gansu & NW Yunnan), and the Himalayas (India, Nepal, Bhutan).

O. sinensis is known in the West as a medicinal mushroom, and its use has a long history in Traditional Chinese medicine as well as Traditional Tibetan medicine.[6] The hand-collected fungus-caterpillar combination is valued by herbalists and as a status symbol;[7] it is used as an aphrodisiac and treatment for ailments such as fatigue and cancer, although such use is mainly based on traditional Chinese medicine and anecdote. Recent research however seems to indicate a variety of beneficial effects in animal testing, including increased physical endurance through heightened ATP production in rats.[8]

Cordyceps ( Dong Chong Cao ) 冬蟲草 Chinese Herbal Articles also known as chong cao, dong chong cao, yarsa gumba (Nepalese name of Tibetan origin), yartsa gunbu (dbyar rtswa dgun ‘bu) Tibetan name 蟲草, 冬蟲草. It belong to the “Ascomycetes or Clavicipitaceae” family.

Cordyceps ( Dong Chong Cao ) 冬蟲草 has a sweet, warm properties. It is use for treating the lung and kidney.

1. Improves auto-immune system.
2. Protects kidneys from toxins.
3. Protects kidneys from exhaustion.
4. Protects liver from toxins and treats and prevents cirrhosis of liver.
5. Protect the heart from the damaging effect of ouabain (C29H44O12.8H2O).
6. Anti-arrhythmia.
7. Anti-rejection effect in cornea transplant.
8. Antibiotic effect.
9. Inhibits contraction of smooth muscles.

Cordyceps ( Dong Chong Cao ) 冬蟲草 use in large dosages and/or long term usage can be toxic to kidneys.

According to the classics Medical Material, “Ben Cao Bei Yao” 本草備要, the best dong chong xia cao 冬蟲夏草, are produced in Sichuan. Today, most of them are produced in Xizang (Tibet) and Qinghai. Because the sizes the larvae are larger, they fetch higher prices.

According to the classics Medical Material, “Ben Cao Bei Yao” 本草備要, the best dong chong xia cao 冬蟲夏草, are produced in Sichuan. Today, most of them are produced in Xizang (Tibet) and Qinghai. Because the sizes the larvae are larger, they fetch higher prices.

Taxonomic History/ Systematics

Caterpillars with emergingOphiocordyceps sinensis

Morphological Features

Similar to other Cordyceps]] species, O. sinensis consists of two parts, a fungal endosclerotium (caterpillar) and stroma.^[2] The stroma is the upper fungal part and is dark brown or black, but can be a yellow color when fresh and, longer than the caterpillar itself, usually 4–10 cm. It grows singly from the larval head, and is clavate, sublanceolate or fusiform and distinct from the stipe.^[9] The stipe is slender, glabrous, and longitudinally furrowed or ridged. The fertile part of the stroma is the head. The head is granular due to the ostioles of the embedded perithecia.^[2] The perithecia are ordinally arranged and ovoid ^[9] The asci are cylindrical or slightly tapering at both ends, and may be straight or curved, with a capitate and hemispheroid apex and may be two to four spored.^[2] Similarly, ascospores are hyaline, filiform, multiseptate at a length of 5-12 um and subattenuated on both sides.^[9] Perithecial, ascus and ascospore characters in the fruiting bodies are the key identification characteristics of O. sinensis. Ophiocordyceps (Petch) Kobayasi species produce whole ascospores and do not separate into part spores which is different from other Cordyceps species, which produce either immersed or superficial perithecia perpendicular to stromal surface and the ascospores at maturity are disarticulated into part spores.^[10] Generally Cordyceps species possess brightly colored and fleshy stromata, but O. sinensis had dark pigments and tough to pliant stromata, a typical characteristic feature of most of the Ophiocordyceps species.^[3]

Important developments in Classification

The species was first described scientifically by Miles Berkeley in 1843 as Sphaeria sinensis;^[11] Pier Andrea Saccardo transferred the species to the genus Cordyceps in 1878.^[12]The scientific name‘s etymology is from the Latin cord ”club”, ceps ”head”, and sinensis ”from China“. The fungus was known as Cordyceps sinensis until 2007, when molecularanalysis was used to emend the classification of the Cordycipitaceae and the Clavicipitaceae, resulting in the naming of a new family Ophiocordycipitaceae and the transfer of several Cordyceps species to Ophiocordyceps.^[10] Based on a molecular phylogenetic study, Sung et al. (2007) separated the megagenus Cordyceps into four genera as it was polyphyletic, viz. Cordyceps (40 spp.), Ophiocordyceps (146 spp.), Metacordyceps (6 spp.) and Elaphocordyceps (21 spp.), while the remaining 175 spp. were left in Cordyceps. As a result, C. sinensis was transferred to Ophiocordyceps, hence renamed as O. sinensis.^[2]

Common Names[edit]

In Tibetan it is known as དབྱར་རྩྭ་དགུན་འབུ་ (ZYPY: yartsa gunbu, Wylie: dbyar rtswa dgun ‘bu, “summer grass winter bug”), which is the source of the Nepali यार्शागुम्बा, yarshagumba,yarchagumba or yarsagumba. The transliteration in Bhutan is Yartsa Guenboob. It is known as keera jhar, keeda jadi, keeda ghas or ‘ghaas fafoond in Hindi. Its name in Chinese Dōng chóng xià cǎo (冬蟲夏草) means “winter worm, summer grass” (i.e., “worm in the winter, [turns to] plant in the summer”). The Chinese name is a literal translation of the original Tibetan name, which was first recorded in the 15th Century by the Tibetan doctor Zurkhar Namnyi Dorje. In colloquial Tibetan Yartsa gunbu is often shortened to simply “bu” or “yartsa”.

In traditional Chinese medicine, its name is often abbreviated as chong cao (蟲草 “insect plant”), a name that also applies to other Cordyceps species, such as C. militaris. InJapanese, it is known by the Japanese reading of the characters for the Chinese name, tōchūkasō (冬虫夏草).

Strangely, sometimes in Chinese English language texts Cordyceps sinensis is referred to as aweto [Hill H. Art. XXXVI: The Vegetable Caterpillar (Cordiceps robertsii). Transactions and Proceedings of the Royal Society of New Zealand 1868-1961. Vol 34, 1901;396-401], which is the Māori name for Cordyceps robertsii, a species from New Zealand.

The English term “vegetable caterpillar” is a misnomer, as no plant is involved. “Caterpillar fungus” is a preferable term.

Nomenclature of the anamorph

Since the 1980s, 22 species in 13 genera have been attributed to the anamorph of O. sinensis. Of the 22 species, Cephalosporium acreomonium is the zygomycetous species ofUmbelopsis, Chrysosporium sinense has very low similarity in RAPD polymorphism, hence it is not the anamorph. Likewise, Cephalosporium dongchongxiacae, C. sp. sensu,Hirsutella sinensis and H. hepiali and Synnematium sinnense are synonymous and only H. sinensis is only validly published in articles. Cephalosporium sinensis possibly might be synonymous to H. sinensis but there is lack of valid information. Isaria farinose is combined to Paecilomyces farinosus and is not the anamorph. Several species like Isaria sp. Verticella sp. Scydalium sp. Stachybotrys sp. were identified only up to generic level, and thus it is dubious that they are anamorph. Mortierella hepiali is discarded as anamorph as it belongs to Zygomycota. Paecilomyces sinensis and Sporothrix insectorum are discarded based on the molecular evidence. P. lingi appeared only in one article and thus is discarded due to incomplete information. Tolypocladium sinense, P. hepiali, and Scydalium hepiali, have no valid information and thus are not considered as anamorph toOphiocordyceps sinensis. V. sinensis is not considered anamorph as there is no valid published information. Similarly, Metarhizium anisopliae is not considered anamorph as it has widely distributed host range, and is not restricted only in high altitude.^[13] Thus Hirsutella sinensis is considered the validly published anamorph of O. sinensis. Cordyceps nepalensis and C. multiaxialis which had similar morphological characteristics to C. sinensis, also had almost identical or identical ITS sequences and its presumed anamorph, H. sinensis. This also confirms H. sinensis to be anamorph of O. sinensis and suggests C. nepalensis and C. multiaxialis are synonyms.^[14] Evidence based on microcyclic conidiation from ascospores and molecular studies ^[2] support H. sinensis as the anamorph of the caterpillar fungus, O. sinensis.

Ecology

The caterpillars prone to infection by O. sinensis generally live 6 inches underground ^[4] in alpine grass and shrub-lands on the Tibetan Plateau and the Himalayas at an altitude between 3,000 and 5,000 m (9,800 and 16,400 ft). The fungus is reported from the northern range of Nepal, Bhutan, and also from the northern states of India, apart from northern Yunnan, eastern Qinghai, eastern Tibet, western Sichuan, southwestern Gansu provinces.^[4] The fungus consumes its host from inside out as they hibernate in alpine meadows. Usually the larvae are more vulnerable after shedding their skin, during late summer. The fungal fruiting body disperses spores which infect the caterpillar. The infected larvae tend to remain vertical to the soil surface with their heads up. The fungus then germinates in the living larva, kills and mummifies it, and then the stalk-like fruiting body emerges from the head and the fungus finally emerges from the soil by early spring.^[15] Fifty-seven taxa from seven genera (1 Bipectilus, 1 Endoclita, 1 Gazoryctra, 12 Hepialus, 2Magnificus, 3 Pharmacis, and 37 Thitarodes ^[3]) are recognized as potential hosts of O. sinensis.

Reproduction Biology

Ophiocordyceps sinensis has both teleomorphic and anamorphic phases. Spending up to five years underground before pupating, the Thitarodes caterpillar is attacked while feeding on roots. It is not certain how the fungus infects the caterpillar; possibly by ingestion of a fungal spore or by the fungus mycelium invading the insect through one of the insect’s breathing pores. The dark brown to black fruiting body (or mushroom) emerges from the ground in spring or early summer, the long, usually columnar fruiting body reaches 5–15 cm above the surface and releases spores.

In late autumn, chemicals on the skin of the caterpillar interact with the fungal spores and release the fungal mycelia, which then infects the caterpillar.^[4] After invading a host larva, the fungus ramifies throughout the host and eventually kills it. Gradually the host larvae become rigid due to the production of fungal sclerotia. Fungal sclerotia are multihyphal structures that can remain dormant and then germinate to produce spores. After over-wintering, the fungus ruptures the host body, forming a sexual sporulating structure (a perithecial stroma) from the larval head in summer that is connected to the sclerotia (dead larva) below ground and grows upward to emerge from the soil.^[16] The slow growing O. sinensis grows at a comparatively low temperature, i.e., below 21^oC. Temperature requirements and growth rates are crucial factors that identify O. sinensis from other similar fungi.^[3]

Use in medicine

It is used as a curative to many diseases, anti- aging,^[17] hypoglycemic,^[18] aphrodisiac and also treatment against cancer. Ophiocordyceps sinensis serves against kidney and lung problems and stimulates the immune system; it is used for treatment of fatigue, night sweating, respiratory disease, hyperglycemia, hyperlipidemia, asthenia after severe illness, arrhythmias and other heart diseases and liver disease.^[4]

Traditional Asian medicines

Weighing the precious Caterpillar fungus in Yushu, Southern Qinghai,China, imported from Nepal

Medicinal use of the caterpillar fungus apparently originated in Tibet and Nepal. So far the oldest known text documenting its use was written in the late fourteen hundreds by the Tibetan doctor Zurkhar Nyamnyi Dorje (Wylie: Zur mkhar mnyam nyid rdo rje)[1439-1475]) in his text: Man ngag bye ba ring bsrel (“Instructions on a Myriad of Medicines”). A translation is available at Winkler.^[19]

The first mention of Ophiocordyceps sinensis in traditional Chinese Medicine was in Wang Ang’s 1694 compendium of materia medica, Ben Cao Bei Yao.^[20] In the 18th Century it was listed in Wu Yiluo‘s Ben cao cong xin (“New compilation of materia medica”).^[21] No sources have been published to uphold widespread claims of “thousands of years of use in Chinese medicine” or use of “chong cao since the 7th Century Tang Dynasty in China”. The ethno-mycological knowledge on caterpillar fungus among the Nepalese people is documented byDevkota(2006) The entire fungus-caterpillar combination is hand-collected for medicinal use.

The fungus is a medicinal mushroom which is highly prized by practitioners of Tibetan medicine, Chinese medicine and traditional Folk medicines, in which it is used as an aphrodisiac and as a treatment for a variety of ailments from fatigue to cancer. In Chinese medicine it is regarded as having an excellent balance of yin and yang as it is apparently both animal and vegetable. Assays have found thatOphiocordyceps species produce many pharmacologically active substances. They are now cultivated on an industrial scale for their medicinal value. However, no one has succeeded so far in growing the larva cum mushroom artificially. The biological process that forms the Ophiocordyceps is still unknown and true cultivation has yet to be realized.^[3] All artificial products are derived from mycelia grown on grains or in liquids.

According to Bensky et al. (2004), laboratory-grown C. sinensis mycelia have similar clinical efficacy and less associated toxicity. He notes a toxicity case of constipation, abdominal distension, and decreased peristalsis, two cases of irregular menstruation, and one case report ofamenorrhea following ingestion of tablets or capsules containing C. sinensis. In Chinese medicine C. sinensis is considered sweet and warm, entering the lung and kidney channels; the typical dosage is 3–9 grams.^[22]

Research

Cordycepin, a compound isolated from the “Caterpillar fungus”.

Some work has been published in which Ophiocordyceps sinensis has been used to protect the bone marrow and digestive systems ofmice from whole body irradiation.^[23] An experiment noted Ophiocordyceps sinensis may protect the liver from damage.^[24] An experiment conducted with mice noted the mushroom may have an anti-depressant effect.^[25] Researchers have noted that the caterpillar fungus has ahypoglycemic effect and may be beneficial for people with insulin resistance.^{[26][27][28][29][30]} There is also experimental evidence of the supposed energizing effect of the fungus, as it has been shown to increase endurance through heightened ATP production in rats.^[8]

A March 2013 study on Cordyceps Sinensis documented the medicinal fungus’ anti-inflammatory properties.^[31] Scientists were able to show Cordyceps Sinensis’ ability to suppress interleukin-1b and interleukin-18 secretion by inhibiting both canonical and non-canonical inflammasomes. Inflammasomes have long been associated with auto-inflammatory diseases, such as gout. The study used a specific anamorphic mycelial form of Cordyceps Sinensis known as Hirsutella Sinensis.

Introduction to the Western world

Ophiocordyceps sinensis

The Western world was largely unaware of Ophiocordyceps prior to 1993. The fungus dramatically caught the world’s eye due to the performance of three female Chinese athletes, Wang Junxia, Qu Yunxia, and Zhang Linli. These athletes broke five world records for 1,500, 3,000 and 10,000 meter dashes at the National Games in Beijing, China. The number of new world records set at a single track event attracted much attention and suspicion. Following the races, the women were expected by some to fail drug tests for anabolic steroids. However, the athletes’ tests revealed no illegal substances, and coach Ma Junren told the reporters that the runners were takingOphiocordyceps sinensis and turtle blood at his request. However for the 2000 Sydney Olympics, Ma Junren withdrew some of his athletes at the last minute. It was speculated that a new doping test would have revealed illegal substances, thus half a dozen Chinese field and track athletes were left at home.

Economics and impact

Many shops in downtown Lanzhouadvertise Dōng chóng xià cǎo (冬虫夏草) among other local specialties.

In rural Tibet, yartsa gunbu has become the most important source of cash income. The fungi contributed 40% of the annual cash income to local households and 8.5% to the GDP in 2004. Prices have increased continuously, especially since the late 1990s. In 2008, one kilogram traded for US$3,000 (lowest quality) to over US$18,000 (best quality, largest larvae). The annual production on the Tibetan Plateau was estimated in 2009 at 80–175 tons.^[32] The Himalayan Ophiocordyceps production might not exceed a few tons.

In 2004 the value of a kilogram of caterpillars was estimated at about 30,000 to 60,000 Nepali rupees in Nepal, and about Rs 100,000 in India.^[33] In 2011 the value of a kilogram of caterpillars was estimated at about 350,000 to 450,000 Nepali rupees in Nepal. A 2012 BBC article indicated that in north Indian villages a single fungus was worth Rs 150 (about £2 or $3), which is more than the daily wage of a manual laborer.^[34]

According to Daniel Winkler, the price of Ophiocordyceps sinensis has risen dramatically on the Tibetan Plateau, basically 900% between 1998 and 2008, an annual average of over 20% (after inflation). However, the value of big sized caterpillar fungus has increased more dramatically than smaller size Cordyceps, regarded as lower quality.^[20]

Because of its high value, inter-village conflicts over access to its grassland habitats has become a headache for the local governing bodies and in several cases people were killed. In November 2011, a court in Nepal convicted 19 villagers over the murder of a group of farmers during a fight over the prized aphrodisiac fungus. Seven farmers were killed in the remote northern district of Manang in June 2009 after going to forage for Yarchagumba. ^[35]

Its value gave it a role in the Nepalese Civil War, as the Nepalese Maoists and government forces fought for control of the lucrative export trade during the June–July harvest season.^[36] Collecting yarchagumba in Nepal had only been legalised in 2001, and now demand is highest in countries such as China, Thailand, Vietnam, Korea and Japan. By 2002, the herb was valued at R 105,000 ($1,435) per kilogram, allowing the government to charge a royalty of R 20,000 ($280) per kilogram.

The search for Ophiocordyceps sinensis is often perceived to pose a threat to the environment of the Tibetan Plateau where it grows. While it has been collected for centuries and is still common in such areas, current collection rates are much higher than in historical times.

Ophiocordyceps producers like to perpetuate the story that unscrupulous harvesters insert twigs into the ascocarps of wild C. sinensis to increase their weight and therefore the price paid. A tiny twig is only used when the ascocarp is broken from the caterpillar, and has nothing to do with artificially increasing weight. Supposedly, at some point in the past, someone inserted lead wires with which to increase weight; however, each year hundreds of millions of specimens are harvested and this appears to have been a one-time occurrence.

Cultivated C. sinensis mycelium is an alternative to wild-harvested C. sinensis, and producers claim it may offer improved consistency. Artificial culture of C. sinensis is typically by growth of pure mycelia in liquid culture (in China) or on grains (in the West). The first time in Vietnam, Prof. Aca. Dr. Dai Duy Ban together with scientists and DAIBIO Company and DAIBIO Great Traditional Medicine Family Clinic discovered the Cordyceps sinensis as Isaria cerambycidae N.SP. to develop Fermentation DAIBIO Cordyceps Sinensis.^[37]Ascocarps are not produced through in vitro cultivation.

References

• Winkler, D. 2005. Yartsa Gunbu – Cordyceps sinensis. Economy, Ecology & Ethno-mycology of a Fungus Endemic to the Tibetan Plateau. In: A.BOESI & F. CARDI (eds.). Wildlife and plants in traditional and modern Tibet: Conceptions, Exploitation and Conservation. Memorie della Società Italiana di Scienze Naturali e del Museo Civico di Storia Naturale di Milano, Vol. 33.1:69–85.
• Zhang Y., Zhang S., Wang M., Bai F. & Liu X. (2010). “High Diversity of the Fungal Community Structure in Naturally-Occurring Ophiocordyceps sinensis“. PLoS ONE 5(12): e15570. doi:10.1371/journal.pone.0015570.

External links

Yartsa Gunbu (Cordyceps sinensis) in Tibet

• Daniel Winkler’s Cordyceps blog
• Nepal’s Nature – The Himalayan Viagra
• Page at Everything2.com
• Image gallery of Cordyceps sinensis
• The first time in Vietnam, Prof.Aca.D.Sc Dai Duy Ban with his scientists discovered Cordyceps Sinensis as Isaria cerambycidae N.SP. and Fermentation Daibio Cordyceps Sinensis by Daibio Great Family Traditional Medicine Clinic Company
• Daibio Cordyceps Sinensis in Vietnam
• An Electronic Monograph of Cordyceps and Related Fungi
• Cordyceps information from Drugs.com
• Cordyceps sinensis (Berk.) Sacc. Medicinal Plant Images Database (School of Chinese Medicine, Hong Kong Baptist University) (English) (traditional Chinese)
• Chinese Caterpiller Fungus Chinese Medicine Specimen Database (School of Chinese Medicine, Hong Kong Baptist University) (English) (traditional Chinese)
• Tibet’s Golden “Worm” August 2012 National Geographic (magazine)

ELLAGIC ACID

476-66-4 

2,3,7,8-Tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione

as a very potent CK2 inhibitor

Ellagic acid is a natural phenol antioxidant found in numerous fruits and vegetables. The antiproliferative and antioxidant properties of ellagic acid have spurred preliminary research into the potential health benefits of ellagic acid consumption.

Ellagic acid is the dilactone of hexahydroxydiphenic acid.

Ellagic acid is an antioxidant and an anti-proliferative compound present in fruits, nuts and vegetables. In spite of evidences for anticancer activity in various cancer cell-lines, human cancer cells, the mechanistic role of ellagic acid is not conclusive enough to be recommended for a clinical use. The present review provides information about the chemopreventive role of ellagic acid in oral cancer and proposes molecular basis for ellagic acid’s inhibitory activity against oral cancer. We show that ellagic acid modulates growth of tumor cells through regulation of multiple cell signaling pathways including cell proliferation pathway (cyclin dependent kinase 2, cyclin A2, cyclin B1, cyclin D1, c-myc, PKCα), cell survival/apoptosis pathway (Bcl-XL, Bax, Caspase 9/3, Akt), tumor suppressor pathway (p53, p21), inflaming Metastasis pathways (IL-1 beta, TNF-α, matrix metalloproteinases 9/3, COX-2), angiogenesis pathways (VEGF), cell immortalization (TERT), NF-κβ.

History

Ellagic acid was first discovered by chemist Henri Braconnot in 1831.^[4]:20 Maximilian Nierenstein prepared this substance fromalgarobilla, dividivi, oak bark, pomegranate, myrabolams, and valonea in 1905.^[4]:20 He also suggested its formation from galloyl-glycine by Penicillium in 1915.^[5] Löwe was the first person to synthesize ellagic acid by heating gallic acid with arsenic acid or silver oxide.^[4]:20 [6]

Natural occurrences

Ellagic acid is found in oaks species like the North American white oak (Quercus alba) and European red oak (Quercus robur).^[7]

The  macrophyte Myriophyllum spicatum produces ellagic acid.^[8]

Ellagic acid can be found in the medicinal mushroom Phellinus linteus.^[9]

In food

The highest levels of ellagic acid are found in blackberries, cranberries, pecans, pomegranates, raspberries, strawberries, walnuts,wolfberries, and grapes.^[10] It is also found in peach^[11] and other plant foods.

Description

Ellagic acid is a phytochemical, or plant chemical, found in raspberries, strawberries, cranberries, walnuts, pecans, pomegranates, and other plant foods.

Biosynthesis

Plants produce ellagic acid from hydrolysis of tannins such as ellagitannin^[1] and geraniin.^[2]:208

Biodegradation

Urolithins are microflora human metabolites of dietary ellagic acid derivatives^[3]

Overview

Research in cell cultures and laboratory animals has found that ellagic acid may slow the growth of some tumors caused by certain carcinogens. While this is promising, at this time there is no reliable evidence available from human clinical studies showing that ellagic acid can prevent or treat cancer. Further research is needed to determine what benefits it may have.

How is it promoted for use?

Ellagic acid seems to have some anti-cancer properties. It can act as an anti-oxidant, and has been found to cause cell death in cancer cells in the laboratory. In other laboratory studies, ellagic acid seems to reduce the effect of estrogen in promoting growth of breast cancer cells in tissue cultures. There are also reports that it may help the liver to break down or remove some cancer-causing substances from the blood.

Some supporters have claimed these results mean that ellagic acid can prevent or treat cancer in humans. This has not been proven. Unfortunately, many substances that show promise against cancer in laboratory and animal studies are not found to be useful in people.

Ellagic acid has also been said to reduce heart disease, birth defects, liver problems, and to promote wound healing.

What does it involve?

The highest levels of ellagic acid are found in raspberries, strawberries, and pomegranates, especially when they are freeze-dried. Extracts from red raspberry leaves or seeds, pomegranates, or other sources are said to contain high levels of ellagic acid and are available as dietary supplements in capsule, powder, or liquid form. The best dose of these preparations is not known.

What is the history behind it?

Ellagic acid was studied in the 1960s mainly for its effects on blood clotting. Early published research on ellagic acid and cancer first appeared in the 1970s and 1980s. With the publication of several small laboratory studies in the mid-1990s, ellagic acid began to be promoted on the Internet and elsewhere as a means of preventing and treating cancer.

What is the evidence?

Almost all studies conducted on ellagic acid to date have been done in cell cultures or laboratory animals. Several animal studies have found that ellagic acid can inhibit the growth of tumors of the skin, esophagus, and lung, as well as other tumors caused by carcinogens. Other studies have also found positive effects. A recent study in cell cultures found that ellagic acid may act against substances that help tumors to form new blood vessels. Further studies are needed to determine whether these results apply to humans.

In the only study reported thus far in humans, Italian researchers found that ellagic acid seemed to reduce the side effects of chemotherapy in men with advanced prostate cancer, although it did not slow disease progression or improve survival. The researchers cautioned that more research would be needed to confirm these results.

The interaction between phytochemicals like ellagic acid and the other compounds in foods is not well understood, but it is unlikely that any single compound offers the best protection against cancer. A balanced diet that includes 5 or more servings a day of fruits and vegetables along with foods from a variety of other plant sources such as nuts, seeds, whole grain cereals, and beans is likely to be more effective in reducing cancer risk than eating one particular food, such as raspberries, in large amounts. However, some studies suggest that foods high in ellagic acid might be useful additions to a balanced diet. For example, one nonrandomized clinical study of men with prostate cancer reported that pomegranate juice slowed the increase in blood levels of prostate-specific antigen, a substance that is routinely measured to estimate growth of prostate cancer.

Are there any possible problems or complications?

Eating berries or other natural sources of ellagic acid is generally considered safe. These foods should be part of a balanced diet that includes several servings of fruits and vegetables each day.

Ellagic acid is available in supplement form. Some reports indicate it may affect certain enzymes in the liver, which could alter the way in which some drugs are absorbed. For this reason, people taking medicines or other dietary supplements should talk with their doctors or pharmacists about all their medicines and supplements before taking ellagic acid. The raspberry leaf, or preparations made from it, should be used with caution during pregnancy because it may initiate labor.

Relying on this type of treatment alone and avoiding or delaying conventional medical care for cancer, may have serious health consequences.

Research into potential medicinal uses

Ellagic acid has antiproliferative and antioxidant properties in a number of in vitro and small-animal models.^[12][13] The antiproliferative properties of ellagic acid may be due to its ability to directly inhibit the DNA binding of certain carcinogens, including nitrosamines^[14][15] and polycyclic aromatic hydrocarbons.^[16] As with other polyphenol antioxidants, ellagic acid has a chemoprotective effect in cellular models by reducing oxidative stress.^[10]

These properties have generated interest in potential human health benefits from the consumption of ellagic acid. However, very little study of these proposed benefits has been reported as of 2010. A small randomized controlled trial involving 19 patients with carotid artery stenosis found that pomegranate juice, which is high in ellagic acid, appeared to reduce blood pressure and carotid artery wall thickness.^[17] A 2005 controlled study of 48 patients undergoing chemotherapy for prostate cancer found that ellagic acid supplementation reduced the rate of chemotherapy-associated neutropenia (though there were no cases of severe neutropenia in either the ellagic acid or control group). Ellagic acid supplementation did not improve overall or progression-free survival of patients with prostate cancer in this trial.^[18]

Despite the very preliminary state of evidence supporting health benefits in humans, ellagic acid has been marketed as a dietary supplement with a range of claimed benefits against cancer, heart disease, and other medical problems. Ellagic acid has been identified by the U.S. Food and Drug Administration as a “Fake Cancer Cure Consumers Should Avoid”.^[19] A number of U.S.-based sellers of dietary supplements have received Warning Letters from the Food and Drug Administration for promoting ellagic acid with claims that violate the Federal Food, Drug, and Cosmetic Act.^[20][21]

Urolithins, such as urolithin A, are microflora human metabolites of dietary ellagic acid derivatives that are under study as anti-cancer agents.^[22]

Oral cancer develops by complex interplay between intrinsic and extrinsic factors playing important role in tumor development from primary lesion. The process of expression of tumorigenesis is based on a tightly controlled sequence of events which are dependent on the proper levels of transcription and translation of certain genes. There is a small subset that seems to be particularly important in the prevention, development, and progression of cancer.

These genes have been found to be either malfunctioning or non-functioning in oral cancer. It is, therefore, logical to believe that success of any therapy will depend on its effectiveness to modulate these genes controlling different pathways to restore homeostasis. Molecular targets of ellagic acid are the key regulators, spread across all cancer hallmarks, which should make it an effective agent for prevention of oral cancer. Ellagic acid is known to modulate key regulators like NF-κβ, p53 and CK2. The versatility of EA to inhibit oral carcinogenesis through multiple pathways makes ellagic acid a potent chemopreventive agent

Ellagic Acid Research

Over the last 30 years there has been a great deal of information accumulated in the literature concerning the carcinogenic process. It has been established that disruption of the delicate balance between cell growth and programmed cell death (apoptosis) in favor of the former by inhibiting or slowing down the latter, results in tumor formation. There are two apoptotic pathways: a) the so called “stress pathway” or intrinsic pathway which is initiated by profound damage to the DNA by either chemotherapy and/or radiation; and b) the extrinsic pathway which is initiated by binding of specific cytokines released by cells to their so called “death receptors” expressed on cancer cells also following heavy damage of the DNA.

The binding of cytokines to their death receptors exposed on the cancer cells will result in programmed cell death. Both apoptotic pathways meet at the caspase level. Caspases are enzymes that initiate and perform apoptosis. There are several big differences between apoptosis in cancer cells and normal cells. A normal cell has a very efficient base excision repair system and any damage to DNA is immediately and efficiently repaired. By definition, a normal cell with irreparable DNA/mutations must undergo apoptosis because of activation of p53 through the intrinsic pathway. Some cells with mutations do not die because they are resistant to apoptosis. These cells become cancer cells. Most of the time, the apoptotic process is inhibited from inside the cell by the inhibitors of apoptosis (IAPs). Normal cells do not have any inhibitors of apoptosis.

Casein kinase 2 (CK2) is a kinase that is important for cell proliferation and is over-expressed in all types of cancer studied thus far. There is an overwhelming amount of data gathered in the literature demonstrating that CK2 is over-expressed in tumor cells and is involved in the apoptosis-resistance phenotype of most malignant cells. The emerging hypothesis is that high concentrations of CK2 generate a cellular environment that is favorable to both the establishment and the development of the tumor phenotype. CK2 is localized in the cytoplasm of normal cells. However, in cancer cells, CK2 is also localized in the nucleus and is deregulated, being elevated 3- to 7- fold. The up-regulation and hyperactivity of CK2 in the nucleus has an anti-apoptotic effect and is correlated with aggressive tumor behavior in a multitude of different cancer cell types examined.

Ellagic acid (abundant in pomegranate and red and black raspberries) has been identified as a very potent CK2 inhibitor. The Ki of ellagic acid for CK2 is 20 nM. It has been shown that ellagic acid alone will kill several cancer cell lines through the apoptotic process. Ellagic acid is found naturally in pomegranate and raspberries and is transformed into urolithins that remains in plasma for a longer time. There are a multitude of publications demonstrating that casein kinase 2 maintains a high concentration of IAPs inside the cancer cells. In contrast, a simple inhibitor of casein kinase 2 will induce cell death through apoptosis in all cancer cells studied thus far, without affecting any other normal cell/tissue.

We determined the content of ellagic acid and derivatives inMeeker Red Raspberry powder and in the Black Raspberry Seed powder. Ellagic acid and its derivatives from the two samples were first separated by high pressure liquid chromatography (HPLC) prior or after acid hydrolysis. It is noteworthy that acid hydrolysis of each powder releases several urolithins which were identified according to their molecular mass. The separated components were identified by mass spectrometry. Each peak was quantified and compared to a standard made of serial dilutions of purified ellagic acid. Under the conditions employed we have determined that the Meeker Red raspberry contains 0.128 ± 0.005 mg of pure ellagic acid per gram of powder, while the Black Raspberry Seed contains 0.38 ± 0.024 mg of pure ellagic acid per gram of powder.

Following acid hydrolysis (2 hrs in 2N HCl, 50% MeOH at 95°C) we have determined that the Meeker Red raspberry powdercontains 23.7 ± 8.32 mg of ellagic acid and derivatives per gram of powder while the Black Raspberry Seed powder contains 41 ± 2.1 mg of ellagic acid and derivatives per gram of powder. The powders are usually dispensed orally with a scoop, provided within the container. The scoop contains 19 grams of powder. Under these conditions, two scoops of Meeker Red raspberry powder or of Black Raspberry Seed powder taken orally (and as a consequence subjected to acid hydrolysis in the stomach), will release approximately 1.1 gr and 1.6 gr respectively of ellagic acid and derivatives. In order to maintain a constant amount of ellagic acid and urolithins in the blood stream this procedure should be repeated daily or at least every other day.

References

DW-116; fandofloxacin

164150-99-6 FREE BASE ,

164151-00-2., 164150-85-0
6-fluoro-1-(5-fluoropyridin-2-yl)-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid

6-Fluoro-1-(5-fluoropyridin-2-yl)-7-(4-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

Dong Wha Pharmaceutical Co Ltd

Molecular Formula: C20H18F2N4O3

Molecular Weight: 400.378726

synthesis………….http://www.drugfuture.com/synth/syndata.aspx?ID=226498

http://www.google.com.mx/patents/WO1995005373A1

synthesis 1

Condensation of ethyl 2,4,5-trifluorobenzoylacetate (I) with triethyl orthoformate in refluxing Ac2O produced the benzoyl ethoxyacrylate (II), which was further condensed with 2-amino-5-fluoropyridine (III) to afford enamine (IV). Cyclization of (IV) in the presence of K2CO3 gave rise to the quinolone (V). The 7-fluoride group of (V) was then displaced by N-methylpiperazine (VI) in cold pyridine to furnish the piperazinyl quinolone (VII). Finally, ester hydrolysis in (VII) under acidic conditions yielded the target compound. In a closely related procedure, ester (V) was hydrolyzed to acid (VIII) using HCl. Subsequent displacement of the 7-fluoride of (VIII) with N-methylpiperazine (VI) provided the desired piperazinyl quinolone.

synthesis 2

Condensation of ethyl 2,4,5-trifluorobenzoylacetate (I) with triethyl orthoformate in refluxing Ac2O produced the benzoyl ethoxyacrylate (II), which was further condensed with 2-amino-5-fluoropyridine (III) to afford enamine (IV). Cyclization of (IV) in the presence of K2CO3 gave rise to the quinolone (V). The 7-fluoride group of (V) was then displaced by N-methylpiperazine (VI) in cold pyridine to furnish the piperazinyl quinolone (VII). Finally, ester hydrolysis in (VII) under acidic conditions yielded the target compound. In a closely related procedure, ester (V) was hydrolyzed to acid (VIII) using HCl. Subsequent displacement of the 7-fluoride of (VIII) with N-methylpiperazine (VI) provided the desired piperazinyl quinolone.

Synthesis, pharmacokinetics, and biological activity of a series of new pyridonecarboxylic acid antibacterial agents bearing a 5-fluoro-2-pyridyl group or a 3-fluoro-4-pyridyl group at N-1
J Heterocycl Chem 1997, 34(3): 1021

PRULIFLOXACIN

(RS)-6-Fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl-1-piperazinyl]-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid

6-Fluoro-1-methyl-7-(4-(5-methyl-2-oxo-1,3-dioxelen-4-yl)methyl-1-piperazinyl)-4-oxo-4H-(1,3)thiazeto(3,2-a)quinoline-3-carboxylic acid

123447-62-1 CAS NO

Launched – 2002 BY NIPPON SHINYAKU

SYNTHESIS…….http://www.drugfuture.com/synth/syndata.aspx?ID=151640

Prulifloxacin is an older synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class^[1][2] undergoing clinical trials prior to a possible NDA (New Drug Application) submission to the U.S. Food and Drug Administration (FDA). It is a prodrug which is metabolized in the body to the active compound ulifloxacin.^[3][4] It was developed over two decades ago by Nippon Shinyaku Co. and was patented in Japan in 1987 and in the United States in 1989.^[5][6]

It has been approved for the treatment of uncomplicated and complicated urinary tract infections, community-acquired respiratory tract infections in Italy and gastroenteritis, including infectious diarrheas, in Japan.^[3][7] Prulifloxacin has not been approved for use in the United States.

Prulifloxacin is a novel fluoroquinolone antibiotic that was launched pursuant to a collaboration between Meiji Seika and Nippon Shinyaku in 2002 for the oral treatment of systemic bacterial infections, including acute upper respiratory tract infection, bacterial pneumonia, prostatitis, cholecystitis, bacterial enteritis, internal genital infections, otitis media, sinusitis and others. It is currently marketed in a tablet formulation. A once-daily formulation to be taken over a three-day period is in phase III clinical trials at Optimer Pharmaceuticals to be used in the treatment of bacterial gastroenteritis, including traveler’s diarrhea. The formulation had been in phase II trials at the company for the treatment of urinary tract infections, however, no recent development for this indication have been reported. The drug has also been studied at Optimer for the treatment of community-acquired respiratory tract infections, but recent progress reports for this indication have not been made available.

Prulifloxacin has in vitro activity against a wide range of gram-negative and gram-positive microorganisms. Its antibacterial action results from inhibition of DNA gyrase and topoisomerase IV, both Type II isomerases. DNA gyrase is an essential enzyme that is involved in the replication, transcription, and repair of bacterial DNA. Topoisomerase IV is an enzyme known to play a key role in the partitioning of the chromosomal DNA during bacterial cell division. Together, the Type II topoisomerases remove the positive supercoils that accumulate ahead of a translocating DNA polymerase, allowing DNA replication to continue unhindered by topological strain. Fluoroquinolones may be active against pathogens that are resistant to penicillins, cephalosporins, aminoglycosides, macrolides and tetracyclines, as they possess a distinct mechanism of action from these antibiotics.

Prulifloxacin was discovered by Nippon Shinyaku and codeveloped with Meiji Seika in Japan. Nippon Shinyaku granted Angelini a manufacturing and marketing license for Italy in 1993. Exclusive Korean manufacturing and commercialization rights were acquired by Yuhan from Nippon Shinyaku in March 2003. In June 2004, Optimer was granted exclusive development and commercialization rights to prulifloxacin in the U.S. from Nippon Shinyaku. Finally, Recordati signed a nonexclusive licensing agreement with Angelini for the marketing and sale of prulifloxacin in Spain in October 2004. In March 2009, the product was licensed to Lee’s Pharmaceuticals by Nippon Shinyaku for marketing in China as an oral treatment of bacterial infection. In 2010, prulifloxacin was licensed to Algorithm by Nippon Shinyaku in North Africa and the Middle East for the development and marketing for the treatment of bacterial infections.

History

In 1987 a European Patent (EP 315828) for prulifloxacin (Quisnon ) was issued to the Japanese based pharmaceutical company, Nippon Shinyaku Co., Ltd (Nippon). Ten years after the issuance of the European patent, marketing approval was applied for and granted in Japan (March 1997). Subsequent to being approved by the Japanese authorities in 1997 prulifloxacin (Quisnon) was co-marketed and jointly developed in Japan with Meiji Seika as licensee (Sword).^[6]

In more recent times, Angelini ACRAF SpA, under license from Nippon Shinyaku, has fully developed prulifloxacin, for the European market.^[8] Angelini is the licensee for the product in Italy. Following its launch in Italy, Angelini launched prulifloxacin in Portugal (January 2007) and it has been stated that further approvals will be sought in other European countries.^[9][10]

Prulifloxacin is marketed in Japan and Italy as Quisnon (Nippon Shinyaku); Sword (Meiji); Unidrox (Angelini) and generic as Pruquin.

In 1989 and 1992 United States patents (US 5086049) were issued to Nippon Shinyaku for prulifloxacin. It was not until June 2004, when Optimer Pharmaceuticals acquired exclusive rights to discover, develop and commercialize prulifloxacin (Pruvel) in the U.S. from Nippon Shinyaku Co., Ltd., that there were any attempts to seek FDA approval to market the drug in the United States. Optimer Pharmaceuticals expects to file an NDA (new drug application) for prulifloxacin some time in 2010. As the patent for prulifloxacin has already expired, Optimer Pharmaceuticals has stated that this may have an effect on the commercial prospects of prulifloxacin within the United States market.^[11]

Licensed uses

Prulifloxacin has been approved in Italy ,Japan,China,India and Greece (as indicated), for treatment of infections caused by susceptible bacteria, in the following conditions:

Italy

• Acute uncomplicated lower urinary tract infections (simple cystitis)
• Complicated lower urinary tract infections
• Acute exacerbation of chronic bronchitis

Japan

• Gastroenteritis, including infectious diarrheas

Other countries

• Prulifloxacin has not been approved for use in the United States, but may have been approved in other Countries, other than that which is indicated above.

Availability

Prulifloxacin is available as:

• Tablets (250 mg, 450 mg or 600 mg)

In most countries, all formulations require a prescription.

Prulifloxacin is chemically known as 6-fluoro-1-methyl-7-{4-[(5-methyl-2-oxo-1 ,3-dioxol- 4-yl)methyl]piperazin-1-yl}-4-oxo-4H-[1 ,3]-thiazeto-[3,2-a]-quinoline-3-carboxylic acid, and it has the structure as shown below as formula I:

FORMULA I

Prulifloxacin has significant antibacterial activity and has been marketed as a synthetic antibacterial agent.

Prulifloxacin was first disclosed in US 5,086,049. The patent discloses a process for the preparation of prulifloxacin by the condensation of ulifloxacin with a 4-halomethyl-5- methyl-1 ,3-dioxolen-2-one of formula III

wherein X is halo selected form chloro, bromo or iodo, in the presence or absence of an aprotic solvent and a base to obtain prulifloxacin free base which is recrytallised with chloroform-methanol. In an exemplified process, ethyl 6,7-difluoro-1-methyl-4-oxo-4H- (1 ,3)-thiazeto-(3,2-a)-quinoline-3-carboxylate is condensed with piperazine in the presence of dimethyl formamide and purified by column chromatography followed by basic hydrolysis to give ulifloxacin, which is then converted to prulifloxacin.

The above process involves column chromatography. Prulifloxacin prepared by this method has a purity of 60-65% containing impurities in unacceptable levels. Removal of these impurities by usual purification procedures, such as recrystallisation, distillation and washing, is difficult and requires extensive and expensive multiple purification processes. This further decreases the overall yield. A method involving column chromatographic purifications and multiple purifications cannot be used for large-scale operations, thereby making the process commercially non-viable.

European Patent No. 315828 disclosed a variety of quinoline carboxylic acid derivatives and pharmaceutically acceptable salts thereof. These compounds are exhibiting antibacterial activity and useful as remedies for various infectious diseases. Among them prulifloxacin, chemically (+)-6-Fluoro- 1 -methyl-7-[4-(5-methyl-2-oxo-1 ,3-dioxolen-4-ylmethyl)-1 -piperazinyl]-4-oxo-4H- [1 ,3]thiazeto[3,2-a]quinoline-3-carboxylic acid is a fluoroquinolone antibacterial prodrug which shows potent and broad-spectrum antibacterial activity both in vitro and in vivo. Prulifloxacin also showed superior activity against strains of Enterobacteriaceae and Pseudomonas aeruginosa. Prulifloxacin is represented by the following structure:

Processes for the preparation of prulifloxacin and related compounds were disclosed in European Patent No. 315828 and UK Patent Application No. GB 2190376.

In – the preparation of prulifloxacin, 6-fluoro-1-methyl-4-oxo-7-(1- piperazinyl)-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylic acid of formula I:

is a key intermediate. According to the UK Patent Application No. GB 2190376, the compound of the formula I was prepared by the reaction of 3,4-difluroaniline with carbon disulfide and triethylamine to give triethylammonium N-(3,4- difluorophenyl)dithio carbamate, which by reaction with ethyl chloroformate and triethylamine in chloroform is converted into 3,4-difluorophenyl isothiocyanate, followed by reaction with diethyl malonate and KOH in dioxane affords the potassium salt, which is then treated with methoxymethyl chloride in dimethylformamide to give diethyl 1-(3,4-difluorophenylamino)-1- (methoxymethylthio)-rnethylene-rnalonate. The cyclization of the thio compound at 240⁰C in diphenyl ether affords ethyl 6,7-difluoro-4-hydroxy-2- methoxymethylthioquinoline-3-carboxylate, which by treatment with HCI in ethanol gives ethyl δy-difluoro^-hydroxy^-mercaptoquinoline-S-carboxylate. The cyclization of the mercapto compound with 1,1-dibromoethane by means of potassium carbonate and potassium iodide in hot dimethylformamide yields ethyl 6,7-difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate, which is condensed with piperazine in dimethylformamide to afford ethyl 6- fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]thiazeto[3,2-a]quinoline-3- carboxylate, which is then subjected to hydrolysis with potassium hydroxide in hot tert-butanol to give the compound of formula I.

The compound of formula I obtained by the process described in the UK Patent Application No. GB 2190376 is not satisfactory from purity point of view, the reaction between ethyl 6,7-difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2- a]quinoline-3-carboxylate and piperazine requires longer time about 48 hours to complete, the yield obtained is not satisfactory, and the process also involves column chromatographic purifications. Methods involving column chromatographic purifications cannot be used for large-scale operations, thereby making the process commercially not viable. According to the European Patent No. 315828, prulifloxacin is prepared by reacting 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]thiazeto[3,2-a] quinoline-3-carboxylic acid with 4-bromomethyl-5-methyl-1 ,3-dioxolen-2-one in presence of potassium bicarbonate in dimethylformamide. However, a need still remains for an improved and commercially viable process of preparing pure prulifloxacin that will solve the aforesaid problems associated with process described in the prior art and will be suitable for large- scale preparation, in terms of simplicity, purity and yield of the product.

Prulifloxacin is chemically 6-fluoro-l-methyl-7-{4-[(5-methyl-2-oxo-l,3-dioxol-4- yl)methyl]piperazin-l-yl}-4-oxo-4H-[l,3]thiazeto[3,2-α]quinoline-3-carboxylic acid of Formula I having the structure as depicted below:

FORMULA I

Prulifloxacin has significant antibacterial activity and has been marketed as a synthetic antibacterial agent. U.S. Patent No. 5,086,049 provides a process for the preparation of prulifloxacin by reacting 6-fluoro-l-methyl-4-oxo-7-piperazin-l-yl-4H- [l,3]thiazeto[3,2-α]quinoline-3-carboxylic acid of Formula II,

FORMULA II and 4-(bromomethyl)-5-methyl-l,3-dioxol-2-one of Formula III,

FORMULA III using N,N-dimethylformamide as a solvent. 4-(Bromomethyl)-5-methyl-l,3-dioxol-2-one of Formula III is used in excess to one mole of the compound of Formula II. The process provided in U.S. Patent No. 5,086,049 further involves concentrating the reaction mixture, pouring the residue into water and isolating prulifloxacin by filtration. The resulting prulifloxacin is recrystallized from chloroform-methanol.

However, U.S. Patent No. 5,086,049 does not provide any method to remove the unreacted or the excess of 4-(bromomethyl)-5-methyl-l,3-dioxol-2-one of Formula III used as a starting material. The present inventors have observed that it is difficult to obtain prulifloxacin with pharmaceutically acceptable purity by following the process provided in U.S. Patent No. 5,086,049, which is typically contaminated by process related impurities including 4-(bromomethyl)-5-methyl-l,3-dioxol-2-one

A need still remains for an improved and commercially-viable process for preparing pure prulifloxacin that will solve the aforesaid problems associated with the process described in the prior art and that will be suitable for large-scale preparation, in terms of simplicity, purity and yield of the product.

EP1626051 A1 mentions that Type I, Type II and Type III crystals of prulifloxacin are obtained by crystallization from acetonitrile as reported in lyakuhin Kenkyu, Vol. 28 (1), (1997), 1-11. However, the conditions of crystallization from acetonitrile for preparing Type I, Type II and Type III crystals are not disclosed in lyakuhin Kenkyu, Vol. 28 (1), (1997), 1-11. EP1626051A1 further mentions that Type III crystals have been marketed by considering the solubility, absorbability, therapeutic effect and the like of the respective crystal forms.

US 2007/0149540 discloses a crystal of prulifloxacin acetonitrile solvate (Compound B) which is an intermediate for producing preferentially the type III crystal of prulifloxacin. A crystal of Compound B can be preferentially precipitated by controlling the supersaturation concentration in crystallization using acetonitrile as a solvent, subsequently; the type III crystal of Compound A can be produced by performing desolvation of the crystal.

WO 2008/111018 discloses processes for the preparation of Type I, Type II and Type III crystals of prulifloxacin. There is disclosed a process for preparing Type I crystals by controlled cooling over a period of 7 to 9 hours and prolonged drying over 24 hours. The inventors of the present invention have found that Type I and Type III crystals prepared according to the WO 2008/111018 process are unstable and the process is non-reproducible.

WO 2010/0084508 discloses processes for the preparation of Type I, Type II and Type III crystals of prulifloxacin.

WO 2008/059512 discloses a process for the preparation of prulifloxacin using novel intermediates.

WO 2008/111016 discloses a process for the preparation of prulifloxacin having purity of about 99% or above. It would be a significant contribution to the art to provide a crystalline form of prulifloxacin, which is consistent and to provide industrially viable methods of preparation, pharmaceutical formulations, and methods of use thereof.

…………………

SYNTHESIS

http://www.google.com/patents/WO2012001357A1?cl=en

Scheme 1.

Formula I

[PRULIFLOXACIN]

Example 1

Preparation of ethyl-6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-(1 ,3)-thiazeto-[3,2-a]- quinoline-3-carboxylate (formula III)

5,6-difluoro-1-methyl-4-oxo-4H-[1 ,3]-thiazeto-[3,2-a]-quinoline-3-carboxylic acid ethyl ester of formula (II) (100 gms, 0.321 moles) was stirred in 500 ml of DMF at room temperature. Piperazine (76 gms, 0.882 moles) was added at room temperature and stirred for 10 minutes. The temperature was slowly raised to 50-55°C and the reaction mass was stirred at 50-55°C for 5 hours. After completion of the reaction, the reaction mass was cooled to 25-30°C and stirred for 2 hours. The reaction mass was further chilled to 10-15°C and stirred for 2 hours. The precipitated solid was filtered, washed of chilled DMF (2 x 50 ml). The solid was slurry washed with water (300 ml), filtered, washed with water ( 2 x 100 ml) and dried under vacuum at 70-75°C to yield the title compound [90 gms, 74 % yield, 95% HPLC purity].

Example 2

Preparation of 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]-thiazeto-[3,2-a]- quinoline-3-carboxylic acid (formula IV)

Ethyl-6-fluoro-1 -methyl-4-oxo-7-(1 -piperazinyl)-4H-(1 ,3)-thiazeto-[3,2-a]-quinoline-3- carboxylate (100 gms, 0.265 moles) was stirred in water (600 ml) at 25-30°C. To this potassium hydroxide solution (50 gms of potassium hydroxide flakes is dissolved in 200 ml of water) was added and the reaction mass was heated to 80-85°C. The contents were stirred for 1 hour and after completion of reaction, the reaction mass was cooled to 25-30°C. The pH of the reaction mass was adjusted to 6.5-7.0 using 1:1 aqueous acetic acid solution. The contents were stirred at room temperature for 1 hour. The precipitated solid was filtered, washed with water (2 x 100 ml). The solid was slurried in methanol (300 ml) for 1 hour at 25-30°C, filtered, washed with methanol (2 x 50 ml) and dried under vacuum at 70-75°C to yield the title compound [90 gms, 97% yield, 96% HPLC purity]. Example 3

Preparation of prulifloxacin

To a solution of 4-(chloromethyl)-5-methyl-1,3-dioxol-2-one (55 gms, 0.371 moles) in 50 ml of DMF at 25-30°C, sodium bromide (77 gms, 0.748 moles) was added and the reaction mass was slowly heated to 40-45°C. The contents were stirred at 40-45°C for 2 hours, acetone ( 500 ml) was added at 40-45°C and stirred for 3 hours. The reaction mass was filtered over hyflo, and the bed washed with acetone (100 ml). The solvent was completely distilled off under vacuum below 45°C to yield 4-(bromomethyl)-5- methyl-1 ,3-dioxol-2-one (formula V).

To a solution of 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]-thiazeto-[3,2-a]- quinoline-3-carboxylic acid of formula IV (100 gms, 0.286 moles) in 4.0 It of acetonitrile, DIPEA (70 ml , 0.402 moles)) was added at room temperature, stirred for 10 minutes. The reaction mass was cooled to 10-15°C and a solution of 4-(bromomethyl)-5-methyl- 1 ,3-dioxol-2-one (formula V) in 500 ml of acetonitrile was slowly added at 10-15°C over a period of 1 hour. The contents were stirred at 25-30°C for 20 hour, filtered over hyflo, and the bed washed with 200 ml of acetonitrile. The solvent was distilled off completely under vacuum below 50°C. Acetonitrile (100 ml) was added at 50°C and the contents were stirred for 30-60 minutes. The reaction mass was slowly chilled to 0-5°C and the precipitated solid was filtered, washed with acetonitrile (25 ml) and dried to yield 65 gms of prulifloxacin. Example 4

Preparation of Type I crystals of prulifloxacin

Prulifloxacin (65 gms) was added to 200 ml of DMF at 25-30°C and heated to 80-85°C for 1 hour. The mixture was then slowly cooled to 25-30°C, stirred for 2 hours, chilled to 0-5°C for 2 hours. The precipitated solid was filtered and dried under vacuum at 70- 75°C to yield Type I crystals of prulifloxacin (55 gms, 99.5 % HPLC purity).

Example 5

Preparation of prulifloxacin

(55 gms, 0.371 moles) of 4-(chloromethyl)-5-methyl-1 ,3-dioxol-2-one is taken in 5.0 ml of DMF at 25-30°C. (77 gms, 0.748 moles) of sodium bromide is added and slowly heated the reaction mass to 40-45°C. The contents are stirred at 40-45°C for 2 hours, 500 ml of acetone is added at 40-45°C and stirred for 3 hours. The reaction mass is clarified over hyflo, and the bed washed with 100 ml of acetone to yield a solution of 4- (bromomethyl)-5-methyl-1 ,3-dioxol-2-one (formula V).

To a solution of 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]-thiazeto-[3,2-a]- quinoline-3-carboxylic acid of formula IV (100 gms, 0.286 moles) in 3.5 Its of acetone was at room temperature DIPEA (70 ml, 0.402 moles) and stirred for 10 minutes. The reaction mass was cooled to 10-15°C and a solution of 4-(bromomethyl)-5-methyl-1 ,3- dioxol-2-one (formula V) in acetone was slowly added to the reaction mass at 10-15°C over a period of 1 hour. The contents were further stirred at 25-30°C for 20 hour, filtered over hyflo and the bed washed with 200 ml of acetone. The solvent was distilled off completely under vacuum below 50°C. Acetonitrile (100 ml) was added at 50°C and the contents were stirred for 30-60 minutes. The reaction mass was further chilled to 0- 5°C and stirred for 2 hours. The precipitated solid was filteredand dried to yield prulifloxacin.

………………….

http://www.google.com/patents/WO2008059512A1?cl=en

novel process for preparing 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]thiazeto [3,2-a]quinoline-3-carboxylic acid of formula I:

which comprises: a) reacting the difluoro-quinoline compound of formula

wherein R represents hydrogen atom or alkyl containing 1 to 4 carbon atoms; with boric acid of formula III:

in presence of acetic anhydride and acetic acid to give borane compound of formula IV:

b) reacting the borane compound of formula IV with piperazine of formula V:

HN NH V

to give piperazine compound of formula Vl:

c) treating the compound of formula Vl with an alkaline metal hydroxide, carbonate or bicarbonate to obtain the compound of formula I.

Prulifloxacin and pharmaceutically acceptable acid addition salts of prulifloxacin can be prepared by using the compound of formula I by known methods for example as described in the European Patent No. 315828. Borane compound of the formula IV and Vl are novel and forms part of the invention. Preferably the reaction in step (a) is carried out at about 30⁰C to reflux temperature more preferably at about 80⁰C to reflux temperature and still more preferably at reflux temperature.

Example 1 Step-I:

Acetic anhydride (24 ml) and acetic acid (11 ml) are added to boric acid (3.5 gm) under stirring at 25 – 30⁰C, the contents are heated to reflux and then stirred for 3 hours at reflux. The reaction mass is cooled to 100⁰C, ethyl 6,7- difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate (20 gm) is added at 100⁰C, the contents are heated to reflux and then refluxed for 2 hours. The reaction mass is cooled to 25 – 35⁰C, toluene (200 ml) is added under stirring, the reaction mass is cooled to 5⁰C and then stirred for 1 hour at 5 – 10⁰C. Filtered the solid, washed with 20 ml of toluene and then dried to give 25.5 gm of 6,7-difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate -O³,0⁴/bis/acetato-0/-borone. Step-I I: Acetonitrile (125 ml), dimethylsulfoxide (125 ml) and piperazine (13.8 gm) are added to 6,7-difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3- carboxylate-0³,0⁴/bis/acetato-0/-borone (25.5 gm, obtained in step-l) under stirring at 25 – 35⁰C, the contents are heated to 85⁰C and then stirred for 3 hours at 80 – 85⁰C to form a clear solution. The solution is cooled to 10⁰C and then stirred for 1 hour at 10 – 15⁰C. Filtered the solid, washed with 25 ml of acetonitrile and then dried to give 26 gm of 6-fluoro-1-methyl-4-oxo-7-(1- piperazinyl)-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylate-0³,0⁴/bis/acetato-0/- borone. Step-Ill: Water (155 ml), potassium hydroxide (17 gm) are added to 6-fluoro-1- methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate- O³,O⁴/bis/ acetato-0/-borone (26 gm, obtained in step-ll) under stirring at 25 – 35⁰C, the contents are heated to 65⁰C and then stirred for 4 hours at 60 – 65⁰C. The reaction mass is cooled to 25⁰C, filtered the undesired solid on hi-flow bed and then pH of the resulting filtrate is adjusted to 7 – 7.5 with 50% HCI solution at 25 – 30⁰C. The separated solid is stirred for 1 hour at 25 – 30⁰C, filtered the solid, washed with 35 ml of water and then dried to give 17 gm of 6-fluoro-1- methyl-4-oxo-7-(1 -piperazinyl)-4H-[1 ,3]thiazeto [3,2-a]quinoline-3-carboxylic acid (HPLC Purity: 98.5%). Example 2 Step-I:

Acetic anhydride (12 ml) and acetic acid (5.5 ml) are added to boric acid (1.25 gm) under stirring at 25 – 30⁰C, the contents are heated to reflux and then stirred for 3 hours at reflux. The reaction mass is cooled to 100⁰C, 6,7-difluoro-1- methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylic acid (10 gm) is added at 100⁰C, the contents are heated to reflux and then refluxed for 3 hours. The reaction mass is cooled to 50⁰C, toluene (100 ml) is added under stirring at 50⁰C, the resulting mass is cooled to 10⁰C and then stirred for 1 hour at 10 – 15⁰C. Filtered the solid, washed with 20 ml of toluene and then dried to give 10 gm of 6,7-difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate -O³ , 0⁴/bis/acetato-0/-borone . Step-I I:

Acetonitrile (50 ml), dimethylsulfoxide (50 ml) and piperazine (5.5 gm) are added to 6,7-difluoro-1-methyl-4-oxo-4H-[1 ,3]thiazeto[3,2-a]quinoline-3- carboxylate-0³,0⁴/bis/acetato-0/-borone (10 gm, obtained in step-l) under stirring at 25 – 35⁰C, the contents are heated to 85⁰C and then stirred for 3 hours at 80 – 85⁰C to form a clear solution. The solution is cooled to 10⁰C and then stirred for 1 hour at 10 – 15⁰C. Filtered the solid, washed with 10 ml of acetonitrile and then dried to give 10.4 gm of 6-fluoro-1-methyl-4-oxo-7-(1- piperazinyl)-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate-0³,0⁴/bis/acetato-0/- borone. Step-Ill :

Water (62 ml), potassium hydroxide (7 gm) are added to 6-fluoro-1-methyl-4- oxo-7-(1-piperazinyl)-4H-[1 ,3]thiazeto[3,2-a]quinoline-3-carboxylate-0³,0⁴/bis/ acetato-OAborone (10.4 gm, obtained in step-ll) under stirring at 25 – 35⁰C, the contents are heated to 65⁰C and then stirred for 4 hours at 60 – 65⁰C. The reaction mass is cooled to 25⁰C, filtered the undesired solid on hi-flow bed and then pH of the resulting filtrate is adjusted to 7 – 7.5 with 50% HCI solution at 25 – 30⁰C. The separated solid is stirred for 30 minutes at 25 – 30⁰C, filtered the solid, washed with 20 ml of water and then dried to give 68 gm of 6-fluoro-1- methyl-4-oxo-7-(1-piperazinyl)-4H-[1 ,3]thiazeto [3,2-a]quinoline-3-carboxylic acid (HPLC Purity: 98.6%). Example 3

Acetonitrile (560 ml) and potassium bicarbonate (8 gm) are added to 6- fluoro-i-methyM-oxo-y-CI-piperazinyO^H-CI .SKhiazetofS^-alquinoline-S- carboxylic acid (14 gm, obtained as per the processes described in examples 1 and 2) under stirring at 25 – 30⁰C, the contents are cooled to 15⁰C and then the solution of 4-bromomethyl-5-methyl-1 ,3-dioxolen-2-one (10 gm) in acetonitrile (140 ml) is added at 15 – 20⁰C for 30 to 45 minutes. The contents are stirred for 25 hours at 25 to 30⁰C, filtered and the resulting filtrate is distilled under vacuum. To the residue added acetonitrile (70 ml), cooled the mass to 20⁰C and then stirred for 1 hour to 1 hour 30 minutes at 20 – 25⁰C. Filtered the solid, washed the solid with 15 ml of chilled acetonitrile and then dried to give 16 gm of prulifloxacin crude (HPLC Purity: 98.8%).

To the prulifloxacin crude (obtained above) added acetonitrile (200 ml) at 25 – 30⁰C, the contents are heated to reflux and then refluxed for 30 minutes. To the reaction mass added activated carbon (5 gm) and refluxed for 15 minutes. The reaction mass is filtered on hi-flo bed, the resulting filtrate is cooled to 20⁰C and then stirred for 3 – 4 hours at 20 – 25⁰C. Filtered the solid, washed with 20 ml of acetonitrile and then dried to give 14 gm of prulifloxacin (HPLC Purity: 99.9%).

…………………

http://www.google.com/patents/WO2008111016A1?cl=en

In a first aspect, a process for the preparation of prulifloxacin is provided, the process comprising: a) reacting a compound of Formula II with a compound of Formula III to obtain prulifloxacin;

FORMULA III

FORMULA II

b) contacting the prulifloxacin obtained in step a) with an acid in a biphasic solvent system, wherein the biphasic solvent system comprises water and a water- immiscible organic solvent; c) separating the aqueous layer from the reaction mixture obtained in step b); d) treating the aqueous layer with a base; and e) isolating prulifloxacin.

The process described in steps b – e above may be carried out with prulifloxacin made from any process however.

The compounds of Formula II and Formula III may be prepared according to the methods provided in U.S. Patent No. 5,086,049.

Example 1: Process for the Preparation of Prulifloxacin:

Step A): A solution of 4-(bromomethyl)-5-methyl-l,3-dioxol-2-one (35.5 g, 0.184 mole) in N,N-dimethylformamide (200 ml) was added dropwise at 0 to 5° C to a stirred solution of 6-fluoro-l-methyl-4-oxo-7-piperazin-l-yl-4H-[l,3]thiazeto[3,2-α]quinoline-3- carboxylic acid (50 g, 0.143 mole and potassium bicarbonate (15.8 g, 0.1578 mole) in N,N-dimethylformamide (200 ml). The resulting mixture was stirred at 25° to 28°C for 3 to 4 hours. After the completion of the reaction, the reaction mixture was poured into water (1250 ml). The solid obtained was filtered, washed with water (100 ml), and subsequently dissolved in a mixture of chloroform: methanol (7:3; 1250 ml). The lower organic layer was separated and water (500 ml) was added to the organic layer. A dilute aqueous solution of hydrochloric acid was added to the biphasic reaction mixture to adjust pΗ to 0.8 to 1.0. The reaction mixture was stirred for 15 minutes, allowed to settle and the upper aqueous layer was separated. The process was repeated twice and the aqueous layers were combined. Activated charcoal (10%) was added to the combined aqueous layer and stirred for 30 minutes, filtered and cooled to 20° to 25° C. The pΗ of the reaction mixture was adjusted to 6.5 to 7.0 by adding an aqueous solution of sodium bicarbonate. The solid obtained was extracted with chloroform (375 ml), stirred for 15 minutes and the organic layer was separated. The aqueous layer was further extracted with a mixture of chloroform: methanol (7:3 ratio; 50 ml). The combined organic layer was distilled under vacuum at 35° to 40° C to recover the solvent up to 125 ml. The reaction mass so obtained was stirred for 3 to 4 hours at 28° to 30° C, filtered and washed with chilled chloroform (50 ml). The wet cake obtained was dried at 45° C for 12 hours to obtain the title compound. Step B): The prulifloxacin (30 g) obtained in Step A) was suspended in a mixture of chloroform: ethanol (10:1, v/v, 585 ml: 58.5 ml) and heated to reflux temperature. Activated carbon (3.9 gm) was added to the partially cleared solution and refluxed for 30 minutes, followed by filtration through Celite bed. The bed was further washed with chloroform: ethanol (10:1, v/v, 585 ml: 58.5 ml). The filtrate so obtained was distilled at atmospheric pressure till to partially remove the solvent. The concentrate so obtained was stirred at about 25° C for 1 hour, and filtered. The solid obtained was washed with chloroform: ethanol (39 ml X 2), dried under vacuum at 45° C for 12 hours to obtain the title compound. Yield: 22 g

HPLC Purity: 99%

………………………….

SEE

Studies on pyridonecarboxylic acids. 1. Synthesis and antibacterial evaluation of 7-substituted-6-halo-4-oxo-4H-[1,3]thiazeto[3, 2-a]quinoline-3-carboxylic acids
J Med Chem 1992, 35(25): 4727

http://pubs.acs.org/doi/pdf/10.1021/jm00103a011

The reaction of 3,4-difluoroaniline (I) with carbon disulfide and triethylamine gives triethylammonium N-(3,4-difluorophenyl)dithiocarbamate (II), which by reaction with ethyl chloroformate and triethylamine in chloroform is converted into 3,4-difluorophenyl isothiocyanate (III). The reaction of (III) with diethyl malonate and KOH in dioxane affords the potassium salt (IV), which is treated with chloromethyl methyl ether in DMF to give the corresponding methoxymethylsulfanyl compound (V). The cyclization of (V) at 240 C in diphenyl ether affords 6,7-difluoro-4-hydroxy-2-(methoxymethylsulfanyl)quinoline-3-carboxylic acid ethyl ester (VI), which by treatment with HCl in ethanol gives the corresponding mercapto compound (VII). The cyclization of (VII) with 1,1-dibromoethane by means of K2CO3 and KI in hot DMF yields 5,6-difluoro-1-methyl-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid ethyl ester (VIII), which is condensed with piperazine (IX) in DMF to afford the corresponding piperazino-derivative (X). The hydrolysis of (X) with KOH in hot tert-butanol gives the corresponding free acid (XI) , which is finally condensed with 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (XII) by means of KHCO3 in DMF.

………………….

Treatment of 3,4-difluoroaniline (I) with CS2 and Et3N gives triethylammonium dithiocarbamate (II), which reacts with ethyl chloroformate in chloroform to yield (III). Isothiocyanate (III) is converted into the potassium salt (IV) by reaction with diethyl malonate and KOH in dioxane and then transformed into methoxymethyl thioether (VI) by means of reagent (V) and Et3N in toluene. Cyclization of (VI) by heating in diphenyl ether affords quinoline (VII), which then reacts with benzoyl chloride (VIII) in pyridine to furnish (IX). Benzoyloxy derivative (IX) is converted into (X) by means of HCl in EtOH, and its reaction with 1-bromo-2-fluoroethane (XI) and NaHCO3 yields compound (XII). Chlorination of (XII) with SO2Cl2 in hexane provides (XIII), which by simultaneous hydrolysis and intramolecular cyclization by means of Et3N /H2O in THF provides the mixture of isomers (XIV). (+)-(XV) is obtained by HPLC chromatography of (XIV) on a chiral stationary phase. Treatment of (+)-(XV) with 1-methylpiperazine (XVI) in DMF provides ethyl ester (+)-(XVII), which is finally hydrolyzed by means of H2SO4 in H2O.

INTERMEDIATES

154330-67-3

Ethyl 6,7-difluoro-2-ethylmercapto-4-hydroxyquinoline-3-carboxylate

154330-68-4

Ethyl 4-acetoxy-6,7-difluoro-2-(ethylthio)quinoline-3-carboxylate

113046-72-3

Ethyl 6,7-difluoro-1-methyl-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylate

113028-17-4

Ethyl 6-fluoro-1-methyl-4-oxo-7-(1-piprazinyl)-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylate

112984-60-8

6-Fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid

REFERENCES

1.  Nelson, Jennifer M.; Chiller, Tom M.; Powers, John H.; Angulo, Frederick J. (2007). “Food Safety: Fluoroquinolone‐Resistant Campylobacter Species and the Withdrawal of Fluoroquinolones from Use in Poultry: A Public Health Success Story”. Clinical Infectious Diseases 44 (7): 977–80. doi:10.1086/512369. PMID 17342653.
2.  Kawahara S (1998). “[Chemotherapeutic agents under study]“. Nippon Rinsho (in Japanese) 56 (12): 3096–9. PMID 9883617.
3.  Fritsche, T. R.; Biedenbach, D. J.; Jones, R. N. (2008). “Antimicrobial Activity of Prulifloxacin Tested against a Worldwide Collection of Gastroenteritis-Producing Pathogens, Including Those Causing Traveler’s Diarrhea”. Antimicrobial Agents and Chemotherapy 53 (3): 1221–4. doi:10.1128/AAC.01260-08. PMC 2650572.PMID 19114678.
4.  Giannarini, Gianluca; Tascini, Carlo; Selli, Cesare (2009). “Prulifloxacin: clinical studies of a broad-spectrum quinolone agent”. Future Microbiology 4 (1): 13–24.doi:10.2217/17460913.4.1.13. PMID 19207096.
5.  JP patent 1294680, Kise Masahiro; Kitano Masahiko; Ozaki Masakuni; Kazuno Kenji; Matsuda Masato; Shirahase Ichiro; Segawa Jun, “Quinolinecarboxylic Acid Derivative”, issued November 28, 1989
6.  Prulifloxacin. Drugfuture.com. Retrieved on 2010-11-03.
7. Anonymous (2002). “Prulifloxacin ['Quisnon'; Nippon Shinyaku] has been approved in Japan”. Inpharma 1 (1362): 22.
8.  Research and Development Department of Angelini. Angelinipharma.com. Retrieved on 2010-11-03.
9.  Nippon Shinyaku, Annual Report 2007
10.  “Prulifloxacin. NAD-441A, NM 441, Quisnon”. Drugs in R&D 3 (6): 426–30. 2002.PMID 12516950.
11.  Annual Report 2008, p. 34

Segawa,J,Mashiko kitano, Kenji Kazuno et al, Studies on Pyridonecarboxylic acids,1.Sythesis and antibacterial Evaluation of 7-substituted-6-halo-4-oxo-4H-[1,3]thiazeto [3,2-]quionoline- 3-caroboxylic acids[J].J Med  Chem. 1992,35(25):4727-4738.

Masato Matsuoka, Jun Segawa, Yoshihiko.et al, Studies on Pyridone Carb oxylic acids. V.A Practial synthesis of Ethyl 6,7–Difuoro-1-methyl-4-oxo-[1,3] Thiazeto [3,2-a]quinoline-3- Caroboxylate a   key  intermediate for the new tricyclic quinolone, prulifloxacin (NM441) and Versatile new  syntheses of the 2-thioquinoline Skeleton[J].J Heterocyclic Chem.1997,34,1773-1779.

EXTRA INFO

http://www.google.com/patents/EP2524922A1?cl=en

•  formula 1 is S-(-)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl-1-piperazinyl]-4-oxo-4H-[1,3]thiazet o[3,2-α]quinoline-3-carboxylic acid (levo-prulifloxacin for short); its stereo configuration is S configuration; it has optical property of levorotatory polarized light:
• S-(-) ulifloxacin (as shown in formula 2 below) as raw material and the compound as shown in the following formula 3 are reacted in organic solvent in the presence of alkaline material. The reaction formula is shown below:

  • S-(-)-ulifloxacin and R-(+)-ulifloxacin are prepared according to the method disclosed in CN101550142A .
  • Japanese scholars Masato Matsuoka et al. have proved the absolute configuration of optically pure prulifloxacin. The study (see the publication Chem. Pharm. Bull. 43(7) 1238-1240 (1995)) verifies that (-)-ulifloxacin is S configuration while (+)-ulifloxacin is the enantiomer of R configuration by applying chemical methods together with single-crystal X-ray diffraction.

   

  • Accordingly, R-prulifloxacin can be prepared from R-(+)-ulifloxacin and the compound of formula 3 by the method described hereinbefore.
  • [0022]
    The reaction formula is depicted below:
  • S-prulifloxacin prepared in accordance with the present invention is determined to be laevorotatory by optical rotation measurement, so it is S-(-)-prulifloxacin. R-prulifloxacin prepared in accordance with the present invention is determined to be dextrorotatory by optical rotation measurement, so it is R-(+)-prulifloxacin.
  • The present invention studied the absorption features of S-(-)-prulifloxacin and R-(+)-prulifloxacin on circular polarized light by circular dichroism spectroscopy. The two spectrograms are mirror images of each other, which proves that S-(-)-prulifloxacin and R-(+)-prulifloxacin are enantiomer of each other.
  • Comparing the circular dichroism spectrogram as depicted in figure 4 with the circular dichroism spectrogram of analogue of the similar structure with known absolute configuration as disclosed in the publication Chem. Pharm. Bull. 47(12) 1765-1773 (1999), it is found that (-)-prulifloxacin has similar Cotton effect to the two analogues reported in the publication, ethyl S-(-)-6,7-difluoro-1-methyl-4-oxo-4H-[1,3] thiazeto[3,2-α]quinoline-3-carboxylate and ethyl S-(-)-6, 7-difluoro-1-fluoromethyl-4-oxo-4H-[1,3]thiazeto[3,2-α]quinoline-3-carboxylate; so does (+)-prulifloxacin. The results also verify on the other hand that the absolute configuration of levo-prulifloxacin of the present invention is S type while the absolute configuration of dextro-prulifloxacin is R type.
  • The compound of the present invention and physiologically acceptable acid can be prepared to salts: dissolving or suspending S-(-)-prulifloxacin in solvent such as chloroform, DMF and the like; adding into acid or acid solution (for example, hydrochloric acid or hydrogen chloride-methanol solution and the like) while stirring; precipitating and filtering to obtain solid salt from the solvent solution, or alternatively removing solvent from the salt solution directly by concentration, spray drying and the like to obtain the salt of S-(-)-prulifloxacin. The obtained solid may be further recrystallized.

  Example 1 Preparation of (S)-(-)-uliflourxacin

  • 105 g of racemic uliflourxacin was dissolved in 1,500 mL of dimethyl sulfoxide. 27 g of D-tartaric acid was dissolved in 405 mL of dimethyl sulfoxide dropwise while stirring. After stirring at room temperature for 20 hours, the precipitate was filtrated. The collected solid was dried under vacuum to obtain 86 g solid, which was recrystallized in dimethyl sulfoxide to obtain 37 g of levoulifloxacin-D-tartrate, with C49.08%, H5.06%, N9.50%, S7.44% shown by elemental analysis (molecular formula: C₁₆H₁₆FN₃O₃S·1/2C₄H₆O₆·H₂O, calculated values: C48.86%, H4.78, N9.50%, S7.25%). Said salt was added into water to obtain a suspension, and the pH value was adjusted to 7-8 with 2% NaOH aqueous solution while stirring. After precipitation, filtration, and drying, 24.5 g of (S)-uliflourxacin was obtained, having a chemical name (S)-(-)-6-fluoro-1-methyl-4-oxo-(1-piperazinyl)-1H,4H-[1,3]thiazeto [3,2-α]quinoline-3-carboxylic acid.
  • Specific rotation [α]²⁰ _D= -133° (c=0.5, 0.1 mol/L methanesulfonic acid); ¹H-NMR (DMSO-d₆) ^δ2.11 (3H, d, j=6.2 Hz), 2.87 (4H, m), 3.19 (4H, m), 6.40 (1H, q, j=6.2 Hz), 6.89 (1H, d, j=7.4Hz), 7.79 (1H, d, j=13.9Hz), optical purity e.e. 96%.

  Example 2 Preparation of (R)-(+)-uliflourxacin

  • 105 g of racemic uliflourxacin was dissolved in 1,500 mL of DMSO. 27 g of L-tartaric acid was dissolved in 405 mL dimethyl sulfoxide dropwise while stirring to allow that the solution became turbid and the precipitation occurred. The solution was stirred at room temperature for 20 hours and then filtered. The collected solid was dried under vacuum to obtain 82 g solid which was recrystallized in dimethyl sulfoxide to obtain 34 g of dextrouliflourxacin-L-tartarte. Said salt was added into water to obtain a suspension, and the pH value was adjusted to 7-8 with 2% NaOH aqueous solution while stirring. After filtration and drying, 22 g of (R)-uliflourxacin was obtained, having a chemical name (R)-(+)-6-fluoro-1-methyl-4-oxo-(1-piperazinyl)-1H,4H-[1,3]thiazeto[3,2-a]quinoline -3-carboxylic acid.
  • Specific rotation [α]²⁰ _D= +132.4° (c=0.5, 0.1 mol/L methanesulfonic acid), optical purity e.e. 96%.

  Example 3 Preparation of S-(-)-prulifloxacin

  • 3.49 g (0.01 mol) of S-(-)-uliflourxacin prepared in Example 1, 2.02 g (0.02 mol) of triethylamine and 20 ml of dimethylformamide (hereinafter referred to as DMF) were mixed and stirred. After the solution was cooled to -5∼5 °C, 0.012 mol of 4-bromomethyl-5-methyl-1,3-dioxolen-2-one (hereinafter referred to as DMDO-Br) in DMF (5 ml) solution was added thereinto, followed by stirring at -5∼5 °C for 3 hours. The reaction solution was poured into 100 ml of ice water, stirred for 30 minutes, and then filtered. The filter residue was washed with water. The solid was collected and dried under vacuum. After recrystallization from acetonitrile, 2.9 g of S-(-)-prulifloxacin was obtained, having a chemical name: S-(-)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl-1-piperazinyl ]-4-oxo-4H-[1,3]thiazeto[3,2-α]quinoline-3-carboxylic acid, with a purity of 98% and a yield rate of 63%. Specific rotation [α]²⁰ _D= -108° (c=0.5, 0.1 mol/L methanesulfonic acid)

  Example 4 Preparation of R-(+)-prulifloxacin

  • R-(+)-prulifloxacin prepared in Example 2 was used as raw material to prepare 2.7 g of target product R-(+)-prulifloxacin in accordance with the method as described in Example 3, with a yield rate of 60.7% and a purity of 98%. Specific rotation [α]²⁰ _D= +108° (c=0.5, 0.1 mol/L methanesulfonic acid).

   

  • Comparing the circular dichroism spectrogram as depicted in Figure 4 with the circular dichroism spectrogram of analogue of the similar structure with known absolute configuration as disclosed in the publication Chem. Pharm. Bull. 47(12) 1765-1773 (1999), it was found that (-)-prulifloxacin has similar Cotton effect with the two analogues reported in the publication, ethyl S-(-)-6,7-difluoro-1-methyl-4-oxo-4H-[1,3] thiazeto[3,2-α]quinoline-3-carboxylate and ethyl S-(-)-6, 7-difluoro-1-fluoromethyl-4-oxo-4H-[1,3]thiazeto[3,2-α]quinoline-3-carboxylate; so does (+)-prulifloxacin. The results also verify on the other hand that the absolute configuration of levo-prulifloxacin of the present invention is S type while the absolute configuration of dextro-prulifloxacin is R type.
  • Conclusion: The absolute configuration of the sample prepared in Example 3 is S configuration, as shown in the formula below:

  Example 5 Preparation of S-(-)-prulifloxacin

  • 3.49 g (0.01 mol) of S-(-)-uliflourxacin, 1.2 g (0.012 mol) of anhydrous potassium bicarbonate and 20 ml of dimethylsulfoxide were mixed and stirred. 0.012 mol of DMDO-Br in DMSO (5 mL) solution was added dropwise at -20 °C. Stirring proceeded at -20 °C for 3 hours. The reaction solution was poured into 100 ml of ice water, and the pH value was adjusted to 7 with 20% acetic acid. The solution was filtered after stirring for 30 minutes. The filter residue was washed with water. The solid was collected and dried under vacuum. After recrystallization from acetonitrile, 2.5 g of the target product levo-prulifloxacin was obtained with a purity of 98% and a yield rate of 54%.
    Specific rotation [α]²⁰ _D= -108° (c=0.5, 0.1 mol/L methanesulfonic acid)

  Example 6 Preparation of S-(-)-prulifloxacin

  • 3.49 g (0.01 mol) of S-(-)-uliflourxacin, 1.04 g (0.008 mol) of N,N-diisopropylethylamine and 20 mL of N,N-dimethylformamide (DMF) was mixed and stirred, 0.008 mol of DMDO-Br in DMF (5 mL) solution was added thereinto. The solution was heating to 60 °C and reacted for 15 minutes. The reaction solution was poured into 100 ml of ice water, and the pH value was adjusted to 7 with 20% acetic acid. The solution was filtered after stirring for 30 minutes. The filter residue was washed with water. The solid was collected and dried under vacuum. After recrystallization from acetonitrile, 2.0 g of the target product levo-prulifloxacin was obtained with a purity of 98% and a yield rate of 43%.
    Specific rotation [α]²⁰ _D= -108° (c=0.5, 0.1 mol/L methanesulfonic acid)

  Example 7 Preparation of S-(-)-prulifloxacin

  • 10 g (0.029 mol) of S-(-)-uliflourxacin, 30 ml of N,N-dimethylacetylamide and 14.7 g (0.145mol) of triethylamine was mixed and cooled to 5~10 °C. 8.5 g (0.03 mol) 4-(p-toluenesulfonic acid-1-methyl ester)-5-methyl-1,3-dioxolen-2-one in 25 ml of N,N-dimethylacetylamide solution was added dropwise while stirring. After addition, the solution was reacted at room temperature for 10 hours. The reaction solution was poured into 200 ml of ice water, and the pH value was adjusted to 7 with 20% acetic acid. The solution was filtered after stirring for 30 minutes. The filter residue was washed with water. The solid was collected and dried under vacuum. After recrystallization from acetonitrile, 7.46 g of the target product levo-prulifloxacin was obtained with a purity of 98% and a yield rate of 57%. Specific rotation [α]²⁰ _D= -108° (c=0.5, 0.1 mol/L methanesulfonic acid).

  Example 8 Preparation of S-(-)-prulifloxacin

  • 3.49 g (0.01 mol) of S-(-)-uliflourxacin, 0.79 g (0.05 mol) of potassium carbonate and 20 ml of dimethylformamide (DMF) was mixed and stirred. 0.012 mol of DMDO-Br in DMF (5ml) solution was added at -10 °C. At the same temperature, the solution was reacted for 2 hours. The reaction solution was poured into 100 ml of ice water, and the pH value was adjusted to 7 with 20% acetic acid. The solution was filtered after stirring for 30 minutes. The filter residue was washed with water. The solid was collected and dried under vacuum. After recrystallization from acetonitrile, 2.2 g of the target product levo-prulifloxacin was obtained with a purity of 98% and a yield rate of 48%. Specific rotation [α]²⁰ _D= -108° (c=0.5, 0.1 mol/L methanesulfonic acid).

  Example 9 Preparation of S-(-)-prulifloxacin

  • 3.49 g (0.01 mol) of S-(-)-uliflourxacin, 0.79 g (0.02 mol) of diisopropylamine and 20 ml of dimethylformamide (DMF) was mixed and stirred. 0.02 mol of DMDO-Br in DMF (5ml) solution was added at 0 °C. At the same temperature, the solution was reacted for 2 hours. The reaction solution was poured into 100 ml of ice water, and the pH value was adjusted to 7 with 20% acetic acid. The solution was filtered after stirring for 30 minutes. The filter residue was washed with water. The solid was collected and dried under vacuum. After recrystallization from acetonitrile, 2.5 g of the target product levo-prulifloxacin was obtained with a purity of 98% and a yield rate of 54%. Specific rotation [α]²⁰_D= -108° (c=0.5, 0.1 mol/L methanesulfonic acid).

  Example 10 Preparation of R-(+)-prulifloxacin

  • In accordance with the method as described in Example 5, the raw material R-(+)-prulifloxacin was prepared to 2.5 g of the target product R-(+)-prulifloxacin with a purity of 98% and a yield rate of 54%. Specific rotation [α]²⁰ _D= +108° (c=0.5, 0.1 mol/L methanesulfonic acid).

  Example 11 Preparation of levo-prulifloxacin hydrochloride

  S-(-)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl-1-piperazinyl] -4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid hydrochloride
  • 0.5 g of S-(-)-prulifloxacin was dissolved in 15 mL of chloroform and then 0.5 mL of 33% (v/v) hydrochloric acid- methanol solution was added while stirring. The solution was filtered and the filtration residue was washed with methanol. The collected solid was dried to obtain 450 mg said compound with a yield rate of 83%. The melting point of the product is higher than 220 °C (the sample became darker during the test).

  Example 12 Preparation of levo-prulifloxacin mesylate

  S-(-)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl-1-piperazinyl] -4- oxo-4H-[1,3]thiazeto[3,2-α]quinoline-3-carboxylic acid mesylate
  • 0.5 g of S-(-)-prulifloxacin was dissolved in 15 mL of chloroform and then 0.5 mL of 50% methanesulfonic acid- methanol solution was added while stirring. The solution was filtered and the filtration residue was washed with methanol. The collected yellow solid was dried with calcium chloride under vacuum for 24 hours and further dried with calcium chloride at 80 °C under vacuum for 5 hours to obtain 470 mg said compound with a yield rate of 78%. The melting point of the product is higher than 220 °C (the sample became darker during the test).

  Example 13 Preparation of levo-prulifloxacin hydrochloride

  • 0.5 g of S-(-)-prulifloxacin was dissolved in 15 mL of chloroform and then 0.5 mL of 33% (v/v) hydrochloric acid- methanol solution was added while stirring. The solution was dried by evaporation. Methanol was added to the residue and stirred for 10 minutes. The solution was filtered and the filtration residue was washed with methanol. The collected solid was dried to obtain 460 mg said compound with a yield rate of 85%.

Clinafloxacin

7-(3-Aminopyrrolidin-1-yl)-8-chloro-1-cyclopropyl-6-fluoro-4-oxoquinoline-3-carboxylic acid

7-(3-Amino-1-pyrrolidinyl)-8-chloro-1-cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-3-quinolinecarboxylic acid

(±)-7-(3-Amino-1-pyrrolidinyl)-8-chloro-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid

105956-99-8  cas no

Clinafloxacin (INN) is a fluoroquinolone antibiotic. Its use is associated with phototoxicity and hypoglycaemia.^[1]

Clinafloxacin is a novel quinolone with wide activity against the plethora of microorganisms encountered in intraabdominal infections.

Clinafloxacin is a chlorofluoroquinolone with excellent bioavailability and activity against gram-positive, gram-negative, and anaerobic pathogens . Typical MICs for α-streptococci are 0.06–0.12 µg/mL . MIC₉₀ values for methicillin-resistant Staphylococcus aureus (MRSA) average 1.0 µg/mL. The MIC₉₀ for enterococci is typically 0.5 µg/mL . Both intravenous and oral formulations have been developed . Several studies have demonstrated the efficacy of clinafloxacin monotherapy for serious infections  Clinafloxacin was also active in animal models of endocarditis, including endocarditis due to ciprofloxacin-resistant S. aureus infection .

preparation process for the compound of the invention.

Example 28 7-(3-Amino-1-pyrrolidinyl)-8-chloro-1-cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-3-quinolinecarboxylic acid
• A mixture of 8-chloro-1-cyclopropyl-6,7-difluoro-1,4-di- hydro-4-oxo-3-quinolinecarboxylic acid (0.6 g), anhydrous acetonitrile (6 ml), 3-aminopyrrolidine (0.35 g) and DBU (0.31 g) was refluxed for an hour. Then, 3-aminopyrrolidine (0.2 g) was more added and further refluxed for 2 hours. After cooling, the resulting precipitate was collected by filtration, dissolved in water (9 ml) containing sodium hydroxide (0.12 g) and neutralized with acetic acid. The resulting precipitate was collected by filtration and washed with water and acetonitrile successively to give the title compound (0.52 g) as colorless powder, mp 237-238 °C (decompd.).
• Analysis (%) for C₁₇H₁₇ClFN₃O₃·H2O, Calcd. (Found): C, 53.20 (52.97); H, 4.99 (4.62); N, 10.95 (10.83).

Example 29 7-(3-Amino-1-pyrrolidinyl)-8-chloro-1-cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride

• To a suspension of 7-(3-amino-1-pyrrolidinyl)-8-chloro-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (100 mg) in ethanol (2 ml) was added 0.2 ml of ethanol solution of hydrogen chloride (7.0 mmol HC1/ml) and then the mixture was concentrated. The resulting residue was recrystallized from methanol to give the title compound (79 mg) as light yellow prisms, mp 263-265 °C (decompd.).
• Analysis (%) for C₁₇H₁₇ClFN₃O₃.HCl, Calcd. (Found): C, 50.76 (50.50); H, 4.51 (4.44); N, 10.45 (10.38).

…………………..

J. Med. Chem., 23, 1358 (1980)

• structural formula D

  may be readily prepared from the known starting material methyl 5-oxo-l-(phenylmethyl)-3-pyrrolidinecarboxylate, A, [J. Org. Chem., 26, 1519 (1961)] by the following reaction sequence.
• The compound wherein R₃ is hydrogen, namely 3-pyrrolidinemethanamine, has been reported in J. Org. Chem., 26, 4955 (1961).

Journal of Medicinal Chemistry, 1988 ,  vol. 31, p. 983 – 991

 

References

	Examples of
	reported trade
	names for products
	containing the 6-
6-Fluoroquinolin-	fluoroquinolin-
4(1H)-one	4(1H)-one	Structure
amifloxacin
balofloxacin
ciprofloxacin	Cipro®, Ciprobay, & Ciproxin
clinafloxacin
danofloxacin	Advocin & Advocid
difloxacin	Dicural® & Vetequinon
enrofloxacin	Baytril®
fleroxacin	Megalone
flumequine	Flubactin
garenoxacin
gatifloxacin	Tequin® & Zymar®
grepafloxacin	Raxar
ibafloxacin
levofloxacin	Levaquin®, Gatigol, Tavanic, Lebact, Levox, & Cravit
lomefloxacin	Maxaquin®
marbofloxacin	Marbocyl® & Zenequin
moxifloxacin	Avelox® & Vigamox®
nadifloxacin	Acuatin, Nadoxia, & Nadixa
norfloxacin	Noroxin®, Lexinor, Quinabic, & Janacin
ofloxacin	Floxin®, Oxaldin, & Tarivid
orbifloxacin	Orbax® & Victas
pazufloxacin
pefloxacin
pradofloxacin
prulifloxacin
rufloxacin	Uroflox
sarafloxacin	Floxasol, Saraflox, Sarafin
sitafloxacin
sparfloxacin	Zagam
temalioxacin	Omniflox

enoxacin	Penetrex & Enroxil
gemifloxacin	Factive
tosufloxacin
trovafloxacin	Trovan

BRUSH UP

AT

http://orgspectroscopyint.blogspot.in/

Ulipristal acetate

17alpha-Acetoxy-11beta-[4-(dimethylamino)phenyl]-19-norpregna-4,9-diene-3,20-dione

(8S,11S,13S,14R,17R)-17-Acetoxy-11-[4-(dimethylamino)phenyl]-19-norpregna-4,9-diene-3,20-dione

EMA:Link, US FDA:link

REVIEW.http://www.fsrh.org/pdfs/ellaOneNewProductReview1009.pdf

126784-99-4  CAS

Ulipristal acetate (trade name EllaOne in the European Union, Ella in the U.S. for contraception,^[1] and Esmya for uterine fibroid) is a selective progesterone receptor modulator (SPRM).

Medical uses

Emergency contraception

For emergency contraception^[2] a 30 mg tablet is used within 120 hours (5 days) after an unprotected intercourse or contraceptive failure.^[3] It has been shown to prevent about 60% of expected pregnancies,^[4] and prevents more pregnancies than emergency contraception with levonorgestrel.^[5] Ulipristal acetate is available by prescription for emergency contraception in over 50 countries, with access through pharmacists without a prescription being tested in the United Kingdom.^[6][7][8][9] Emergency contraception (EC) is a woman’s second chance for primary prevention of pregnancy.

A reproductive-age woman is a candidate for emergency contraception if she seeks care within 120 hours of unprotected intercourse (UPI), which is the window of pregnancy risk associated with a given act of intercourse based upon the estimated lifespan of sperm in the genital tract (Wilcox et al, 1995). Current hormonal methods of emergency contraception prevent at least half of expected pregnancies if taken within 72 hours of UPI (Von Hertzen et al, 1998).

Levonorgestrel at a total dose of 1.5 mg (taken in a single dose or two 0.75 mg doses 12 hours apart) is the current standard for hormonal emergency contraception and is licensed for use up to 72 hours after UPI. Clinical trials involving levonorgestrel used for emergency contraception more than 72 hours after intercourse do not conclusively establish efficacy rates because of insufficient sample size. Nevertheless, these studies reveal a trend towards markedly higher failure rates when levonorgestrel is taken 48 hours or more after unprotected intercourse (von Hertzen et al, 1998; Von Hertzen et al, 2002).

This trend may be explained by levonorgestrel mode of action for emergency contraception. Levonorgestrel acts by interfering with the LH peak but does not appear to interfere with the ovulatory process when taken close to ovulation, a time when intercourse is most likely to lead to fertilization (Croxatto et al, 2004; Marions et al, 2004; Wilcox et al, 2004). For a woman who presents for emergency contraception more than 72 hours after intercourse, the only currently available method proven to be highly effective is insertion of a copper contraceptive intra-uterine device (IUD). However, IUDs are not widely available in many countries and insertion can only be performed by a trained clinician. Furthermore, many women decline IUD insertion as a method of emergency contraception because the procedure is invasive, is relatively expensive and has a risk of complications including uterine perforation on insertion (Grimes et al, 2004). Additionally, many women seeking emergency contraception are not seeking a long acting contraceptive method.

There is, therefore, a need for a new hormonal emergency contraceptive that can be used and is highly effective up to 120 hours after UPI. Ulipristal acetate (also known as CDB-2914) is a selective progesterone receptor modulator that inhibits or delays ovulation in a dose-dependent fashion (Stratton et al, 2000). In a double-blind non-inferiority trial, ulipristal acetate was shown to be as efficacious as levonorgestrel for preventing pregnancy when used within 72 hours of UPI (Creinin et al, 2006). Moreover, study data suggest improved efficacy in preventing pregnancy from 48 to 72 hours when levonorgestrel efficacy markedly wanes. ulipristal acetate for use in providing post coital contraception in a female subject between about 3 to about 5 days, or between about 72 to about 120 hours, after unprotected intercourse.

A subject of the invention is thus a method for providing post coital contraception in a female subject, comprising providing the subject with a therapeutically effective amount of ulipristal acetate, between about 3 to about 5 days, or between about 72 to about 120 hours, after unprotected intercourse. It is further provided a kit comprising i) a dosage form comprising ulipristal acetate and ii) a printed matter stating that ulipristal acetate may be taken within 120 hours or 5 days after unprotected intercourse   Any woman of reproductive age may need post-coital or emergency contraception at some point to avoid an unintended pregnancy. It is meant to be used in situations of unprotected intercourse, such as: when no contraceptive has been used;

when there is a contraceptive failure or incorrect use, including: – condom breakage, slippage, or incorrect use; – non-compliance with dosage regimen for combined oral contraceptive pills; – non-compliance with dosage regimen for progestogen-only pill (minipill); – more than two weeks late for a progestogen-only contraceptive injection (depot- medroxyprogesterone acetate or norethisterone enanthate); – more than seven days late for a combined estrogen-plus-progestogen monthly injection; – dislodgment, delay in placing, or early removal of a contraceptive hormonal skin patch or ring; – dislodgment, breakage, tearing, or early removal of a diaphragm or cervical cap; – failed coitus interruptus (e.g., ejaculation in vagina or on external genitalia); – failure of a spermicide tablet or film to melt before intercourse; – miscalculation of the periodic abstinence method or failure to abstain on fertile day of cycle; – IUD expulsion; or in cases of sexual assault when the woman was not protected by an effective contraceptive method.

Uliprisnil acetate, originally developed at the Research Triangle Institute, is a selective progesterone receptor modulator (SPRM) first launched in the E.U. in 2009 by HRA Pharma as emergency contraception within 120 hours (5 days) of unprotected sexual intercourse or contraceptive failure. The company filed for approval of this indication in the U.S. in 2009 and approval was obtained in 2010. In 2012, the product was approved in the E.U. for the pre-operative treatment of moderate to severe symptoms of uterine fibroids in adult women of reproductive age. First E.U. commercialization took place in Germany in March 2012 followed by the U.K. in April. The compound is being developed in phase II clinical trials at the National Institutes of Health (NIH) for the treatment of uterine fibroids and premenstrual syndrome (PMS). Two formulations of uliprisnil are in early clinical trials at the Population Council for the prevention of pregnancy: a vaginal ring and an intrauterine delivery system (IUS). Watson conducted phase III clinical studies for the treatment of women with anemia associated with uterine leiomyoma, however the development has been discontinued.

Uliprisnil acetate is a well-known steroid that possesses antiprogestational and antiglucocorticoid activity. In preclinical studies, the growth of lead follicles exposed to a midfollicular dose of the compound was delayed in a dose-related fashion, indicating that the compound may have an additional mechanism of action involving progesterone or estrogen antagonism.

In 2007, uliprisnil acetate was licensed to PregLem by HRA Pharma in Europe for the treatment of gynecological disorders excluding contraception. A license for North American was granted to HRA in 2010. In 2010, the compound was licensed to Watson (now Actavis) by HRA Pharma for the commercialization in the U.S. for use as emergency contraception. Also in 2010, Watson (now Actavis) obtained a license to uliprisnil for the treatment of uterine fibroids. In 2011, the product was licensed to Gedeon Richter by HRA Pharma for marketing and distribution in China, Russia and (Commonwealth of Independent States) CIS republics for the treatment of uterine myoma.

Treatment of uterine fibroids

Ulipristal acetate is used for pre-operative treatment of moderate to severe symptoms of uterine fibroids in adult women of reproductive age in a daily dose of a 5 mg tablet.^[10] Treatment of uterine fibroids with ulipristal acetate for 13 weeks effectively controlled excessive bleeding due to uterine fibroids and reduced the size of the fibroids.^[11][12][13] Two intermittent 3-month treatment courses of ulipristal acetate 10 mg resulted in amenorrhea at the end of the first treatment course in 79.5%, at the end of the second course in 88.5% of subjects. Mean myoma volume reduction observed during the first treatment course (−41.9%) was maintained during the second one (−43.7%).^[10

^]

Adverse effects

Common side effects include abdominal pain and temporary menstrual irregularity or disruption. Headache and nausea were observed under long-term administration (12 weeks), but not after a single dose.^[3]

Interactions

Ulipristal acetate is metabolized by CYP3A4 in vitro. Ulipristal acetate is likely to interact with substrates of CYP3A4, like rifampicin, phenytoin, St John’s wort, carbamazepine or ritonavir, therefore concomitant use with these agens is not recommended.^[10][14] It might also interact with hormonal contraceptives and progestogens such as levonorgestrel and other substrates of the progesterone receptor, as well as with glucocorticoids.^[10]

Contraindications

Ulipristal acetate should not be taken by women with severe liver diseases^[3] because of its CYP mediated metabolism. It has not been studied in women under the age of 18.^[15]

Pregnancy

Unlike levonorgestrel, and like mifepristone, ulipristal acetate is embryotoxic in animal studies.^[16] Before taking the drug, a pregnancy must be excluded.^[3] The EMA proposed to avoid any allusion to a possible use as an abortifacient in the package insert to avert off-label use.^[17] It is unlikely that ulipristal acetate could effectively be used as an abortifacient, since it is used in much lower doses (30 mg) than the roughly equipotent mifepristone (600 mg), and since mifepristone has to be combined with a prostaglandin for the induction of abortion.^[18] However, data on embryotoxicity in humans are very limited, and it is not clear what the risk for an abortion or for teratogenicity (birth defects) is. Of the 29 women studied who became pregnant despite taking ulipristal acetate, 16 had induced abortions, six had spontaneous abortions, six continued the pregnancies, and one “was lost to follow-up“.^[19]

Lactation

It is not recommended to breast feed within 36 hours of taking the drug since it is not known whether ulipristal acetate or its metabolites are excreted into the breast milk.^[3][20]

Pharmacokinetics

In animal studies, the drug was quickly and nearly completely absorbed from the gut. Intake of food delays absorption, but it is not known whether this is clinically relevant.^[21] Ulipristal acetate is metabolized in the liver, most likely by CYP3A4, and to a small extent by CYP1A2 and CYP2D6. The two main metabolites have been shown to be pharmacologically active, but less than the original drug. The main excretion route is via the faeces.^[22]

Pharmacodynamics

As a SPRM, ulipristal acetate has partial agonistic as well as antagonistic effects on the progesterone receptor. It also binds to the glucocorticoid receptor, but has no relevant affinity to the estrogen, androgen and mineralocorticoid receptors.^[23] Phase II clinical trials suggest that the mechanism might consist of blocking or delaying ovulation and of delaying the maturation of the endometrium.^[24]

History

Ulipristal acetate was granted marketing authorization by the European Medicines Agency (EMA) in March 2009.^[25] The U.S. Food and Drug Administration approved the drug for use in the United States on 13 August 2010,^[26] following the FDA advisory committee’s recommendation.^[27][28] Watson Pharmaceuticals announced the availability of ulipristal acetate in the United States on 1 December 2010, in retail pharmacies, clinics, and one on-line pharmacy, KwikMed.^[29] Amorphous ulipristal acetate. ASKA is developing ulipristal acetate in Japan under license from HRA Pharma for the treatment of uterine fibroids and for emergency contraception. In March 2014, it was in phase II for both indications (in Japan). Also see the co-published WO2014050106 and WO2014050107. Crystalline polymorphic form C of ulipristal acetate.

Also claims its method of preparation. Appears to be the first filing from the assignee on this API, which was developed by HRA Pharma under license from the RTI, indicated in the US as an emergency contraceptive for prevention of pregnancy. In May 2011, ASKA signed an exclusive licensing agreement with HRA Pharma to develop and commercialize the API . In November 2013, ASKA had begun phase II development for emergency contraception  and uterine fibroids [1339186] in Japan. Also see concurrently published WO2014050105 and WO2014050107. Crystalline polymorphic form B of ulipristal acetate. Also claims process for the preparation and composition comprising the same. Useful for the treatment of uterine leiomyoma.

Appears to be the first filing from the assignee on this API, see concurrently published WO2014050105 and WO2014050106. The drug was developed by HRA Pharma under license from the RTI, indicated in the US as an emergency contraceptive for prevention of pregnancy. In May 2011, ASKA signed an exclusive licensing agreement with HRA Pharma to develop and commercialize the API  In November 2013, ASKA had begun phase II development in Japan for emergency contraception  and uterine fibroids Buccal forms or devices are also useful, such as those described in U.S. patent application 20050208129 , herein incorporated by reference. U.S. patent application 20050208129 describes a prolonged release bioadhesive mucosal therapeutic system containing at least one active principle, with an active principle dissolution test of more than 70% over 8 hours and to a method for its preparation.

Said bioadhesive therapeutic system comprises quantities of natural proteins representing at least 50% by weight of active principle and at least 20% by weight of said tablet, between 10% and 20% of a hydrophilic polymer, and compression excipients, and comprising between 4% and 10% of an alkali metal alkylsulphate to reinforce the local availability of active principle and between 0.1 % and 1% of a monohydrate sugar.

Ulipristal acetate, formerly known as CDB-2914, designates within the context of this application 17α-acetoxy-11β-[4-N,N-dimethylamino-phenyl)-19-norpregna-4,9-diene-3,20-dione, represented by formula I:

Ulipristal acetate, and methods for its preparation, are described e.g., in U.S. Pat. Nos. 4,954,490; 5,073,548, and 5,929,262, as well as in international patent applications WO2004/065405 and WO2004/078709. Ulipristal acetate possesses antiprogestational and antiglucocorticoidal activity, and has been proposed for contraception, in particular for emergency contraception, and for the therapy of various hormonal diseases.

 (Steroids, 2000,65, 395 ~ 400; US5929262A; CN1298409A; CN101466723A). Reaction is as follows:

Properties of this compound are further described in Blithe et al, Steroids. 2003 68(10-13):1013-7. So far, clinical trials have been conducted using oral capsules of ulipristal acetate (Creinin et al, Obstetrics & Gynecology 2006; 108:1089-1097; Levens et al, Obstet Gynecol. 2008, 111(5):1129-36). In order to increase the properties and clinical benefit of this molecule, there is a need for improved formulations thereof

• Ulipristal acetate, formerly known as CDB-2914, is 17α-acetoxy-11β-[4-N, N-dimethylamino-phenyl)-19-norpregna- 4, 9-diene-3, 20-dione, represented by formula I:
• It is a well-known steroid, more specifically a 19-norprogesterone, which possesses antiprogestational and antiglucocorticoidal activity. This compound, and methods for its preparation, are described in U. S. Patent Nos. 4,954, 490,5 , 073,548 , and 5,929, 262 , and international patent applications WO2004/065405 and WO2004/078709 . Properties of this compound are further described in Blithe et al, 2003.
• Metabolites of CDB-2914, include those described in Attardi et al, 2004 , e.g. monodemethylated CDB-2914 (CDB-3877) ;didemethylated CDB-2914 (CDB-3963) ; 17alpha-hydroxy CDB-2914 (CDB-3236) ; aromatic A-ring derivative of CDB-2914 (CDB-4183).
• It is now proposed to use ulipristal acetate or a metabolite thereof for treating uterine fibroids, more particularly for reducing or stopping bleeding in a patient afflicted with uterine fibroids, reducing the size of uterine fibroids and/or reducing uterine volume More particularly the inventors have shown in a randomized, placebo-controlled, double blinded, parallel trial, that ulipristal acetate significantly reduces fibroid volume after 3 months, and stops bleeding
• Ulipristal acetate or a metabolite thereof alleviates symptoms of uterine fibroids, including bleeding, pelvic pain, pressure.
• Ulipristal acetate or a metabolite thereof is useful for preventing or treating anemia in patients afflicted with uterine fibroids.
• It is also useful for preventing or treating leiomyosarcomas and for preventing dissemination of uterine fibroids to other organs.

..................

synthesis

http://www.google.com.br/patents/US5929262

CA2216737A1, EP0817793A2, WO1996030390A2, WO1996030390A3

The United States Of America As Represented By The Department Of Health And Human Services EXAMPLE 7 The Preparation of the Compound of Formula (I) (17α-Acetoxy-11β-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione) From the Compound of Formula (VIII) 340 mL of acetic acid (5.92 mol) were added to a well stirred mixture containing 834 mL of trifluoroacetic anhydride (5.92 mol) in 2,300 mL of methylene chloride under argon. After stirring for 30 minutes at room temperature, 51.3 g of p-toluenesulfonic acid (0.26 mol) were added, and the mixture was chilled to 0 methylene chloride solution containing 128.3 g of the compound of formula (VIII) (0.30 mol) were added, and the reaction mixture was stirred at 0 cautious addition of a 4.5N potassium carbonate solution until the pH was in the range of 7.0-7.5. The reaction mixture was diluted with water and extracted with methylene chloride. The methylene chloride extracts were washed with water and brine, combined, and dried over sodium sulfate.

Evaporation of the solvent gave the acetate of formula (I) as a thick syrup. The above syrup was dissolved in 300 mL of isopropyl alcohol and evaporated. The dissolution and evaporation were repeated three times. Finally, the remaining solid, which retained isopropyl alcohol as solvent of recrystallization, was dissolved in ethyl acetate and evaporated to give a stable foam. The foam was quickly dissolved in ether, and this solution was set aside to crystallize. The solid that formed was collected by filtration, washed with ether, and dried in vacuo to yield 105.7 g of the compound of formula (I) as yellow crystals in 75% yield;

m.p. 183-185 1735 and 1714(--C═O), 1664 and 1661 (conjugated --C═O), 1563, 1518, 1441, 1351, 1305, 1252, 1203, 1171; NMR (CDCl.sub.3) δ0.38 (s, 18-CH.sub.3), 2.10 (s, 17-OAc), 2.14 (s, 21-CH.sub.3), 2.92 (s, --N(CH.sub.3).sub.2, 4.44 (d, C-11 H), 5.83 (br. s, C-4 H), 6.71 and 7.07 (d, aromatic H); MS(EI) m/z (relative intensity) 475(M.sup.+, 41), 134(18), 121 (100). Analysis calculated for C.sub.30 H.sub.37 NO.sub.4 : C, 75.76; H, 7.84; N, 2.94. Found. C, 75.80; H 7.96; N, 3.09.

.................

SYNTHESIS http://www.google.com/patents/WO2004065405A1?cl=en

CA2514169A1, CA2514169C, CN1753905A, CN100354300C, EP1602662A1, EP2348033A2, EP2348033A3

EXAMPLE 1 Preparation of 17α-aceto-d-llβ-(4-N, N-dimetüaminofeniI)-19-norpregna-4 ,9-dien-3,20-dione [VA-2914] Raw Were charged 38.5 g of 3,3 – (l ,2-etanodioxi)-5α-hydroxy-llβ-(4-N, N-dimethylaminophenyl)-17α-acetoxy-19-norpregna-9-en-20-one [carbinol acetate] purified in a flask under nitrogen atmosphere at a temperature between 20 ° C and 22 ° C, and added 385 ml of deionized water and 17.91 g of HKSO. The resulting suspension was stirred until complete dissolution, for about 4 hours. The end of the reaction was determined by thin layer chromatography (TLC). Then added 3.85 g of neutral Al ₂ O _3, stirred for 30 minutes, the suspension was filtered and the insolubles were washed with 38.5 ml of deionized water. To the filtrate were added 325 ml of ethyl acetate and the pH was adjusted to a constant value between 7.0 and 7.2 with sodium bicarbonate solution to 7% w / v. The phases were allowed to decant for 15 minutes and, after checking the absence of the final product therein by means of TLC, the phases were separated, discarding the aqueous phase. The resultant organic phase was added 192.5 ml of deionized water, stirred for 10 minutes and the phases were allowed to decant for 15 minutes.

After verifying the absence of aqueous phase final product by TLC, the phases were separated, discarding the aqueous phase. The resulting organic phase was concentrated under vacuum to a residue and obtained approximately 28 g of 17α-acetoxy-llβ-(4-N, N-dimethylaminophenyl)-19-norpregna-4,9-dien-3 ,20-dione [NA -2914] raw. EXAMPLE 2 Isopropanol hemisolvate obtaining 17α-acetoxy-llβ-(4-Ν, Ν-dimethylaminophenyl)-19-norpregna-4 ,9-dien-3 ,20-dione The crude 17α-acetoxy-l lβ-(4-Ν, Ν-dimethylaminophenyl)-19-norpregna-4 ,9-dien-3,20-dione obtained in Example 1 was added 2 x 38.5 ml isopropanol concentrating vacuum to a residue both times. The finally obtained solid was added 77 ml of isopropanol and heated until dissolved. Then allowed to cool to a temperature between 0 ° C and 5 ° C, and the temperature was maintained for 1 h. The resulting suspension was filtered and the cake washed with cold isopropanol.

The yield achieved was 96% molar (5.5% isopropanol content). Isopropanol hemisolvate obtemdo NA-2914 has been characterized by IR spectroscopy, DSC and XRD, as indicated in the description, and has the characteristics indicated therein and shown in Figures 1-3.

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A new and efficient method for the synthesis of Ulipristal acetate

http://dx.doi.org/10.1016/j.steroids.2014.03.009

http://www.sciencedirect.com/science/article/pii/S0039128X14000634

In this study, we describe another new and efficient route for preparing Ulipristal acetate. The 1,4-addition compound 5 was greatly improved after the starting material ketone 1 was underwent epoxidation, cyanation, hydroxyl group protection and Grignard addition. The synthetic procedure is only 6 steps and the total yield is about 27.4%, which is much suitable for industrial process.

We have succeeded in finding another convenient and efficient synthetic route for the synthesis of Ulipristal acetate with a good yield.

•The yield of 11β-substituted isomer was greatly improved.

•The 17β-carbonitrile compound was obtained with high purity after the reaction.

•The yield of once Grignard addition dione was greatly improved.

•These synthetic procedures are much suitable for industrial process.

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Volume 78, Issues 12–13, 11 December 2013, Pages 1293–1297

http://www.sciencedirect.com/science/article/pii/S0039128X13002122

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http://www.google.com/patents/CN103145787A?cl=en

Reaction is as follows:

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WO2013063859A1

http://www.google.com/patents/WO2013063859A1?cl=en

Preparation of related reports Uli Division acetate compounds as follows:

1, U.S. Patent US4954490 methods, (see Reaction Scheme 1),

The method is based on the 3 – methoxy -19 – norpregn-1, 3,5 (10), 17 (20) – tetraene as a starting material, in turn by the addition, oxidation, reduction, hydrolysis, addition and elimination, oxidation of 17-hydroxy-19 – norpregn left the -4,9 – 31 women -3, 20 – dione (Compound V2), and then condensed by ethylene glycol, epoxidized-chloroperbenzoic acid, Bonus format, acid hydrolysis, acetylation of 10-step reaction by Uli acetate SECRETARY (Compound 1), and a melting point of 118-121 ° C the product was obtained by recrystallization with methanol ^. Due to the method, the step length, but difficult to obtain a starting material, the complexity of the reaction conditions, the required intermediate product was purified by column chromatography, the total yield is only 0.62%, Gao costs, the instability of the resulting product is not suitable pharmaceutically acceptable. And is not suitable for industrial production.

Reaction Formula I:

2, U.S. Patent No. US5929262 discloses his method Another method for preparing acetic acid Uli Division (see Reaction Scheme II), the reaction of formula II:

The method is based on 3,3 – ethylenedioxy-17) 8 – cyano -19 – norpregn -5 (10)-9 (11) – dien-17 alcohol (compound III) as a starting material, , first with dimethyl chloromethyl silane protected hydroxy, and then at the cryogenic-70Ό obtained by acid hydrolysis with the DBB / LI reagents After the reaction, a condensation reaction with ethylene glycol ketal, epoxy reaction, then the format of the reaction, The acid hydrolysis reaction and the acetylation reaction to obtain the target object and sequentially by treatment with isopropanol, ethyl acetate and crystallized from ether to obtain a yellow product with a melting point of 183-185Ό. The method expensive starting materials prices, harsh reaction conditions, need to be ultra-low temperature and water and oxygen reaction, high cost of low yield (total yield of about 14%), and therefore not suitable for industrial production.

3, World Patent WO2004078709 discloses a method for preparing (see Reaction Scheme III), the method the Πα hydroxy _19_ norpregn _ _4, 9 ₍₁₀₎ _ diene-_ _ _{3, 17-dione} (Compound _{V2 ),} followed by acetylation of 3 – bit carbonyl condensation, epoxy, Bonus format, acid hydrolyzed to give the target. Although the steps are shorter, but a starting material is from Compound VI was prepared by hydrolysis under acidic conditions to obtain a total yield of about 11.8% (starting from the compound VI operator), the actual reaction step is longer, lower yield, higher cost not suitable for industrial production.

In this method, 3,3 – ethylenedioxy -19 – norpregn -5 (10), 9 (11) – dien-17 – one (referred to as 3 – ketal compound II) as a starting material, by the addition of acetylene, benzene sub-sulfonyl chloride, and then by hydrolysis of sodium methoxide, acid hydrolysis, condensation of ethylene glycol, epoxy, Grignard reaction, acid hydrolysis and acetylation reaction of 9-step reaction to obtain a target object, isopropoxy alcohol crystallization with ethanol and water was heated at 70 ° C after 14h excluding solvate crystal. The method uses a greater risk of acetylene and odor of benzene times sulfonyl chloride, especially benzene times, unstable sulfonyl chloride, easy storage, decomposition of impurities involved in the reaction leads to a low yield, and benzene of times sulfonyl chloride of environmental pollution Further crystallization prolonged heating will produce new impurities, the total yield of 13.8% -15.8%, high cost, is not suitable for industrial production.

The existing methods, the methods 1, 2 and 4 are related to the preparation of compound VI, and also the starting materials in Method 3 Hydrolysis of compound VI is obtained. SUMMARY OF THE INVENTION

Technical problems to be solved by the present invention is to overcome these drawbacks,, study design Uli acetate Secretary industrialization production methods.

WO2013063859A1

http://www.google.com/patents/WO2013063859A1?cl=en

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Relugolix (TAK-385)

1-[4-[1-(2,6-Difluorobenzyl)-5-(dimethylaminomethyl)-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea

N-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N’-methoxyurea

CAS NO 737789-87-6

Relugolix (TAK-385)

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http://www.google.co.in/patents/EP1591446A1?cl=en

(Production Method 1)

(Production method 2)

Example 83
http://www.google.co.in/patents/EP1591446A1?cl=en

Production of N-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N’-methoxyurea

• The similar reaction as described in Example 4 by using the compound (100 mg, 0.164 mmol) obtained in Reference Example 54 and methyl iodide (0.010 ml, 0.164 mmol) gave the title compound (17.3 mg, 17 %) as colorless crystals.
  ¹ H-NMR(CDCl₃) δ: 2.15 (6H, s), 3.6-3.8 (2H, m), 3.82 (3H, s), 4.18 (3H, s), 5.35 (2H, s), 6.92 (2H, t, J = 8.2 Hz), 7.12 (1H, d, J = 8.8 Hz), 7.2-7.65 (7H, m), 7.69 (1H, s).

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Discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (TAK-385) as a potent, orally active, non-peptide antagonist of the human gonadotropin-releasing hormone receptor
J Med Chem 2011, 54(14): 4998. http://pubs.acs.org/doi/full/10.1021/jm200216q

1-{4-[1-(2,6-Difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (16b)

tak 385

http://pubs.acs.org/doi/suppl/10.1021/jm200216q/suppl_file/jm200216q_si_001.pdf

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new patent

WO-2014051164

Method for the production of TAK-385 or its salt and crystals starting from 6-(4-aminophenyl)-1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl) thieno[2,3-d] pyrimidine-2,4 (1H,3H)-dione or its salt. Takeda Pharmaceutical is developing relugolix (TAK-385), an oral LHRH receptor antagonist analog of sufugolix, for the treatment of endometriosis and uterine fibroids. As of April 2014, the drug is in Phase 2 trails. See WO2010026993 claiming method for improving the oral absorption and stability of tetrahydro-thieno[2,3-d]pyrimidin-6-yl]-phenyl)-N’-methoxy urea derivatives.

references

Discovery of TAK-385, a thieno[2,3-d]pyrimidine-2,4-dione derivative, as a potent and orally bioavailable nonpeptide antagonist of gonadotropin releasing hormone (GnRH) receptor
238th ACS Natl Meet (August 16-20, Washington) 2009, Abst MEDI 386

Discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (TAK-385) as a potent, orally active, non-peptide antagonist of the human gonadotropin-releasing hormone receptor
J Med Chem 2011, 54(14): 4998. http://pubs.acs.org/doi/full/10.1021/jm200216q

Buserelin

57982-77-1  cas no

D-Ser(Tbu)6EA10LHRH

(2S)-N-[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-5-(diaminomethylideneamino)-1-[(2S)-2-(ethylcarbamoyl)pyrrolidin-1-yl]-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-[(2-methylpropan-2-yl)oxy]-1-oxopropan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]-5-oxopyrrolidine-2-carboxamide

6-[O-(1,1-dimethylethyl)-D-serine]-9-(N-ethyl-L-prolinamide)-10-deglycinamideluteinizing hormone-releasing factor (pig)

Buserelin is a luteinizing hormone-releasing hormone (LHRH) agonist, a synthetic hormone which stimulates the pituitary gland’s gonadotrophin-releasing hormone receptor (GnRHR). It is used in prostate cancer treatment.

Buserelin stimulates the pituitary gland’s gonadotrophin-releasing hormone receptor (GnRHR). Buserelin desensitizes the GnRH receptor, reducing the amount of LH and testosterone. However, there is a concomitant surge in LH and testosterone levels with the decrease in androgens, so antiandrogens must administered.

buserelin

Buserelin is a Gonadotropin-releasing hormone agonist (GnRH agonist). The drug’s effects are dependent on the frequency and time course of administration. GnRH is released in a pulsatile fashion in the postpubertal adult. Initial interaction of any GnRH agonist, such as buserelin, with the GnRH receptor induces release of FSH and LH by gonadotrophes. Long-term exposure to constant levels of buserelin, rather than endogenous pulses, leads to downregulation of the GnRH receptors and subsequent suppression of the pituitary release of LH and FSH.

Like other GnRH agonists, buserelin may be used in the treatment of hormone-responsive cancers such as prostate cancer or breast cancer, estrogen-dependent conditions (such as endometriosis or uterine fibroids), and in assisted reproduction.

It is normally delivered via a nasal spray, but is also available as an injection.

Buserelin acetate is marketed by Sanofi-Aventis under the brand name Suprefact and a generic form of Buserelin is now produced by CinnaGen under the brand name CinnaFact.

Buserelin is also marketed under the brand name Metrelef. Metrelef is approved to treat patients with endometriosis by suppression of ovarian hormone production. In ovulation induction Metrelef is used as a pituitary blockade as an adjunct togonadotrophin administration.

Buserelin, a synthetic gonadotropin-releasing hormone (GRH) agonist, specifically binds to GRH receptor presented at anter iorpituitary and increases or decreases the number of receptors in hypophysis through auto- regulation mechanism (G. Tolis et al., Tumor Growth Inhibition in Patients with Prostatic Carcinoma Treated with Luteinizing Hormone-Feleasing Hormone Agonists, Proc. Natl. Acad. Sci. , 79, pl658, 1982).

<5> The synthetic methods for preparing peptides are divided into two methods, i.e., liquid phase synthesis and solid phase synthesis. The liquid phase peptide synthesis of which all the reagents reacts together under the solution phase by being dissolved in the solution, has been reported to show rapid reaction rate however it has disadvantages such as the difficulty in separating and purification of the products. In a while, solid phase peptide synthesis which have been developed based on the theory of R. B. Merrifield, has been reported to have various advantages comparing with the former method for example, convenient to isolation and purification, the ‘applicability to automation (Bodanszky et al, In Peptide Synthesis, John Wiley & Sons, 1976). Lots of peptide synthetic resins have been developed to synthesize various peptides after the publication of the theory of R. B. Merrifield till now. For example, chloromethyl polystyrene resin had been developed by Merrifield and Wang resin having 4-alkoxybenzyl alcohol had been developed with modifying the former resin to overcome the disadvantages thereof at the early stage. Various resins to improve the disadvantages of conventional resins have been developed after then and the representative resins among those resins are trityl group introduced 2-chlorotrityl resin and rink amide resin which can provide amide group from the carboxyl terminal of peptide under mild cleavage condition, respectively.

<6> At the early stage, the simple structured type-peptides have been synthesized using by the resins however the complex structured type peptides showing various physiological activities have been synthesized mainly. The peptides comprising unnatural amino acids have been synthesized by chemical synthetic method since the peptides could not be prepared by enzymatic synthesis. Among them, the peptides comprising D-amino acid or aza-amino acid have been reported to have potent physiological activities and further to be developed as a medicine (USP Nos. 6,624,290; 6,069,163; 5,965,538; and 4,634,715). However, the novel method for preparing LH-RH such as goserelin or GnRH peptides using by solid phase synthesis has been still need till now since previously known methods, for example, the methods disclosed in USP No. 5,602,231; EP No. 0518655; USP No. 6,879,289; and USP No. 3,914,412, have been reported to have unsolved problems such as a limit to obtain pure product etc.

http://www.google.com/patents/WO2008044890A1?cl=en

Example 4: Preparation of buserelin

<98> Ig of 2-chlroro trityl chloride resin showing 0.9 mM/g of substitution rate was swollen with 10ml of DMF and the reaction mixture mixed with 768 mg of Fmoc-Arg (N0₂)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together. The resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc-Leu-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 670 mg of Fmoc-D- SeKtBu)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.

<99> The resin was washed again and 2ml of 1% TFA (Trifluoroacetic acid)/DCM (dichloromethane) per 70mg of peptide resin was added to the resin, eluted to release the peptide from the resin and the elute was collected with 200 microliter of pyridine. The above-described step was repeated five times. The resin was washed with DCM (dichloromethane) and methanol and the elute was collected with the former elute. The elute was concentrated with evaporation and ether was added thereto to obtain the precipitated peptide. The precipitated peptide was performed to coupling reaction with 305 mg of Pro- NH-CH₂CH₃ (2.4mM) and 303mg of DIC (2.4 niM) in the presence of DCM

(dichloromethane) solvent. The solution was subjected to concentration with evaporator. The resulting concentrate was dissolved in EtOAc, washed with saturated NaHCOs solution, distilled water, 5% citrate solution and dried with anhydrous MgS(V The remaining MgS04 was discarded with filtration and the filtrate was concentrated with evaporation. The benzyl group and Cbz group among the side chain protecting group in the peptide were removed through catalytic hydrogen transfer reaction using by Pd/C and ammonium formate in the presence of methanol. The resulting peptide was purified with reverse phase column chromatography (Shimadzu H-kit, acetonitrile^water= 22:78 → 32:68, 1% increase/min) to isolate pure buserelin (Yield: 40%).

new patent

WO-2014047822

Solid state method for the preparation of buserelin, an LHRH analog useful for the treatment of sexual dysfunction, ovulation, puberty retardation and cancer. Method is under basic conditions and increases yield and purity. This appears to be the first PCT application from Hybio with this target, however several Chinese national filings have been published. Pan, Ma and Yuan are named on several previous solid phase synthesis PCT applications, most recently WO2013117135.

Cuban doctors have filed Wednesday in Havana, the result of 14 years of research, a solution of antitumor peptides whose natural analogue is able to offer positive dynamics in cancer treatments

http://youthandeldersja.wordpress.com/2014/03/22/cuba-may-have-found-cure-for-cancer/

Dilip sanghvi, sun pharma promoter

The move will make the company the largest pharma firm in India, while Daiichi Sankyo – majority owner of Ranbaxy – will become the second largest shareholder in Sun Pharma with a 9% stake and the right to nominate one director to Sun Pharma’s Board of Directors. http://www.pharmatimes.com/Article/14-04-07/Sun_buys_Ranbaxy_for_4_billion.aspx

Read more at: http://www.pharmatimes.com/Article/14-04-07/Sun_buys_Ranbaxy_for_4_billion.aspx#ixzz2yGIjkMob

Dilip Shanghvi, Managing Director of Sun Pharma said in a release, “Ranbaxy has a significant presence in the Indian pharma market and in the US where it offers a broad portfolio of ANDAs and first-to-file opportunities. In high-growth emerging markets, it provides a strong platform which is highly complementary to Sun Pharma’s strengths,”

Under the agreement, Ranbaxy shareholders will get 0.8 shares of Sun Pharma for each Ranbaxy share.

Arun Sahwney, managing director and chief executive officer of Ranbaxy said in a statement, “Sun Pharma has a proven track record of creating significant long-term shareholder value and successfully integrating acquisitions into its growing portfolio of assets,”

Who Will Benefit?

Daiichi Sankyo Co. Ltd is the parent company of Ranbaxy as they acquired it from previous promoters and investors. As soon as Ranbaxy was acquired, their plants came under a scanner from US Food and Drug Administration (FDA), which troubled Daiichi as their own reputation was under stake.

Now, they will be the most relived entity as Sun Pharma will manage all such cases pertaining to Ranbaxy. Daiichi will now control 9% of Sun Pharma as a result of the current acquisition.

Insiders are claiming that Daiichi will sell this 9% stake as well and come out of the business all together.

Ranbaxy shareholders have cheered this latest development as their shares have gained since the announcement of this deal.

ioforminol

Ioforminol [INN], UNII-95FNF21CDN, 1095110-48-7, FEK-256-062

5-[formyl-[3-[formyl-[3,5-bis(2,3-dihydroxypropylcarbamoyl)-2,4,6- triiodophenyl]amino]-2-hydroxypropyl]amino]-N,N’-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene- 1 ,3-dicarboxamide
1,3-Benzenedicarboxamide, 5,5′-[(2-hydroxy-1,3-
propanediyl)bis(formylimino)]bis[N1,N3-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-

All-ambo-5,5'-[2-hydroxypropane-1,3-diylbis(formylazanediyl)]bis[N,N'-bis(2,3-
dihydroxypropyl)-2,4,6-triiodobenzene-1,3-dicarboxamide]

https://download.ama-assn.org/resources/doc/usan/x-pub/ioforminol.pdf

MOLECULAR FORMULA C33H40I6N6O15

MOLECULAR WEIGHT 1522.1

SPONSOR GE HealthCare Ltd

CODE DESIGNATION FEK-256-062

CAS REGISTRY NUMBER 1095110-48-7

WHO NUMBER 9245

Visualisation of anatomical structures of the body during computed tomography for diagnostic purposes

All diagnostic imaging is based on the achievement of different signal levels from different structures within the body. Thus, in X-ray imaging for example, for a given body structure to be visible in the image, the X-ray attenuation by that structure must differ from that of the surrounding tissues. The difference in signal between the body structure and its surroundings is frequently termed contrast and much effort has been devoted to means of enhancing contrast in diagnostic imaging since the greater the contrast between a body structure and its surroundings the higher the quality of the images and the greater their value to the physician performing the diagnosis. Moreover, the greater the contrast the smaller the body structures that may be visualized in the imaging procedures, i.e. increased contrast can lead to increased spatial resolution. The diagnostic quality of images is strongly dependent on the inherent noise level in the imaging procedure, and the ratio of the contrast level to the noise level can thus be seen to represent an effective diagnostic quality factor for diagnostic images.

For the last 50 years the field of X-ray contrast agents has been dominated by soluble iodine containing compounds. Commercial available contrast media containing iodinated contrast agents are usually classified as ionic monomers such as diatrizoate (Gastrografen™), ionic dimers such as ioxaglate (Hexabrix™), nonionic monomers such as iohexol (Omnipaque™), iopamidol (Isovue™), iomeprol (lomeron™) and the non-ionic dimer iodixanol (Visipaque™). The most widely used commercial non-ionic X-ray contrast agents such as those mentioned above are considered safe. Contrast media containing iodinated contrast agents are used in more than 20 million of X-ray examinations annually in the USA and the number of adverse reactions is considered acceptable. However, since a contrast enhanced X- ray examination will require up to about 200 ml contrast media administered in a total dose, there is a continuous drive to provide improved contrast media.

Achieving improvement in such a diagnostic quality factor has long been and still remains an important goal.

In techniques such as X-ray, one approach to improve the diagnostic quality factor has been to introduce contrast enhancing materials formulated as contrast media into the body region being imaged. Thus for X-ray, early examples of contrast agents were insoluble inorganic barium salts which enhanced X-ray attenuation in the body zones into which they distributed. For the last 50 years the field of X-ray contrast agents has been dominated by soluble iodine containing compounds.

Commercial available contrast media containing iodinated contrast agents are usually classified as ionic monomers such as diatrizoate (marketed e.g. under the trade mark Gastrografen™), ionic dimers such as ioxaglate (marketed e.g. under the trade mark Hexabrix™), nonionic monomers such as iohexol (marketed e.g. under the trade mark Omnipaque™), iopamidol (marketed e.g. under the trade mark Isovue™), iomeprol (marketed e.g. under the trade mark Iomeron™) and the non-ionic dimer iodixanol (marketed under the trade mark Visipaque™). The clinical safety of iodinated X-ray contrast media has continuously been improved over the recent decades through development of new agents; from ionic monomers (Isopaque™) to non-ionic monomers (e.g. Omnipaque™) and non-ionic dimers (e.g. Visipaque™).

The utility of the contrast media is governed largely by its toxicity, by its diagnostic efficacy, by adverse effects it may have on the subject to which the contrast medium is administered, but also by the ease of production, storage and administration. The toxicity and adverse biological effects of a contrast medium are contributed to by the components of the formulation medium, i.e. of the diagnostic composition, e.g. the solvent or carrier as well as the contrast agent itself and its components such as ions for the ionic contrast agents and also by its metabolites.

The manufacture of non-ionic X-ray contrast media involves the production of the active

pharmaceutical ingredient (API), i.e. the contrast agent prepared in the primary production, followed by the formulation into the drug product, herein denoted the X-ray composition, prepared in the secondary production. In the preparation of an X-ray composition, the contrast agent is admixed with additives, such as salts, optionally after dispersion in a physiologically tolerable carrier. The contrast agent has to be completely solved in the carrier when additives are included and the composition is prepared. A well-known process for preparing X-ray compositions includes heating the contrast agent in the carrier, such as water for injection, to ensure complete dissolution. For instance, for the contrast media Visipaque™ the secondary production process includes dissolution of the contrast agent iodixanol in water for injection and heating to about 98 °C. Heating at this temperature for an adequate period of time ensures that the contrast agent is completely dissolved.

However, different X-ray contrast agents have different solubility. For instance WO 2009/008734 of GE Healthcare AS discloses a new class of compounds and their use as X-ray contrast agents. The compounds are dimers containing two linked iodinated phenyl groups. Compound I, now called

Ioforminol, falling within the formula I of WO2009/008734, has been found by the applicant to have particularly favourable properties. Ioforminol is supersaturated at the relevant storage conditions.

Compound I, Ioforminol:

5-[formyl-[3-[formyl-[3,5-bis(2,3-dihydroxypropylcarbamoyl)-2,4,6- triiodophenyl]amino]-2-hydroxypropyl]amino]-N,N’-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene- 1 ,3-dicarboxamide.

A solution in which the concentration of the solute (API) exceeds the equilibrium solute concentration at a given temperature is said to be supersaturated. This is possible because the solute does not precipitate immediately when the solution is cooled below the saturation temperature. Such solutions are denoted supersaturated.

As the solubility of Ioforminol decreases with decreasing temperature, the supersaturation increases. At room temperature the solubility of Ioforminol is limited. To achieve solutions with a concentration higher than the thermodynamic equilibrium concentration, at room temperature, Ioforminol is dissolved at a temperature above room temperature. When a clear solution has been achieved the solution is cooled and enters a state defined as supersaturated.

Supersaturated solutions are thermodynamically unstable and prone to nucleate and therefore to precipitate on storage. Among several factors, the onset of the precipitation depends on the degree of supersaturation, presence of the crystals of the solute and foreign particles such as dust or other impurities, i.e. purity, and storage temperature of the solution.

The injection solution of Ioforminol, i.e. the administrable X-ray composition, is highly supersaturated. The nucleation (precipitation) in the injection solution at storage conditions is strongly undesirable. The physical stability of the solution, i.e. prevention of the nucleation for a certain time at storage conditions, may be improved substantially by heat treatment of the solution well above its saturation temperature for a sufficiently long period of time.

WO2011/117236 of the applicant is directed to a process involving hea treatment at low pH to avoid degradation and precipitation of an X-ray contrast agent composition. However, a high heat load is needed to obtain a seed- free solution. This heat load causes a greater degradation of the product and a lower pH in the final product resulting in liberation of iodine. This sets a restriction to the total heat load that may be given to the formulated solution.

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WO2014052091A1

X-ray contrast media containing a chemical compound as the active pharmaceutical ingredient(s) having two triiodinated phenyl groups linked by a linking group are usually referred to as dimeric contrast agents or dimers. During the years a wide variety of iodinated dimers have been proposed. Currently, one contrast medium having an iodinated non-ionic dimer as the active pharmaceutical ingredient is on the market_^ the product Visipaque™ containing the compound iodixanol. In WO2009/008734 of the applicant a novel dimeric contrast agent named loforminol is disclosed.

The properties of this is described in more detail in the publications Chai et al. “Predicting cardiotoxicity propensity of the novel iodinated contrast medium GE-145: ventricular fibrillation during left coronary arteriography in pigs”, Acta Radiol, 2010, and in Wistrand, L.G., et al “GE-145, a new low-osmolar dimeric radiographic contrast medium”, Acta Radiol, 2010. loforminol (GE-145) is named Compound 1 herein and has the following structure:

Compound 1 :

5,5′-(2-Hydroxypropane-1 ,3-diyl)bis(formylazanediyl)bis(N¹ ,N³-bis(2,3- dihydroxypropyl)-2,4,6-triiodoisophthalamide)

The manufacture of non-ionic X-ray contrast media involves the production of the chemical drug, the active pharmaceutical ingredient (API), i.e. the contrast agent, followed by the formulation into the drug product, herein denoted the X-ray composition. WO2009/008734 of the applicant provides a synthetic route for preparing the API loforminol.

loforminol can e.g., as provided by the general preparation description and Example 1 of WO2009/008734, be synthesized from 5- amino-N,N’-bis-(2,3-dihydroxy-propyl)-2,4,6-triiodo-isophthalamide (compound (4)), which is commercially available. The preparation of this compound is known from the synthesis of both iohexol and iodixanol and can also be prepared from 5- nitroisophthalic acid for instance as described in WO2006/016815, including hydrogenation and subsequent iodination e.g. by iodine chloride, I CI. Alternatively,

5-amino-2,4,6-triiodoisophthalic acid may be used, which is commercially available precursor, e.g. from Sigma-Aldrich. The free amino group of the isophthalamide compound (compound (4)) is then acylated and the hydroxyl groups in the substituents may also be protected by acylation. The protecting groups may be removed for example by hydrolysis to give N¹ ,N³-bis(2,3-dihydroxypropyl)-5- formylamino-2,4,6-triiodoisophthalamide.

In a dimerization step this is reacted e.g. with epichlorohydrin to provide the loforminol contrast agent compound. The state of the art synthesis of loforminol, as disclosed in examples 1 and 2 of WO2009/008734, is shown in Scheme 1 below.

Scheme 1 .

As described in WO2009/008734 compound 3 is a mixture comprising 1 – formylamino-3,5-bis(2,3-bis(formyloxy)propan-1 -ylcarbamoyl)-2,4,6-trioodobenzene, and X is then a formyl group. In each synthetic step it is important to optimize the yield and minimize the production of impurities. The problem to be solved by the present invention may be regarded as the provision of optimizing the process for preparation of compound mixture (3) of scheme 1 , i.e. a mixture comprising 1 -formylamino-3,5-bis(2,3- bis(formyloxy)propan-1 -ylcarbamoyl)-2,4,6-trioodobenzene.

The process is hence directed to the preparation of compound mixture (3) by the formylation of the amino function of 5-amino-N¹ ,N³-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide (4), including a work-up procedure.

Examples

Example 1 : Preparation of compound mixture (3) comprising 1-formylamino- 3,5-bis(2,3-bis(formyloxy)propan-1-ylcarbamoyl)-2,4,6-trioodobenzene

5-amino-N1 ,N3-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide (compound (4)) (7.5 kg, 10.6 moles) was dissolved in formic acid (4.9 I) and heated to 45 until a clear solution was obtained (~4 hours), then the thick amber solution was cooled to 10 °C.

Formic acid (9.4 I) was charged into a different reactor and cooled to 10 ^<€, after reaching the target temperature acetic anhydride was added at such a rate that the temperature did not exceeded 15 ^<€.

After 2.5 hours all acetic anhydride was added to the formic acid and the mixed anhydride solution was added drop wise to the compound (4) solution. The rate of addition was adjusted so that the temperature never exceeded 20 °C. After 2 hours all mixed anhydride had been added and the reaction was left stirring at 15 °C for additional 1 hour. Isopropanol (4.9 I) was added carefully and the suspension became noticeable thicker and was left stirring at ambient temperature. After 16 hours the reaction slurry was filtered on a vacuum nutch and washed with isopropanol (3 ^* 1 .5 I) to give compound mixture (3) comprising 1 -formylamino-3,5- bis(2,3-bis(formyloxy)propan-1 -ylcarbamoyl)-2,4,6-trioodobenzene as a dense white powder (7.98kg). The quantitative yield with regards to N-formylation was > 99 %.

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WO2009008734A2

Preparation of intermediates (when not commercially available)

The precursors to the compounds of formulas (IVa) and (IVb), the tri-iodinated phenyl groups having a free amino group are commercially available or can be produced following procedures described or referred to e.g. in WO95/35122 and WO98/52911. 5-amino-2,4,6-triiodo-isophtalic acid for example is available e.g. from Aldrich and 5-amino-2,4,6-triiodo-N,N’-bis(2,3-dihydroxypropyl)-isophtalamide is commercially available e.g. from Fuji Chemical Industries, Ltd.

Examples of commercial available precursors of the compounds of formulas (IVa) and (IVb), either commercially available or previously described in the literature include:

5-Amino-N,N’-bis-(2,3-dihydroxy-propyl)-2,4,6-triiodo-isophthalamide

5-Amino-N-(2,3-dihydroxy-propyl)-N’-(2-hydroxy-1-hydroxymethyl-ethyl)- 2,4,6-triiodo-isophthalamide (WO2002044125)

5-Amino-N,N’-bis-(2,3-dihydroxy-propyl)-2,4,6-triiodo-N,N’-dimethyl- isophthalamide

5-Amino-N-(2,3-dihydroxy-propyl)-N’-(2-hydroxy-ethyl)-2,4,6-triiodo-is ophthalamide (WO 8700757)

The compounds of formulas (IVa) and (IVb), may be prepared by acylation of the corresponding compounds having free amino groups. In this reaction, hydroxyl groups in the substituents R may also be protected by acylation.

Acylation may be effected by any convenient method, e.g. by use of activated formic acid such as mixed anhydrides which can prepared by a variety of methods described in the literature.

A convenient method of preparing mixed anhydrides is to add a carboxylic acid anhydride to an excess of formic acid under controlled temperature. It is also possible to make mixed anhydrides by addition of a carboxylic acid chloride to a solution of a formic acid salt. Formyl-mixed anhydrides may include acetyl, isobutyryl, pivaloyl, benzoyl etc.

In the present implementation acetic-formic mixed anhydride is employed. To an excess of cooled pre-prepared acetic-formic mixed anhydride is added a 5-amino- monomer and the mixture is stirred overnight. The mixture is concentrated in vacuo and may be used directly in the alkylation step as described in the experimental section (procedure B) or alternatively the O-acylated groups may be hydrolysed prior to alkylation as described in the experimental section (procedure A). Hydrolysis is conveniently performed in aqueous basic media as exemplified in the experimental section or may alternatively be effected by alcoholysis e.g. as described in WO1997000240.

It is also possible to dissolve the 5-aminomonomer in formic acid and subsequently add the carboxylic acid anhydride but in order to reduce unwanted acylation it is preferred to prepare the mixed anhydride separately and subsequently mix this with the 5-aminomonomer as described above.

Experimental

Example 1

5,5′-(2-hvdroxypropane-1 ,3-diyl)bis(formylazanediyl)bis(N¹,N³-bis(2,3- dihvdroxypropyl)-2.4,6-triiodoisophthalamide)

Procedure A:

1 a) N,N’-Bis-(2₁3-dihvdroxy-propyl)-5-formylamino-2,4,6-triiodo-isophthalamide Formic acid (300 ml) was charged in a dry 1000 ml flask fitted with a dropping funnel, stir bar, thermometer and a gas inlet. The acid was cooled on an ice bath under a nitrogen blanket and acetic anhydride (144.8 g, 1.418 mol) was added drop wise at a rate so that the temperature did not exceed 2.5 C. After complete addition, the ice bath was removed and the temperature was allowed to reach 10 °C. The mixture was again ice cooled and 5-amino-N,N’-bis(2,3-dihydroxypropyl)-2,4,6- triiodo-isophthalamide (100 g, 141.8 mmol) was added over 5 minutes and the mixture was left stirring over night while attaining ambient temperature. The mixture was evaporated to dryness and methanol (300 ml) and water (300 ml) was added. 2 M potassium hydroxide was added until all material was in solution and a stable pH 12.5 was attained. The methanol was removed in vacuo. The mixture was neutralized with 4 M HCI and a slow precipitation started. 300 ml water was added and the product was precipitated over night. The precipitate was collected and rinsed with a small amount of water and dried on filter to a moist cake and further dried in vacuo to yield 84.8 g ( 81.5 %) of N,N’-bis-

(2,3-dihydroxy-propyl)-5-formylamino-2,4,6-triiodo-isophthalamide.

¹H-NMR 500 MHz (solvent: D₂O, ref. H₂O=4.8 ppm, 25 ⁰C): 8.35 and 8.05 ppm (2s,

1 H), 3.94 ppm (m, 2H), 3.67 ppm (m, 2H), 3.55 ppm (m, 2H), 3.45 ppm (m, 2H),

3.34 ppm (m, 2H).

LC-MS (column Agilent Zorbax SB-Aq 3.5 μm 3.0 x 100 mm, solvents: A = water/ 0.1 % formic acid and B = acetonitrile/ 0.1% formic acid; gradient 0-30 % B over 20 min; flow 0.3 ml/ min, UV detection at 214 and 254 nm, ESI-MS) gave two peaks centred at 5.5 minutes with m/z (M + H+) 733.828, m/z (M + NH₄+) 750.855, m/z (M + Na+) 755.817 corresponding to the structure.

1 b) 5,5′-(2-hvdroxypropane-1 ,3-diyl)bis(formylazanediyl)bis(N¹,N³-bis(2,3- dihvdroxypropyl)-2,4,6-triiodoisophthalamide)

Potassium hydroxide (1.07 g) was dissolved in water (6.9 ml) and methanol (3.4 ml) in a 50 ml round bottomed flask fitted with a magnetic stir bar. Boric acid (0.41 g, 6.6 mmol) and N,N’-bis-(2,3-dihydroxy-propyl)-5-formylamino-2,4,6-triiodo- isophthalamide (7.0 g, 9.56 mmol) was added to the stirred solution.. Epichlorohydrin (260 ul, 3.32 mmol) was added to the solution and a pH electrode was fitted in the flask and the pH was maintained at pH 12.7 by drop wise addition of 4 M potassium hydroxide for 4 h. At this point, the mixture was left stirring over night. The pH was adjusted with 4 M hydrochloric acid to pH 4 and the methanol was removed in vacuo. The remaining aqueous solution was diluted with water (75 ml) and treated with ion exchangers (AMB200C and IRA67) to zero conductivity. The ion exchangers were removed by filtration and rinsed with water and the combined aqueous filtrates were freeze dried. The crude product was purified by preparative HPLC (column Phenomenex Luna C18 10 μm solvents: A = water and B = acetonitrile; gradient 05-20 % B over 60 min. After freeze drying 3.80 g of 5,5′- (2-hydroxypropane-1 ,3-diyl)bis(formylazanediyl)bis(N¹,N³-bis(2,3-dihydroxypropyl)- 2,4,6-triiodoisophthalamide) (74.8 % yield) was obtained.

¹H-NMR 500 MHz (solvent: D₂O, ref. H₂O=4.8 ppm, 25 ⁰C): 8.34 and 8.08 ppm (m, 2 H), 2.80-4.80 ppm (m 26 H). LC-MS TOF; 1522.68 m/z (M + H⁺), 1544.66 m/z (M + Na⁺).

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 New patent

WO-2014052091

Process for the preparation of 1-formylamino-3,5-bis(2,3-bis(formyloxy)propan-1-ylcarbamoyl)-2,4,6-trioodobenzene, used as a key intermediate in the preparation of ioforminol. Also claims a process for the preparation of ioforminol, useful in X-ray imaging. GE Healthcare is developing ioforminol (GE-145; AN-113111) as an iv contrast agent (Phase 2). See WO2013104690 claiming X-ray imaging contrast media with low iodine concentration and X-ray imaging process. Also see concurrently published WO2014052092 claiming preparation of ioforminol. Appears to be the first filing from Medi-Physics on this compound.

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The most preferred iodinated agents are;

Diatrizoic acid

loxaglinic acid

loversol

lodixanol

lomeprol

lobitriol

The most preferred chelates are:

Gadopentetate

Ňadoversetamide

Gadoxetinic acid