PRULIFLOXACIN by Nippon Shinyaku Co.

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Apr 062014

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

NM 441, Quisnon, Pruvel, Sword, Prulifloxacin [INN], 123447-62-1, NM-441, CCRIS 7686, NCGC00164615-01NAD-441A
OPT-99

Molecular Formula: C₂₁H₂₀FN₃O₆S

Molecular Weight: 461.463403

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.

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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%).

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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

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.
Kawahara S (1998). “[Chemotherapeutic agents under study]”. Nippon Rinsho (in Japanese) 56 (12): 3096–9. PMID 9883617.
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.
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.
JP patent 1294680, Kise Masahiro; Kitano Masahiko; Ozaki Masakuni; Kazuno Kenji; Matsuda Masato; Shirahase Ichiro; Segawa Jun, “Quinolinecarboxylic Acid Derivative”, issued November 28, 1989
Prulifloxacin. Drugfuture.com. Retrieved on 2010-11-03.
Anonymous (2002). “Prulifloxacin [‘Quisnon’; Nippon Shinyaku] has been approved in Japan”. Inpharma 1 (1362): 22.
Research and Development Department of Angelini. Angelinipharma.com. Retrieved on 2010-11-03.
Nippon Shinyaku, Annual Report 2007
“Prulifloxacin. NAD-441A, NM 441, Quisnon”. Drugs in R&D 3 (6): 426–30. 2002.PMID 12516950.
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.

US5498422	3-13-1996	Sustained release capsule
US5456923	10-11-1995	Method of manufacturing solid dispersion
US5086049	2-5-1992	7(4-(5 METHYL-2-OXO-1,3-DIOXALEN-4-YL)METHYL 1-PIPERZINYL)-4-OXO-4H-(1,3)THIAZETO(3,2-A)QUINOLINE-3-CARBOXYLIC ACIDS


US2011159049	6-31-2011	PHARMACEUTICAL COMPOSITION
US2011034690	2-11-2011	PROCESS FOR THE PREPARATION OF PURE PRULIFLOXACIN
US2010197910	8-6-2010	PROCESS FOR PREPARATION OF PRULIFLOXACIN USING NOVEL INTERMEDIATES
US2010113783	5-7-2010	PROCESS FOR THE PREPARATION OF CRYSTALS OF PRULIFLOXACIN
US2010087409	4-9-2010	COMPOSITION COMPRISING AN ANTIBIOTIC AND A CORTICOSTEROID
US2009306035	12-11-2009	Compounds and Methods for modulating the Silencing of a Polynucleotide of Interest
US2007196504	8-24-2007	PHARMACEUTICAL COMPOSITION
US2007148235	6-29-2007	PHARMACEUTICAL COMPOSITION
US2005152975	7-15-2005	Pharmaceutical composition
US2004022848	2-6-2004	Medicinal composition

WO2008059512A1	Nov 17, 2006	May 22, 2008	Hetero Drugs Ltd	Process for preparation of prulifloxacin using novel intermediates
WO2008111016A1	Mar 14, 2008	Sep 18, 2008	Ranbaxy Lab Ltd	Process for the preparation of pure prulifloxacin
WO2008111018A2	Mar 14, 2008	Sep 18, 2008	Ranbaxy Lab Ltd	Process for the preparation of crystals of prulifloxacin
WO2010084508A2	Dec 10, 2009	Jul 29, 2010	Elder Pharmaceuticals Ltd.	Process for the preparation of type i, type ii and type iii crystalline prulifloxacin
EP0315828A1 *	Oct 26, 1988	May 17, 1989	Nippon Shinyaku Company, Limited	Quinolinecarboxylic acid derivatives
EP1626051A1	Apr 28, 2004	Feb 15, 2006	Nippon Shinyaku Co., Ltd.	Crystals of quinolinecarboxylic acid derivative solvate
US5086049	Apr 8, 1991	Feb 4, 1992	Nipponshinyaku Co., Ltd.	7[4-(5 methyl-2-oxo-1,3-dioxalen-4-yl)methyl 1-piperzinyl]-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acids
US20070149540	Apr 28, 2004	Jun 28, 2007	Nippon Shinyaky Co., Ltd.	Crystals of quinolinecarboxylic acid derivative solvate

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
- 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
- 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 from kyorin

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Apr 052014

File:Clinafloxacin.png

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 .

Clinafloxacin HCl, CI-960 HCl, 105956-99-8, Clinafloxacin hydrochloride (USAN), Clinafloxacin hydrochloride [USAN], AC1L1SJB,

Molecular Formula: C₁₇H₁₈Cl₂FN₃O₃ Molecular Weight: 402.247523

……………………………………..

EP 0195316

http://www.google.com/patents/EP0195316A1?cl=en

preparation process for the compound of the invention.

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

Rubinstein, E. (2001). “History of quinolones and their side effects.”. Chemotherapy. 47 Suppl 3: 3–8; discussion 44–8.doi:10.1159/000057838. PMID 11549783.

EP0106489A2 *	Sep 6, 1983	Apr 25, 1984	Warner-Lambert Company	Antibacterial agents
EP0153163A2 *	Feb 15, 1985	Aug 28, 1985	Warner-Lambert Company	7-Substituted-1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids; 7-substituted-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acids; their derivatives; and a process for preparing the compounds
BE899399A1 *				Title not available
GB2057440A *				Title not available




	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

Cenicriviroc in Phase 2 for HIV by Takeda/Tobira

Uncategorized Comments Off

Apr 032014

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, Cenicriviroc (USAN/INN), TAK652, TBR652, , 497223-25-3, D09878

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

References

Klibanov, Olga M.; Williams, Shannon H.; Iler, Cameron A (2010). “Cenicriviroc, an orally active CCR5 antagonist for the potential treatment of HIV infection”. Current Opinion in Investigational Drugs 11 (8): 940–950. PMID 20721836.
Baba, Masanori; Takashima, Katsunori; Miyake, Hiroshi; Kanzaki, Naoyuki; Teshima, Koichiro; Wang, Xin; Shiraishi, Mitsuru; Iizawa, Yuji (2005). “TAK-652 inhibits CCR5-mediated human immunodeficiency virus type 1 infection in vitro and has favorable pharmacokinetics in humans”. Antimicrobial Agents and Chemotherapy 49 (11): 4584–4591. doi:10.1128/AAC.49.11.4584-4591.2005. PMC 1280155. PMID 16251299.
C. Reviriego (2011). Drugs of the Future 36 (7): 511–517. doi:10.1358/dof.2011.36.7.1622066.
“Tobira Therapeutics Initiates Phase 2b Trial of Cenicriviroc”. The Body. July 5, 2011.
CROI 2013: CCR5/CCR2 Inhibitor Cenicriviroc Has Both Anti-HIV and Anti-inflammatory Effects. Highleyman, Liz. HIVandHepatitis.com. 7 March 2013.


22931505	11-26-2012	Chemokine receptor antagonists.	Journal of medicinal chemistry
21317794	6-1-2011	Safety, efficacy, and pharmacokinetics of TBR-652, a CCR5/CCR2 antagonist, in HIV-1-infected, treatment-experienced, CCR5 antagonist-naive subjects.	Journal of acquired immune deficiency syndromes (1999)
20721836	8-1-2010	Cenicriviroc, an orally active CCR5 antagonist for the potential treatment of HIV infection.	Current opinion in investigational drugs (London, England : 2000)
19064629	3-1-2009	The relative activity of “function sparing” HIV-1 entry inhibitors on viral entry and CCR5 internalization: is allosteric functional selectivity a valuable therapeutic property?	Molecular pharmacology
17116673	2-1-2007	Isolation and characterization of human immunodeficiency virus type 1 resistant to the small-molecule CCR5 antagonist TAK-652.	Antimicrobial agents and chemotherapy
17037325	9-10-2006	[Progress in AIDS therapy].	Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine
16539392	3-23-2006	Highly potent and orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activities of 1-benzazocine derivatives containing a sulfoxide moiety.	Journal of medicinal chemistry
16251299	11-1-2005	TAK-652 inhibits CCR5-mediated human immunodeficiency virus type 1 infection in vitro and has favorable pharmacokinetics in humans.	Antimicrobial agents and chemotherapy
15658852	1-27-2005	Stereoselective synthesis of [L-Arg-L/D-3-(2-naphthyl)alanine]-type (E)-alkene dipeptide isosteres and its application to the synthesis and biological evaluation of pseudopeptide analogues of the CXCR4 antagonist FC131.	Journal of medicinal chemistry
16430202	1-1-2005	TAK-652, a novel CCR5 inhibitor, has favourable drug interactions with other antiretrovirals in vitro.	Antiviral therapy

……………….

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

RANBEZOLID FROM RANBAXY

Uncategorized Comments Off

Apr 022014

Ranbezolid

392659-39-1 hydrochloride

392659-38-0 (free base)

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

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

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

Molecular Formula: C₂₁H₂₄FN₅O₆ Molecular Weight: 461.443563

Ranbaxy Lab Ltd ORIGINATOR

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

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

Ranbezolid hydrochloride, RBx-7644

US2005209248

9-23-2005

Plymorphic forms of phenyl oxazolidinone derivatives

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

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

…………….

synthesis

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

………………….

polymorphs

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

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

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

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

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

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

………………

Synthesis and SAR of novel oxazolidinones: Discovery of ranbezolid

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

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

Synthesis and SAR of novel oxazolidinones: Discovery of ranbezolid

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

Graphical abstract

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

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

Scheme 5.

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

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

References

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

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

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

MK 2048 an HIV integrase inhibitor from Merck

Uncategorized Comments Off

Apr 022014

MK 2048

Molecular Formula: C₂₁H₂₁ClFN₅O₄ Molecular Weight: 461.873943

869901-69-9, 3oyl, 3oyn

Merck & Co., Inc.

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

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

US7538112

5-27-2009

Hiv Integrase Inhibitors

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

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

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

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

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

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

Structure of MK-2048 with important pharmacophore highlighted

…………………..

Synthesis

WO2005110415A1

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

335

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

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

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

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

Step 12:

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

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

MK 2048

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

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

MK 2048sodium salt

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

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

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

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

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

SL-J006-2

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

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

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

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

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

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

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

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

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

……………..

US 7538112

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

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

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

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

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

References

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

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Mar 302014

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

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

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

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

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

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

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

Serotonin system, contrasted with thedopamine system

Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

http://www.depression.org/

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

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

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

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

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

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

Serotonin Lysergic acid diethylamide

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

Serotonin LSD

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

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

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

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

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

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

www.findthelight.net/Depression/the_chemistry_of_dep.htm

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

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

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

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

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

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

The cancer fighting benefits of whey protein.lactoferrin

cancer Comments Off

Mar 292014

lactoferrin

Mon. Mar. 24, 2014 by Dr. Matthew Roe

http://www.naturalhealth365.com/food_news/0943_whey_protein.html

(NaturalHealth365) There is overwhelming evidence to suggest that whey protein can support the immune system, while killing cancer cells. Whey has multi-factorial benefits for cancer patients according to validated studies. It has a broad spectrum of compounds, which protect healthy cells and suppresses cancer cells.

Why should cancer patients consider whey protein?

Simply put, whey’s lactoferrin is a cancer killer. Lactoferrin activates the innate immune system cells like the neutrophils, macrophages and T-cells. These are the first line of defense against harmful pathogens – including cancer cells.

You see, cancer cells have a highly negative membrane charge which attracts lactoferrin, while healthy normal cells have a neutral charge. Lactoferrin is attracted to the cancer cells, attaches to them and triggers a process that kills the cancer cell; as well as blocking angiogenesis – the growth of blood vessels that feed cancer cells.

– See more at: http://www.naturalhealth365.com/food_news/0943_whey_protein.html

Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as milk, saliva, tears, andnasal secretions. Lactoferrin is also present in secondary granules of PMN and is secreted by some acinar cells. Lactoferrin can be purified from milk or produced recombinantly. Human colostrum (“first milk”) has the highest concentration, followed by human milk, then cow milk (150 mg/L).

Lactoferrin is one of the components of the immune system of the body; it has antimicrobial activity (bacteriocide, fungicide) and is part of the innate defense, mainly at mucoses. In particular, lactoferrin provides antibacterial activity to human infants. Lactoferrin interacts withDNA and RNA, polysaccharides and heparin, and shows some of its biological functions in complexes with these ligands.

FINAFLOXACIN IN PHASE II for the treatment of ear infections

Phase 3 drug Comments Off

Mar 292014

FINAFLOXACIN

(S-cyano-1-cyclopropyl-ό-fluoro-T-^aS, 7aS)-hexahydropyrrolo [3,4- b]-1,4-oxazin-6(2H)-yl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid)

7-[(4aS,7aS)-3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazin-6-yl]-8-cyano-1-cyclopropyl-6-fluoro-4-oxoquinoline-3-carboxylic acid |

BAY-35-3377
BY-377

CAS Registry Number: 209342-40-5

HYD SALT

(-)-(4aS,7aS)-8-Cyano-1-cyclopropyl-6-fluoro-4-oxo-7-(perhydropyrrolo[3,4-b]-1,4-oxazin-6-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride

209342-41-6,

C20 H19 F N4 O4 . Cl H
MW	434.849

Synonyms: Finafloxacin, UNII-D26OSN9Q4R,

MerLion Pharmaceuticals (Singapore)…POSTER…….http://www.merlionpharma.com/sites/default/files/file/PPS/F1-2036_Wohlert.pdf

H. pylori, Broad-Spectrum

Finafloxacin is a novel fluoroquinolone being developed by MerLion Pharmaceuticals. Under neutral pH conditions (pH 7.2–7.4), the compound has shown in vitro activity equivalent to that of ciprofloxacin. However, under slightly acidic pH5.8 the compound shows enhanced potency.

Other marketed fluoroquinolones, such as ciprofloxacin, levofloxacin and moxifloxacin, exhibit reduced activity at slightly acidic pH 5.0–6.5. This feature of finafloxacin makes the compound suitable for use in the treatment of infections in acidic foci of infections such as urinary tract infections

Finafloxacin hydrochloride, a novel highly potent antibiotic, is in phase III clinical trials at Alcon for the treatment of ear infections. MerLion Pharmaceuticals is evaluating the product in phase II clinical trials at for the treatment of Helicobacter pylori infection and for the treatment of lower uncomplicated urinary tract infections in females.

A quinolone, finafloxacin holds potential for the treatment of Helicobacter pylori infection and urinary tract infection. Unlike existing antibiotics, finafloxacin demonstrates a unique acid activated activity whereby it becomes increasingly active under acidic conditions.

In 2009, a codevelopment agreement was signed between Chaperone Technologies and MerLion Pharmaceuticals. In 2011, finafloxacin hydrochloride was licensed to Alcon by MerLion Pharmaceuticals in North America for the treatment of ear infections.

MerLion Pharmaceuticals has announced that the FDA has granted a Qualified Infectious Disease Product Designation and Fast Track Status for finafloxacin. The company is currently recruiting patients for the Phase II clinical trial of the compound for the treatment of complicated urinary tract infections (cUTI) and/or acute pyelonephritis compared to ciprofloxacin

Finafloxacin and derivatives thereof can be synthesized according to the methods described in U.S. Patent No. 6,133,260 to Matzke et al., the contents of which are herein incorporated by reference in their entirety. The compositions of the invention are particularly directed toward treating mammalian and human subjects having or at risk of having a microbial tissue infection. Microbial tissue infections that may be treated or prevented in accord with the method of the present invention are referred to in J. P. Sanford et al., “The Sanford Guide to Antimicrobial Therapy 2007” 37 Edition (Antimicrobial Therapy, Inc.). Particular microbial tissue infections that may be treatable by embodiments of the present invention include those infections caused by bacteria, protozoa, fungi, yeast, spores, and parasites.

SYNTHESIS

WO1998026779A1

http://www.google.sc/patents/WO1998026779A1 COPY PASTE ON BROWSER

8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5 ,8-di-azabicyclo [4.3.0] non-8-yl)-l, 4-dihydro-4-oxo-3-quinolinecarboxylic acid.

The compounds, which are suitable for use in the invention are known already to some extent in EP-A-0350733, EP-A-0550903 as well as from DE-A-4329600 or can be prepared according to the processes described in .

If, for example 9,10-difluoro-3 ,8-dimethyl-7-oxo-2 ,3-dihydro-7H-pyrido [l ,2,3-d, e] [l, 3,4] benzoxadiazine-6 -carboxylic acid and 2-oxa-5 ,8-diazabicyclo [4.3.0] nonane, the reaction can be represented by the following equation:

The 7-halo-quinolonecarboxylic acid derivatives used for preparing the compounds of Fomel (I) of the invention are known or can be prepared by known methods. Thus, the 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid, or of the 7-chloro-8-cyano-l-cyclopropyl-6-fluoro- l been ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester described in EP-A-0 276 700th The corresponding 7-fluoro derivatives can be, for example, via the following reaction sequence to build:

An alternative process for preparing the intermediate compound 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride as the starting material for the preparation of 7-chloro-

8-cyano-1-cyclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid is used (EP-A-0276700) and in the 3-cyano-2 ,4,5-trifluoro- benzoyl can be converted, is based on 5-fluoro-l ,3-xylene, 5-fluoro-l ,3-xylene, in the presence of a catalyst under ionic conditions in the nucleus disubstituted to 2,4-dichloro-5-fluoro-l ,3-dimethylbenzene, and this is subsequently chlorinated chlorinated under free radical conditions in the side chains of 2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloro-methylbenzene. This is the 2,4-dichloro-5-fluoro-3-dichloromethyl-benzoic acid to give 2,4-dichloro-5-fluoro-3-formyl-benzoic acid, and then hydrolyzed to 2,4-dichloro-5-fluoro-3 N-hydroxyiminomethyl acid implemented. By treatment with thionyl chloride, 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride is obtained, which can still be ,4,5-trifluoro-ben-zoylfluorid converted by a chlorine / fluorine exchange on-3-cyano-2 .

The amines used for the preparation of compounds of formula (I) according to the invention are known from EP-A-0550903, EP-A-0551653 as well as from DE-A-4 309 964th

An alternative to the synthesis of lS, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane-dihydro-drobromid or the free base 1 S, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0 ] nonane and the corresponding IR, 6R enantiomer provides the following path represents:

Starting material for this synthesis is the cis-l ,4-dihydroxy-2-butene, which is converted to the bis-mesylate with mesylation tosylamide for 1-tosylpyrrolidine. This is converted into the epoxide m-chloroperbenzoic. The ring opening of the epoxide by heating in isopropanol with ethanolamine to trans-3-hydroxy-4 – (2-hydroxy-ethylamino)-l-(toluene-4-sulfonyl)-pyrrolidine in 80% yield. Tetrahydrofuran is then in pyridine / reacted with tosyl chloride, with cooling to Tris-tosylate, which as a crude product in a mixture with some tetra-tosyl derivative with basichen reaction conditions to give the racemic trans-5 ,8-bis-tosyl-2-oxa-5, 6 – diazabicyclo [4.3.0] nonane is cylisiert. At this stage occurs with high selectivity of a chromatographic resolution kieselgelgebundenem poly (N-methacryloyl-L-leucine-d menthylamide) as the stationary phase. The desired enantiomer, (lS, 6S) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane, is of a purity of

> 99% ee. Cleavage of the p-tosyl protecting groups is carried out with HBr-acetic acid to the lS, 6S-2-Oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide, the one with a base such as sodium or potassium hydroxide or with the aid of ion exchanger can be converted into the free base. The analogous sequence may be used for the preparation of lR, 6R-2-Oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide.

HBr / AcOH

Synthesis of lS, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane

Examples of compounds of the invention are mentioned in addition to the compounds listed in the preparation examples, the compounds listed in Table 1 below, which can be used both in racemic form as well as enantiomerically pure or diastereomerically pure compounds. Table 1:

Example 1 Z

8-cyano-1-cyclopropyl-6 ,7-difluoro-1 ,4-dihydro-4-oxo-3-quinoline-carboxylic acid ethyl ester

a 3-bromo-2 ,4,5-trifluoro-benzoate

To a mixture of 1460 ml of methanol and 340 g of triethylamine, 772 g of 3-bromo-2 ,4,5-trifluoro-benzoyl fluoride was added dropwise under ice cooling. There is one

Stirred for an hour at room temperature. The Reaktionsgemsich is concentrated, the residue dissolved in water and methylene chloride, and the aqueous phase was extracted with methylene chloride. After drying the organic phase over sodium sulfate, concentrated, and the residue was distilled in vacuum. This gives 752.4 g of 3-bromo-2 ,4,5-trifluoro-benzoic acid methyl ester of boiling point 122 ° C/20 mbar.

b. 3-Cyano-2 ,4,5-trifluoro-benzoic acid methyl ester:

269 g of 3-bromo-2 ,4,5-trifluoro-benzoic acid methyl ester and 108 g of copper cyanide are heated to reflux in 400 ml of dimethylformamide for 5 hours. , All volatile components of the reaction mixture are then distilled off in vacuo. The distillate was then fractionated on a column. This gives 133 g of 3-cyano-2 ,4,5-trifluoro-benzoate of boiling point 88-89 ° C / 0.01 mbar.

c. 3-Cyano-2 ,4,5-trifluoro-benzoic acid

A solution of 156 g of 3-cyano-2 ,4,5-trifluoro-benzoate in 960 ml of glacial acetic acid, 140 ml of water and 69 ml concentrated sulfuric acid is heated for 8 hours under reflux. Then the acetic acid is distilled off under vacuum and the residue treated with water. Of failed-ne solid is filtered off, washed with water and dried. Obtained

118.6 g of 3-cyano-2 ,4,5-trifluoro-benzoic acid as a white solid, mp 187-190 ° C.

d 3-cyano-2 ,4,5-trifluoro-benzoyl chloride:

111 g of 3-cyano-2 ,4,5-trifluoro-benzoic acid and 84 g of oxalyl chloride are stirred in 930 ml of dry methylene chloride with the addition of a few drops of dimethylformamide for 5 hours at room temperature. The methylene chloride is evaporated and the residue distilled in vacuo. This gives 117.6 g of 3-cyano-2 ,4,5-trifluoro-benzoyl chloride as a yellow oil.

e 2 – (3-cyano-2 ,4,5-trifluoro-benzoyl)-3-dimethylamino-acrylic acid ethyl ester:

To a solution of 36.5 g of 3-dimethylamino-acrylate and 26.5 g of triethylamine in 140 ml toluene, a solution of 55 g 3-cyano-2, 4,5 – trifluoro-benzoyl chloride are added dropwise in 50 ml of toluene so that the temperature 50-55 ° C remains. Then stirred for 2 hours at 50 ° C.

The reaction mixture is concentrated in vacuo and used without further

Processing used in the next step. f 2 – (3-cyano-2 ,4,5-trifluoro-benzoyl)-3-cyclopropylamino-acrylic acid ethyl ester:

To the reaction product of step e 30 g of glacial acetic acid are added dropwise at 20 ° C. A solution of 15.75 g of cyclopropyl amine in 30 ml of toluene is added dropwise. The mixture is stirred at 30 ° C for 1 hour. Are then added 200 ml of water, stirred 15 minutes, the organic phase is separated off and shakes it again with 100 ml of water. The organic phase is dried over sodium sulfate and concentrated in vacuo. The crude product thus obtained is a set-without further purification in the next step.

g 8-cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester:

The reaction product from stage f and 27.6 g of potassium carbonate are stirred in 80 ml dimethylformamide for 16 hours at room temperature. The reaction mixture is then poured into 750 ml ice water, the solid filtered off with suction and washed with 80 ml cold methanol. After drying, 47 g of 8 – cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinoline carboxylic acid ethyl ester, mp 209-211 ° C.

Example 2 Z

2,4-dichloro-5-fluoro-l ,3-dimethylbenzene

a solvent-free

In 124 g of 3,5-dimethyl-fluorobenzene 1 g of anhydrous iron (III) chloride are pre-loaded and launched with the speed of chlorine (about 4 h), with which the reaction. This is initially slightly exothermic (temperature increase from 24 to 32 ° C) and is maintained by cooling below 30 ° C. After addition of 120 g of chlorine, the mixture is determined. According to GC analysis are 33.4% monochloro compound, formed 58.4% desired product and 5%> overchlorinated connections. The hydrogen chloride is removed and the reaction mixture is then distilled in a column in a water jet vacuum:

In the run 49 g of 2-chloro-5-fluoro-l ,3-dimethylbenzene obtained at 72-74 ° C/22 mbar. After 5 g of an intermediate fraction proceed at 105 ° C/22 mbar 75 g of 2,4 – dichloro-5-fluoro-l ,3-dimethylbenzene via, Melting range: 64 – 65 ° C.

b in 1,2-dichloroethane

1 kg of 3,5-dimethyl-fluorobenzene and 15 g of anhydrous iron (III) chloride are placed in 1 1 1 ,2-dichloroethane and chlorine is introduced in the same extent as the reaction proceeds (about 4 h). The reaction is initially exothermic (temperature rise from 24 to 32 ° C) and is kept below 30 ° C by cooling. After the introduction of 1200 g of chlorine are according to GC analysis 4% monochloro compound, 81.1% and 13.3% desired product overchlorinated connections emerged. After distilling off the solvent and the hydrogen chloride is distilled in a column in a water jet vacuum:

In the run 40 g of 2-chloro-5-fluoro-l ,3-dimethylbenzene receive. After some intermediate run going at 127-128 ° C/50 mbar 1115 g of 2,4-dichloro-5-fTuor-l ,3-dimethyl-ethylbenzene over.

Example 3 Z

2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloromethylbenzene

In a photochlorination using chlorine inlet and outlet for the hydrogen chloride to a scrubber and a light source in the vicinity of the chlorine inlet tube, 1890 g of 2,4-dichloro-5-fluoro-l ,3-dimethylbenzene pre-loaded and at 140 to 150 ° C. Chlorine metered. Within 30 hours 3850 g of chlorine are introduced. The content of the desired product according to GC analysis is 71.1% and the proportion of connections minderchlorierten 27.7%. The DestiUaton a 60 cm column with Wilson spirals provides a flow of 1142 g, which can be reused in the chlorination. The main fraction at 160-168 ° C / 0.2 mbar gives 2200 g of 2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloro-methyl benzene having a melting range of 74-76 ° C. After one recrystallization

Sample from methanol, the melting point 81-82 ° C.

Example Z 4

2,4-dichloro-5-fluoro-3-formyl-benzoic acid

In a 2500 ml stirred apparatus with gas discharge are presented 95% sulfuric acid at 70 ° C. and under stirring, 500 g of molten added dropwise 2,4-dichloro-5-fluoro-3-dichloromethyl-1 trichloromethylbenzene. It is after a short while hydrochloric development. Is metered during a 2 h and stirred until the evolution of gas after. After cooling to 20 ° C., the mixture is discharged ice to 4 kg and the precipitated solid is filtered off with suction. The product is after-washed with water and dried.

Yield: 310 g, melting range: 172-174 ° C

Example Z 5

2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl-benzoic acid

In a stirred reactor 80 g of hydroxylamine hydrochloride in 500 ml of ethanol are charged and added dropwise 200 ml of 45% strength sodium hydroxide solution and then with 40 – 200 g of 2,4-dichloro-5-fluoro-3-formyl-benzoic acid added 45.degree.The reaction is slightly exothermic and it is stirred for 5 h at 60 ° C. After cooling to

Room temperature is provided by the dropwise addition of hydrochloric acid to pH <3, the product taken up in tert-butyl methyl ether, the organic phase separated and the solvent distilled off. The residue obtained 185 g of 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl benzoic acid, melting range: 190 – 194 ° C.

Example No. 6

2,4-dichloro-3-cyano-5-benzoyl-fιuor

In a stirred vessel with metering and gas outlet via a reflux condenser to a scrubber 600 ml of thionyl chloride are introduced and registered at 20 ° C. 210 g of 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl benzoic acid in the proportion as hydrochloric developed and sulfur dioxide. After the addition the mixture is heated until the gas evolution under reflux. Mixture is then distilled, and boiling in the range of 142-145 ° C/10 mbar, 149 g of 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride (98.1% purity by GC) Melting range: 73-75 ° C.

Example No. 7

3-Cyano-2 ,4,5-trifluoro-benzoyl

50 g of potassium fluoride are suspended in 120 ml of tetramethylene sulfone and at 15 mbar for drying distilled (ca. 20 mL).Then, 50.4 g of 2,4 – dichloro-3-cyano-5-fluoro-benzoyl chloride was added and stirred at an internal temperature with exclusion of moisture for 12 hours at 180 ° C. Are removed by vacuum distillation to 32.9 g of 3-cyano-2 ,4,5-trifluoro-benzoyl fluoride in the boiling range of 98 –

Obtain 100 ° C/12 mbar.

Example No. 8

3-Cyano-2 ,4,5-trifluoro-benzoyl chloride

76.6 g of 3-cyano-2 ,4,5-trifluoro-benzoyl fluoride together with 1 g of anhydrous

Aluminum chloride introduced at 60-65 ° C and then added dropwise 25 g of silicon tetrachloride gas in the course of development. After the evolution of gas at 65 ° C is distilled in a vacuum. Boiling range 120-122 ° C/14 mbar, 73.2 g of 3 – cyano-2 ,4,5-trifluoro-benzoyl chloride over.

Example No. 9

1 – (toluene-4-sulfonyl-pyrroline

In a 20 1 HC4-HWS boilers are 2.016 kg (17.6 mol)

Submitted methanesulfonyl chloride in dichloromethane and 12 1 at -10 ° C internal temperature under strong cooling (-34 ° C) solution of 705 g (8.0 mol) of 2-butene-l ,4-diol in 1.944 kg (2.68 1 , 19.2 mol) of triethylamine was added dropwise over 30 minutes. A yellow suspension stirred for 1 hour at -10 ° C and then treated with 4 1 of water, the temperature rises to 0 ° C.The suspension is warmed to room temperature, stirred for 10 minutes at room temperature and then fed in a 30 1 separating funnel. The phases are stirred separately (good phase separation) and the aqueous phase extracted with 2 1 of dichloromethane. The combined dichloromethane phases are presented in a pre-cooled 20 1 HC4 vessel and kept at 0 ° C.

In another 20-1 HC4 boiler distillation 1.37 kg (8.0 mol) toluenesulfonamide be submitted in 6 1 toluene. It is mixed with 3.2 kg of 45% sodium hydroxide solution, 0.8 1 of water and 130.5 g Tetrabutylammomiimhydrogensulfat, heated to 40 ° C maximum temperature inside and creates a vacuum. Then, the previously obtained

Dichloromethane (15.2 1) was added dropwise over 1.5 hours while the dichloromethane was removed by distillation at 450 mbar (bath temperature: 60 ° C). During the distillation is foaming. In the end, a solution is available at an internal temperature of 33-40 ° C. After the addition of dichloromethane is distilled off, until barely distillate is (duration: about 85 minutes; internal temperature 40 ° C at 60 ° C bath temperature at the end). The vessel contents will be warm transferred to a separating funnel and rinsed the tank with water and 5 1 2 1 toluene at 50 ° C. Before phase separation, the solids are extracted in the intermediate phase and washed with 0.5 1 of toluene. The organic phase is extracted with 2.4 1 of water, separated and evaporated to dryness on a rotary evaporator. The solid residue (1758 g) is suspended in 50 ° C bath temperature in 1.6 1 of methanol, the suspension is transferred into a 10 1-flanged flask and the flask rinsed with diisopropyl 2,4 1. The mixture is heated to reflux temperature (59 ° C) and stirred for 30 minutes under reflux. The suspension is cooled to 0 ° C., stirred at 0 ° C for 1 hour and extracted with 0.8 1 of a cold mixture of ether Methanol/Diisopropyl-: washed (1 1.5). The crystals are dried under a nitrogen atmosphere at 50 ° C/400 mbar.

Yield: 1456 g (81.5% of theory)

Example Z 10

3 – (toluene-4-sulfonylV6-oxa-3-aza-bicvclo [3.1.0] hexane

^{o “|” h “CH3}

334.5 g (1.5 mol) of l-(toluene-4-sulphonyl)-pyrroline are dissolved in 1.5 1 of dichloromethane at room temperature and over 15 minutes with a suspension of 408 g (approx. 1.65 to 1, 77 mol) of 70-75% m-chloroperbenzoic acid in 900 ml of dichloromethane (cools added in manufacturing from). The mixture is heated under reflux for 16 hr (test for

Peroxide with KI / starch paper shows yet to peroxide), the suspension was cooled to 5 ° C, sucks the precipitated m-chlorobenzoic acid and washed with 300 ml of dichloromethane (peroxide with Precipitation: negative; precipitate was discarded). The filtrate is to destroy excess peroxide with 300 ml of 10% sodium sulfite solution, washed twice (test for peroxide runs now negative), extracted with 300 ml of saturated sodium bicarbonate solution, washed with water, dried with sodium sulfate and about a quarter of the volume evaporated. Again on test peroxide: negative. The mixture is concentrated and the solid residue is stirred with ice cooling, 400 ml of isopropanol, the precipitate filtered off and dried at 70 ° C in vacuum.

Yield: 295 g (82.3%),

Mp: 136-139 ° C,

TLC (dichloromethane methanol 98:2): 1 HK (Jodkammer)

Example CLOSED

^{trans-3-Hydroxy-4-(2-hydroxy-ethylamino-l-(‘toluene-4-sulfonyl’)} pyrrolidine

643.7 g (2.65 mol) 3 – (Toluoι-4-sulfonyl)-6-oxa-3-aza-bicyclo [3.1.0] hexane to 318.5 ml with ethanolamine in 4 1 of isopropanol at reflux for 16 hours cooked. After TLC monitoring, further 35.1 ml (total 5.86 mol) of ethanolamine added to the mixture and boiled again until the next morning. The mixture is filtered hot with suction and the filtrate concentrated on a rotary evaporator to 3.5 ltr. After seeding and stirring at room temperature for 3.5 1 diisopropyl ether are added, and stirred at 0 ° C for 6 hours. The precipitated crystals are filtered off, with 250 ml of a mixture of isopropanol / diisopropyl ether (1: 1) and washed 2 times with 300 ml of diisopropyl ether and dried overnight under high vacuum.

Yield: 663.7 g (83% of theory), content: 96.1% (area% by HPLC). Example Z 12

trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l-(toluene-4-sulfonyl)-pyrrolidin-3-yl] – ftoluol-4-sulfonyl)-amino]-ethyl ester)

552 g (1.837 mol) of trans-3-hydroxy-4-(2-hydroxy-ethylamino)-l-(toluene-4-sulfonyl) – pyrrolidine are dissolved under argon in 1.65 1 tetrahydrofuran and 0.8 1 of pyridine dissolved and at -10 ° C in portions 700 g (3.675 mol) p-toluenesulfonyl chloride are added thereto. The mixture is then stirred at this temperature for 16 hours. The work is done by adding 4.3 18.5 1% aqueous hydrochloric acid, extraction twice with dichloromethane (3 1, 2 1), washing the combined organic phases with saturated Natriurnhydrogencarbonatlösung (3 1, 2 1), drying over sodium sulfate, extracting and distilling off the solvent in vacuo. The residue is dried overnight at the oil pump and crude in the next reaction. There were 1093 g as a hard foam (content [area% by HPLC]: 80% Tris-tosyl-product and 13% tetra-tosyl-product, yield see next step). Example Z 13

rac. trans-5 ,8-bis-tosyl-2-oxa-5 .6-diazabicyclor4 .3.01 nonane

1092 g of crude trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l-(toluene-4-sulfonyl) – pyrrolidin-3-yl] – (toluene-4-sulfonyl)-amino]-ethyl} were dissolved in tetrahydrofuran and 9.4 1 at 0-3 ° C with 1.4 1 of a 1.43 molar solution of sodium hydroxide in

Methanol reacted. After half an hour at this temperature, 2.1 1 of water and 430 ml of diluted (2:1) was added to the mixture and acetic acid with previously isolated crystals of trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l – (toluene-4-sulfo-phenyl)-pyrrolidin-3-yl] – (toluene-4-sulfonyl)-amino] ethyl}-seeded. The suspension is stirred overnight at 0 to -4 ° C. The next morning, the crystals are filtered off, washed twice with 400 ml of cold mixture of tetrahydrofuran / water (4:1) and dried at 3 mbar at 50 ° C overnight.

Yield: 503 g of white crystals (62.7%> of theory over 2 steps), content: 99.7% (area% by HPLC). Example Z 14

Preparative chromatographic resolution of racemic rac. trans-5.8-bis-tosyl-2-oxa-5.6-diazabicyclor4.3.0] nonane

The chromatography of the racemate at room temperature in a column (inner diameter 75 mm), which with 870 g of a chiral stationary phase (kie-selgelgebundenes poly (N-methacryloyl-L-leucine-d menthylamide) based on the mer captomodifizierten silica Polygosil 100 , 10 microns; see EP-A 0 379 917) is filled (bed height: 38 cm). Detection is carried out using a UV detector at 254 nm

For the sample application using a solution of a concentration of 100 g of rac. trans-5 ,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane in 3000 ml of tetrahydrofuran. A Trenncyclus is carried out under the following conditions: with the aid of a pump is required for 2 min at a flow of 50 ml / min, a part of the sample solution and the same time at a flow rate of 50 ml / min, pure n-heptane to the column.

Thereafter eluted at a flow rate of 100 ml / min 18 minutes with a mixture of n-Heptan/Tetrahydrofuran (3/2 vol / vol). This is followed for 3 minutes at a flow of 100 ml / min elution with pure tetrahydrofuran. Thereafter, further eluted with n-Heptan/Tetrahydro-furan (3/2 vol / vol). This cycle is repeated several times.

The first eluted enantiomer is the (lS, 6R) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo-[4.3.0] nonane, which is isolated by concentration. The eluate of the more retarding enantiomers is largely evaporated in vacuo, and the precipitated crystals are filtered off with suction and dried. From the separation of 179 g of racemate in this

As 86.1 g (96.2% of theory) of the enantiomer (lS, 6S) -5,8-bis-tosyl-2-oxa-5, 6 – diazabicyclo [4.3.0] nonane having a purity of> 99 % ee. Example Z 15

(LR, 6R-2-oxa-5.6-diazabicvclo [4.3.0] nonane dihydrobromide

38.3 g (87 mmol) of (lS, 6R) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane in 500 ml of 33 -% HBr / glacial acetic acid 10 g added anisole and heated for 4 hours at 60 ° C (bath). After standing overnight, the suspension is cooled, the precipitate filtered, with

100 ml of abs. Ethanol and dried at 70 ° C under high vacuum.

Yield: 23.5 g (93%) of white solid product, mp 309-310 ° C (dec.), DC (dichloromethane/methanol/17% aq ammonia 30:8:1.): 1 HK

[Α] _D: + 0.6 ° (c = 0.53, H ₂ O) (fluctuating).

Example Z 16

(LS.6S-2-oxa-5.6-diazabicvclor4.3.01nonan-Dihvdrobromid

Z is analogous to Example 15 from (lS, 6S) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] no-nan (1S, 6S)-2-oxa-5, 6-diazabicyclo [4.3.0] nonane dihydrobromide receive. Example Z 17

(1 R.6R-2-oxa-5.8-diazabicvclo [4.3.Olnonan

1 Method: 5,8 g (20 mmol) of (lS, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydro-drobromid are suspended in 100 ml of isopropanol at room temperature with 2.4 g ( 42.9 mmol) and powdered potassium hydroxide while leaving about 1 hour in an ultrasonic bath. The suspension is cooled in an ice bath, filtered, washed with isopropanol and the undissolved salt, the filtrate was concentrated and distilled in a Kugelrohr oven at 150-230 ° C oven temperature and 0.7 mbar. Obtained 2.25 g (87.9% of theory) of a viscous oil which crystallizes. [Α] _D -21.3 ° (c = 0.92, CHC1 ₃₎ Accordingly, this reaction can be carried out in ethanol.

2 Method: A homosexual genie catalyzed mixture of (lR, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide and 620 mg (11 mmol) of powdered potassium hydroxide is dry in a Kugelrohr apparatus at 0.2 mbar and increasing oven temperature to 250 ° C distilled. Obtained 490 mg (76.6% of theory) of (lR, 6R) -2 – oxa-5 ,8-diazabicyclo [4.3.0] nonane as a viscous oil which slowly crystallized.

3 Method: 100 g of moist, pretreated cation exchanger (Dowex 50WX, H ⁺ – form, 100-200 mesh, capacity: 5.1 meq / g of dry or 1.7 meq / mL) are charged into a column with about 200 ml 1 N HC1 activated and washed neutral with water 3 1. A solution of 2.9 g (10 mmol) of (lS, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane

Dihydrobromide in 15 ml of water is added to the ion exchanger, and then washed with 2 1 water, and eluted with approximately 1 1 1 N ammonia solution. The eluate is evaporated. concentrated. Yield: 1.3 g of a viscous oil (quantitative), DC (dichloromethane/methanol/17% NH ₃ 30:8:1): 1 HK, GC: 99.6% (area).

Example Z 18

(LS.6SV2-oxa-5.8-diazabicvclor4.3.01nonan

Z is analogous to Example 17 from (lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane-di-hydrobromide the free base (lS, 6S)-2-oxa-5 ,8-diazabicyclo [ 4.3.0] nonane made.

Example Z 19

2 – (2,4-dichloro-3-cyano-5-fluoro-benzoyl)-3-dimethylamino-acrylic acid ethyl ester

To a solution of 626 g (4.372 mol) of 3-dimethylamino-acrylate and 591 g (4.572 mol) of ethyl-diisopropyl-amine (Hunigs base) in 1060 ml of dichloromethane, a solution of 1075 g starting at room temperature 2,4-dichloro -3-cyano-5-fluoro-benzoyl chloride (94% pure, corresponding to 1010.5 g = 4.00 mol) was dropped in 850 ml of dichloromethane. The temperature rises to 50-55 ° C (dropwise addition about 90 minutes). Then stirred for 2 hours at 50 ° C and the reaction mixture was used without further purification in the next step.

Example Z 20

2 – (2,4-dichloro-3-Cyano-5-fluoro-benzoyl-3-cvclopropylamino-acrylate

To the reaction mixture from the above step 306 g (5.1 mol) of glacial acetic acid are added dropwise under cooling at about 15 ° C. Then, with further cooling at 10-15 ° C. 267.3 g (4.68 mol) of cyclopropyl amine is added dropwise. Immediately after which the reaction mixture is mixed with 1300 ml of water under ice-cooling and 15 minutes stirred well. The dichloromethane layer was separated and used in the next step.

Example 21 Z

7-chloro-8-cyano-1-cyclopropyl-6-fluoro-1.4-dihydro-4-oxo-3-chinolincarbonsäureethyl ester

To a heated to 60-70 ° C suspension of 353 g (2.554 mol) of potassium carbonate in 850 ml of N-methylpyrrolidone, the dichloromethane phase is dropped from the precursor (about 90 minutes). During the addition of the dichloromethane at the same time

Reaction mixture was distilled off. Then the reaction mixture for 5 Vz hours at 60-70 ° C is well stirred. The mixture is cooled to about 50 ° C. and distilled under a vacuum of about 250 mbar residual dichloromethane from. At room temperature is added dropwise 107 ml 30% hydrochloric acid under ice cooling, then to obtain a pH of 5-6 is set. Then, 2,200 ml of water are added under ice cooling. The reaction mixture is thoroughly stirred for 15 minutes, the solid was then filtered off and washed on the filter twice with 1000 ml of water and extracted three times with 1000 ml of ethanol and then dried in a vacuum oven at 60 ° C.

Yield: 1200 g (89.6% of theory).

This product can be purified, if desired by, the solid is stirred in 2000 ml of ethanol for 30 minutes at reflux. You filtered hot with suction, washed with 500 ml of ethanol and dried at 60 ° C in vacuum. Melting point: 180-182 ° C.

Η-NMR (400 MHz, CDC1 _3): d = 1.2 to 1.27 (m, 2H), 1.41 (t, 3H), 1.5-1.56 (m, 2H), 4, 1 to 4.8 (m, 1H), 4.40 (q, 2H), 8.44 (d, J = 8.2 Hz, H), 8.64 (s, 1H) ppm.

Example Z 22

7-chloro-8-cyano-1-cvclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid

33.8 g (0.1 mol) of 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylate dissolved in a mixture of 100 ml of acetic acid, 20 ml water and 10 ml concentrated sulfuric acid was heated for 3 hours under reflux. After cooling, the mixture is poured onto 100 ml of ice water, the precipitate filtered off, washed with water and ethanol and dried at 60 ° C in vacuum.

Yield: 29.6 g (96% of theory),

Mp 216-21 C. (with decomposition)

Example 1

A 8-Cyano-l-cvclopropyl-6-fluoro-7-((lS.6S-2-oxa-5.8-diazabicvclo [4.3.0] non-8-yl – 1 ,4-dihydro-4-oxo-3 -quinoline carboxylic acid

1.00 g (3.26 mmol) of 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid are heated with 501 mg (3.91 mmol) of ( lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane and 0.9 ml of triethylamine in 30 ml of acetonitrile was stirred at 40-45 ° C under argon for 25 hours. All volatile components in vacuo. removed and the residue recrystallized from ethanol. Yield: 1.22 g (94%)

Melting point: 294 ° C. (with decomposition)

B) ^{8-Cyano-l-cyclopropyl-6-fluoro-7-(‘(lS.6S-2-oxa-5} ,8-diazabicvclo [4.3.01nonan-8-YLV 1.4-dihydro-4-oxo-3- quinoline carboxylic acid Hvdrochlorid

200 mg (0.63 mmol) of 8-cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester to be 97 mg (0.75 mmol) of (lS, 6S)-2-oxa-5, 8 – diazabicyclo [4.3.0] nonane and 0.17 ml of triethylamine in 3 ml of acetonitrile was stirred at 40-45 ° C for 2 hours under argon. All volatile components in vacuo. removed, the residue treated with water, insolubles filtered off and the filtrate was extracted with dichloromethane. The organic phase is dried over sodium sulfate and then concentrated under reduced pressure. a. The resulting residue is dissolved in 6 ml of tetrahydrofuran and 2 ml of water and 30 mg (0.72 mmol) of lithium hydroxide monohydrate was added. After 16 hours of stirring at room temperature, acidified with dilute hydrochloric acid and the resulting precipitate was filtered off with suction and dried. Yield: 155 mg (57%) Melting point:> 300 ° C

C) 8-Cyano-l-cvclopropyl-6-fluoro-7-((lS, 6S-2-oxa-5.8-diazabicvclo [4.3.01non-8 yiyi.4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride

1 g (2.5 mmol) of 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] non-8-yl )-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid is suspended in 20 ml of water was added to the suspension, 10 ml hydrochloric acid and stirred for In at room temperature for 3 hours. The resulting precipitate is filtered off, washed with ethanol and dried at 80 ° C under high vacuum.

Yield: 987 mg (90.6% of theory), Melting point: 314-316 ° C. (with decomposition).

D) 8-Cyano-l-cvclopropyl-6-fluoro-7-(iS, 6S)-2-oxa-5.8-diazabicyclo [4.3.0] non-8-YLV 1 ,4-dihydro-4-oxo-3 -quinoline carboxylic acid hydrochloride

86.4 g (217 mmol) of 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5, 8 – diazabicyclo [4.3.0] non-8-yl) – l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid are dissolved at room temperature in 963 ml of water and 239 ml of 1 N aqueous sodium hydroxide solution. After filtration and washing with 200 ml of water is added to 477 ml in aqueous hydrochloric acid and the precipitated crystals placed at 95 ° C to 100 ° C in solution. The solution is cooled overnight, the precipitated crystals are filtered off with suction and washed three times with 500 ml of water and dried in vacuum.

Yield 90 g (94.7% of theory), content:> 99% (area% by HPLC) 99.6% ee. [] _D ^23: -112 ° (c = 0.29, N NaOH).

……………….

Tetrahedron Lett 2009, 50(21): 2525

A novel approach to Finafloxacin hydrochloride (BAY35-3377)

Pages 2525-2528
Jian Hong, Zonghua Zhang, Huoxing Lei, Haiying Cheng, Yufang Hu, Wanliang Yang, Yinglin Liang, Debasis Das, Shu-Hui Chen, Ge Li

Graphical abstract

Finafloxacin hydrochloride, an important clinical compound was synthesized by a novel synthetic approach. An active intermediate ethyl 7-chloro-8-cyano-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate 19 was prepared by a new route. The chiral (S,S′)-N-Boc 10 was derived from protected pyrrolidine and the absolute stereochemistry was established by X-ray analysis.

http://www.sciencedirect.com/science/article/pii/S0040403909005875

……………….

Durata Therapeutics, Inc. Finafloxacin for the treatment of cUTI and/or acute pyelonephritis. Available online: http://www.clinicaltrials.gov/ct2/show/NCT01928433 (accessed on 28 September 2013).
Merlion Pharma. A multi-dose, double-blind, double-dummy, active control, randomized clinical (Phase II) study of two dosing regimens of finafloxacin for the treatment of cUTI and/or acute pyelonephritis.Available online: http://www.clinicaltrialsregister.eu/ctr-search/trial/2011–006041–14/PL/ (accessed on 14 April 2013).
Pharma, M. FDA Grants Qualified Infectious Disease Product Designation and Fast Track Status for MerLion Pharma’s Lead Antibacterial Candidate Finafloxacin; Merlion Pharma: Singapore, 2013; Volume 2013.
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Finafloxacin hydrochlorideDrugs Fut 2009, 34(6): 451
A novel approach to finafloxacin hydrochloride (BAY35-3377)Tetrahedron Lett 2009, 50(21): 2525
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EP0350733A2 *	30 Jun 1989	17 Jan 1990	Bayer Ag	7-(1-Pyrrolidinyl)-3-quinolone- and -naphthyridone-carboxylic-acid derivatives, method for their preparation and for substituted mono- and bi-cyclic pyrrolidine intermediates, and their antibacterial and feed additive compositions
EP0550903A1 *	28 Dec 1992	14 Jul 1993	Bayer Ag	Quinolone- and naphthyridone carboxylic acid derivatives as antibacterial agents
EP0603887A2 *	23 Dec 1993	29 Jun 1994	Daiichi Pharmaceutical Co., Ltd.	Bicyclic amine derivatives
EP0676199A1 *	23 Mar 1995	11 Oct 1995	Pfizer Inc.	Use of trovafloxacin or derivatives thereof for the manufacture of a medicament for the treatment of H. pylori infections
GB2289674A *				Title not available

This Little Known Chinese Herb Kills 12,000 Cancer Cells For Every Healthy Cell

Ayurveda Comments Off

Mar 272014

WORMWOOD PLANT

This Little Known Chinese Herb Kills 12,000 Cancer Cells For Every Healthy Cell

collective-evolution.com

Today, odds are that you have had/have cancer, or know somebody who does. In Canada, approximately one million Canadians that were alive at…

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http://peoplestrusttoronto.wordpress.com/2014/03/26/this-little-known-chinese-herb-kills-12000-cancer-cells-for-every-healthy-cell/

A little known Chinese herb might be eligible for the growing list of cancer killers via alternative methods of treatment. According to studies published in Life Sciences, Cancer Letters and Anticancer Drugs, artemesinin, a derivative of the wormwood plant commonly used in Chinese medicine, can kill off cancer cells, and do it at a rate of 12,000 cancer cells for every healthy cell.

Artemisinin is currently FDA approved for the treatment of malaria, it’s very safe and easy to use. It’s inexpensive and works on all cancers but has yet to find it’s way into the mainstream. It’s really time to move beyond just radiation, surgery and chemotherapy for the treatment of cancer.

artemisinin

“Artemisinin reacts with iron to form free radicals that kill cells. Since cancer cells uptake relatively larger amounts of iron than normal cells, they are more susceptible to the toxic effect of artemisinin. In previous research, we have shown that artemisinin is more drawn to cancer cells than to normal cells. In the present research, we covalently attached artemisinin to the iron-carying plasma glycoprotein transferrin.Transferrin is transported into the cells via receptor-mediated endocytosis and cancer cells express significantly more transferrin receptors on their cell surface and endocytose more transferrin than normal cells. Thus, we hypothesize that by tagging artemisinin to transferrin, both iron and artemisinin would be transported into cancer cells in one package. Once inside a cell, iron is released and can readily react with artemisinin close by tagged to the transferrin. This would enhance the toxicity and selectivity of artemisinin towards cancer cells. We found that holotransferrin-tagged artemisinin, when compared with artemisinin, was very potent and selective in killing cancer cells. Thus, this ‘tagged-compound’ could potentially be developed into an effective chemotherapeutic agent for cancer treatment.”

Wormwood

Other common name(s): absinthium, absinth wormwood

Scientific/medical name(s): Artemisia absinthium

Description

Wormwood is a shrubby perennial plant whose upper shoots, flowers, and leaves are used in herbal remedies and as a bitter flavoring for alcoholic drinks. It is native to Europe, northern Africa, and western Asia, and now also grows in North America.

Overview

Available scientific evidence does not support claims that wormwood is effective in treating cancer, the side effects of cancer treatment, or any other conditions. The plant contains a volatile oil with a high level of thujone (see Thuja). There are reports that taking large doses of wormwood internally can cause serious problems with the liver and kidneys. It can also cause nausea, vomiting, stomach pain, headache, dizziness, seizures, numbness of the legs and arms, delirium, and paralysis.

Wormwood, or Artemisia absinthium, should not be confused with sweet wormwood, or Artemisia annua. Although wormwood is related to sweet wormwood, they are used in different ways. Extracts of sweet wormwood have been used in traditional herbal medicine, and an active ingredient, artemisinin, is now used in conventional medical treatment of malaria.

How is it promoted for use?

Wormwood is promoted as a sedative and anti-inflammatory. There are also claims that it can treat loss of appetite, stomach disorders, and liver and gallbladder complaints. In folk medicine it is used for a wide range of stomach disorders, fever, and irregular menstruation. It is also used to fight intestinal worms. Externally, it is applied to poorly healing wounds, ulcers, skin blotches, and insect bites. It is used in Moxibustion treatments for cancer (seeMoxibustion). Available scientific evidence does not support these claims.

What does it involve?

Wormwood is taken in small doses for a short period of time, usually a maximum of 4 weeks. It is available as a capsule and as a liquid that can be added to water to make a tincture. The whole herb is sometimes brewed as a tea. Wormwood oil, washes, or poultices can also be used on the skin. Although pure wormwood is not available, “thujone-free” wormwood extract has been approved by the US Food and Drug Administration (FDA) for use in foods and as a flavoring in alcoholic drinks such as vermouth.

What is the history behind it?

Artemisia absinthium was used by Hippocrates, and the earliest references to wormwood in Western civilization can be found in the Bible. Extract of wormwood was also used in ancient Egypt. The herb is mentioned often in first-century Greek and Roman writings and reportedly was placed in the sandals of Roman soldiers to help soothe their sore feet. It was taken as a treatment for tapeworms as far back as the Middle Ages.

In 1797, Henri Pernod developed absinthe, an alcoholic drink containing distilled spirits of wormwood, fennel, anise and sometimes other herbs. Absinthe became very popular in Europe and the United States in the nineteenth century. It was eventually banned in several countries in the early twentieth century due to its purported ill effects and addictive qualities. More recent analysis has suggested that, when properly prepared and distilled, the thujone content in these drinks was very low. It appears more likely that the addictiveness and other ill effects of absinthe were due to its alcohol content, which is around 60% to 85%. Varying additives or impurities from different distillers may have also produced some of these effects. Even though absinthe is illegal in some countries, various types can be found in some European countries. However, their thujone content is strictly limited. Wormwood is also an ingredient in vermouth and other drinks.

What is the evidence?

Available scientific studies do not support the use of wormwood for the treatment of cancer or the side effects of conventional cancer treatment. There is not enough evidence available to support its use for other conditions. Wormwood oil has been tested in laboratory studies and appears to inhibit the growth of some fungi. However, human tests have not been completed.

Some derivatives of Artemisia annua, or sweet wormwood, a relative of wormwood, have been shown to be effective in the treatment of malaria. In fact, the World Health Organization approved artemisinin for use against malaria in Africa in 2004. These extracts also show some promise in laboratory studies as cancer treatment drugs. Further studies are required to find out whether the anti-cancer results apply to people. It is important to remember that extracted compounds are not the same as the whole herb, and study results are not likely to show the same effects.

Are there any possible problems or complications?

This product is sold as a dietary supplement in the United States. Unlike companies that produce drugs (which must be tested before being sold), the companies that make supplements are not required to prove to the Food and Drug Administration that their supplements are safe or effective, as long as they don’t claim the supplements can prevent, treat, or cure any specific disease.

Some such products may not contain the amount of the herb or substance that is on the label, and some may include other substances (contaminants). Actual amounts per dose may vary between brands or even between different batches of the same brand. In 2007, the FDA wrote new rules to improve the quality of manufacturing for dietary supplements and the proper listing of supplement ingredients. But these rules do not address the safety of the ingredients or their effects on health.

Most such supplements have not been tested to find out if they interact with medicines, foods, or other herbs and supplements. Even though some reports of interactions and harmful effects may be published, full studies of interactions and effects are not often available. Because of these limitations, any information on ill effects and interactions below should be considered incomplete.

Wormwood should be avoided, especially by women who are pregnant or breast-feeding, by people who have had seizures, and by those with ulcers or stomach irritation. Thujone, a component of wormwood, is known to cause muscle spasms, seizures, and hallucinations if taken internally. In high doses it is known to damage the liver and the kidneys.

Because of its thujone content, large doses of wormwood taken internally can lead to vomiting, stomach and intestinal cramps, headaches, dizziness, nervous system problems, and seizures. Wormwood can also lead to liver failure. The New England Journal of Medicine reported that a man who ordered essential oil of wormwood over the Internet, thinking he had purchased absinthe, suffered liver failure shortly after drinking the oil. Wormwood may also make seizures more likely and may interfere with the anti-convulsant effects of medicines such as phenobarbital.

The plant is a relative of ragweed and daisies. Those with allergies to these types of plants may also be allergic to wormwood. Contact with wormwood can cause rash in some people.

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

SUROTOMYCIN for Clostridium difficile-associated diarrhea

Phase 3 drug Comments Off

Mar 272014

Surotomycin

http://www.ama-assn.org/resources/doc/usan/surotomycin.pdf

N-[(2E)-3-(4-Pentylphenyl)-2-butenoyl]-D-tryptophyl-D-asparaginyl-N-[(3S,6S,9R,15S,18R,21S,24S,30S,31R)-3-[2-(2-aminophenyl)-2-oxoethyl]-24-(3-aminopropyl)-15,21-bis(carboxymethyl)-6-[(2R)-1-carboxy-2 -propanyl]-9-(hydroxymethyl)-18,31-dimethyl-2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28-nonaazacyclohentriacontan-30-yl]-L-α-asparagine

MOLECULAR FORMULA C77H101N17O26

MOLECULAR WEIGHT 1680.7

SPONSOR Cubist Pharmaceuticals, Inc.

CODE DESIGNATION CB-183,315

CB-315, CB-183315, CB-183,315

CAS REGISTRY NUMBER 1233389-51-9

U.S. – Fast Track (Treat Clostridium difficile-associated diarrhea (CDAD));
U.S. – Qualified Infectious Disease Program (Treat Clostridium difficile-associated diarrhea (CDAD))

Company	Cubist Pharmaceuticals Inc.
Description	Oral antibacterial lipopeptide
Therapeutic Modality	Macrocycle

Latest Stage of Development	Phase III
Standard Indication	Diarrhea (infectious)
Indication Details	Treat Clostridium difficile-associated diarrhea (CDAD)

EMEA……..

Name
P/0096/2013: EMA decision of 29 April 2013 on the agreement of apaediatric investigation plan and on the granting of a deferral for surotomycin (EMEA-001226-PIP01-11)

Surotomycin is an investigational oral antibiotic. This antibiotic is under investigation for the treatment of life-threatening Diarrhea, commonly caused by the bacteria Clostridium difficile.^[1]

CB-183315 is an investigational antibacterial drug candidate in phase III clinical trials at Cubist for the treatment of Clostridium difficile-associated diarrhea. It is a potent, oral, cidal lipopeptide. In 2012, Qualified Infectious Disease Product Designation was assigned in the U.S. for the treatment of clostridium difficile-associated diarrhea (CDAD).

Surotomycin (CB-315)

Surotomycin Overview

Surotomycin Fact Sheet

Surotomycin is an antibacterial lipopeptide discovered by Cubist scientists in our research laboratories in Lexington, Massachusetts. Surotomycin is both bactericidal against Clostridium difficile and more potent than vancomycin in vitro. Surotomycin stays at the site of infection in the bowel, with minimal systemic absorption and it does not interfere with normal bowel flora. Based on its features and its preclinical safety profile, Cubist filed an Investigational New Drug (IND) Application for surotomycin in December 2008.

Following safety and pharmacokinetic studies in healthy human volunteers, Cubist began a Phase 2 study in April 2010 to assess the safety and efficacy of surotomycin in patients with CDAD, in particular to assess its ability to reduce relapse rates. In this trial of 209 patients, two different doses of surotomycin were studied and compared with oral vancomycin. The higher dose demonstrated a high clinical cure rate as evidenced by resolution of diarrhea, comparable to oral vancomycin. The most interesting results in this study, however, relate to recurrence rates. The percent of patients who had an initial response to treatment but who subsequently had a recurrence or relapse was 36 percent in the oral vancomycin arm and was 17 percent in the surotomycin 250mg treatment group — about a 50% reduction in relapse rate, which was statistically significant. In this trial, 32% of patients were infected with the hypervirulent NAP-1 strain of C. difficile. The clinical response rate in the subset of patients infected with the NAP-1 strain was comparable across the surotomycin and oral vancomycin groups. Though not statistically significant, there was a modest reduction in the relapse rates in the subset of surotomycin patients infected with NAP-1 strains.

The ability to reduce relapses is important to both patients and health care providers. In the Phase 2 study we assessed the impact of surotomycin and oral vancomycin on normal bowel flora. Treatment with surotomycin had a very minimal impact on levels of Bacteroides, a key normal bowel bacterial species, compared to oral vancomycin which resulted in a marked depletion of stool levels of these bacteria during treatment. Why does this matter? The reason is — bowel flora like Bacteroides are critical in providing a competitive environment in the bowel that prevents C. difficile overgrowth. We believe that it is this difference in impact on normal bowel flora that helps explain the differences seen in recurrence rates following treatment with Surotomycin versus oral vancomycin.

Surotomycin’s Phase 3 program includes two identical global, randomized, double-blind, active-controlled, multi-center trials. The primary objective is to demonstrate non-inferiority of surotomycin versus the comparator, oral vancomycin, in clinical response at the end of treatment in adult subjects with CDAD, using a non-inferiority margin of 10%. We also have designed this trial to allow us to demonstrate that sustained clinical response to surotomycin at the end of the study is superior to oral vancomycin. Also, we will fully evaluate the safety of surotomycin in the study subjects.

In late 2012 Cubist received from the FDA a Qualified Infectious Disease Product (QIDP) designation for surotomycin. Additionally, in early 2013 Cubist was granted Fast track status for surotomycin. The QIDP designation and subsequent granting of Fast Track status was made possible by the GAIN Act, Title VIII (Sections 801 through 806) of the Food and Drug Administration Safety and Innovation Act. The GAIN Act provides pharmaceutical and biotechnology companies with incentives to develop new antibacterial and antifungal drugs for the treatment of life-threatening infectious diseases caused by drug resistant pathogens. Qualifying pathogens are defined by the GAIN Act to include multi-drug resistant Gram-negative bacteria, including Pseudomonas, Acinetobacter, Klebsiella, and Escherichia coli species; resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus; multi-drug resistant tuberculosis; and Clostridium difficile.

About CDAD

CDAD is a disease caused by an overgrowth of, and subsequent toxin production by, C. difficile, a resident anaerobic spore-forming Gram-positive bacterium of the lower gastrointestinal tract. This overgrowth is caused by the use of antibiotics for the treatment of common community and hospital acquired infections (HAIs). Although they treat the underlying infection, many antibiotics disrupt the natural gut flora and allow C. difficile to proliferate. C. difficile produces enterotoxin and cytotoxin, which can lead to severe diarrhea, sepsis and even death. While most types of HAIs are declining, the infection caused by C. difficile remains at historically high levels. According to the latest data from the Centers for Disease Control, C. difficile continues to be the leading cause of death associated with gastroenteritis in the US. For CDAD alone, there was more than a five-fold increase in deaths between 1999 and 2007. C. difficile causes diarrhea linked to 14,000 American deaths each year. About 25% of C. difficile infections first show symptoms in hospital patients; 75% first show in nursing home patients or in people recently cared for in doctors’ offices and clinics. C. difficile infections cost at least $1 billion in extra health care costs annually.

ChemSpider 2D Image | Surotomycin | C77H101N17O26 SUROTOMYCIN

CB-183,315 is a cyclic lipopeptide antibiotic currently in Phase III clinical trials for the treatment of Clostridium difficile-associated disease (CDAD). As disclosed in International Patent Application WO 2010/075215, herein incorporated by reference in its entirety, CB-183,315 has antibacterial activity against a broad spectrum of bacteria, including drug-resistant bacteria and C. difficile. Further, the CB-183,315 exhibits bacteriacidal activity.

CB-183,315 (Figure 1) can be made by the deacylation of BOC-protected daptomycin, followed by acylation and deprotection as described in International Patent Application WO 2010/075215.

During the preparation and storage of CB-183,315, the CB-183,315 molecule can convert to structurally similar compounds as shown in Figures 2-4, leading to the formation of anhydro-CB-183,315 (Figure 3) and beta-isomer of CB-183,315 (“B- isomer CB183,315” in Figure 2). Accordingly, one measure of the chemical stability of CB- 183 ,315 is the amount of CB- 183 ,315 (Figure 1 ) present in the CB- 183 ,315 composition relative to the amount of structurally similar compounds including anhydro-CB-183,315 (Figure 3) and beta-isomer of CB-1 83,315 (Figure 2). The amount of CB-183,315 relative to the amount of these structurally similar compounds can be measured by high performance liquid chromatography (FIPLC) after reconstitution in an aqueous diluent (e.g., as described in Example 10). In particular, the purity of CB-183,315 and amounts of structurally similar compounds (e.g., Figures 2, 3 and 4) can be determined from peak areas obtained from HPLC (e.g., according to Example 10 herein), and measuring the rate of change in the amounts of CB-183,315 over time can provide a measure of CB-183,315 chemical stability in a solid form.

There is a need for solid CB-183,315 compositions with improved chemical stability in the solid form (i.e., higher total percent CB-183,315 purity over time), providing advantages of longer shelf life, increased tolerance for more varied storage conditions (e.g., higher temperature or humidity) and increased chemical stability.

……………..

WO2010075215A1

http://www.google.com/patents/WO2010075215A1?cl=en ………… copy paste link

Example 1

Preparation of N-{1 -[(E)-3-(4-pentylphenyl)but-2-enoyl]}-L-tryptophyl-D- asparaginyl-L-α-aspartyl-L-threonylglycyl-L-ornithyl-L-α-aspartyl-D-alanyl-L-α- aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2-diamino-γ- oxobenzenebutanoic acid (13→4)-lactone (49).

1003 1004

Step 1 : Preparation of (E)-ethyl 3-(4-pentylphenyl) but-2-enoate (1002).

A mixture of commercially available 1-(4-pentylphenyl)ethanone (5 g, 26.3 mmol) and (ethoxycarbonylmethylene)-triphenylphosphorane (18.3 g, 52.5 mmol) was stirred at 150 ⁰C for 48 hours under a nitrogen atmosphere. The reaction mixture was cooled to ambient temperature and diluted with ethyl acetate (50 ml_) and petroleum ether (200 ml_). The suspension was filtered through a fritted funnel. The concentrated filtrate was purified by flash column chromatography with silica gel (petroleum ether : ethyl acetate = 80:1 ) to give the title compound (1.6 g) having the following physical data: ¹H NMR (300 MHz, δ, CDCI₃) 0.90 (br, 3H), 1.36 (br, 7), 1.63 (br, 2H), 2.58 (s, 3H), 2.63 (br, 2H), 4.22 (q, 2H), 6.15 (s, 1 H), 7.20 (d, 2H), 7.41 (d, 2H).

Step 2: Preparation of (E)-3-(4-pentylphenyl) but-2-enoic acid (1003).

A solution of compound 1002 (1.5 g, 5.77 mmol) in ethanol (50 ml_) and 3N potassium hydroxide (25 ml_) was stirred at 45 ⁰C for 3 hours. The reaction mixture was concentrated and the resulting residue was diluted with water (50 ml_). The aqueous solution was acidified to pH 2 with 1 N hydrochloric acid and extracted with EtOAc (2 * 30 ml_). The combined organic layers were dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (silica gel, petroleum ether : ethyl acetate = 10:1) to afford the title compound (0.95 g) having the following physical data: 1 H NMR (300 MHz, δ, CDCI3) 0.90 (br, 3H), 1.33 (br, 4H), 1.62 (br, 2H), 2.60 (br, 5H), 6.18 (s, 1 H), 7.18 (d, 2H), 7.42 (d, 2H).

Step 3: Preparation of (E)-3-(4-pentylphenyl)but-2-enoyl chloride (1004).

Oxalyl chloride (3.2 mL, 36.60 mmol) and DMF (50 μl_) were added drop wise to a solution of compound 1003 (5.0 g, 21.52 mmol) in dichloromethane (100 mL) at 0 ⁰C. The reaction solution was warmed up to room temperature and stirred for 4 hours. The reaction mixture was concentrated in vacuum and the residue was dried under hi-vacuum for 3 hours. The crude product was used in the next step without further purification.

Step 4: Preparation of N-{1 -[(E)-3-(4-pentylphenyl)but-2-enoyl]}-L-tryptophyl-D- asparaginyl-L-α-aspartyl-L-threonylglycyl-L-[(N-tert-butoxycarbonyl)-ornithyl]-L-α- aspartyl-D-alanyl-L-α-aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2- diamino-γ-oxobenzenebutanoic acid (13-→4)-lactone (1005).

Deacylated BOC-protected daptomycin (3.5Og, 2.23 mmol) and sodium bicarbonate (1.13 g, 61.0 mmol) were dissolved in THF (130 mL) and water (50 mL). The deacylated BOC-protected daptomycin sodium bicarbonate solution was cooled to 0 ⁰C. and a solution of compound 1004 (1.96 g, 7.82 mmol) in THF (20 mL) was then introduced. The reaction mixture was warmed to room temperature and stirred for 4 hours. The mixture was concentrated in vacuum to remove THF. The remaining aqueous solution was loaded on a C18 flash chromatography column (35mηn^χ 300mm, Bondesil HF C18 resin purchased from Varian). The column was first washed with water to remove salt and then with methanol to wash out product. Crude compound 1005 (3.46 g) was afforded as a white solid after removal of methanol. MS m/z 1780.8 (M + H)⁺.

Steps 5-6: Preparation of N-{1-[(E)-3-(4-pentylphenyl)but-2-enoyl]}-L-tryptophyl- D-asparaginyl-L-α-aspartyl-L-threonylglycyl-L-ornithyl-L-α-aspartyl-D-alanyl-L-α- aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2-diamino-γ- oxobenzenebutanoic acid (13→4)-lactone (49).

TFA (10 ml_) was added to a solution of compound 1005 (3.46 g) in DCM (50 mL) at room temperature. The reaction mixture was stirred vigorously for 45 minutes and added slowly to vigorously stirring diethyl ether (100 mL). The resulting yellow precipitation was collected by filtration. The crude product was purified by Preparative HPLC to afford the TFA salt of compound 6 (0.75 g). MP carbonate resin (purchased from Biotage) was added to the solution of compound 6 TFA salt (0.70 g, 0.39 mmol) in anhydrous methanol (30.0 mL). The mixture was stirred at room temperature for 4 hours. The resins were removed by filtration and rinsed with methanol. The methanol solution was concentrated under vacuum to give product as off-white solid (408 mg). MS m/z 1680.7 (M + H)⁺.

Example 1 b

Alternative preparation of N-{1-[(E)-3-(4-pentylphenyl)but-2-enoyl]}- L-tryptophyl-D-asparaginyl-L-α-aspartyl-L-threonylglycyl-L-ornithyl-L-α-aspartyl-D- alanyl-L-α-aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2-diamino-γ- oxobenzenebutanoic acid (13→4)-lactone (49).

daptomycin,

1003

A solution of (E)-3-(4-pentylphenyl)but-2-enoic acid (1 100 g, 4.73 mol), Λ/-Ethyl-Λ/’-(3-dimethylaminopropyl)carbodiimide hydrochloride (907 g, 4.73 mol), HOBT (640 g, 4.73 mol) and 4-(dimethylamino)pyridine (22 g, 0.18 mol) in DMF (11 L) was stirred at room temperature for 4 hours at which point the activation of the (E)-3-(4-pentylphenyl)but-2-enoic acid was deemed complete by HPLC.

This reaction mixture was added to a suspension of Deacylated BOC- protected daptomycin (2600 g, 1.66 mol), sodium bicarbonate (804 g, 9.57 mol) in water (11.25 L) and 1 ,4-dioxane (33.75 L). The mixture was stirred at room temperature for 2.5 hours at which time HPLC indicated complete consumption of Deacylated BOC-protected daptomycin. The reaction mixture was diluted with water (22.5 L) and cooled with an ice bath. Concentrated hydrochloric acid (5.25 L) was added while maintaining the internal temperature below 30 ⁰C. After the addition, the solution was stirred at room temperature for 5 days at which time HPLC indicated complete consumption of the Boc protected intermediate.

The reaction mixture was washed with methyl terf-butyl ether (90 L then approximately 60 L then approximately 45 L then approximately 45 L) to remove 1 ,4-dioxane. The remaining solution (approximately 44 L) was adjusted to pH 2.69 with 2N sodium hydroxide (11.3 L) and water (53.4 L). This material was processed by Tangential Flow Filtration (TTF) with a 1 K membrane until the total volume was reduced to 54 L.Water (120 L) was added in two portions and the solution was concentrated to 52 L by continued TTF. The aqueous solution (30 L of 52 L) was purified by chromatography using the following protocol: The aqueous solution was brought to three times of its volume (30 L→90l_) with 20% IPA in aqueous ammonium acetate solution (50 mM). The diluted solution was applied to a 38 L HP20SS resin column at 1.5 L/min. The column was eluted with IPA solution in aqueous 50 mM ammonium acetate (25%→30%→35%, 60 L each concentration).

Fractions (approximately 11 L) were collected and analyzed by HPLC. The fractions with HPLC purity less than 80% were combined and purified again using the same method. The key fractions from both chromatographic separations (with HPLC purity >80%) were combined and acidified with concentrated HCI to pH 2-3. The resulting solution was desalted on an ion exchange column (HP20SS resin, 16 L) which was eluted with WFI (until conductivity = 4.8 μS) followed by IPA in WFI (36 L 10%→ 40 L 60%). The yellow band which was eluted with 60% IPA (approximately 19L) was collected, adjusted to pH 2-3 with concentrated HCI and lyophilized to yield 636.5 g of Compound 49 (HPLC purity of 87.0%). MS m/z 1680.7 (M + H)⁺.

……………………………..

see formulation

WO2012162567A1

May 24, 2012

Nov 29, 2012

Cubist Pharmaceuticals, Inc.

Cb-183,315 compositions and related methods

References

http://www.cubist.com/downloads/Surotomycin-Fact-Sheet-13013.pdf
1. Cubist Pharmaceuticals. Cubist products and pipeline. Available online: http://www.cubist.com/products/(accessed on 15 April 2013).
2. Cubist Pharmaceuticals. Study of CB-183,315 in patients with Clostridium difficile associated diarrhea.Available online: http://www.clinicaltrials.gov/ct2/show/NCT01597505 (accessed on 15 April 2013).
3. Cubist Pharmaceuticals. A study of CB-183,315 in patients with Clostridium difficile associated diarrhea.Available online: http://www.clinicaltrials.gov/ct2/show/NCT01598311 (accessed on 15 April 2013).
4. Mascio, C.T.M.; Mortin, L.I.; Howland, K.T.; van, P.A.D.G.; Zhang, S.; Arya, A.; Chuong, C.L.; Kang, C.; Li, T.; Silverman, J.A. In vitro and in vivo characterization of CB-183,315, a novel lipopeptide antibiotic for treatment of Clostridium difficile. Antimicrob. Agents Chemother. 2012, 56, 5023–5030, doi:10.1128/AAC.00057-12.
5. WO2012162567A1 May 24, 2012 Nov 29, 2012 Cubist Pharmaceuticals, Inc. Cb-183,315 compositions and related methods

WO2001097851A2 *	Jun 18, 2001	Dec 27, 2001	Cubist Pharm Inc	Compositions and methods to improve the oral absorption of antimicrobial agents
WO2010075215A1	Dec 18, 2009	Jul 1, 2010	Cubist Pharmaceuticals, Inc.	Novel antibacterial agents for the treatment of gram positive infections
WO2011063419A2 *	Nov 23, 2010	May 26, 2011	Cubist Pharmaceuticals Inc.	Lipopeptide compositions and related methods