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Dacomitinib in phase 3 for lung (non-small cell) (NSCLC) Cancer

April 9, 2014 4:04 am / Leave a comment

Dacomitinib

(2E)-N-{4-[(3-Chloro-4-fluorophenyl)amino]-7-methoxy-6-quinazolinyl}-4-(1-piperidinyl)-2-butenamide

4-Piperidin-1-yl-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-7-methoxy-quinazolin-6-yl]-amide

1042385-75-0

pf299804…… pfizer

EGFR (HER1; erbB1) Inhibitors
HER4 (erbB4) Inhibitors
HER2 (erbB2) Inhibitors

Molecular formula:C24H25ClFN5O2
Molecular mass:469.95

Dacomitinib (PF-00299804) is an experimental drug candidate under development by Pfizer for the treatment of non-small-cell lung carcinoma. It is a selective and irreversible inhibitor of EGFR.^[1]

Dacomitinib has advanced to several Phase III clinical trials. The results of the first trials were disappointing, with a failure to meet the study goals,^[2]^[3]^[4] Additional Phase III trials are ongoing.^[2]

Dacomitinib is a HER (erbB) inhibitor in clinical trial development at Pfizer for the treatment of advanced non-small cell lung cancer (NSCLC) and for the treatment of relapsed/recurrent glioblastoma.

No recent development has been reported for research into the treatment of recurrent and/or metastatic head and neck squamous cell cancer. In 2012, Pfizer and SFJ Pharmaceuticals signed a codevelopment agreement for dacomitinib for the treatment of patients with locally advanced or metastatic NSCLC with activating mutations of epidermal growth factor receptor.

Substituted 4-phenylamino-quinazolin-6-yl-amides useful in the treatment of cancer have been described in the art, including those of U.S. Pat. No. 5,457,105 (Barker), U.S. Pat. No. 5,760,041 (Wissner et al.), U.S. Pat. No. 5,770,599 (Gibson), U.S. Pat. No. 5,929,080 (Frost), U.S. Pat. No. 5,955,464 (Barker), U.S. Pat. No. 6,251,912 (Wissner et al.), U.S. Pat. No. 6,344,455 (Bridges et al.), U.S. Pat. No. 6,344,459 (Bridges et al.), U.S. Pat. No. 6,414,148 (Thomas et al.), U.S. Pat. No. 5,770,599 (Gibson et al.), U.S. patent application 2002/0173509 (Himmelsbach et al.), and U.S. Pat. No. 6,323,209 (Frost).

Dacomitinib is a pan-human epidermal growth factor receptor (pan-HER) inhibitor developed by Pfizer, as ー small molecules targeting ffiR-1, HER-2 and HER-4 tyrosine kinase inhibitor by irreversibly binding to HER-l, HER-2, HER-4 and anti-tumor effect. Ni-line treatment of non-small cell lung cancer (NSCLC) display, Dacomitinib in non-small cell lung cancer Dinner erlotinib compared to some extend on progression-free survival and quality of life have mentioned the smell.

_4] Structural formula for Dacomitinib

[0005] U.S. patent US7772243 Dacomitinib first proposed a synthesis method, first, a fluorine-2_ _4_ amino acid and formamidine ring closure reaction to give 7 – fluoro-4 – quinazolinone, nitration and then successively chlorination reaction, to give 4 – chloro-7 – fluoro-6 – nitro-quinazoline; another aspect ー 3 – chloro-4 – amino-substituted on a fluoroaniline to give 3 – chloro – # – (3,4 – ni section yl methoxy)-4_ fluoro-aniline, obtained after the coupling of both an amino-protected N-(3 – chloro-4 – fluorophenyl)-7 – fluoro-6 – nitro-quinazoline -4 – amine, protected amino N-(3 – chloro-4 – fluorophenyl

Yl)-7_ fluoro-6 – nitro-quinazolin-4 – amine is of formula

Followed by a methoxy group, an amidation reaction and hydrogenation, the final deprotection ko under the action of trifluoroacetic acid to give the final product Dacomitinib. Throughout the reaction as follows:

synthesis

http://www.google.com/patents/CN103304492A?cl=en

Synthesis ー kind EGFR inhibitors Dacomitinib, synthetic route for

A synthetic method EGFR inhibitors Dacomitinib, concrete steps are as follows:

Step I, 7 – fluoro-4 – Synthesis of quinazolinone:

30 g (0.1934mol) 2 – fluoro-amino acid was dissolved in 250 ml _4_ formamide among the reaction was heated to 150 ° C for 6 inch, TLC plates to determine the point of completion of the reaction. The reaction was poured hot into 2000 ml of ice water, filtered, the filter cake was washed with water, vacuum dried at 50 ° C for 14 hours to give a pale brown solid powder 7 – fluoro-4 – quinazolinone, 28 g, yield 88%.

[0021] 2 walk 7 – fluoro-6 – nitro-4_ (hydrogen) _ Synthetic quinazolinones of:

Concentrated sulfuric acid (50 ml) and fuming nitric acid (50 ml) mixture was cooled with an ice bath to (TC hereinafter under stirring slowly added 25 g (0.1523mol) 7 – fluoro-4 – quinazolinone , the addition was complete, the reaction mixture was stirred at room temperature for I hour and then the reaction was heated to 110 ° C for 2 inch, TLC plates to determine the point of completion of the reaction the reaction was cooled to room temperature, 300 ml of ice water, the precipitated solid was stirred for 30 minutes , filtered, the filter cake was washed with water, vacuum dried at 50 ° C in 14 hours to give a yellow solid powder 7 – fluoro-6 – nitro-4 – (hydrogen) – quinazolinone, 26 g, yield 82%.

[0022] Step 3 6 – amino-7 – fluoro-4 – (hydrogen) – quinazolinone Synthesis:

24 g (0.1148mol) 7 – fluoro-6 – nitro _4_ (hydrogen) – quinazolinone was dissolved in 400 ml of methanol was added 2 g of palladium / carbon catalyst was added 8 ml of concentrated hydrochloric acid, and hydrogen was 2 small inch atmospheric reaction, TLC plates to determine the point of the reaction is complete. The catalyst was removed by suction filtration through celite, washed several fitness methanol, and the filtrate was concentrated by rotary evaporation to dryness to give 6 – amino-7_ fluoro-4 – (hydrogen) – quinazolinone, yellow powder, 20 g, yield 97%.

[0023] 4 walk, ⑶ -4 – (piperidin – Suites yl) -2 – butene acid methyl ester synthesis:

18 g (0.1006mol) 4 – bromo-methyl crotonate dissolved in 180 ml of methylene chloride ni added 27.9 g (0.2019mol) potassium carbonate, cooled to ice-bath (TC, was slowly added dropwise 10 ml (0.1012mol ) piperidine, (I reaction was stirred under a small inch TC, TLC plates to determine the point of completion of the reaction was concentrated by rotary evaporation to dryness, to give (E) -4 – (piperidin-1 – yl) – 2 – butenoic acid methyl Cool as a yellow solid, 17.1 g, yield 93%.

[0024] 5th walk, Buddhist) -4 – (piperidin-1 – yl) -2 – butene acid hydrochloride synthesis:

16 g (0.0873mol) of W) -4 – (piperidin-_1_ yl) -2 – butenyl acetate and 80 ml of concentrated hydrochloric acid was added to 250 ml of 1,4 – ni oxygen dioxane, heated under reflux 20 hours inch, TLC plate point the reaction was determined complete, the reaction solution was concentrated by rotary evaporation to dryness surplus was recrystallized from isopropanol to give a pale yellow solid, Buddhist) _4-(piperidin-1 – yl) -2 – butene acid hydrochloride, 14.5 g, yield 81%.

[0025] Step 6, (E) -4 – (piperazine Jie fixed -1 – yl) – 2 – butenyl chloride synthesis:

13 g (0.0632mol) of (K) ~ 4 ~ (piperidin-1 – yl) -2 – butene acid hydrochloride was dissolved in 750 ml of methylene chloride ni, 5 ml of DMF, was slowly added dropwise 8 ml ( 0.0933mol) of oxalyl chloride, the reaction was stirred at room temperature for I h, TLC plates to determine the point of completion of the reaction, the reaction solution was concentrated to dryness by rotary evaporation to give a pale yellow oil, Buddhist) _4-(piperidin-1 – yl) -2 – butyl allyl chloride, 11.8 g, yield 99%.

[0026] Step 7 (cargo) – # – (7 – fluoro-4 – oxo-3 ,4 – ni hydrogen quinazolin-6 – yl) -4 – (piperidin-1 – yl) -2 – butene amide Synthesis:

11 g (0.0586mol) of the) -4 – (piperidin-1 – yl) – 2 – butenyl chloride ni chloride (50 ml) was slowly added dropwise to 6 – amino-1 – fluoro-4 – ( hydrogen) – quinazolinone (7 g, 0.0391mmol), three ko amine (14 ml) and the mixture was ni chloride (200 ml), the reaction mixture was stirred at room temperature for 2 hours the reaction inch, TLC determined the completion of reaction points board , was added 800 liters of halo ni halo chloroformate and 500 liters of burning the separated organic phase was washed with 500 liters of halo, halo and then with 500 liters of brine, dried over magnesium sulfate, and concentrated by rotary evaporation to dryness was subjected to silica gel surplus Column chromatography (30% acid ko ko acetate / hexane) to give (M)-N-(7 – fluoro-4 – oxo-3 ,4 – ni hydrogen quinazolin-6 – yl) -4 – (piperidin-1 – yl) -2 – butenamide, as a pale yellow solid, 12.3 g, yield 95%.

Step 8 [0027] (2 ^) – # – (7 – methoxy – 4 – oxo _3, 4_ ni hydrogen quinazolinyl _6_ yl)-4_ (piperidin-1 – yl) – Synthesis 2_ butenamide:

I ^ xN MeONa N. Under nitrogen atmosphere, to 100 ml of anhydrous methanol was slowly added 1.52 g of sodium metal (0.0661mol), stirred for 10 minutes to dissolve all of the sodium metal to the completion of the reaction, to obtain a freshly prepared solution of sodium methoxide, and the The sodium methoxide solution was added 11 g (0.0333mol) of (receive) (7 – fluoro-4 – oxo-3 ,4 – ni hydrogen quinazolin-6 – yl) -4 – (piperidin-1 – yl) 2_ butene-amide, the reaction was heated to reflux for 3 inch, TLC plates to determine completion of the reaction point, cooled to room temperature, acidified with 2N hydrochloric acid solution to pH = 3 ~ 4, and concentrated by rotary evaporation to dryness, the residue was washed with water beating, filtration, The filter cake was washed with water, vacuum dried at 50 ° C in 14 hours to give (article) – # – (7 – methoxy – 4 – oxo – ni hydrogen quinazolin-6 – yl) – 4_ (piperidin-1 – yl)-2_ butenamide yellow solid, 10.6 g, yield 93%.

[0028] Step 9, {W,-N-(4 – chloro-7 – methoxy-quinazoline _6_ yl)-4_ (piperidin _1_ yl)-amide <EMI butene 2_:

9 g (0.0263mol) of (receive) – # – (7 – methoxy _4_ oxo – ni hydrogen quinazolin-6 – yl)-4_ (piperidin-1 – yl) – 2_ butenamide were added to 40 ml of phosphorus oxychloride was heated under reflux for 2 inch, TLC plates to determine the point of completion of the reaction, the reaction solution was concentrated to dryness by rotary evaporation, ice water was added surplus, beating, filtered, the cake washed with washed with water, vacuum dried at 50 ° C in 14 hours to give {W,-N-(4 – chloro-7 – methoxy-quinazolin-6 – yl) -4 – (piperidin-1 – yl) – 2 – butene amide as a yellow solid, 7 g, yield 74%

(2E)-N-(4 – chloro-7 – methoxy-quinazolin-6 – yl) -4 – (piperidin-1 – yl) -2 – butene amide (6 g,

0.0166mol), 3 – chloro-4-fluoro-aniline (2.6 g, 0.0179mol) and three ko amine (2.6 ml, 0.0186mol) was added to 140 ml of isopropanol and the reaction was heated to reflux for 3 inch, TLC plates to determine the point completion of the reaction, cooled to room temperature, filtered, the filter cake washed with methanol, vacuum dried at 50 ° C in 14 hours to give the final product Dacomitinib, a yellow solid, 6.6 g, yield 84%.

/////////////////////////

synthesis

US7772243
http://www.google.com/patents/US7772243

Scheme 1, wherein the 4-position aniline group is represented a 4-fluoro-3-chloro aniline group.

4-Chloro-7-fluoro-6-nitroquinazoline (7) can be prepared by methods similar to those described in J.Med. Chem. 1996, 39, 918-928. Generally, 2-amino-4-fluoro-benzoic acid (1) can be reacted with formamidine (2) and acetic acid (3) in the presence of 2-methoxyethanol to provide 7-Fluoro-3H-quinazolin-4-one (4). The 7-fluoro-3H-quinazolin-4-one (4) can be nitrated to 7-fluoro-6-nitro-3H-quinazolin-4-one (5), which can be treated with thionyl chloride to yield 4-chloro-6-nitro-7-fluoro-3H-quinazoline (6). The 4-chloro-quinazoline compound (6) can be combined with a desirably substituted aniline, represented above by 4-fluoro-3-chloro-aniline, in the presence of a tertiary amine and isopropanol to provide the 4-anilino-6-nitro-7-fluoro-quinazoline (7).

The 4-anilino-6-nitro-7-fluoro-quinazoline (7) may be reacted with an alcohol of the formula R₃OH, wherein R₃is as defined above, to yield the 7-alkoxylated compound (8). Reduction of the 6-nitro compound (8) provides the 6-amino analog (9).

The 6-position amino compound (9) may be reacted with a haloalkenoyl chloride (12), such as a 4-bromo-but-2-enoyl chloride, 5-bromo-pent-2-enoyl chloride, 4-chloro-but-2-enoyl chloride, or 5-chloro-pent-2-enoyl chloride, to provide an alkenoic acid[4-anilino]-7-alkoxylated-quinazolin-6-yl-amide (13). Haloalkenoyl chloride agents useful in this scheme may be prepared by methods known in the art, such as the treatment of a relevant haloalkenoic acid, represented by bromoalkenoic acid ester (10), with a primary alcohol, yielding the corresponding haloalkenoic acid (11), which may in turn be treated with oxalyl chloride to provide the desired haloalkenoyl chloride (12).

Finally, the quinazoline-6-alkanoic acid compound (13) may be treated with a cyclic amine, such as piperidine, piperazine, etc., to provide the desired final compound (14).

EXAMPLE 2

4-Piperidin-1-yl-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-7-methoxy-quinazolin-6-yl]-amide (Synthetic Route No. 1)

The title compound and other 7-methoxy analogs of this invention can be prepared as described in Example 1 by replacing the 2-fluoroethanol used in Example 1 with stoichiometric amount of methanol.

EXAMPLE 3 4-Piperidin-1 -yl-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-7-methoxv -quinazolin-6-yl]-amide (Synthetic Route No. 2)

An alternative synthetic route for compounds of this invention involves preparing the 6-position substituent chain as a Het-alkenoyl chloride as depicted in Scheme 2, below.

It will be understood that other compounds within this invention may be prepared using Het-butenoyl halide, Het-pentenoyl halide and Het-hexenoyl halide groups of the formula:

wherein R₄is as described herein and halo represents F, Cl, Br or I, preferably Cl or Br. One specific group of these Het-alkenoyl halides includes those compounds in which halo is Cl or Br, R₄is —(CH₂)_m-Het, m is an integer from 1 to 3, and Het is piperidine or the substituted piperidine moieties disclosed above.

EXAMPLE 4 4-Piperidin-1-yl-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-7-methoxy-quinazolin-6-yl]-amide (Synthetic Route No. 3)

3-Chloro-4-fluoro-phenylamine 15 (50.31, 345.6 mmole) and 3,4-Dimethoxy-benzaldehyde 16 (57.43 g, 345.6 mmole) were mixed in 500 ml of IPA and cooled in an ice-water. The glacial acetic acid was added (20.76 g, 345.6 mole) and then sodium cyanoborohydride in one portion. The reaction was stirred at room temperature (RT) for 24 hrs. 250 mL of 10% NaOH was added dropwise at RT after the reaction was completed. The mixture was stirred for ½ hr. The slurry was then filtered and washed with IPA and dried in vacuo. The mass weight 88.75 g (17, 87%).

Compounds 6 (3 g, 13.18 mmole) and 17 (3.9 g, 13.18 mmole) were combined in CH₃CN (25 mL) and heated for one hr. Mass spectroscopy indicated no starting material. Saturated K₂CO₃was added and the reaction was extracted 3× with EtOAc. The organic layers were combined, washed with brine and concentrated in vacuo to give 6.48 g of 7 (78.4%).

Compound 7 (72.76 g, 149.4 mmole) was added to a cool solution of NaOMe in 1.5 L of dry MeOH under N₂. The cooling bath was removed and the mixture was heated to reflux and stirred for 1 hr. The reaction was cooled to room temperature and quenched with water until the product precipitated out. The solid was filtered and washed with water and hexanes. The product was slurred in refluxing EtOAc and filtered hot to provide 68.75 g of yellow soled 8 (73%).

Compound 8 (63.62 g, 127.5 mole) was hydrogenated using Raney/Ni as catalyst to obtain 43.82 g of 9 (100%). Oxalyl chloride (6.5 g, 51.18 mmole) was added slowly to a suspension of 13 (10.5 g, 51.2 mmole) in 200 ml of dichloromethane containing 8 drops of DMF, after the reaction become homogeneous, the solvent was removed and the residual light yellow solid was slurred in 200 ml of DMAC and 9 (20 g, 42.65 mmole) was added gradually as a solid. The reaction was stirred for 15 min. and poured slowly into 1N NaOH. The mixture was extrated 3× EtOAc. The combined organic layers were washed with brine, filtered and concentrated in vacuo to obtain 28.4 g (100%) 10.

Compound 10(13.07 g, 21.08 mmole) was dissolved in trifluoroacetic acid (TFA) (74 g, 649 mmole) and heated to 30° C. for 24 hrs. The reaction was cooled to RT and poured gradually into a cooled 1 N NaO H-brine solution. Precipitate formed and was filtered and washed with 3X water then dried. The precipitate was recrystallized from toluene to obtain pure 4-Piperidin-1-vl-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino) -7-methoxv-puinazolin-6-yl]-amide (9.90 g, 89%).

Example 1 is similar but not same…caution

EXAMPLE 1 4-Piperidin-1-yl-but-2-enoic acid [4-(3-chloro-4-fluoro-phenylamino)-7-(2-fluoro-ethoxy)-quinazolin-6-yl]-amide

7-fluoro-6-nitro-4-chloroquinazoline (14.73,g, 65 mmol) was combined with 3-choro-4-fluoroaniline (9.49 g, 65 mmol) and triethylamine (10 mL, 72 mmol) in 150 mL of isopropanol. The reaction was stirred at room temperature for 1.5 hours, resulting in a yellow slurry. The solid was collected by filtration, rinsing with isopropanol and then water. The solid was dried in a 40° C. vacuum oven overnight to give 19.83 g (91%) of the product as an orange solid.

MS (APCI, m/z, M+1): 337.0

NaH (60% in mineral oil, 3.55 g, 88 mmol) was added, in portions, to a solution of 2-fluoroethanol (5.19 g, 80 mmol) in 200 mL THF. The reaction was stirred for 60 minutes at room temperature. To the reaction was added 7-fluoro-6-nitro-4-(3-chloro-4-fluoroaniline)quinazoline (18.11 g, 54 mmol) as a solid, rinsing with THF. The reaction was heated to 65° C. for 26 hours. The reaction was cooled to room temperature and quenched with water. THF was removed in vacuo. The resulting residue was sonicated briefly in water then the solid collected by filtration. The solid was triturated with MeOH, filtered and dried in a 40° C. vacuum oven overnight to 12.63 g of the product. Additional product was obtained by concentrating the MeOH filtrate to dryness and chromatography eluting with 50% EtOAc/hex. The isolated material was triturated with MeOH (2×), filtered and dried. 3.90 g

Total yield: 16.53 g, 81%

MS (APCI, m/z, M+1): 381.0

7-(2-fluoroethoxy)-6-nitro-4-(3-chloro-4-fluoroaniline)quinazoline (0.845 g, 2.2 mmol) in 50 mL THF was hydrogenated with Raney nickel (0.5 g) as the catalyst over 15 hours. The catalyst was filtered off and the filtrate was evaporated to give 0.77 g of product. (99%)

MS (APCI, m/z, M+1): 351.2

Methyl 4-bromocrotonate (85%, 20 mL, 144 mmol) was hydrolyzed with Ba(OH)₂in EtOH/H₂O as described in J.Med.Chem. 2001, 44(17), 2729-2734.

MS (APCI, m/z, M−1): 163.0

To a solution of 4-bromocrotonic acid (4.17 g, 25 mmol) in CH₂Cl₂(20 mL) was added oxalyl chloride (33 mL, 38 mmoL) and several drops of DMF. The reaction was stirred at room temperature for 1.5 hours. The solvent and excess reagent was removed in vacuo. The resulting residue was dissolved in 10 mL THF and added to a 0° C. mixture of 6-amino-7-(2-fluoroethoxy)-4-(3-chloro-4-fluoroaniline)quinazoline (5.28 g, 15 mmol) and triethylamine (5.2 mL, 37 mmol). The reaction was stirred at 0° C. for 1 hour. Water was added to the reaction and the THF removed in vacuo. The product was extracted into CH₂Cl₂(400 mL). The organic layer was dried over MgSO₄, filtered and concentrated. The crude material was chromatographed on silica gel eluting with 0-4% MeOH/CH₂Cl₂. An isolated gold foam was isolated. Yield: 4.58 g, 61%

MS (APCI, m/z, M−1): 497.1

Piperidine (0.75 mL, 6.7 mmol) was added to a solution of the above compound (3.35 g, 6.7 mmol) and TEA (2.80 mL, 20 mmol) in 10 mL DMA at 0° C. The reaction was stirred at 0° C. for 17 hours. Water was added to the reaction until a precipitate was evident. The reaction was sonicated for 40 minutes and the liquid decanted. The residue was dissolved in CH₂Cl₂, dried over MgSO₄, filtered and concentrated. The material was chromatographed on silica gel eluting with 4-10% MeOH/CH₂Cl₂. The isolated residue was triturated with acetonitrile (2×) and collected by filtration. Impurity found: Michael addition of piperidine (2.2% in first trituration of acetonitrile). Additional material can be obtained from the acetonitrile filtrates.

Yield: 0.95 g, 27%

MS (APCI, m/z, M+1): 502.3

……………

US 20050250761 A1,

References

“Dacomitinib”. NCI Drug Dictionary.
Zosia Chustecka (January 27, 2014). “Dacomitinib Fails in Pretreated Non-small Cell Lung Cancer”. Medscape.
“Blow to Pfizer as dacomitinib fails in lung cancer trials”. pmlive.com. 28th January 2014.
“Pfizer Announces Top-Line Results From Two Phase 3 Trials Of Dacomitinib In Patients With Refractory Advanced Non-Small Cell Lung Cancer”. Pfizer Press Release. January 27, 2014.
Tyrosine kinase inhibitors.17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(Phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides baring additional solubilizing functions
J Med Chem 2000, 43(7): 1380

Dacomitinib Fact Sheet, Pfizer

WO1996033980A1 *	23 Apr 1996	31 Oct 1996	Keith Hopkinson Gibson	Quinazoline derivatives
WO1997038983A1 *	8 Apr 1997	23 Oct 1997	Alexander James Bridges	Irreversible inhibitors of tyrosine kinases
WO2002050043A1 *	12 Dec 2001	27 Jun 2002	Boehringer Ingelheim Pharma	Quinazoline derivatives, medicaments containing said compounds, their utilization and method for the production thereof
WO2004069791A2 *	3 Feb 2004	19 Aug 2004	Hubert Gangolf Klemens Barth	Preparation of substituted quinazolines
US5760041 *	21 Jan 1997	2 Jun 1998	American Cyanamid Company	4-aminoquinazoline EGFR Inhibitors

Ozenoxacin in phase 3……topical formulation in the treatment of impetigo

March 28, 2014 4:59 am / 2 Comments on Ozenoxacin in phase 3……topical formulation in the treatment of impetigo

1-cyclopropyl-8-methyl-7-[5-methyl-6-(methylamino)-3-pyridinyl]-4-oxo-1 ,4-dihydro-3- quinolinecarboxylic acid

1-cyclopropyl-8-methyl-⁷-{5-methyl-6-[(methylamino)methyl]-3-pyridyl}-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid.

Ferrer Internacional (Spain), phase 3 Gram-positive

Ferrer Internacional has completed one Phase III clinical trial to evaluate the topical formulation of ozenoxacin in the treatment of impetigo [

Ozenoxacin
CAS Number: 245765-41-7Molecular Formula: C₂₁H₂₁N₃O₃
Molecular Weight: 363.41 g.mol^-1

poster……http://landing.quotientbioresearch.com/blog/bid/50380/Ozenoxacin-Activity-against-Atypical-Bacteria

Ozenoxacin is active against a great number of pathogens, such as Propionibacterium acnes, Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) including ciprofloxacin-resistant strains, methicillin-susceptible Staphylococcus epidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis (MRSE), Streptococcus pyogenes, Group G Streptococci, penicillin-resistant Streptococcus pneumoniae, Beta-lactamase positive Haemophilus influenzae, non-typeable strains of Haemophilus influenzae, Beta-lactamase positive Moraxella catarrhalis, Neisseria meningitides, Legionella pneumophila, Mycoplasma pneumoniae, Legionella pneumophila, Mycobacterium tuberculosis, Streptococcus agalactiae group B, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum Helicobacter pylori, Bacteroides fragilis, Clostridium perfringens, Escherichia coli, quinolone-resistant Escherichia coli, Salmonella spp., Shigella spp., ciprofloxacin-susceptible Pseudomonas aeruginosa, Clostridium difficile, and Listeria monocytogenes.

Ozenoxacin is a novel non-fluorinated quinolone antibacterial agent. It is currently in late stage phase 3 trials for the topical treatment of impetigo. The bacterial action of ozenoxacin is through the dual inhibition of DNA gyrase and topoisomerase IV. Excellent in vitro and in vivo antibacterial activity has been demonstrated in pre-clinical and clinical studies against a broad range of bacterial organisms. This includes organisms with emerging resistance to quinolones. Phase I and II clinical trials have also shown that ozenoxacin is a safe and effective antibacterial agent. No evidence of adverse effects as linked to topically formulated halogenated quinolones has been shown.

Ozenoxacin (I) was firstly disclosed in US6335447, and equivalent patents. Its chemical name is 1-cyclopropyl-8-methyl-7-[5-methyl-6-(methylamino)-3-pyridinyl]-4-oxo-1 ,4-dihydro-3- quinolinecarboxylic acid. Its chemical formula is: H

Ozenoxacin (I)

Topical application of antimicrobial agents is a useful tool for therapy of skin and skin structures infections, sexually transmitted diseases and genital tract infections and some systemic infections susceptible to topical treatment. Topical antimicrobial therapy has several potential advantages compared with systemic therapy.

Firstly, it can avoid an unnecessary exposure of the gut flora which may exert selection for resistance. Secondly, it is expected that the high local drug concentration in topical application and the negligible systemic absorption should overwhelm many mutational resistances. Thirdly, topical applications are less likely than systemic therapy to cause side effects. Accordingly, some topical compositions comprising ozenoxacin have been reported in the art.

JP2002356426A discloses ointments and gels for skin. An ointment comprising ozenoxacin 1%, N-methyl-2-pyrrolidone 8%, propylene glycol 14.9%, oleic acid 0.9%, diisopropanolamine 2.3%, polyethylene glycol 400 20.2%, polyethylene glycol 4000 50.2%, and water 3.2% is reported in Example 2.

JP2003226643A discloses aqueous solutions comprising ozenoxacin, cyclodextrin, and a viscous agent.

EP1731138A1 discloses fine particle dispersion liquid comprising ozenoxacin to be used in the manufacture of pharmaceutical compositions.

WO2007015453A1 discloses lotions comprising ozenoxacin.

JP2007119456A discloses aqueous suspensions containing nanoparticles and solution granules of ozenoxacin to be used in the manufacture of pharmaceutical compositions. Ophthalmic solutions are mentioned preferably. A combined use of ozenoxacin, magnesium ions, and hydroxypropyl-β-cyclodextrin specially for ophthalmic use is disclosed in Yamakawa, T. et al., Journal of Controlled Release (2003), 86(1 ), 101-103.

Semisolid topical compositions are useful alternatives to liquid compositions, because of their better manipulation and consequent patient preferences. However, in spite of the great diversity of components present in the semisolid compositions disclosed in the art, no quantitative stability studies are available for them.

Thus, there is a need of proved stable semisolid topical compositions comprising ozenoxacin as active ingredient, wherein microbiological and therapeutic activities are warranted because of demonstrated durable and prolonged pharmaceutical stability.

Synthesis

US6335447

http://www.google.co.in/patents/US6335447

EXAMPLE 5

To a solution of 0.80 g of 7-[6-({[(benzyloxy)-carbonyl] (methyl)amino}methyl)-5-methyl-3-pyrdyl]-1-cyclo-propyl-8-methyl-4-oxo-1,4-dihydro-3-quinoline-carboxylic acid in 16 ml of acetic acid was added 0.20 g of 5% (w/w) palladium-carbon and the mixture was stirred at ambient temperature and atmospheric pressure for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and the solvent was evaporated under reduced pressure. The obtained residue was dissolved in a mixed solvent consisting of 3.8 ml of ethanol and 3.8 ml of water. After adding 3.8 ml of an aqueous 1 mol/l sodium hydroxide solution thereto and adjusting the solution to pH 5.5with 1 mol/l hydrochloric acid, 10 ml of chloroform was added thereto. An organic layer was separated and dried over anhydrous magnesium sulfate and the solvent was evaporated under reduced pressure. Addition of diethyl ether to the obtained residue and filtration of crystals afforded 0.25 g of colorless crystals of 1-cyclopropyl-8-methyl-⁷-{5-methyl-6-[(methylamino)methyl]-3-pyridyl}-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid.

IR (KBr) cm⁻¹: 3322, 1721; NMR(d₁-TFA) δ: 1.2-1.9 (4H, m), 2.94 (3H, s), 3.05 (3H, s), 3.29 (3H, s), 4.6-5.0 (1H, m), 5.12 (2H, s), 7.91 (1H, d, J=8.5 Hz), 8.6-9.0 (2H, m), 9.0-9.3 (1H, brs), 9.75 (1H, s). Melting point: 199° C.

Ferrer Group. Key development projects. Available online: http://www.ferrergrupo.com/Innovation_Innovacion-Pipeline-de-proyectos-ENG (accessed on 15 April 2013).
Yamakawa, T.; Mitsuyama, J.; Hayashi, K. In vitro and in vivo antibacterial activity of T-3912, a novel non-fluorinated topical quinolone. J. Antimicrob. Chemother. 2002, 49, 455–465, doi:10.1093/jac/49.3.455.
Ferrer Internacional. Efficacy and safety of ozenoxacin 1% cream versus placebo in the treatment of patients with impetigo. Available online: http://clinicaltrials.gov/ct2/show/NCT01397461 (accessed on 13 April 2013).

SUROTOMYCIN for Clostridium difficile-associated diarrhea

March 27, 2014 7:40 am / Leave a comment

Surotomycin

Click to access 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

Aldoxorubicin…….Treatment of cancer …HIV-derived Kaposi’s Sarcoma, pancreatic cancer and for the treatment of soft tissue sarcoma.

March 27, 2014 4:23 am / 4 Comments on Aldoxorubicin…….Treatment of cancer …HIV-derived Kaposi’s Sarcoma, pancreatic cancer and for the treatment of soft tissue sarcoma.

Aldoxorubicin-INNO206 structure

Aldoxorubicin

Click to access aldoxorubicin.pdf

in phase 3

(E)-N’-(1-((2S,4S)-4-(((2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide hydrochloride

1H-Pyrrole-1-hexanoic acid, 2,5-dihydro-2,5-dioxo-, (2E)-2-[1-[(2S,4S)-4-[(3-amino-
2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-
7-methoxy-6,11-dioxo-2-naphthacenyl]-2-hydroxyethylidene]hydrazide

N’-[(1E)-1-{(2S,4S)-4-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-2,5,12-
trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl}-2-
hydroxyethylidene]-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanohydrazide
MOLECULAR FORMULA C37H42N4O13

MOLECULAR WEIGHT 750.7

SPONSOR CytRx Corp.

CODE DESIGNATION INNO-206

CAS REGISTRY NUMBER 1361644-26-9

CAS: 151038-96-9 (INNO-206); 480998-12-7 (INNO-206 HCl salt), 1361644-26-9

hydrochloride

CAS: 151038-96-9

Chemical Formula: C37H42N4O13

Exact Mass: 750.27484

Molecular Weight: 750.75

Certificate of Analysis:	View current batch of CoA
QC data:	View NMR, View HPLC, View MS
Safety Data Sheet (MSDS):	View Material Safety Data Sheet (MSDS)

In vitro protocol:

Clin Cancer Res. 2012 Jul 15;18(14):3856-67

In vivo protocol:

Clin Cancer Res. 2012 Jul 15;18(14):3856-67.

Invest New Drugs. 2010 Feb;28(1):14-9.

Invest New Drugs. 2012 Aug;30(4):1743-9.

Int J Cancer. 2007 Feb 15;120(4):927-34.

Clinical study:

Expert Opin Investig Drugs. 2007 Jun;16(6):855-66.

Aldoxorubicin (INNO-206): Aldoxorubicin, also known as INNO-206, is the 6-maleimidocaproyl hydrazone derivative prodrug of the anthracycline antibiotic doxorubicin (DOXO-EMCH) with antineoplastic activity. Following intravenous administration, doxorubicin prodrug INNO-206 binds selectively to the cysteine-34 position of albumin via its maleimide moiety. Doxorubicin is released from the albumin carrier after cleavage of the acid-sensitive hydrazone linker within the acidic environment of tumors and, once located intracellularly, intercalates DNA, inhibits DNA synthesis, and induces apoptosis. Albumin tends to accumulate in solid tumors as a result of high metabolic turnover, rapid angiogenesis, hyervasculature, and impaired lymphatic drainage. Because of passive accumulation within tumors, this agent may improve the therapeutic effects of doxorubicin while minimizing systemic toxicity.

“Aldoxorubicin has demonstrated effectiveness against a range of tumors in both human and animal studies, thus we are optimistic in regard to a potential treatment for Kaposi’s sarcoma. The current standard-of-care for severe dermatological and systemic KS is liposomal doxorubicin (Doxil®). However, many patients exhibit minimal to no clinical response to this agent, and that drug has significant toxicity and manufacturing issues,” said CytRx President and CEO Steven A. Kriegsman. “In addition to obtaining valuable information related to Kaposi’s sarcoma, this trial represents another opportunity to validate the value and viability of our linker technology platform.” The company expects to announce Phase-2 study results in the second quarter of 2015.

Kaposi’s sarcoma is an orphan indication, meaning that only a small portion of the population has been diagnosed with the disease (fewer than 200,000 individuals in the country), and in turn, little research and drug development is being conducted to treat and cure it. The FDA’s Orphan Drug Act may grant orphan drug designation to a drug such as aldoxorubicin that treats a rare disease like Kaposi’s sarcoma, offering market exclusivity for seven years, fast-track status in some cases, tax credits, and grant monies to accelerate research

INNO-206 is an anthracycline in early clinical trials at CytRx Oncology for the treatment of breast cancer, HIV-related Kaposi’s sarcoma, glioblastoma multiforme, stomach cancer and pancreatic cancer. In 2014, a pivotal global phase 3 clinical trial was initiated as second-line treatment in patients with metastatic, locally advanced or unresectable soft tissue sarcomas. The drug candidate was originally developed at Bristol-Myers Squibb, and was subsequently licensed to KTB Tumorforschungs. In August 2006, Innovive Pharmaceuticals (acquired by CytRx in 2008) licensed the patent rights from KTB for the worldwide development and commercialization of the drug candidate. No recent development has been reported for research that had been ongoing for the treatment of small cell lung cancer (SCLC).

INNO-206 is a doxorubicin prodrug. Specifically, it is the 6-maleimidocaproyl hydrazone of doxorubicin. After administration, the drug candidate rapidly binds endogenous circulating albumin through the acid sensitive EMCH linker. Circulating albumin preferentially accumulates in tumors, bypassing uptake by other non-specific sites including the heart, bone marrow and the gastrointestinal tract. Once inside the acidic environment of the tumor cell, the EMCH linker is cleaved and free doxorubicin is released at the tumor site. Like other anthracyclines, doxorubicin inhibits DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, thus preventing the replication of rapidly-growing cancer cells. It also creates iron-mediated free oxygen radicals that damage the DNA and cell membranes. In 2011, orphan drug designation was assigned in the U.S. for the treatment of pancreatic cancer and for the treatment of soft tissue sarcoma.

CytRx Corporation (NASDAQ:CYTR) has announced it has initiated a pivotal global Phase 3 clinical trial to evaluate the efficacy and safety of aldoxorubicin as a second-line treatment for patients with soft tissue sarcoma (STS) under a Special Protocol Assessment with the FDA. Aldoxorubicin combines the chemotherapeutic agent doxorubicin with a novel linker-molecule that binds specifically to albumin in the blood to allow for delivery of higher amounts of doxorubicin (3.5 to 4 times) without several of the major treatment-limiting toxicities seen with administration of doxorubicin alone.

According to a news from Medicalnewstoday.com; CytRx holds the exclusive worldwide rights to INNO-206. The Company has previously announced plans to initiate Phase 2 proof-of-concept clinical trials in patients with pancreatic cancer, gastric cancer and soft tissue sarcomas, upon the completion of optimizing the formulation of INNO-206. Based on the multiple myeloma interim results, the Company is exploring the possibility of rapidly including multiple myeloma in its INNO-206 clinical development plans.

According to CytRx’s website, In preclinical models, INNO-206 was superior to doxorubicin with regard to ability to increase dosing, antitumor efficacy and safety. A Phase I study of INNO-206 that demonstrated safety and objective clinical responses in a variety of tumor types was completed in the beginning of 2006 and presented at the March 2006 Krebskongress meeting in Berlin. In this study, doses were administered at up to 4 times the standard dosing of doxorubicin without an increase in observed side effects over historically seen levels. Objective clinical responses were seen in patients with sarcoma, breast, and lung cancers.

INNO-206 – Mechanism of action:

According to CytRx’s website, the proposed mechanism of action is as the follow steps: (1) after administration, INNO-206 rapidly binds endogenous circulating albumin through the EMCH linker. (2) circulating albumin preferentially accumulates in tumors, bypassing uptake by other non-specific sites including heart, bone marrow and gastrointestinal tract; (3) once albumin-bound INNO-206 reaches the tumor, the acidic environment of the tumor causes cleavage of the acid sensitive linker; (4) free doxorubicin is released at the site of the tumor.

INNO-206 – status of clinical trials:

CytRx has announced that, in December 2011, CytRx initiated its international Phase 2b clinical trial to evaluate the preliminary efficacy and safety of INNO-206 as a first-line therapy in patients with soft tissue sarcoma who are ineligible for surgery. The Phase 2b clinical trial will provide the first direct clinical trial comparison of INNO-206 with native doxorubicin, which is dose-limited due to toxicity, as a first-line therapy. (source:http://cytrx.com/inno_206, accessed date: 02/01/2012).

Results of Phase I study:

In a phase I study a starting dose of 20 mg/m2 doxorubicin equivalents was chosen and 41 patients with advanced cancer disease were treated at dose levels of 20–340 mg/m2 doxorubicin equivalents . Treatment with INNO-206 was well tolerated up to 200 mg/m2 without manifestation of drug-related side effects which is a ~3-fold increase over the standard dose for doxorubicin (60 mg/kg). Myelosuppression and mucositis were the predominant adverse effects at dose levels of 260 mg/m2 and became dose-limiting at 340 mg/m2. 30 of 41 patients were assessable for analysis of response. Partial responses were observed in 3 patients (10%, small cell lung cancer, liposacoma and breast carcinoma). 15 patients (50%) showed a stable disease at different dose levels and 12 patients (40%) had evidence of tumor progression. (source: Invest New Drugs (2010) 28:14–19)

References

1: Kratz F, Azab S, Zeisig R, Fichtner I, Warnecke A. Evaluation of combination therapy schedules of doxorubicin and an acid-sensitive albumin-binding prodrug of doxorubicin in the MIA PaCa-2 pancreatic xenograft model. Int J Pharm. 2013 Jan 30;441(1-2):499-506. doi: 10.1016/j.ijpharm.2012.11.003. Epub 2012 Nov 10. PubMed PMID: 23149257.

2: Walker L, Perkins E, Kratz F, Raucher D. Cell penetrating peptides fused to a thermally targeted biopolymer drug carrier improve the delivery and antitumor efficacy of an acid-sensitive doxorubicin derivative. Int J Pharm. 2012 Oct 15;436(1-2):825-32. doi: 10.1016/j.ijpharm.2012.07.043. Epub 2012 Jul 28. PubMed PMID: 22850291; PubMed Central PMCID: PMC3465682.

3: Kratz F, Warnecke A. Finding the optimal balance: challenges of improving conventional cancer chemotherapy using suitable combinations with nano-sized drug delivery systems. J Control Release. 2012 Dec 10;164(2):221-35. doi: 10.1016/j.jconrel.2012.05.045. Epub 2012 Jun 13. PubMed PMID: 22705248.

4: Sanchez E, Li M, Wang C, Nichols CM, Li J, Chen H, Berenson JR. Anti-myeloma effects of the novel anthracycline derivative INNO-206. Clin Cancer Res. 2012 Jul 15;18(14):3856-67. doi: 10.1158/1078-0432.CCR-11-3130. Epub 2012 May 22. PubMed PMID: 22619306.

5: Kratz F, Elsadek B. Clinical impact of serum proteins on drug delivery. J Control Release. 2012 Jul 20;161(2):429-45. doi: 10.1016/j.jconrel.2011.11.028. Epub 2011 Dec 1. Review. PubMed PMID: 22155554.

6: Elsadek B, Kratz F. Impact of albumin on drug delivery–new applications on the horizon. J Control Release. 2012 Jan 10;157(1):4-28. doi: 10.1016/j.jconrel.2011.09.069. Epub 2011 Sep 16. Review. PubMed PMID: 21959118.

7: Kratz F, Fichtner I, Graeser R. Combination therapy with the albumin-binding prodrug of doxorubicin (INNO-206) and doxorubicin achieves complete remissions and improves tolerability in an ovarian A2780 xenograft model. Invest New Drugs. 2012 Aug;30(4):1743-9. doi: 10.1007/s10637-011-9686-5. Epub 2011 May 18. PubMed PMID: 21590366.

8: Boga C, Fiume L, Baglioni M, Bertucci C, Farina C, Kratz F, Manerba M, Naldi M, Di Stefano G. Characterisation of the conjugate of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin with lactosaminated human albumin by 13C NMR spectroscopy. Eur J Pharm Sci. 2009 Oct 8;38(3):262-9. doi: 10.1016/j.ejps.2009.08.001. Epub 2009 Aug 18. PubMed PMID: 19695327.

9: Graeser R, Esser N, Unger H, Fichtner I, Zhu A, Unger C, Kratz F. INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin), shows superior antitumor efficacy compared to doxorubicin in different tumor xenograft models and in an orthotopic pancreas carcinoma model. Invest New Drugs. 2010 Feb;28(1):14-9. doi: 10.1007/s10637-008-9208-2. Epub 2009 Jan 8. PubMed PMID: 19148580.

10: Kratz F. Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release. 2008 Dec 18;132(3):171-83. doi: 10.1016/j.jconrel.2008.05.010. Epub 2008 May 17. Review. PubMed PMID: 18582981.

11: Unger C, Häring B, Medinger M, Drevs J, Steinbild S, Kratz F, Mross K. Phase I and pharmacokinetic study of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin. Clin Cancer Res. 2007 Aug 15;13(16):4858-66. PubMed PMID: 17699865.

12: Lebrecht D, Walker UA. Role of mtDNA lesions in anthracycline cardiotoxicity. Cardiovasc Toxicol. 2007;7(2):108-13. Review. PubMed PMID: 17652814.

13: Kratz F. DOXO-EMCH (INNO-206): the first albumin-binding prodrug of doxorubicin to enter clinical trials. Expert Opin Investig Drugs. 2007 Jun;16(6):855-66. Review. PubMed PMID: 17501697.

14: Kratz F, Ehling G, Kauffmann HM, Unger C. Acute and repeat-dose toxicity studies of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH), an albumin-binding prodrug of the anticancer agent doxorubicin. Hum Exp Toxicol. 2007 Jan;26(1):19-35. PubMed PMID: 17334177.

15: Lebrecht D, Geist A, Ketelsen UP, Haberstroh J, Setzer B, Kratz F, Walker UA. The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage. Int J Cancer. 2007 Feb 15;120(4):927-34. PubMed PMID: 17131338.

16: Di Stefano G, Lanza M, Kratz F, Merina L, Fiume L. A novel method for coupling doxorubicin to lactosaminated human albumin by an acid sensitive hydrazone bond: synthesis, characterization and preliminary biological properties of the conjugate. Eur J Pharm Sci. 2004 Dec;23(4-5):393-7. PubMed PMID: 15567293.

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Delamanid……….an experimental drug for the treatment of multi-drug-resistant tuberculosis.

March 26, 2014 3:54 am / 1 Comment on Delamanid……….an experimental drug for the treatment of multi-drug-resistant tuberculosis.

Delamanid

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

(2R)-2-Methyl-6-nitro-2-[(4-{4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl}phenoxy)methyl]-2,3-dihydroimidazo[2,1-b][1,3]oxazole

2(R)-Methyl-6-nitro-2-[4-[4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl]phenoxymethyl]-2,3-dihydroimidazo[2,1-b]oxazole

(R) -2-methyl-6-nitro-2- { 4- [4- (4- trifluoromethoxyphenoxy) piperidin-l-yl] phenoxymethyl } -2 , 3- dihydroimidazo [2 , 1-b] oxazole

Imidazo[2,1-b]oxazole, 2,3-dihydro-2-methyl-6-nitro-2-[[4-[4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl]phenoxy]methyl]-, (2R)-

(R)-2-methyl-6-nitro-2-{4-[4-(4-trifluoromethoxyphenoxy)piperidin-1-yl]phenoxymethyl}-2,3-dihydroimidazo[2,1-b]oxazole

681492-22-8 cas no

Delamanid, 681492-22-8, Delamanid (JAN/USAN), Delamanid [USAN:INN],UNII-8OOT6M1PC7,

OPC 67683
OPC-67683
UNII-8OOT6M1PC7

Molecular Formula: C₂₅H₂₅F₃N₄O₆

Molecular Weight: 534.48441

Clinical trials……….http://clinicaltrials.gov/search/intervention=OPC+67683+OR+Delamanid

CLINICAL TRIALS

Trial Name: A Placebo-Controlled, Phase 2 Trial to Evaluate OPC 67683 in Patients With Pulmonary Sputum Culture-Positive, Multidrug-Resistant Tuberculosis (TB)
Primary Sponsor: Otsuka Pharmaceutical Development & Commercialization, Inc.
Trial ID / Reg # / URL: http://clinicaltrials.gov/ct2/show/NCT00685360

Delamanid (USAN, codenamed OPC-67683) is an experimental drug for the treatment of multi-drug-resistant tuberculosis. It works by blocking the synthesis of mycolic acids in Mycobacterium tuberculosis, the organism which causes tuberculosis, thus destabilising its cell wall.^[1]^[2]^[3]

In phase II clinical trials, the drug was used in combination with standard treatments, such as four or five of the drugs ethambutol, isoniazid,pyrazinamide, rifampicin, aminoglycoside antibiotics, and quinolones. Healing rates (measured as sputum culture conversion) were significantly better in patients who additionally took delamanid.^[3]^[4]

The European Medicines Agency (EMA) recommended conditional marketing authorization for delamanid in adults with multidrug-resistant pulmonary tuberculosis without other treatment options because of resistance or tolerability. The EMA considered the data show that the benefits of delamanid outweigh the risks, but that additional studies were needed on the long-term effectiveness.^[5]

Delamanid, an antibiotic active against Mycobacterium tuberculosis strains, has been filed for approval in the E.U. and by Otsuka for the treatment of multidrug-resistant tuberculosis. In 2013, a positive opinion was received in the E.U. for this indication. Phase III trials for treatment of multidrug-resistant tuberculosis are under way in the U.S. Phase II study for the pediatric use is undergone in the Europe.

The drug candidate’s antimycobacterial mechanism of action is via specific inhibition of the synthesis pathway of mycolic acid, which is a cell wall component unique to M. tuberculosis.

In 2008, orphan drug designation was received in Japan for the treatment of pulmonary tuberculosis.

Tuberculosis (TB), an airborne lung infection, still remains a major public health problem worldwide. It is estimated that about 32% of the world population is infected with TB bacillus, and of those, approximately 8.9 million people develop active TB and 1.7 million die as a result annually according to 2004 figures. Human immunodeficiency virus (HIV) infection has been a major contributing factor in the current resurgence of TB. HIV-associated TB is widespread, especially in sub-Saharan Africa, and such an infectious process may further accelerate the resurgence of TB.

Moreover, the recent emergence of multidrug-resistant (MDR) strains ofMycobacterium tuberculosis that are resistant to two major effective drugs, isonicotinic acid hydrazide (INH) and rifampicin (RFP), has further complicated the world situation.

The World Health Organization (WHO) has estimated that if the present conditions remain unchanged, more than 30 million lives will be claimed by TB between 2000 and 2020. As for subsequent drug development, not a single new effective compound has been launched as an antituberculosis agent since the introduction of RFP in 1965, despite the great advances that have been made in drug development technologies.

Although many effective vaccine candidates have been developed, more potent vaccines will not become immediately available. The current therapy consists of an intensive phase with four drugs, INH, RFP, pyrazinamide (PZA), and streptomycin (SM) or ethambutol (EB), administered for 2 months followed by a continuous phase with INH and RFP for 4 months. Thus, there exists an urgent need for the development of potent new antituberculosis agents with low-toxicity profiles that are effective against both drug-susceptible and drug-resistant strains of M. tuberculosis and that are capable of shortening the current duration of therapy.

………………………

US20060094767

(R)-2-bromo-4-nitro-1-(2-methyl-2-oxiranylmethyl)imidazole

4-[4-(4-Trifluoromethoxyphenoxy)piperidin-1-yl]phenol

ARE THE INTERMEDIATES

Example 1884

Production of (R)-2-methyl-6-nitro-2-{4-[4-(4-trifluoromethoxyphenoxy)piperidin-1-yl]phenoxymethyl}-2,3-dihydroimidazo[2,1-b]oxazole

4-[4-(4-Trifluoromethoxyphenoxy)piperidin-1-yl]phenol (693 mg, 1.96 mmol) was dissolved in N,N′-dimethylformamide (3 ml), and sodium hydride (86 mg, 2.16 mmol) was added while cooling on ice followed by stirring at 70-75° C. for 20 minutes. The mixture was cooled on ice. To the solution, a solution prepared by dissolving (R)-2-bromo-4-nitro-1-(2-methyl-2-oxiranylmethyl)imidazole (720 mg, 2.75 mmol) in N,N′-dimethylformamide (3 ml) was added followed by stirring at 70-75° C. for 20 minutes. The reaction mixture was allowed to return to room temperature, ice water (25 ml) was added, and the resultant solution was extracted with methylene chloride (50 ml) three times. The organic phases were combined, washed with water 3 times, and dried over magnesium sulfate. After filtration, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (methylene chloride/ethyl acetate=3/1). Recrystallization from ethyl acetate/isopropyl ether gave (R)-2-methyl-6-nitro-2-{4-[4-(4-trifluoromethoxyphenoxy)piperidin-1-yl]phenoxymethyl}-2,3-dihydroimidazo[2,1-b]oxazole (343 mg, 33%) as a light yellow powder.

…………………………

WO 2010021409 AND http://worldwide.espacenet.com/publicationDetails/biblio?CC=IN&NR=203704A1&KC=A1&FT=D

FOR 2, 4 DINITROIMIDAZOLE

…………………………………………

WO2011093529A1

These patent literatures disclose Reaction Schemes A and B below as the processes for producing the aforementioned 2, 3-dihydroimidazo [2, 1-b] oxazole compound.

Reaction Scheme A:

wherein R¹ is a hydrogen atom or lower-alkyl group; R² is a substituted pxperidyl group or a substituted piperazinyl group; and X¹ is a halogen atom or a nitro group.

Reaction Scheme B:

wherein X² is a halogen or a group causing a substitution reaction similar to that of a halogen; n is an integer from 1 to 6; and R¹, R² and X¹ are the same as in Reaction Scheme A.

An oxazole com ound represented by Formula (la) :

, i.e., 2-methyl-6-nitro-2-{4- [4- (4- trifluoromethoxyphenoxy) piperidin-l-yl] phenoxymethyl }-2, 3- dihydroimidazo [2, 1-b] oxazole (hereunder, this compound may be simply referred to as “Compound la”) is produced, for example, by the method shown in the Reaction Scheme C below (Patent

Literature 3) . In this specification, the term “oxazole compound’ means an oxazole derivative that encompasses compounds that contain an oxazole ring or an oxazoline ring (dihydrooxazole ring) in the molecule.

Reaction Scheme C:

However, the aforementioned methods are unsatisfactory in terms of the yield of the objective compound. For example, the method of Reaction Scheme C allows the objective oxazole Compound (la) to be obtained from Compound (2a) at a yield as low as 35.9%. Therefore, alternative methods for producing the compound in an industrially advantageous manner are desired. Citation List

Patent Literature

PTL 1: WO2004/033463

PTL 2: WO2004/035547

PTL 3: WO2008/140090

Example 9

Production of (R) -2-methyl-6-nitro-2- { 4- [4- (4- trifluoromethoxyphenoxy) piperidin-l-yl] phenoxymethyl } -2 , 3- dihydroimidazo [2 , 1-b] oxazole

{R) -1- [ – {2 , 3-epoxy-2-methylpropoxy ) phenyl] -4- [4- ( trifluoromethoxy ) phenoxy ] piperidine (10.0 g, 23.6 mmol, optical purity of 94.3%ee), 2-chloro-4-nitroimidazole (4.0 g, 27.2 mmol), sodium acetate (0.4 g, 4.9 mmol), and t- butyl acetate (10 ml) were mixed and stirred at 100°C for 3.5 hours. Methanol (70 ml) was added to the reaction mixture, and then a 25% sodium hydroxide aqueous solution (6.3 g, 39.4 mmol) was added thereto dropwise while cooling with ice. The resulting mixture was stirred at 0°C for 1.5 hours, and further stirred at approximately room

temperature for 40 minutes. Water (15 ml) and ethyl acetate (5 ml) were added thereto, and the mixture was stirred at 45 to 55°C for 1 hour. The mixture was cooled to room temperature, and the precipitated crystals were collected by filtration. The precipitated crystals were subsequently washed with methanol (30 ml) and water (40 ml) . Methanol (100 ml) was added to the resulting

crystals, followed by stirring under reflux for 30 minutes. The mixture was cooled to room temperature. The crystals were then collected by filtration and washed with methanol (30 ml) . The resulting crystals were dried under reduced pressure, obtaining 9.3 g of the objective product (yield: 73%) .

Optical purity: 99.4%ee.

……………….

Synthesis and antituberculosis activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles
J Med Chem 2006, 49(26): 7854

http://pubs.acs.org/doi/abs/10.1021/jm060957y

(R)-2-Methyl-6-nitro-2-{4-[4-(4-trifluoromethoxyphenoxy)piperidin-1-yl]phenoxymethyl}-2,3-dihydroimidazo[2,1-b]oxazole (19, DELAMANID).

To a mixture of 27 (127.56 g, 586.56 mmol) and 4-[4-(4-trifluoromethoxyphenoxy)piperidin-1-yl]phenol (28g) (165.70 g, 468.95 mmol) in N,N-dimethylformamide (1600 mL) was added 60% sodium hydride (22.51 g, 562.74 mmol) at 0 °C portionwise. After the mixture was stirred at 50 °C for 2 h under a nitrogen atmosphere, the reaction mixture was cooled in an ice bath and carefully quenched with ethyl acetate (230 mL) and ice water (50 mL). The thus-obtained mixture was poured into water (3000 mL) and stirred for 30 min. The resulting precipitates were collected by filtration, washed with water, and dried at 60 °C overnight. This crude product was purified by silica gel column chromatography using a dichloromethane and ethyl acetate mixture (5/1) as solvent. The appropriate fractions were combined and evaporated under reduced pressure. The residue was recrystallized from ethyl acetate (1300 mL)−isopropyl alcohol (150 mL) to afford 19 (119.11 g, 48%) as a pale yellow crystalline powder.

Mp 195−196 °C.

¹H NMR (CDCl₃) δ 1.77 (3H, s), 1.87−2.16 (4H, m), 2.95−3.05 (2H, m), 3.32−3.41 (2H, m), 4.02 (1H, d, J = 10.2 Hz), 4.04 (1H, d, J = 10.2 Hz), 4.18 (1H, J = 10.2 Hz), 4.36−4.45 (1H, m), 4.49 (1H, d, J = 10.2 Hz), 6.76 (2H, d, J = 6.7 Hz), 6.87−6.94 (4H, m), 7.14 (2H, d, J = 8.6 Hz), 7.55 (1H, s).

[α −9.9° (c 1.01, CHCl₃).

MS (DI) m/z 535 (M⁺ + 1). Anal. (C₂₅H₂₅F₃N₄O₆) C, H, N.

http://pubs.acs.org/doi/suppl/10.1021/jm060957y/suppl_file/jm060957ysi20061113_095044.pdf

References

Matsumoto, M.; Hashizume, H.; Tomishige, T.; Kawasaki, M.; Tsubouchi, H.; Sasaki, H.; Shimokawa, Y.; Komatsu, M. (2006). “OPC-67683, a Nitro-Dihydro-Imidazooxazole Derivative with Promising Action against Tuberculosis in Vitro and in Mice”. PLoS Medicine 3 (11): e466.doi:10.1371/journal.pmed.0030466. PMC 1664607. PMID 17132069. edit
Skripconoka, V.; Danilovits, M.; Pehme, L.; Tomson, T.; Skenders, G.; Kummik, T.; Cirule, A.; Leimane, V.; Kurve, A.; Levina, K.; Geiter, L. J.; Manissero, D.; Wells, C. D. (2012). “Delamanid Improves Outcomes and Reduces Mortality for Multidrug-Resistant Tuberculosis”. European Respiratory Journal41 (6): 1393–1400. doi:10.1183/09031936.00125812. PMC 3669462. PMID 23018916. edit
H. Spreitzer (18 February 2013). “Neue Wirkstoffe – Bedaquilin und Delamanid”. Österreichische Apothekerzeitung (in German) (4/2013): 22.
Gler, M. T.; Skripconoka, V.; Sanchez-Garavito, E.; Xiao, H.; Cabrera-Rivero, J. L.; Vargas-Vasquez, D. E.; Gao, M.; Awad, M.; Park, S. K.; Shim, T. S.; Suh, G. Y.; Danilovits, M.; Ogata, H.; Kurve, A.; Chang, J.; Suzuki, K.; Tupasi, T.; Koh, W. J.; Seaworth, B.; Geiter, L. J.; Wells, C. D. (2012). “Delamanid for Multidrug-Resistant Pulmonary Tuberculosis”. New England Journal of Medicine 366 (23): 2151–2160. doi:10.1056/NEJMoa1112433.PMID 22670901. edit
Drug Discovery & Development. EMA Recommends Two New Tuberculosis Treatments. November 22, 2013.
Synthesis and antituberculous activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles
45th Intersci Conf Antimicrob Agents Chemother (ICAAC) (December 16-19, Washington DC) 2005, Abst F-1473

17181168	12-28-2006	Synthesis and antituberculosis activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles.	Journal of medicinal chemistry
17132069	11-1-2006	OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice.	PLoS medicine

18691051

1-1-2008

New anti-tuberculosis drugs with novel mechanisms of action.

Current medicinal chemistry

21069962

11-11-2010

Synthesis and Structure-Activity Relationships of Aza- and Diazabiphenyl Analogues of the Antitubercular Drug (6S)-2-Nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824).

Journal of medicinal chemistry

22421275	5-1-2012	Tuberculosis: the drug development pipeline at a glance.	European journal of medicinal chemistry
22148391	1-12-2012	Structure-activity relationships for amide-, carbamate-, and urea-linked analogues of the tuberculosis drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824).	Journal of medicinal chemistry

US2009227630	9-11-2009	Pharmaceutical Composition Achieving Excellent Absorbency of Pharmacologically Active Substance
US2009018169	1-16-2009	Sulfonamide Derivatives for the Treatment of Bacterial Infections

WO2004033463A1	Oct 10, 2003	Apr 22, 2004	Otsuka Pharma Co Ltd	2,3-DIHYDRO-6-NITROIMIDAZO[2,1-b]OXAZOLES
WO2004035547A1	Oct 14, 2003	Apr 29, 2004	Otsuka Pharma Co Ltd	1-substituted 4-nitroimidazole compound and process for producing the same
WO2008140090A1	May 7, 2008	Nov 20, 2008	Otsuka Pharma Co Ltd	Epoxy compound and method for manufacturing the same
JP2009269859A *				Title not available

It is estimated that a third of the world’s population is currently infected with tuberculosis, leading to 1.6 million deaths annually. The current drug regimen is 40 years old and takes 6-9 months to administer. In addition, the emergence of drug resistant strains and HIV co-infection mean that there is an urgent need for new anti-tuberculosis drugs. The twenty-first century has seen a revival in research and development activity in this area, with several new drug candidates entering clinical trials. This review considers new potential first-line anti-tuberculosis drug candidates, in particular those with novel mechanisms of action, as these are most likely to prove effective against resistant strains.

From among acid-fast bacteria, human Mycobacterium tuberculosis has been widely known. It is said that the one-third of the human population is infected with this bacterium. In addition to the human Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis have also been known to belong to the Mycobacterium tuberoculosis group. These bacteria are known as Mycobacteria having a strong pathogenicity to humans.

Against these tuberculoses, treatment is carried out using three agents, rifampicin, isoniazid, and ethambutol (or streptomycin) that are regarded as first-line agents, or using four agents such as the above three agents and pyrazinamide.

However, since the treatment of tuberculosis requires extremely long-term administration of agents, it might result in poor compliance, and the treatment often ends in failure.

Moreover, in respect of the above agents, it has been reported that: rifampicin causes hepatopathy, flu syndrome, drug allergy, and its concomitant administration with other drugs is contraindicated due to P450-associated enzyme induction; that isoniazid causes peripheral nervous system disorder and induces serious hepatopathy when used in combination with rifampicin; that ethambutol brings on failure of eyesight due to optic nerve disorder; that streptomycin brings on diminution of the hearing faculty due to the 8th cranial nerve disorder; and that pyrazinamide causes adverse reactions such a hepatopathy, gouty attack associated with increase of uric acid level, vomiting (A Clinician’s Guide To Tuberculosis, Michael D. Iseman 2000 by Lippincott Williams & Wilkins, printed in the USA, ISBN 0-7817-1749-3, Tuberculosis, 2nd edition, Fumiyuki Kuze and Takahide Izumi, Igaku-Shoin Ltd., 1992).

Actually, it has been reported that cases where the standard chemotherapy could not be carried out due to the adverse reactions to these agents made up 70% (approximately 23%, 52 cases) of the total cases where administration of the agents was discontinued (the total 228 hospitalized patients who were subject to the research) (Kekkaku, Vol. 74, 77-82, 1999).

In particular, hepatotoxicity, which is induced by rifampicin, isoniazid, and ethambutol out of the 5 agents used in combination for the aforementioned first-line treatment, is known as an adverse reaction that is developed most frequently. At the same time, Mycobacterium tuberculosis resistant to antitubercular agents, multi-drug-resistant Mycobacterium tuberculosis, and the like have been increasing, and the presence of these types of Mycobacterium tuberculosismakes the treatment more difficult.

According to the investigation made by WHO (1996 to 1999), the proportion ofMycobacterium tuberculosis that is resistant to any of the existing antitubercular agents to the total types of Mycobacterium tuberculosis that have been isolated over the world reaches 19%, and it has been published that the proportion of multi-drug-resistant Mycobacterium tuberculosis is 5.1%. The number of carriers infected with such multi-drug-resistant Mycobacterium tuberculosis is estimated to be 60,000,000, and concerns are still rising that multi-drug-resistantMycobacterium tuberculosis will increase in the future (April 2001 as a supplement to the journal Tuberculosis, the “Scientific Blueprint for TB Drug Development.”)

In addition, the major cause of death of AIDS patients is tuberculosis. It has been reported that the number of humans suffering from both tuberculosis and HIV reaches 10,700,000 at the time of year 1997 (Global Alliance for TB drug development). Moreover, it is considered that the mixed infection of tuberculosisand HIV has an at least 30 times higher risk of developing tuberculosis than the ordinary circumstances.

Taking into consideration the aforementioned current situation, the profiles of the desired antitubercular agent is as follows: (1) an agent, which is effective even for multi-drug-resistant Mycobacterium tuberculosis, (2) an agent enabling a short-term chemotherapy, (3) an agent with fewer adverse reactions, (4) an agent showing an efficacy to latent infecting Mycobacterium tuberculosis (i.e., latentMycobacterium tuberculosis), and (5) an orally administrable agent.

Examples of bacteria known to have a pathogenicity to humans include offending bacteria of recently increasing MAC infection (Mycobacterium avium—intracellulare complex infection) such as Mycobacterium avium andMycobacterium intracellulare, and atypical acid-fast bacteria such asMycobacterium kansasii, Mycobacterium marinum, Mycobacterium simiae, Mycobacterium scrofulaceum, Mycobacterium szulgai, Mycobacterium xenopi, Mycobacterium malmoense, Mycobacterium haemophilum, Mycobacterium ulcerans, Mycobacterium shimoidei, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium smegmatis, and Mycobacterium aurum.

Nowadays, there are few therapeutic agents effective for these atypical acid-fast bacterial infections. Under the presence circumstances, antitubercular agents such as rifampicin, isoniazid, ethambutol, streptomycin and kanamycin, a newquinolone agent that is a therapeutic agent for common bacterial infections, macrolide antibiotics, aminoglycoside antibiotics, and tetracycline antibiotics are used in combination.

However, when compared with the treatment of common bacterial infections, the treatment of atypical acid-fast bacterial infections requires a long-term administration-of agents, and there have been reported cases where the infection is changed to an intractable one, finally leading to death. To break the afore-mentioned current situation, the development of an agent having a stronger efficacy is desired.

For example, National Publication of International Patent Application No. 11-508270 (WO97/01562) discloses that a 6-nitro-1,2,3,4-tetrahydro[2,1-b]-imidazopyran compound has a bactericidal action in vitro to Mycobacterium tuberculosis (H37Rv strain) and multi-drug-resistant Mycobacterium tuberculosis, and that the above compound has a therapeutic effect to a tuberculosis-infected animal model when it is orally administered and thus useful as antitubercular agent.

Zabofloxacin

March 25, 2014 7:21 am / 1 Comment on Zabofloxacin

zabofloxacin, 219680-11-2

UNII-LV66BA6V2G, DW-224a

Molecular Formula: C₁₉H₂₀FN₅O₄

Molecular Weight: 401.391603

DONG WHA PHARMA SOUTH KOREA in phase 3

1-Cyclopropyl-6-fluoro-7-[8-(methoxyimino)-2,6-diazaspiro[3.4]oct-6-yl]-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid

poster…..http://www.jmilabs.com/data/posters/ICID2008%5C22008.pdf

Zabofloxacin is being developed as a new fluoroquinolone antibiotic that is a potent and selective inhibitor of the essential bacterial type II topoisomerases and topoisomerase IV. Zabofloxacin is indicated for community-acquired respiratory infections due to Gram-positive bacteria. The aim of this study was to compare the pharmacokinetics (PK) of the zabofloxacin hydrochloride 400 mg capsule (DW224a, 366.7 mg aszabofloxacin) with the PK of the zabofloxacin aspartate 488 mg tablet (DW224aa, 366.5 mg as zabofloxacin) in healthy Korean male volunteers to assess the bioequivalence between the two drug formulations

Zabofloxacin hydrochloride is a fluoroquinolone antibiotic with enhanced in vitro activity against Streptococcus pneumoniae, including strains resistant to other antibiotics. The spectrum of activity of zabofloxacin includes bacterial strains that are responsible for most community-acquired respiratory infections. Phase III clinical studies are currently ongoing at Dong-Wha for the treatment of patients with acute bacterial exacerbation of chronic obstructive pulmonary disease. Phase II trials had been ongoing at IASO; however no recent developments have been reported.The product candidate was originated by Dong Wha. In 2007, Dong Wha granted PB BioSciences worldwide exclusive development and marketing rights, except in Japan, Korea, China, Taiwan, Singapore, Indonesia, India, Thailand, Malaysia, Vietnam, Hong Kong, Australia and New Zealand.

Zabofloxacin was separated using an isocratic elution on a Capcell Pak C18 column using an acetonitrile–methanol–phosphate buffer (1 g of KH₂PO₄ and 1 g of heptane sulfonic acid sodium salt in 720 mL of purified water) and a 1 M tetrabutylammonium dihydrogenphosphate solution (18.5:8.5:72:1, by volume) as a mobile phase at a flow rate of 0.25 mL/min with UV detection at 275 nm. The lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) were 100 ng/mL and 20000 ng/mL, respectively, with acceptable linearity in the range from 100 to 20000 ng/mL (R > 0.999). The intra- and inter-day accuracy (RE) ranged from −8.2% to 1.8% and the intra- and inter-day precision (CV) ranged from 3.8% to 10.6% for zabofloxacin. In addition, stock solution stability, recovery, freeze–thaw effects, and short-term and long-term stability met the acceptance criteria.

…………………………

WO1999000393A1

Example 1. l-Cyclopropyl-6-fluoro-7-[8-(methoxyimino)-2,6-diazaspiro[3,4]oct-6-yl]-4- oxo-l,4-dihydro[l,8]naphthyridine-3-carboxylic acid methanesulfonate

30 350mg of

7-[2-(t-buthoxycarbonyl)-8-(methoxyimino)-2,6-diazaspiro[3.4]oct-6-yl]-l- cyclopropyl-6-fluoro-4-oxo-l,4-dihydro[l,8]naphthyridine-3-carboxylic acid was dissolved in 5ml of dichloromethane and thereto 0.6ml of trifluoroacetic acid was dropped. The mixture was stirred for 5 hours at room temperature and thereto 10ml (if ethylether was added. It was stirred additionally for 1 hour and thus precipitated solid was filtered, dissolved in 5ml of diluted NaOH and neutralized with diluted hydrochloric acid. The precipitate thus obtained was filtered and dried. The resulting solid was added to 5ml of lN-methanesulfonic acid in ethanol and stirred for 1 hour. Thus obtained precipitate was filtered and dried to give 185g of the titled compound(yield : 47.8%). m.p. : 228- 229 ^°C

1H-NMR(DMSO-dG+CF₃COOD, ppm): 0.97(s, 2H), 1.14(d, 2H), 2.48(s, 3H), 3.57(bs, IH), 3.88(s, 3H), 4.06-4.17(m, 411), 4.40(s, 2H), 4.49(s, 2H), 7.88(d, Hi, J=12.67Hz), 8.49(s, IH).

………………………………..

US20100184795

aspartate of 1-cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid comprises a step of reacting 1-cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid with aspartic acid in a solvent. The method can be represented by Scheme 1.

Example 1 Preparation of the D-Aspartic Acid Salt of 1-cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid

1-Cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid (5.0 g) was added to 50% ethanol (80 mL), and then the mixture was stirred at 50° C. for 10 minutes. D-Aspartic acid (2.0 g) was added and then the mixture was stirred at 50° C. for 1 hour. The mixture was cooled to room temperature, and then the resulting solid was collected by filtration. Ethanol (100 mL) was added to the filtrate, and then the mixture was stirred for 30 minutes. The resulting solid was collected by filtration to obtain a total of 5.55 g of the target compound (yield: 83%). Melting point: 200-201° C. ¹H NMR (D₂O): δ 0.97 (bs, 2H), 1.27 (d, 2H), 2.00 (dd, 1H, J=8.8, 17.6 Hz), 2.77 (dd, 1H, J=3.3, 17.0 Hz), 3.53 (bs, 1H), 3.84 (dd, 1H, J=3.3, 8.78 Hz), 4.01 (s, 3H), 4.31-4.45 (m, 8H), 7.46 (d, 1H, J=12.2 Hz), 8.42 (s, 1H).

Example 2 Preparation of L-Aspartic Acid Salt of 1-cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid

1-Cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid (500 mg) was added to 50% ethanol (20 mL), and then the mixture was stirred at 50° C. for 10 minutes. L-Aspartic acid (174 mg) was added and then the mixture was stirred at 50° C. for 1 hour. The mixture was cooled to room temperature. Ethanol (20 mL) was added to the reaction mixture, and then the mixture was stirred for 30 minutes. The resulting solid was collected by filtration to obtain 550 mg of the target compound (yield: 82%). Melting point: 205-206° C. ¹H NMR (d₆-DMSO): δ 0.93 (d, 2H, J=3.5 Hz), 1.20 (d, 2H, J=6.8 Hz), 2.42 (dd, 1H, J=9.2, 17.3 Hz), 2.59 (dd, 1H, J=3.3, 17.2 Hz), 3.50 (m, 1H), 3.59 (1H, dd, J=3.1, 9.1 Hz), 3.91 (s, 3H), 4.24 (m, 6H), 4.41 (br, 2H), 7.59 (d, 1H, J=12.4 Hz), 8.41 (s, 1H).

Example 3 Preparation of Hydrochloric Acid Salt, Phosphate Salt, and Formate Salt of 1-cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid

3-1 Hydrochloric Acid Salt

Ethanol (3 mL) was cooled to 0° C. and acetyl chloride (1.13 mL) was added, and then the mixture was stirred for 30 minutes. 1-Cyclopropyl-6-fluoro-7-(8-methoxyimino-2,6-diaza-spiro[3.4]oct-6-yl)-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic acid (800 mg) was added to the reaction mixture, and then stirred at 0° C. for 30 minutes. Tetrahydrofuran (4 mL) was added, and then the mixture was stirred for 30 minutes. The resulting solid was collected by filtration and dried to obtain 776 mg of the target compound (yield: 89%). Melting point: 244-245° C. ¹H NMR (d₆-DMSO): δ 1.07 (d, 2H, J=4.7 Hz), 1.21 (d, 2H, J=6.8 Hz), 3.68 (m, 1H), 3.94 (s, 3H), 4.17 (m, 2H), 4.40 (s, 2H), 4.53 (s, 2H), 8.03 (d, 1H, J=12.5 Hz), 8.59 (s, 1H).

ref

Subacute toxicity and toxicokinetics of a new antibiotic, DW-224a, after single and 4-week repeated oral administration in dogs.

Han J, Kim JC, Chung MK, Kim B, Choi DR.

Biol Pharm Bull. 2003 Jun;26(6):832-9

Fluorescence detection of Zabofloxacin, a novel fluoroquinolone antibiotic, in plasma, bile, and urine by HPLC: the first oral and intravenous applications in a pharmacokinetic study in rats.

Jin HE, Kang IH, Shim CK.

J Pharm Pharm Sci. 2011;14(3):291-305.

Determination of zabofloxacin in rat plasma by liquid chromatography with mass spectrometry and its application to pharmacokinetic study.

Jin HE, Lee KR, Kang IH, Chung SJ, Shim CK.

J Pharm Biomed Anal. 2011 Mar 25;54(4):873-7. doi: 10.1016/j.jpba.2010.11.001. Epub 2010 Nov 9.

Kosowska-Shick, K.; Credito, K.; Pankuch, G.A.; Lin, G.; Bozdogan, B.; McGhee, P.; Dewasse, B.;
Choi, D.-R.; Ryu, J.M.; Appelbaum, P.C. Antipneumococcal activity of DW-224a, a new
quinolone, compared to those of eight other agents. Antimicrob. Agents Chemother. 2006, 50,
2064–2071.

Park, H.-S.; Kim, H.-J.; Seol, M.-J.; Choi, D.-R.; Choi, E.-C.; Kwak, J.-H. In vitro and in vivo
antibacterial activities of DW-224a, a new fluoronaphthyridone. Antimicrob. Agents Chemother.
2006, 50, 2261–2264.

Dong Wha Pharmaceutical Co. Ltd. A study to evaluate efficacy and safety profile of
Zabofloxacin tablet 400 mg and moxifloxacin tablet 400 mg. Available online:
http://www.clinicaltrials.gov/ct2/show/NCT01658020 (accessed on 15 July 2013).

Dong Wha Pharmaceutical Co. Ltd. A new quinolone antibiotic. Available online:
http://www.dong-wha.co.kr/english/rnd/rnd02_03.asp (accessed on 15 April 2013).

US4957922 *	Mar 29, 1989	Sep 18, 1990	Bayer Aktiengesellschaft	Infusion solutions of 1-cyclopropyl-6-fluoro-1,4-di-hydro-4-oxo-7-(1-piperazinyl)-quinoline-3-carboxylic acid
US5563149 *	Aug 8, 1994	Oct 8, 1996	Cheil Foods & Chemicals, Inc.	Aqueous solutions of pyridone carboxylic acids
US6552196 *	Sep 6, 2001	Apr 22, 2003	Dong Wha Pharmaceutical Industrial Co., Ltd.	Quinolone carboxylic acid derivatives

US2010184795

7-23-2010

ASPARTATE OF 1-CYCLOPROPYL-6-FLUORO-7-(8-METHOXYIMINO-2,6-DIAZA-SPIRO[3.4]OCT-6-YL)-4-OXO-1,4-DIHYDRO-[1,8]NAPHTHYRIDINE-3-CARBOXYLIC ACID, METHOD FOR PREPARING THE SAME, AND ANTIMICROBIAL PHARMACEUTICAL COMPOSITION COMPRISING THE SAME

PRINOMASTAT

March 25, 2014 5:16 am / Leave a comment

PRINOMASTAT

Molecular Formula: C₁₈H₂₁N₃O₅S₂
Molecular Weight: 423.5064
CAS No: 192329-42-3
IUPAC Name: 2-[(Hydroxyamino)methyl]-5,6-dimethyl-4-(4-pyridin-4-yloxyphenyl)sulfonylmorpholine-3-thione

3-Thiomorpholinecarboxamide,N-hydroxy-2,2-dimethyl-4-[[4-(4-pyridinyloxy)phenyl]sulfonyl]-, (S)-; AG 3340;KB-R 9896; Prinomastat

Prinomastat, AG-3362(maleate), AG-3354(HCl), AG-3340

Agouron (Originator)

Prinomastat (AG-3340) is a matrix metalloprotease (MMP) inhibitor with specific selectivity for MMPs 2, 3, 9, 13, and 14. Investigations have been carried out to determine whether the inhibition of these MMPs is able to block tumour metastasis by preventing MMP degradation of the extracellular matrix proteins and angiogenesis.

Prinomastat is a synthetic hydroxamic acid derivative with potential antineoplastic activity. Prinomastat inhibits matrix metalloproteinases (MMPs) (specifically, MMP-2, 9, 13, and 14), thereby inducing extracellular matrix degradation, and inhibiting angiogenesis, tumor growth and invasion, and metastasis. As a lipophilic agent, prinomastat crosses the blood-brain barrier.

Prinomastat underwent a Phase III trial to investigate its effectiveness against non-small cell lung cancer (nsclc), in combination with gemcitabine chemotherapy. However, it was discovered that Prinomastat did not improve the outcome of chemotherapy in advanced Non-Small-Cell Lung Cancer. ^[1] ^[2]

Matrix metalloproteinases (“MMPs”) are a family of enzymes, including, collagenases, gelatinases, matrilysin, and stromelysins, that are involved in the degradation and remodeling of connective tissues. These enzymes are contained in a number of cell types that are found in or are associated with connective tissue, such as fibroblasts, monocytes, macrophages, endothelial cells and metastatic tumor cells. They also share a number of properties, including zinc and calcium dependence, secretion as zymogens, and, 40-50% amino acid sequence homology.

Matrix metalloproteinases degrade the protein components of the extracellular matrix, i.e., the protein components found in the linings of joints, interstitial connective tissue, basement membranes, cartilage and the like. These proteins include collagen, proteoglycan, fibronectin and lamanin.

In a number of pathological disease conditions, however, deregulation of matrix metalloproteinase activity leads to the uncontrolled breakdown of extracellular matrix. These disease conditions include arthritis (e.g., rheumatoid arthritis and osteoarthritis), periodontal disease, aberrant angiogenesis, tumor metastasis and invasion, tissue ulceration (e.g., comeal ulceration, gastric ulceration or epidermal ulceration), bone disease, HIV-infection and complications from diabetes.

Administration of matrix metalloproteinase inhibitors has been found to reduce the rate of connective tissue degradation, thereby leading to a favorable therapeutic effect. For example, in Cancer Res., 53, 2087 (1993), a synthetic matrix metalloproteinase inhibitor was shown to have in vivo efficacy in a murine model for ovarian cancer with an apparent mode of action consistent with inhibition of matrix remodeling. The design and uses of MMP inhibitors are reviewed, for example, in J. Enzyme Inhibition, 2, 1-22 (1987); Progress in Medicinal Chemistry, 29, 271-334 (1992); Current Medicinal Chemistry, 2, 743-762 (1995); Exp. Opin. Ther. Patents, 5, 12871296 (1995); and Drug Discovery Today, 1, 16-26 (1996).

Matrix metalloproteinase inhibitors are also the subject of numerous patents and patent applications, including: U.S. Pat. Nos. 5,189,178; 5,183,900; 5,506,242; 5,552,419; and 5,455,258; European Patent Application Nos. EP 0 438 223 and EP 0 276 436; International Publication Nos. WO 92/21360; WO 92/06966; WO 92/09563; WO 96/00214; WO 95/35276; and WO 96/27583.

Further, U.S. patent application Ser. Nos. 6,153,757 and 5,753,653 relate to prinomistat and its synthesis, the disclosures of each are incorporated herein by reference in their entireties.

Prinomastat, shown below, is a potent inhibitor of certain metalloproteinases (MMP), particularly matrix metalloproteinases and tumor necrosis factor-α convertase. International Publication No. WO 97/208824 discloses the chemical structure of prinomastat, its pharmaceutical composition, as well as pharmaceutical uses, methods of its preparation and intermediates useful in its synthesis.

Until now, metabolites of prinomastat have not been identified, isolated, purified or synthesized. Further, it is shown that some of these metabolites are potent matrix metalloproteinase inhibitors

The sulfonation of 4-chlorodiphenyl ether (I) with chlorosulfonic acid in dichloromethane gives the 4-(4-chlorophenoxy)benzenesulfonic acid (II), which is treated with oxalyl chloride and DMF in the same solvent yielding the sulfonyl chloride (III).

The reduction of (III) with trimethyl phosphite and KOH in toluene affords the methylsulfanyl derivative (IV), which is chlorinated with SO2Cl2 in dichloromethane to give the chloromethylsulfanyl derivative (V). The condensation of (V) with the silylated enol ether (VI) by means of ZnCl2 and KOH in refluxing dichloromethane yields 4-[4-(4-chlorophenoxy)phenylsulfanylmethyl]tetrahydropyran-4-carboxylic acid (VII), which is treated with oxalyl chloride affording the corresponding acyl chloride (VIII).

The reaction of (VIII) with NH2OH in dichloromethane provides the carbohydroxamic acid (IX), which is finally oxidized with oxone (potassium peroxymonosulfate) in N-methyl-2-pyrrolidone/H2O to furnish the target sulfone.

The cyclization of D-penicillamine (I) with 1,2-dichloroethane by means of DBU and TMS-Cl in DMF gives 2,2-dimethylthiomorpholine-3(S)-carboxylic acid (XV), which is treated with isobutylene (XVI) and sulfuric acid in dioxane to yield the corresponding tert-butyl ester (XVII). The sulfonation of (XVII) with the sulfonyl chloride (VI) as before affords 2,2-dimethyl-4-[4-(4-pyridyloxy)phenylsulfonyl]thiomorpholine-3(S)-carboxylic acid tert-butyl ester (XVIII), which is finally treated with HCl in refluxing dioxane to give the previously reported free acid intermediate (XIV).

The cyclization of D-penicillamine methyl ester (XIX) with 1,2-dibromoethane by means of DBU in DMF gives 2,2-dimethylthiomorpholine-3(S)-carboxylic acid methyl ester (XX), which is sulfonated with the sulfonyl chloride (VI) as before, affording 2,2-dimethyl-4-[4-(4-pyridyloxy)phenylsulfonyl]thiomorpholine-3(S)-carboxylic acid methyl ester (XXI). Finally, this compound is hydrolyzed with refluxing aqueous HCl to yield the previously reported intermediate (XIV).

The silylation of D-penicillamine (I) with dimethylhexylsilyl chloride (Dmhs-Cl) and DBU gives the ester (XI), which is cyclized with 1,2-dichloroethane and DBU in DMF, yielding 2,2-dimethylthiomorpholine-3(S)-carboxylic acid dimethylhexylsilyl ester (XII).

The sulfonation of (XII) with the sulfonyl chloride (VI) as before affords 2,2-dimethyl-4-[4-(4-pyridyloxy)phenylsulfonyl]thiomorpholine-3(S)-carboxylic acid dimethylhexylsilyl ester (XIII), which is desilylated in refluxing methanol to give the free acid (XIV) Finally, this compound is treated with oxalyl chloride and hydroxylamine in dichloromethane.

References

Hande, Kenneth R; Mary Collier, Linda Paradiso, Jill Stuart-Smith, Mary Dixon, Neil Clendeninn, Geoff Yeun, Donna Alberti, Kim Binger and George Wilding (2004). “Phase I and Pharmacokinetic Study of Prinomastat, a Matrix Metalloprotease Inhibitor”. Journal of Drugs in Dermatology: JDD 3 (4): 393–7. PMID 15303783.
Bissett, K Donald; en J. O’Byrne, J. von Pawel, Ulrich Gatzemeier, Allan Price, Marianne Nicolson, Richard Mercier, Elva Mazabel, Carol Penning, Min H. Zhang, Mary A. Collier, Frances A. Shepherd (2005). “Phase III Study of Matrix Metalloproteinase Inhibitor Prinomastat in Non–Small-Cell Lung Cancer”.Journal of Clinical Oncology 10: 909. doi:10.1158/1078-0432.CCR-0981-3.

clinical trial results

1. Phase II, prinomastat in patients with esophageal adenocarcinoma.

All patients, regardless of treatment arm, were able to successfully undergo neoadjuvant combined modality therapy and esophagectomy. However, early closure of the study due to unexpected thrombo-embolic events precluded any conclusions regarding clinical activity of prinomastat in locally advanced esophageal cancer patients.

2. Phase III study of prinomastat in non-small-cell lung cancer.

Prinomastat does not improve the outcome of chemotherapy in advanced NSCLC.

Vatiquinone, バチキノン

March 19, 2014 3:59 am / Leave a comment

ChemSpider 2D Image | Vatiquinone | C29H44O3

Vatiquinone

バチキノン

Vatiquinone; Alpha-Tocotrienol quinone; EPI-743; UNII-6O85FK9I0X; 1213269-98-7; Vincerenone

Molecular Formula:	C₂₉H₄₄O₃
Molecular Weight:	440.668 g/mol

2-[(3R,6E,10E)-3-hydroxy-3,7,11,15-tetramethylhexadeca-6,10,14-trienyl]-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione

2-((R,6E,10E)-3-hydroxy-3,7,11,15-tetramethylhexadeca-6,10,14-trien-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione

2-[(3R,6E,10E)-3-hydroxy-3,7,11,15-tetramethylhexadeca-6,10,14-trien-1-yl]-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione

6O85FK9I0X

9604

Research Code：EPI-743; ATQ-3, BioE-743

MOA：Mitochondria

Originator Edison Pharmaceuticals
Developer Edison Pharmaceuticals; Sumitomo Dainippon Pharma; University of Florida; Yale University
Class Alkadienes; Benzoquinones; Cyclohexenes; Small molecules
Mechanism of Action Antioxidants; NQO1 modulators

Orphan Drug Status Yes – Mitochondrial disorders; Leigh disease; Friedreich’s ataxia
New Molecular Entity Yes

Highest Development Phases

Phase III Leigh disease
Phase II Friedreich’s ataxia; Methylmalonic acidaemia; Mitochondrial disorders; Noise-induced hearing loss; Parkinson’s disease; Rett syndrome
No development reported Gilles de la Tourette’s syndrome

Most Recent Events

04 Nov 2017 No recent reports of development identified for phase-I development in Gilles-de-la-Tourette’s-syndrome in USA (PO)
01 Apr 2017 Efficacy data from a phase II trial in Friedreich’s ataxia presented at the 69^th Annual Meeting of the American Academy of Neurology (AAN- 2017)
16 Apr 2016 Initial efficacy and safety data from a phase IIa trial in Parkinson’s disease presented at the 68th Annual Meeting of the American Academy of Neurology (AAN – 2016)

Vatiquinone is in phase II/III clinical trials for the treatment of leigh syndrome in JP. Phase II clinical trials is also ongoing for Friedreich’s ataxia, Parkinson’s disease, Pearson syndrome, cobalamin C deficiency syndrome, hearing loss and Rett’s syndrome.

Vatiquinone was originally developed by Edison Pharmaceuticals, then licensed to Sumitomo Dainippon Pharma in Japan in 2013.

Orphan drug designations for the treatment of Friedreich’s, Leigh syndrome and Rett’s syndrome were granted to the compound by FDA in 2014.
In 2013, the compound was licensed to Sumitomo Dainippon Pharma by Edison Pharmaceuticals in Japan for development and commercialization for the treatment of pediatric orphan inherited mitochondrial and adult central nervous system diseases.

On 17 January 2018, orphan designation (EU/3/17/1971) was granted by the European Commission to Edison Orphan Pharma BV, The Netherlands, for vatiquinone (also known as alpha-tocotrienol quinone) for the treatment of RARS2 syndrome.

http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/orphans/2018/03/human_orphan_002075.jsp&mid=WC0b01ac058001d12b

Vatiquinone, also known as EPI 743, is an orally bioavailable para-benzoquinone being developed for inherited mitochondrial diseases. The mechanism of action of EPI-743 involves augmenting the synthesis of glutathione, optimizing metabolic control, enhancing the expression of genetic elements critical for cellular management of oxidative stress, and acting at the mitochondria to regulate electron transport.

Vatiquinone has been investigated for the treatment and prevention of Retinopathy, Rett Syndrome, Genetic Disease, Noise-induced Hearing Loss, and Methylmalonic Aciduria and Homocystinuria,Cblc Type.

EPI-743 (vatiquinone) is a compound being developed by BioElectron (previously known as Edison Pharmaceuticals) to treat Friedreich’s ataxia (FA), a rare, autosomal recessive genetic disorder. The disorder is caused by mutations in the FXN gene, which encodes for a protein called frataxin. Frataxin is required for the normal functioning of mitochondria, or the energy factories of the cells. Decreased levels of frataxin, as observed in patients with FA, disrupts the normal function of mitochondria and leads to the gradual development of symptoms associated with the disease: impairment of muscle coordination, loss of muscle strength and sensation, and impaired speech, vision, and hearing.

Currently, there are no drugs available that could cure or help to effectively manage the condition, although a large number of potential treatments are in the pipeline.

How EPI-743 works

EPI-743 is a drug belonging to the class of para-benzoquinones, a group of potent antioxidants. The regulation of oxidative stress is disturbed in people with FA. EPI-743 targets an enzyme called NADPH quinone oxidoreductase 1 (NQO1), helping to increase the biosynthesis of glutathione, a compound essential for the control of oxidative stress. The drug does not target any FA-specific biochemical pathways directly, but helps to improve the regulation of cellular energy metabolism in general. Due to its non-specific mechanism, the drug can be used in a variety of disorders where mitochondrial function is affected.

EPI-743 in clinical trials

In December 2012, Edison Pharmaceuticals started a placebo-controlled Phase 2 study (NCT01728064) to examine the safety and efficacy of EPI-743 on visual and neurological function in FA patients. The study was completed in February 2016. The results indicated no significant differences in visual function at six months between patients treated with EPI-743 and those who received a placebo. However, researchers reported a trend toward improvement in neurological function.

In October 2013, the University of South Florida started a small Phase 2 study (NCT01962363) to evaluate the effects of EPI-743 in patients with rare point mutations leading to FA. The study investigated whether treatment with EPI-743 has a discernible impact on neurological function. The results announced in April 2016 demonstrated significant improvements in neurological functions over 18 months. However, the trial only included three participants.

Currently, no further trials testing EPI-743 in FA patients is taking place. However, the drug is in clinical trials for several other disorders that affect the functions of mitochondria, including Leigh syndrome, mitochondrial respiratory chain disease, Pearson syndrome, and others.

Other information

In February 2014, the U.S. Food and Drug Administration (FDA) granted orphan drug status to EPI-743, which allows a more expedited drug approval process. The FDA also granted fast track status to EPI-743 for the treatment of FA in March 2014.

ADDITIONAL INFORMATION

Edison Pharmaceuticals is developing vatiquinone, which was awarded Fast Track status for Friedreich’s ataxia in March 2014.

Reference

Bioorg. Med. Chem. Lett. 2011, 21, 3693-3698.

https://www.sciencedirect.com/science/article/pii/S0960894X11005440

Reference

WO2013041676A1 / US9045402B2.

It is known that a-tocotrienol quinones are pharmaceutically active.

US 201 1 /0172312 A1 discloses that tocotrienol quinones are used in treating Leight Syndrome. WO 2010/126909 A1 and US 2006/0281809 A1 disclose that tocotrienol quinones can be used for treating ophthalmic diseases and mitochondrial diseases. US 5,318,993 discloses the activity of tocotrienol quinones as cholesterol suppression. W.D. Shrader et al., Bioorganic & Medical Chemistry Letters 21 (201 1 ), 3693-3698 disclose that the R-isomer of a-tocotrienol quinone is a metabolite of α-tocotrienol and is a potent cellular protectant against oxidative stress and ageing. The R-isomer of α-tocotrienol used for this study has been extracted from Elaeis guineensis. All these documents either use tocotrienol from natural sources or do not disclose the source of tocotrienol respectively tocotrienol quinones or disclose very specific complex synthesis thereof. These methods are very expensive and limited in producing industrial amounts of the desired products.

It is well known that from vitamin E the tocopherols and tocotrienols having the R-configuration have a significantly higher bioactivity (biopotency) than the corresponding S-isomer. This is also the case for the corresponding R-isomers of tocotrienol quinones.

Synthetic pathways to produce the R-isomer of tocotrienol quinones in a stereospecific way are very expensive and therefore only of limited interest.

The synthesis of a-tocotrienol is known from Kabbe and Heitzer, Synthesis 1978, 888-889, however, no indication of chirality whatsoever is indicated.

The synthesis of tocotrienol from the corresponding 4-oxo-chromanol-derivative is known from US 6,096,907, however, no indication of chirality is indicated.

J. Org. Chem. 1981 , 46, 2445-2450 and CH 356754 disclose the chemical transformation of a-tocopherol to a-tocopheryl quinone and to a-tocopherylhydro-quinone, however, neither tocotrienols nor tocotrienol quinones are mentioned.

Separation of chiral compounds by chromatography is principally known. However, it is also known that the quantitative separation is very often very difficult to achieve.

Due to the importance of these substances, there exists a high interest in a process which would produce R-tocotrienol quinones in a large scale in an easy and economic way.

Examples

The present invention is further illustrated by the following experiments.

1 . Chromatographic separation

Starting materials:

Solvents and reagents used as received were heptane (Fluka, 51750), ethanol (Merck, 1 .00983), isopropanol (Sigma-Aldrich, 59300) and acetic acid (Fluka, 45730).

Chromatography:

Preparative separations were performed on an Agilent 1 100 series hplc system consisting of an Agilent 1 100 degasser, Agilent 1 100 preparative pump, Agilent 1 100 diode array detector, Agilent 1 100 MPS G2250A autosampler/fraction collector controlled by chemstation/CC-mode software package.

HPLC conditions for preparative separation:

Column: Daicel Chiracel® OD-H, 250 mm x 20 mm; eluent 0.5% isopropanol, 0.2 % acetic acid in n-heptane; flow 13 ml/min; detection 220 nm, 400 μΙ injection.

Separation of (R)-6-hydroxy-2,5,7,8-tetramethyl-2-((3E,7E)-4,8, 12-trimethyl-trideca-3,7, 11-trienyl) chroman-4-one and (S)-6-hydroxy-2,5,7,8-tetramethyl-2-((3E, 7E)-4,8, 12-trimethyltrideca-3, 7, 11-trienyl) chroman-4-one

Example 1 :

6-Hydroxy-2,5,7,8-tetramethyl-2-((3E,7E)-4,8,12-trimethyltrideca-3,7,1 1 -trienyl) chroman-4-one was prepared according to the example 6a in Kabbe and Heitzer, Synthesis 1978, 888-889.

The product was analyzed by HPLC (Column: Daicel Chiracel® OD-H, 250 mm x 4.6 mm; eluent 1 % ethanol in n-hexane; flow 1 ml/min; detection 220 nm, 2 μΙ injection). Figure 9 b) shows this chromatogram. It shows that the product is a 49.5 : 50.5 mixture (Retention time 13.2 and 14.2 min.)

87.5 mg of this product in heptane was injected and the two peaks with retention time at maximum 35.4 min. (1 ) (50.9%) resp. 43.5 min. (2) (49.1 %) were se-parated by the preparative HPLC separation. Figure 9 a) shows the chromatogram of the preparative HPLC separation.

After evaporation to dryness and dissolution the two collected fractions have been reanalysis on an analytical column (Daicel Chiracel® OD-H, 250 mm x 4.6 mm; eluent 1 % ethanol in n-hexane; flow 1 ml/min; detection 220 nm, 2 μΙ injection). Figure 9 c), respectively Figure 9 d), show the chromatogram of the first fraction, respectively the second fraction. The separation of the two isomers (Retention time 13.2 min, resp. 14.2 min) in the two fraction shows to be 94.9 : 5.1 (Figure 9 c)) resp. 7.1 : 92.9 (Figure 9 d)). Hence, the two isomers have been separation by preparative chromatography almost completely.

Patent

WO2010126909

The active component of the formulation of the present invention is selected from alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, and mixtures thereof. In one embodiment, the formulation of the present invention comprises alpha-tocotrienol quinone as the active component. In other embodiments, the formulations of the present invention comprise one or more tocotrienol quinones of Formula I or mixtures thereof, in a pharmaceutically acceptable vehicle, and in other embodiments, the formulations of the present invention comprise alpha-tocotrienol quinone in a pharmaceutically acceptable vehicle. In other particular embodiments, the formulations are administered orally. In other embodiments, the formulations of the present invention comprise one or more tocotrienol quinones of Formula I or mixtures thereof, in an ophthalmically acceptable vehicle for topical, periocular, or intraocular administration, and in other embodiments, the formulations of the present invention comprise alpha-tocotrienol quinone in an ophthalmically acceptable vehicle.

[0120] The formulations of the present invention comprise tocotrienol quinones which can be produced synthetically from the respective tocotrienol by oxidation with suitable oxidizing agents, as for example eerie ammonium nitrate (CAN). Particularly, the formulations of the present invention comprise alpha-tocotrienol quinone (CAS Reg. No. 1401-66-7) produced by oxidation of alpha-tocotrienol. A preferred process for the production of alpha-tocotrienol has been described in co-owned US provisional application USAN 61/197,585 titled “Process for Enrichment and Isolation of alpha-Tocotrienol from Natural Extracts”.

[0121] Syntheses of various members of the tocotrienol family in the d,l- or (RS)-form have been published, see for example Schudel et al, HeIv. Chim. Acta (1963) 46, 2517-2526; H. Mayer et al, HeIv. Chim. Acta (1967) 50, 1376-11393; H.-J. Kabbe et al, Synthesis (1978), 888-889; M. Kajiwara et al, Heterocycles (1980) 14, 1995-1998; S. Urano et al, Chem. Pharm. Bull. (1983) 31, 4341-4345, Pearce et al, J. Med Chem. (1992), 35, 3595-3606 and Pearce et al, J. Med. Chem. (1994). 37, 526-541. None of these reported processes lead to the natural form of the tocotrienols, but rather produces racemic mixtures. Syntheses of natural form d-tocotrienols have been published. See for example. J. Scott et al, HeIv. CMm. Acta (1976) 59, 290-306, Sato et al. (Japanese Patent 63063674); Sato et al. (Japanese Patent NoJP 01233278) and Couladouros et al. (US Patent No. 7,038,067).

[0122] While synthetic and natural tocopherols are readily available in the market, the natural tocotrienol supply is limited, and generally comprises a mixture of tocotrienols. Crude palm oil which is rich in tocotrienols (800-1500 ppm) offers a potential source of natural tocotrienols. Carotech, Malaysia is able to extract and concentrate tocotrienols from crude palm oil, by a process patented in U.S. Pat. No. 5,157,132. Tocomin®-50 typically comprises about 25.32% mixed tocotrienols (7.00% alpha-tocotrienol, 14.42% gamma-tocotrienol, 3.30% delta-tocotrienol and 0.6% beta-tocotrienol ), 6.90% alpha-tocopherol and other phytonutrients such as plant squalene, phytosterols, co-enzyme QlO and mixed carotenoids.

[0123] Other methods for isolation or enrichment of tocotrienol from certain plant oils and plant oil by-products have been described in the literature. For some examples of such isolation and purification processes, see for instance Top A. G. et al, U.S. Pat. No. 5,190,618; Lane R et al, U.S. Pat No. 6,239,171; Bellafiore, L. et al. U.S. Pat. No.6,395,915; May, CY et al, U.S. Pat. No.6,656,358; Jacobs, L et al, U.S. Pat. No. 6,838,104; Sumner, C et al. Int. Pat. Pub. WO 99/38860, or Jacobs, L, Int. Pat. Pub. WO 02/500054. The compounds for use in the present invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions for use in the present invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with other therapeutic agents, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

REFERENCES

1: Peragallo JH, Newman NJ. Is there treatment for Leber hereditary optic neuropathy? Curr Opin Ophthalmol. 2015 Nov;26(6):450-7. doi: 10.1097/ICU.0000000000000212. PubMed PMID: 26448041; PubMed Central PMCID: PMC4618295.

2: Miller DK, Menezes MJ, Simons C, Riley LG, Cooper ST, Grimmond SM, Thorburn DR, Christodoulou J, Taft RJ. Rapid identification of a novel complex I MT-ND3 m.10134C>A mutation in a Leigh syndrome patient. PLoS One. 2014 Aug 12;9(8):e104879. doi: 10.1371/journal.pone.0104879. eCollection 2014. PubMed PMID: 25118196; PubMed Central PMCID: PMC4130626.

3: Strawser CJ, Schadt KA, Lynch DR. Therapeutic approaches for the treatment of Friedreich’s ataxia. Expert Rev Neurother. 2014 Aug;14(8):949-57. doi: 10.1586/14737175.2014.939173. Epub 2014 Jul 18. PubMed PMID: 25034024.

4: Enns GM. Treatment of mitochondrial disorders: antioxidants and beyond. J Child Neurol. 2014 Sep;29(9):1235-40. doi: 10.1177/0883073814538509. Epub 2014 Jun 30. PubMed PMID: 24985754.

5: Avula S, Parikh S, Demarest S, Kurz J, Gropman A. Treatment of mitochondrial disorders. Curr Treat Options Neurol. 2014 Jun;16(6):292. doi: 10.1007/s11940-014-0292-7. PubMed PMID: 24700433; PubMed Central PMCID: PMC4067597.

6: Hargreaves IP. Coenzyme Q10 as a therapy for mitochondrial disease. Int J Biochem Cell Biol. 2014 Apr;49:105-11. doi: 10.1016/j.biocel.2014.01.020. Epub 2014 Feb 2. Review. PubMed PMID: 24495877.

7: Chicani CF, Chu ER, Miller G, Kelman SE, Sadun AA. Comparing EPI-743 treatment in siblings with Leber’s hereditary optic neuropathy mt14484 mutation. Can J Ophthalmol. 2013 Oct;48(5):e130-3. doi: 10.1016/j.jcjo.2013.05.011. PubMed PMID: 24093206.

8: Pastore A, Petrillo S, Tozzi G, Carrozzo R, Martinelli D, Dionisi-Vici C, Di Giovamberardino G, Ceravolo F, Klein MB, Miller G, Enns GM, Bertini E, Piemonte F. Glutathione: a redox signature in monitoring EPI-743 therapy in children with mitochondrial encephalomyopathies. Mol Genet Metab. 2013 Jun;109(2):208-14. doi: 10.1016/j.ymgme.2013.03.011. Epub 2013 Mar 24. PubMed PMID: 23583222.

9: Sadun AA, La Morgia C, Carelli V. Mitochondrial optic neuropathies: our travels from bench to bedside and back again. Clin Experiment Ophthalmol. 2013 Sep-Oct;41(7):702-12. doi: 10.1111/ceo.12086. Epub 2013 Apr 11. Review. PubMed PMID: 23433229.

10: Kerr DS. Review of clinical trials for mitochondrial disorders: 1997-2012. Neurotherapeutics. 2013 Apr;10(2):307-19. doi: 10.1007/s13311-013-0176-7. Review. PubMed PMID: 23361264; PubMed Central PMCID: PMC3625388.

11: Blankenberg FG, Kinsman SL, Cohen BH, Goris ML, Spicer KM, Perlman SL, Krane EJ, Kheifets V, Thoolen M, Miller G, Enns GM. Brain uptake of Tc99m-HMPAO correlates with clinical response to the novel redox modulating agent EPI-743 in patients with mitochondrial disease. Mol Genet Metab. 2012 Dec;107(4):690-9. doi: 10.1016/j.ymgme.2012.09.023. Epub 2012 Sep 28. PubMed PMID: 23084792.

12: Martinelli D, Catteruccia M, Piemonte F, Pastore A, Tozzi G, Dionisi-Vici C, Pontrelli G, Corsetti T, Livadiotti S, Kheifets V, Hinman A, Shrader WD, Thoolen M, Klein MB, Bertini E, Miller G. EPI-743 reverses the progression of the pediatric mitochondrial disease–genetically defined Leigh Syndrome. Mol Genet Metab. 2012 Nov;107(3):383-8. doi: 10.1016/j.ymgme.2012.09.007. Epub 2012 Sep 10. PubMed PMID: 23010433.

13: Büsing A, Drotleff AM, Ternes W. Identification of α-tocotrienolquinone epoxides and development of an efficient molecular distillation procedure for quantitation of α-tocotrienol oxidation products in food matrices by high-performance liquid chromatography with diode array and fluorescence detection. J Agric Food Chem. 2012 Aug 29;60(34):8302-13. doi: 10.1021/jf301137b. Epub 2012 Aug 16. PubMed PMID: 22747466.

14: Sadun AA, Chicani CF, Ross-Cisneros FN, Barboni P, Thoolen M, Shrader WD, Kubis K, Carelli V, Miller G. Effect of EPI-743 on the clinical course of the mitochondrial disease Leber hereditary optic neuropathy. Arch Neurol. 2012 Mar;69(3):331-8. doi: 10.1001/archneurol.2011.2972. PubMed PMID: 22410442.

15: Enns GM, Kinsman SL, Perlman SL, Spicer KM, Abdenur JE, Cohen BH, Amagata A, Barnes A, Kheifets V, Shrader WD, Thoolen M, Blankenberg F, Miller G. Initial experience in the treatment of inherited mitochondrial disease with EPI-743. Mol Genet Metab. 2012 Jan;105(1):91-102. doi: 10.1016/j.ymgme.2011.10.009. Epub 2011 Oct 21. PubMed PMID: 22115768.

16: Shrader WD, Amagata A, Barnes A, Enns GM, Hinman A, Jankowski O, Kheifets V, Komatsuzaki R, Lee E, Mollard P, Murase K, Sadun AA, Thoolen M, Wesson K, Miller G. α-Tocotrienol quinone modulates oxidative stress response and the biochemistry of aging. Bioorg Med Chem Lett. 2011 Jun 15;21(12):3693-8. doi: 10.1016/j.bmcl.2011.04.085. Epub 2011 Apr 24. PubMed PMID: 21600768.

17: Gagnon KT. HD Therapeutics – CHDI Fifth Annual Conference. IDrugs. 2010 Apr;13(4):219-23. PubMed PMID: 20373247.

18: Bidichandani SI, Delatycki MB. Friedreich Ataxia. 1998 Dec 18 [updated 2014 Jul 24]. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Fong CT, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from http://www.ncbi.nlm.nih.gov/books/NBK1281/ PubMed PMID: 20301458.

19: Yu-Wai-Man P, Chinnery PF. Leber Hereditary Optic Neuropathy. 2000 Oct 26 [updated 2013 Sep 19]. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Fong CT, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016. Available from http://www.ncbi.nlm.nih.gov/books/NBK1174/ PubMed PMID: 20301353.

バチキノン
Vatiquinone

C₂₉H₄₄O₃ : 440.66
[1213269-98-7]

Patent ID	Title	Submitted Date	Granted Date
US9162957	METHODS FOR SELECTIVE OXIDATION OF ALPHA TOCOTRIENOL IN THE PRESENCE OF NON-ALPHA TOCOTRIENOLS	2012-07-19	2014-09-04
US9670545	METHODS AND KITS FOR TREATING AND CLASSIFYING INDIVIDUALS AT RISK OF OR SUFFERING FROM TRAP1 CHANGE-OF-FUNCTION	2014-06-11	2016-06-30
US2017297991	METHODS FOR SELECTIVE OXIDATION OF ALPHA TOCOTRIENOL IN THE PRESENCE OF NON-ALPHA TOCOTRIENOLS	2017-01-20
US2014221674	PROCESS FOR THE PRODUCTION OF ALPHA-TOCOTRIENOL AND DERIVATIVES	2013-09-26	2014-08-07
US8575369	Process for the production of alpha-tocotrienol and derivatives	2012-01-25	2013-11-05

Patent ID	Title	Submitted Date	Granted Date
US2017037023	PROCESS FOR THE PRODUCTION OF ALPHA-TOCOTRIENOL AND DERIVATIVES	2016-03-11
US9670170	RESORUFIN DERIVATIVES FOR TREATMENT OF OXIDATIVE STRESS DISORDERS	2014-03-14	2016-02-11
US9296712	RESORUFIN DERIVATIVES FOR TREATMENT OF OXIDATIVE STRESS DISORDERS	2013-03-15	2014-09-18
US8106223	PROCESS FOR THE PRODUCTION OF ALPHA-TOCOTRIENOL AND DERIVATIVES	2010-04-29	2012-01-31
US9567279	METHODS FOR SELECTIVE OXIDATION OF ALPHA TOCOTRIENOL IN THE PRESENCE OF NON-ALPHA TOCOTRIENOLS	2015-09-10	2016-01-07

////////////orphan drug status, EPI-743, fast track, EPI743, EPI-743, EPI 743, Vatiquinone; alpha-Tocotrienol quinone, Vincerenone, バチキノン , BioE-743

CC1=C(C(=O)C(=C(C1=O)C)CCC(C)(CCC=C(C)CCC=C(C)CCC=C(C)C)O)C

Biogen Idec, Atlas Venture Pump $17M into Ataxion

Biogen Idec and Atlas Venture have agreed to invest a combined $17 million of Series A financing in a nearly-year-old drug developer focused on hereditary ataxias. Biogen Idec is separately providing R&D and other funding to the company, called Ataxion. The biotech giant has the option to acquire Ataxion to continue development of the program upon completion of a Phase I multiple ascending dose (MAD) study at pre-negotiated terms, including undisclosed upfront and milestone payments. Earlier this month, Edison Pharmaceuticals won FDA “fast-track” designation for its own Fredrich’s ataxia drug, the company’s lead drug candidate EPI-743, now in Phase II trials. And on February 12, the developer of a preclinical gene therapy for Friedrich’s ataxia, Voyager Therapeutics, was launched by Third Rock Ventures with $45 million in Series A financing. read at http://www.genengnews.com/gen-news-highlights/biogen-idec-atlas-venture-pump-17m-into-ataxion/81249632/
EPI-743 is being developed at Edison Pharmaceuticals in phase II clinical trials for several indications; Leigh syndrome, Friedreich’s ataxia, Parkinson’s disease, Pearson syndrome, cobalamin C deficiency syndrome and Rett’s syndrome. The licensee, Dainippon Sumitomo is developing the product in phase II/III study for the treatment of Leigh syndrome in children. Preclinical studies are also underway for the treatment of Huntington’s disease. In 2011, an orphan drug designation was assigned by the FDA for the treatment of inherited mitochondrial respiratory chain diseases and by the EMA for the treatment of Leigh syndrome, and in 2014, the FDA assigned another orphan drug for the treatment of Friedreich’s ataxia. In 2014, the product was granted fast track designation for this indication. In 2013, the compound was licensed to Dainippon Sumitomo Pharma by Edison Pharmaceuticals in Japan for development and commercialization for the treatment of pediatric orphan inherited mitochondrial and adult central nervous system diseases.
OLD ARTICLE

19 February 2013 EPI-743 Vatiquinone is a new drug that is based on vitamin E. Tests have shown that it can help improve the function of cells with mitochondrial problems. It may be able to treat people with genetic disorders that affect metabolism and mitochondria Edison Pharmaceuticals and Bambino Gesu Children’s Hospital have announced the commencement of EPI-743 Phase 2 cobalamin C deficiency syndrome trial. EPI-743 is an orally bioavailable small molecule and a member of the para-benzoquinone class of drugs. The trial’s principal investigator, Bambino Gesu Children’s Hospital, division of metabolism Professor Carlo Dionisi-Vici said, “Given the central role of glutathione in cellular redox balance and antioxidant defense systems, we are eager to explore whether a therapeutic that increases glutathione such as EPI-743 will provide clinical benefit.” Improvement in visual function is the primary endpoint of the placebo-controlled study while secondary outcome measurements assess neurologic and neuromuscular function, glutathione biomarkers, quality of life, in addition to safety parameters. The investigation is aimed at assessing the efficacy of EPI-743 in disorders of intermediary metabolism that also result in redox disturbances. EPI-743 is an orally absorbed small molecule that readily crosses into the central nervous system. It works by targeting the enzyme NADPH quinone oxidoreductase 1 (NQO1). Its mode of action is to synchronize energy generation in mitochondria with the need to counter cellular redox stress Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative and cardiodegenerative disorder caused by decreased levels of the protein frataxin. The disease causes the progressive loss of voluntary motor coordination (ataxia) and cardiac complications. Symptoms typically begin in childhood, and the disease progressively worsens as the patient grows older; patients eventually become wheelchair-bound due to motor disabilities. Patients with Friedreich’s ataxia develop loss of visual acuity or changes in color vision. Most have jerky eye movements (nystagmus), but these movements by themselves do not necessarily interfere with vision. ……………… Bioorg Med Chem Lett 2011, 21(12): 3693 http://www.sciencedirect.com/science/article/pii/S0960894X11005440We report that α-tocotrienol quinone (ATQ3) is a metabolite of α-tocotrienol, and that ATQ3 is a potent cellular protectant against oxidative stress and aging. ATQ3 is orally bioavailable, crosses the blood–brain barrier, and has demonstrated clinical response in inherited mitochondrial disease in open label studies. ATQ3 activity is dependent upon reversible 2e-redox-cycling. ATQ3 may represent a broader class of unappreciated dietary-derived phytomolecular redox motifs that digitally encode biochemical data using redox state as a means to sense and transfer information essential for cellular function.

Figure 1.

The conversion of α-tocotrienol to α-tocotrienol quinone.

Figure 1.

The conversion of α-tocotrienol to α-tocotrienol quinone.

Amgen Drug Evolocumab Hits Endpoint of Cholesterol Reduction

March 19, 2014 3:17 am / 2 Comments on Amgen Drug Evolocumab Hits Endpoint of Cholesterol Reduction

Amgen announced that the Phase 3 TESLA (Trial Evaluating PCSK9 Antibody in Subjects with LDL Receptor Abnormalities) trial evaluating evolocumab met its primary endpoint of the percent reduction from baseline at week 12 in low-density lipoprotein cholesterol (LDL-C). The percent reduction in LDL-C, or “bad” cholesterol, was clinically meaningful and statistically significant………….read at

http://www.dddmag.com/news/2014/03/amgen-drug-hits-endpoint-cholesterol-reduction?et_cid=3829907&et_rid=523035093&type=cta

Evolocumab
Monoclonal antibody
Source	Human
Target	PCSK9
Clinical data
Legal status	?
Identifiers
CAS number	1256937-27-5
ATC code	None
Chemical data
Formula	C₆₂₄₂H₉₆₄₈N₁₆₆₈O₁₉₉₆S₅₆
Mol. mass	141.8 kDa

Evolocumab^[1] is a monoclonal antibody designed for the treatment of hyperlipidemia.^[2] Evolocumab is a fully human monoclonal antibody that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9).

PCSK9 is a protein that targets LDL receptors for degradation and thereby reduces the liver’s ability to remove LDL-C, or “bad” cholesterol, from the blood.

Evolocumab, being developed by Amgen scientists, is designed to bind to PCSK9 and inhibit PCSK9 from binding to LDL receptors on the liver surface. In the absence of PCSK9, there are more LDL receptors on the surface of the liver to remove LDL-C from binding to LDL receptors on the liver surface. In the absence of PCSK9, there are more LDL receptors on the surface of the liver to remove LDL-C from the blood.

On 23 January 2014 Amgen announced that the Phase 3 GAUSS-2 (Goal Achievement After Utilizing an Anti-PCSK9 Antibody in Statin Intolerant Subjects-2) trial evaluating evolocumab in patients with high cholesterol who cannot tolerate statins met its co-primary endpoints: the percent reduction from baseline in low-density lipoprotein cholesterol (LDL-C) at week 12 and the mean percent reduction from baseline in LDL-C at weeks 10 and 12. The mean percent reductions in LDL-C, or “bad” cholesterol, compared to ezetimibe were consistent with results observed in the Phase 2 GAUSS study.^[3]

The GAUSS-2 trial evaluated safety, tolerability and efficacy of evolocumab in 307 patients with high cholesterol who could not tolerate effective doses of at least two different statins due to muscle-related side effects. Patients were randomized to one of four treatment groups: subcutaneous evolocumab 140 mg every two weeks and oral placebo daily; subcutaneous evolocumab 420 mg monthly and oral placebo daily; subcutaneous placebo every two weeks and oral ezetimibe 10 mg daily; or subcutaneous placebo monthly and oral ezetimibe 10 mg daily.

Safety was generally balanced across treatment groups. The most common adverse events (> 5 percent in evolocumab combined group) were headache (7.8 percent evolocumab; 8.8 percent ezetimibe), myalgia (7.8 percent evolocumab; 17.6 percent ezetimibe), pain in extremity (6.8 percent evolocumab; 1.0 percent ezetimibe), and muscle spasms (6.3 percent evolocumab; 3.9 percent ezetimibe).

Evolocumab, a PCSK9 inhibitor, was safe and effective at lowering low-density lipoprotein cholesterol (LDL-C) after one year of treatment, according to a study published online Nov. 19 inCirculation and presented simultaneously at the American Heart Association scientific session in Dallas.

The Open-Label Study of Long-term Evaluation Against LDL-C (OSLER) trial took place at 156 study centers around the world that participated in at least one of four phase 2 studies of between October 2011 and June 2012. Evolocumab is a PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitor made by Amgen.

Investigators led by Michael J. Koren, MD, of the Jacksonville Center for Clinical Research in Florida, randomized 1,104 participants in a 2:1 ratio to receive either evolocumab (420 mg every four weeks) plus standard-of-care therapy (based on guidelines for treatment of hypercholesterolemia) or evolocumab alone, which served as the control. After 12 weeks, lipid results were unblinded and investigators were able to adjust standard-of-care therapy in either group.

The main efficacy objective was to determine the effects of longer-term evolocumab therapy on cholesterol levels and the main safety endpoints included incidence of adverse events, serious adverse events and adverse events resulting in discontinuation of the drug.

Patients who received evolocumab for the first time in the OSLER study had an average LDL-C reduction of 52.3 percent at one year. Patients previously dosed with evolocumab in a prior trial and were in the evolocumab and standard-of-care group in OSLER had an average LDL-C reduction of 52.1 percent at the end of the study compared with 50.4 percent at baseline. Patients who terminated evolocumab when they entered OSLER had their LDL-C levels returned to around their baseline.

Adverse events occurred in 73.1 percent of the standard-of-care group and 81.4 percent of the evolocumab plus standard-of-care group. The researchers determined that 5.6 percent of adverse events were related to evolocumab. Serious adverse events occurred in 6.3 percent of the control group and 7.1 percent in the combination group.

The authors explained that their findings offer more insight into the use of this class of drugs to lower LDL-C in at-risk patients.

“Challenging patients such as those who fail to reach current lipid goals despite maximum doses of highly effective statin agents or those with well-documented statin intolerance are thus logical populations for treatment with PCSK9 inhibitors,” they concluded.

References

Nemonoxacin….TaiGen’s pneumonia antibiotic Taigexyn 奈诺沙星 gets marketing approval in Taiwan

March 18, 2014 4:07 am / Leave a comment

Nemonoxacin 奈诺沙星

378746-64-6 CAS

TG-873870

C20-H25-N3-O4

371.4345

WARNER CHILCOTT ORIGINATOR

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

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid

7-[3(S)-Amino-5(S)-methylpiperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
Taigexyn has been approved in Taiwan IN 2014

太景生物

“TAIPEI, MARCH 13, 2014 /PRNEWSWIRE/ — TAIGEN BIOTECHNOLOGY …”
13.03.14 |

TaiGen Biotechnology Receives Marketing Approval from the Taiwan Food and Drug Administration for Taigexyn in Taiwan

TAIPEI, March 13, 2014 /PRNewswire/ — TaiGen Biotechnology Company, Limited (“TaiGen”) today announced that the Taiwan Food and Drug Administration (TFDA) has approved the new drug application (NDA) of Taigexyn® (nemonoxacin) oral formulation (500 mg) for the treatment of community-acquired bacterial pneumonia (CAP). With this NDA approval, Taiwan is the first region to grant marketing approval to Taigexyn®. An NDA for Taigexyn® was also submitted to China FDA (CFDA) in April 2013 and is currently under review.

http://www.ad-hoc-news.de/taigen-biotechnology-receives-marketing-approval-from-the-taiwan-food-and-drug-administration-for-taigexyn-in-taiwan–/de/News/35824729

Nemonoxacin is a novel non-fluorinated quinolone antibiotic undergoing clinical trials.

Taigexyn Granted QIDP and Fast Track Designations

TaiGen Biotechnology announced that the FDA has granted nemonoxacin (Taigexyn) Qualified Infectious Disease Product (QIDP) and Fast Track designations for community-acquired bacterial pneumonia (CAP) and acute bacterial skin and skin structure infections (ABSSSI).

Safety and clinical pharmacokinetics of nemonoxacin, a novel non-fluorinated quinolone, in healthy Chinese volunteers following single and multiple oral doses

Nemonoxacin is a novel non-fluorinated quinolone broad spectrum antibiotic available in both oral and intravenous formulations. Nemonoxacin demonstrates activity against gram-positive and gram-negative bacteria and atypical pathogens. Nemonoxacin also possesses activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant pathogens.

Nemonoxacin is a novel non-flourinated quinolone antibiotic registered in Taiwan for the oral treatment of community-acquired pneumonia. Clinical trials are in development at TaiGen Biotechnology for the treatment of diabetic foot infections and for the treatment of moderate to severe community-acquired pneumonia with an intravenous formulation. The drug is thought to accomplish its antibacterial action through topoisomerase inhibition.

Originally developed at Procter & Gamble, nemonoxacin was the subject of a strategic alliance formed in January 2005 between P&G and TaiGen to further the development and commercialization of nemonoxacin. In 2012, the product was licensed by TaiGen Biotechnology to Zhejiang Medicine in China for manufacturing, sales and marketing. In 2014, TaiGen out-licensed the exclusive rights of the product in Russian Federation, Commonwealth Independent States and Turkey to R-Pharm.

TaiGen has completed two Phase 2 clinical studies, one in CAP and the other in diabetic foot infections with demonstrated efficacy and safety. In the clinical trials conducted to date, nemonoxacin has shown activity against drug-resistant bacteria such as MRSA, quinolone-resistant MRSA, as well as quinolone-resistant Streptococcus pneumoniae.

Malate salt

Nemonoxacin malate anhydrous
951163-60-3 CAS NO, MW: 505.5209

Nemonoxacin malate hemihydrate
951313-26-1, MW: 1029.0566

Chemical structure of nemonoxacin as a malate salt (C20H25N3O4·C4H6O5·H2O). Nemonoxacin is the free base, and its molecular mass is 371.44 g/mol. The molecular mass of the salt, nemonoxacin malate, is 514.53 g/mol.

……………………..

isomeric compounds are:

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD1…….DESIRED

(3S,5R)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD 1’….NOT DESIRED

EP2303271A1

Example 1

Malate salts of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and (3S,5R)-7- [3-ammo-5-methyl-piperidinyl]- 1 -cyclopropyl- 1 ,4-dihydro-8-methoxy-4-oxo-3- quinolinecarboxylic acid (Compound 1′) were synthesized as follows:

(A) Synthesis of (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (Compound 9) and (3S,5R)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (Compound 9′): Compound 9′ was synthesized as shown in Scheme 1 below:

Scheme 1

3 4 Boc

A 50-L reactor was charged with Compound 2 (5.50 kg, 42.60 mol), methanol (27 L) and cooled to 10-15⁰C. Thionyl chloride (10.11 kg, 2.0 equiv.) was added via an addition funnel over a period of 65 min, with external cooling to keep temperature below 30°. The resulting solution was stirred at 25⁰C for 1.0 hour, after which methanol was removed under reduced pressure. The oily residue was azeotroped with ethyl acetate (3 x 2.5 L) to remove residual methanol, dissolved in ethyl acetate (27.4 L), charged into a 50 L reactor, and neutralized by slow addition of triethylamine (3.6 kg) below 3O⁰C. The resulting suspension was filtered to remove triethylamine hydrochloride.

The filtrate was charged to a 50 L reactor, along with DMAP (0.53 kg). Di- fert-butyl dicarbonate (8.43 kg) was added via hot water heated addition funnel, over a period of 30 min at a temperature of 20-30⁰C. The reaction was complete after 1 hour as determined by TLC analysis. The organic phase was washed with ice cold IN HCl (2 x 7.5 L), saturated sodium bicarbonate solution (1 x 7.5 L), dried over magnesium sulfate, and filtered. After ethyl acetate was removed under reduced pressure, crystalline slurry was obtained, triturated with MTBE (10.0 L), and filtered to afford Compound 3 as a white solid (5.45 kg, 52.4%).

Anal. Calcd for C_HH_I7NO₅ : C, 54.3; H, 7.04; N, 5.76. Found: C, 54.5; H, 6.96; N, 5.80. HRMS (ESI⁺) Expected for C_HH_I8NO₅, [M+H] 244.1185. Found

244.1174; ¹H NMR (CDCl₃, 500 MHz):δ=4.54 (dd, J= 3.1, 9.5 Hz, IH), 3.7 (s, 3H), 2.58-2.50 (m, IH), 2.41 (ddd, IH, J= 17.6, 9.5, 3.7), 2.30-2.23 (m, IH), 1.98-1.93 (m, IH), 1.40 (s, 9H); ¹³C NMR (CDCl₃, 125.70 MHz) δ 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5. Mp 70.2⁰C.

A 50-L reactor was charged with Compound 3 (7.25 kg, 28.8 mol), DME (6.31 kg), and Bredereck’s Reagent (7.7 kg, 44.2 mole). The solution was agitated and heated to 75⁰C + 5⁰C for three hours. The reaction was cooled to O⁰C over an hour, during which time a precipitate formed. The mixture was kept at O⁰C for an hour, filtered, and dried in a vacuum oven for at least 30 hours at 3O⁰C + 5⁰C to give compound 4 as a white crystalline solid (6.93 kg, 77.9%).

Anal. Calcd for Ci₄H₂₂N₂O₅: C, 56.4; H, 7.43; N, 9.39. Found C, 56.4; H, 7.32; N, 9.48; HRMS (ESI⁺) Expected for Ci₄H₂₂N₂O₅, [M+H] 299.1607. Found 299.1613; ¹H NMR (CDCl₃, 499.8 MHz) δ = 7.11 (s, IH), 4.54 (dd, IH, J= 10.8, 3.6), 3.74 (s, 3H), 3.28-3.19 (m, IH), 3.00 (s, 6H), 2.97-2.85 (m,lH), 1.48 (s, 9H); ¹³C NMR (CDCl₃, 125.7 MHz) δ = 172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. MP 127.9⁰C. A 10-gallon Pfaudler reactor was charged with ESCAT 142 (Engelhard Corp.

N.J, US) 5% palladium powder on carbon (50% wet, 0.58 kg wet wt), Compound 4 (1.89 kg, 6.33 mol), and isopropanol (22.4 Kg). After agitated under a 45-psi hydrogen atmosphere at 45⁰C for 18 hrs, the reaction mixture was cooled to room temperature and filtered though a bed of Celite (0.51 kg). The filtrate was evaporated under reduced pressure to give a thick oil, which was solidified on standing to afford Compound 5 (1.69 kg, 100%) as a 93:7 diastereomeric mixture.

A sample of product mixture was purified by preparative HPLC to give material for analytical data. Anal. Calcd for Ci₂Hi₉NO₅: C, 56.0; H, 7.44; N, 5.44. Found C, 55.8; H, 7.31; N, 5.44; MS (ESI⁺) Expected for Ci₂Hi₉NO₅, [M+H] 258.1342. Found 258.1321; ¹H NMR (CDCl₃, 499.8 MHz) δ = 4.44 (m, IH), 3.72 (s, 3H), 2.60-2.48 (m, 2H), 1.59-1.54 (m, IH), 1.43 (s, 9H), 1.20 (d, j = 6.8 Hz,3H); ¹³C NMR (CDCl₃, 125.7 MHz) δ = 175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Mp 89.9⁰C.

A 50-L reactor was charged with Compound 5 (3.02 kg, 11.7 mol), absolute ethanol (8.22 kg), and MTBE (14.81 kg). Sodium borohydride (1.36 kg, 35.9 mol) was added in small portions at 0⁰C + 5⁰C. A small amount of effervescence was observed. The reaction mixture was warmed to 1O⁰C + 5⁰C and calcium chloride dihydrate (2.65 kg) was added in portions at 1O⁰C + 5⁰C over an hour. The reaction was allowed to warm to 2O⁰C + 5⁰C over one hour and agitated for an additional 12 hours at 20⁰C + 5⁰C. After the reaction was cooled to -5⁰C + 5⁰C, ice-cold 2N HCl (26.9 kg) was added slowly at of O⁰C + 5⁰C. Agitation was stopped. The lower aqueous phase was removed. The reactor was charged with aqueous saturated sodium bicarbonate (15.6 kg) over five minutes under agitation. Agitation was stopped again and the lower aqueous phase was removed. The reactor was charged with magnesium sulfate (2.5 kg) and agitated for at leastlO minutes. The mixture was filtered though a nutsche filter, and concentrated under reduced pressure to afford Compound 6 (1.80 kg, 66%). Anal. Calcd for CnH₂₃NO₄: C, 56.6 H, 9.94; N, 6.00. Found C, 56.0; H, 9.68;

N, 5.96; HRMS (ESI⁺) Expected for CnH₂₄NO₄, [M+H] 234.1705. Found 234.1703; ¹H NMR (CDCl₃, 500 MHz) δ = 6.34 (d, J= 8.9 Hz, IH, NH), 4.51 (t, J= 5.8, 5.3 Hz, IH, NHCHCH₂OH), 4.34 (t, J= 5.3, 5.3 Hz, IH, OBCHCH₂OH), 3.46-3.45, (m, IH, NHCH), 3.28 (dd, J= 10.6, 5.3 Hz, NHCHCHHOH), 3.21 (dd, J= 10.2, 5.8 Hz , IH, CH₃CHCHHOH), 3.16 (dd, J = 10.2, 6.2 Hz, IH, NHCHCHHOH), 3.12 (dd, J= 10.6, 7.1 Hz , IH, CH₃CHCHHOH), 1.53-1.50 (m, IH, CH₃CHCHHOH), 1.35 (s, 9H, 0(CH_B)₃, 1.30 (ddd, J = 13.9, 10.2, 3.7 Hz, IH, NHCHCHHCH), 1.14 (ddd, J= 13.6, 10.2, 3.4 Hz, IH, NHCHCHHCH), 0.80 (d, J= 6.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125.7 MHz) δ 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Mp 92.1⁰C. A 50 L reactor was charged with a solution of Compound 6 (5.1 kg) in isopropyl acetate (19.7 kg). The reaction was cooled to 15⁰C + 5°C and triethylamine (7.8 kg) was added at that temperature. The reactor was further cooled to O⁰C + 5⁰C and methanesulfonyl chloride (MsCl) (6.6 kg) was added. The reaction was stirred for a few hours and monitored for completion by HPLC or TLC. The reaction was quenched by saturated aqueous bicarbonate solution. The organic phase was isolated and washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution, and finally saturated aqueous brine solution. The organic phase was dried, filtered, and concentrated in vacuo below 55⁰C + 5⁰C to afford compound 7 as a solid/liquid slurry, which was used in the subsequent reaction without further purification.

After charged with 9.1 kg of neat benzylamine, a 50 L reactor was warmed to 55⁰C, at which temperature, a solution of compound 7 (8.2 kg) in 1,2- dimethoxyethane (14.1 kg) was added. After the addition, the reaction was stirred at 6O⁰C + 5⁰C for several hours and monitored for completion by TLC or HPLC. The reaction was cooled to ambient temperature and the solvent was removed under vacuum. The residue was diluted with 11.7 kg of 15% (v/v) ethyl acetate/hexanes solution and treated, while agitating, with 18.7 kg of 20% (wt) aqueous potassium carbonate solution. A triphasic mixture was obtained upon standing. The upper organic layer was collected. The isolated middle layer was extracted twice again with 11.7 kg portions of 15% (v/v) ethyl acetate/hexanes solution. The combined organic layers were concentrated under vacuum to give an oily residue. The residue was then purified by chromatography to afford Compound 8 as an oil. A 40 L pressure vessel was charged with 0.6 kg 50% wet, solid palladium on carbon (ElOl, 10 wt. %) under flow of nitrogen. A solution of Compound 8 (3.2 kg) in 13.7 kg of absolute ethanol was then added to the reactor under nitrogen. The reactor was purged with nitrogen and then pressurized with hydrogen at 45 psi. The reaction was then heated to 45°C. It was monitored by TLC or LC. Upon completion, the reaction was cooled to ambient temperature, vented, and purged with nitrogen. The mixture was filtered through a bed of Celite and the solid was washed with 2.8 kg of absolute ethanol. The filtrate was concentrated under vacuum to afford Compound 9 as a waxy solid.

TLC R/(Silica F₂₅₄, 70:30 v/v ethyl acetate-hexanes, KMnO₄ stain) = 0.12; ¹H NMR (300 MHz, CDCl₃) δ 5.31 (br s, IH), 3.80-3.68 (m, IH), 2.92 (d, J=I 1.4 Hz,

IH), 2.77 (AB quart, J_AB=12.0 Hz, v=50.2 Hz, 2H), 2.19 (t, J=10.7 Hz, IH), 1.82-1.68 (m, 2H), 1.54 (br s, IH), 1.43 (s, 9H), 1.25-1.15 (m, IH), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ: 155.3, 78.9, 54.3, 50.8, 45.3, 37.9, 28.4, 27.1, 19.2; MS (ESI+) m/z 215 (M+H), 429 (2M+H). Similarly, (3S,5R)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester

(Compound 9′) was synthesized as shown in Scheme 2.

Scheme 2

_HN Boc HN Boc

NaBH₄,EtOH _w – ^“ MsCI₁TEA . „ _. – – _. „ Benzyl Amine

THF EA₁CoId

(B) Synthesis of l-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-l,4-dihydro-quinoline-3- carboxylic acid (Compound 10): Compound 10 was prepared according to the method described in U.S. Patent

6,329,391.

(C) Synthesis of borone ester chelate of l-Cyclopropyl-7-fluoro-8-methoxy-4-oxo- l,4-dihydro-quinoline-3-carboxylic acid (Compound 11):

Scheme 3

Toluene, tert-Butylmethyl ether 20-50⁰C, filter

A reactor was charged with boron oxide (2.0 kg, 29 mol), glacial acetic acid (8.1 L, 142 mol), and acetic anhydride (16.2 L, 171 mol). The resulting mixture was refluxed at least 2 hours, and then cooled to 40⁰C, at which temperature, 7- fluoroquinolone acid compound 10 (14.2 kg, 51 mol) was added. The mixture was refluxed for at least 6 hours, and then cooled to about 90⁰C. Toluene (45 L) was added to the reaction. At 5O⁰C, terϊ-butylmethyl ether (19 L) was added to introduce precipitation. The mixture was then cooled to 20⁰C and filtered to isolate the precipitation. The isolated solid was then washed with teτt-butylmethyl ether (26 L) prior to drying in a vacuum oven at 4O⁰C (50 torr) to afford Compound 11 in a yield of 86.4%. Raman (cm ¹): 3084.7, 3022.3, 2930.8, 1709.2, 1620.8, 1548.5, 1468.0, 1397.7, 1368.3, 1338.5, 1201.5, 955.3, 653.9, 580.7, 552.8, 384.0, 305.8. NMR (CDCl₃, 300 MHz) δ (ppm): 9.22 (s, IH), 8.38-8.33 (m, IH), 7.54 (t, J=9.8 Hz, IH), 4.38-4.35 (m, IH), 4.13 (s, 3H), 2.04 (s, 6H), 1.42-1.38 (m, 2H), 1.34-1.29 (m, 2H). TLC (Whatman MKC18F Silica, 6θA, 200 μm), Mobile Phase: 1 :1 (v/v) CH₃CN : 0.5N NaCl (aq), UV (254/366 nm) visualization; R^O.4-0.5. (D) Synthesis of malate salt of (3S,5S)-7-[3-amino-5-methyl-piperidmyl]-l- cyclopropyl-l,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and malate salt of (3S,5R)-7-[3-amino-5-methyl-piperidmyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1′)

Compound 1 was synthesized from compound 9 as shown in Scheme 4 below:

Scheme 4

5O⁰C 3 d

a 6 0 N HCI (aq) CH₂CI₂ 35°40°C 12 h t> Extract pH ad]ust to ~7-8 50″-65″C filter

A reactor was charged with Compound 11 (4.4 kg, 10.9 mol), Compound 9 (2.1 kg, 9.8 mol), triethylamine (TEA) (2.1 L, 14.8 mol), and acetonitrile (33.5 L, 15.7 L/kg). The resulting mixture was stirred at approximately 50⁰C till completion of the reaction, as monitored by HPLC or reverse phase TLC. It was cooled to approximately 35°C and the reaction volume was reduced to approximately half by distillation of acetonitrile under vacuum between 0-400 torr. After 28.2 kg of 3.0 N NaOH (aq) solution was added, the reaction mixture was warmed to approximately 4O⁰C, distilled under vacuum until no further distillates were observed, and hydro lyzed at room temperature. Upon completion of hydrolysis, which was monitored by HPLC or reverse phase TLC, 4-5 kg of glacial acetic acid was added to neutralize the reaction mixture.

The resulting solution was extracted 3 times with 12.7 kg (9.6 L) of dichloromethane. The organic layers were combined and transferred to another reactor. The reaction volume was reduced to approximately a half by evaporation at 40⁰C. After 20.2 Kg 6.0N HCl (aq) solution was added, the reaction mixture was stirred for at least 12 hours at 35°C. After the reaction was completed as monitored by HPLC or reverse phase TLC, agitation was discontinued to allow phase separation. The organic phase was removed and the aqueous layer was extracted with 12.7 kg (9.6 L) of dichloromethane. The aqueous layer was diluted with 18.3 kg distilled water and warmed to approximately 50⁰C. Dichloromethane was further removed by distillation under vacuum (100-400 torr).

The pH of the aqueous solution was then adjusted to 7.8-8.1 by adding about 9.42 kg of 3.0 N NaOH (aq) below 65°C. The reaction mixture was stirred at 50⁰C for at least an hour and then cooled to room temperature. The precipitate was isolated by suction filtration, washed twice with 5.2 kg of distilled water, and dried with suction for at least 12 hours and then in a convection oven at 55°C for additional 12 hours. Compound 12 (3.2 kg, 79%) was obtained as a solid.

A reactor was charged with 3.2 kg of Compound 12 and 25.6 kg of 95% ethanol. To the reactor was added 1.1 kg of solid D,L-malic acid. The mixture was refluxed temperature (~80°C). Distilled water (-5.7 L) was added to dissolve the precipice and 0.2 kg of activated charcoal was added. The reaction mixture was passed through a filter. The clear filtrate was cooled to 45°C and allowed to sit for at least 2 hours to allow crystallization. After the reaction mixture was further cooled to 5°C, the precipitate was isolated by suction filtration, washed with 6.6 kg of 95% ethanol, and dried with suction for at least 4 hours. The solid was further dried in a convection oven at 45⁰C for at least 12 hours to afford 3.1 kg of Compound 1 (yield: 70%). NEMONOXACIN

NMR (D₂O, 300 MHz) δ (ppm): 8.54 (s, IH), 7.37 (d, J=9.0 Hz, IH), 7.05 (d, J=9.0 Hz, IH), 4.23-4.18 (m, IH), 4.10-3.89 (m, IH), 3.66 (br s, IH), 3.58 (s, 3H), 3.45 (d, J=9.0 Hz, IH), 3.34 (d, J=9.3 Hz, IH), 3.16 (d, J=12.9 Hz, IH), 2.65 (dd, J=16.1, 4.1 Hz, IH), 2.64-2.53 (m, IH), 2.46 (dd, J=16.1, 8.0 Hz, IH), 2.06 (br s, IH), 1.87 (d, J=14.4 Hz, IH), 1.58-1.45 (m, IH), 1.15-0.95 (m, 2H), 0.91 (d, J=6.3 Hz, 3H), 0.85-0.78 (m, 2H).

Similarly, Compound 1′ was synthesized from Compound 9′ as shown in Scheme 5 below:

Scheme 5

(3S,5R)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD 1’….NOT DESIRED

…………………

US20070232650

US2007/232650 A1,

malate salts of

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (hereinafter Compound I, see also intermediate (23) in Section D, of Detailed Description of the Invention).

EXAMPLES Example 1 Synthesis of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid and malate salt thereof A. Synthesis of (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8)

(2S)-1-(1,1-Dimethylethyl)-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester, (2). A 50-L reactor is charged with compound (1) (5.50 Kg, 42.60 mol), methanol (27 L) and cooled to 10-15° C. Thionyl chloride (10.11 Kg, 2.0 equiv.) is added via addition funnel over a period of 65 min, with external cooling to maintain temperature at <30°. The resulting solution is stirred at 25° C.+5° C. for 1.0 hour, after which the methanol is distilled off under reduced pressure. The resulting thick oil is azeotroped with ethyl acetate (3×2.5 L) to remove residual methanol. The residue is dissolved in ethyl acetate (27.4 L), charged into a 50 L reactor, and neutralized by the addition of triethylamine (3.6 Kg) from an addition funnel over 30 minutes. The temperature of the neutralization is maintained below 30° C. via external cooling. The resulting suspension of triethylamine hydrochloride is removed by filtration, and the clarified mother liquor solution is charged to a 50 L reactor, along with DMAP (0.53 Kg). Di-tert-butyl dicarbonate (8.43 Kg) is added via hot water heated addition funnel, over a period of 30 min with external cooling to maintain temperature at about 20-30° C. The reaction is complete after 1 hour as determined by TLC analysis. The organic phase is washed with ice cold 1N HCl (2×7.5 L), saturated sodium bicarbonate solution (1×7.5 L), and dried over magnesium sulfate. The mixture is filtered through a nutsche filter and ethyl acetate is removed under reduced pressure to yield a crystalline slurry that is triturated with MTBE (10.0 L) and filtered to afford intermediate (2) as a white solid (5.45 Kg, 52.4%). Anal. Calcd for C₁₁H₁₇NO₅: C, 54.3; H, 7.04; N, 5.76. Found: C, 54.5; H, 6.96; N, 5.80. HRMS (ESI⁺) Expected for C₁₁H₁₈NO₅, [M+H] 244.1185. Found 244.1174; ¹H NMR (CDCl₃, 500 MHz): δ=4.54 (dd, J=3.1, 9.5 Hz, 1H), 3.7 (s, 3H), 2.58-2.50 (m, 1H), 2.41 (ddd, 1H, J=17.6, 9.5, 3.7), 2.30-2.23 (m, 1H), 1.98-1.93 (m, 1H), 1.40 (s, 9H); ¹³C NMR (CDCl₃, 125.70 MHz) δ 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5; Mp 70.2° C.

(2S,4E)-1-(1,1-Dimethylethyl)-4-[(dimethylamino)methylene]-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester (3). A 50-L reactor is charged with intermediate (2) (7.25 Kg, 28.8 mol), DME (6.31 Kg), and Bredereck’s Reagent (7.7 Kg, 44.2 mole). The solution is agitated and heated to 75° C.±5° C. for at least three hours. The progress of the reaction is monitored by HPLC. The reaction is cooled to 0° C.±5° C. over on hour during which time a precipitate forms. The mixture is held at 0° C.±5° C. for one hour and filtered though a nutsche filter and the product dried in a vacuum oven for at least 30 hours at 30° C.±5° C. to give intermediate (3) as a white crystalline solid (6.93 Kg, 77.9%). Anal. Calcd for C₁₄H₂₂N₂O₅: C, 56.4; H, 7.43; N, 9.39. Found C, 56.4; H, 7.32; N, 9.48; HRMS (ESI⁺) Expected for C₁₄H₂₂N₂O₅, [M+H] 299.1607. Found 299.1613; ¹H NMR(CDCl₃, 499.8 MHz)δ=7.11 (s, 1H), 4.54 (dd, 1H, J=10.8, 3.6), 3.74 (s, 3H), 3.28-3.19 (m, 1H), 3.00 (s, 6H), 2.97-2.85 (m, 1H), 1.48 (s, 9H); ¹³C NMR (CDCl₃, 125.7 MHz) δ=172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. Mp 127.9° C.

(2S,4S)-1-(1,1-Dimethylethyl)-4-methyl-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester (4). A 10-gallon Pfaudler reactor is inerted with nitrogen and charged with ESCAT 142 5% palladium powder on carbon (50% wet, 0.58 Kg wet wt.), intermediate (3) (1.89 Kg, 6.33 mol) and isopropanol (22.4 Kg). The reaction mixture is agitated under a 45-psi hydrogen atmosphere at 45° C. for 18 hrs. The reaction mixture is then cooled to room temperature and filtered though a bed of Celite (0.51 Kg) in a nutsche filter to remove catalyst. The mother liquor is evaporated under reduced pressure to give a thick oil that crystallizes on standing to afford 4 (1.69 Kg, 100%) as a 93:7 diastereomeric mixture. A sample of product mixture is purified by preparative HPLC to give material for analytical data. Anal. Calcd for C₁₂H₁₉NO₅: C, 56.0; H, 7.44; N, 5.44. Found C, 55.8; H, 7.31; N, 5.44; MS (ESI⁺) Expected for C₁₂H₁₉NO₅, [M+H] 258.1342. Found 258.1321; ¹H NMR (CDCl₃, 499.8 MHz) δ=4.44 (m, 1H), 3.72 (s, 3H), 2.60-2.48 (m, 2H), 1.59-1.54 (m, 1H), 1.43 (s, 9H), 1.20 (d, j=6.8 Hz,3H); ¹³C NMR (CDCl₃, 125.7 MHz) δ=175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Mp 89.9° C.

(1S,3S)-(4-Hydroxyl-1-hydroxymethyl-3-methyl-butyl)-carbamic acid tert-butyl ester (5). A 50-L reactor is charged with intermediate (4) (3.02 Kg, 11.7 mol), absolute ethanol (8.22 Kg), and MTBE (14.81 Kg). The solution is agitated and cooled to 0° C.±5° C. and sodium borohydride (1.36 Kg, 35.9 mol) is added in small portions so as to maintain reaction temperature at 0° C.±5° C. A small amount of effervescence is observed. The reaction mixture is warmed to 10° C.±5° C. and calcium chloride dihydrate (2.65 Kg) is added portion wise at a slow rate over an hour so as to maintain a reaction temperature of 10° C.±5° C. The reaction is allowed to warm to 20° C.±5° C. over one hour and agitated for an additional 12 hours at 20° C.±5° C. The reaction is cooled to −5° C.±5° C., ice-cold 2N HCl (26.9 Kg) is added at a rate to maintain a reaction temperature of 0° C.±5° C. Agitation is stopped to allow phases to separate. The lower aqueous phase (pH=1) is removed. The reactor is charged with aqueous saturated sodium bicarbonate (15.6 Kg) over five minutes. Agitation is stopped to allow phases to separate. The lower aqueous phase (pH=8) is removed. The reactor is charged with magnesium sulfate (2.5 Kg) and agitated for at least 10 minutes. The mixture is filtered though a nutsche filter, and condensed under reduced pressure to afford intermediate (5) (1.80 Kg, 66%). Anal. Calcd for C₁₁H₂₃NO₄: C, 56.6; H, 9.94; N, 6.00. Found C, 56.0; H, 9.68; N, 5.96; HRMS (ESI⁺) Expected for C₁₁H₂₄NO₄, [M+H] 234.1705. Found 234.1703; ¹H NMR (CDCl₃, 500 MHz)δ=6.34(d, J=8.9 Hz, 1H, NH), 4.51 (t, J=5.8, 5.3 Hz, 1H, NHCHCH₂OH), 4.34 (t, J=5.3, 5.3 Hz, 1H, CH3CHCH₂OH), 3.46-3.45, (m, 1H, NHCH), 3.28 (dd, J=10.6, 5.3 Hz, NHCHCHHOH), 3.21 (dd, J=10.2, 5.8 Hz, 1H, CH₃CHCHHOH), 3.16 (dd, J=10.2, 6.2 Hz, 1H, NHCHCHHOH), 3.12 (dd, J=10.6, 7.1 Hz, 1H, CH₃CHCHHOH), 1.53-1.50 (m, 1H, CH₃CHCHHOH), 1.35 (s, 9H, O(CH ₃)₃, 1.30 (ddd, J=13.9, 10.2, 3.7 Hz, 1H, NHCHCHHCH), 1.14 (ddd, J=13.6, 10.2, 3.4 Hz, 1H, NHCHCHHCH), 0.80 (d, J=6.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125.7 MHz) δ 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Mp 92.1° C.

(2S,4S)-Methanesulfonic acid 2-tert-butoxycarbonylamino-5-methanesulfonyloxy-4-methyl-pentyl ester (6). A 50 L reactor is charged with a solution of intermediate (5) (5.1 Kg) in isopropyl acetate (i-PrOAc) 11.8 Kg followed by a rinse with an additional 7.9 Kg i-PrOAc. The reaction is cooled to 15° C.±5° C. and triethylamine (TEA) (7.8 Kg) is added while maintaining the set temperature. The reactor is further cooled to 0° C.±5° C. and methanesulfonyl chloride (MsCl) (6.6 Kg) is added to the reaction solution while maintaining the set temperature. The reaction is stirred for a few hours and monitored for completion by HPLC or TLC. The reaction is quenched by the addition of a saturated aqueous bicarbonate solution and the resulting isolated organic phase is washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution, and finally saturated aqueous brine solution. The organic phase is dried, filtered, and concentrated in vacuo below 55° C.±5° C. until a solid/liquid slurry containing intermediate (6) is obtained. The slurry is used crude in subsequent reaction without further characterization.

(3S,5S)-(1-Benzyl-5-methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (7). A 50 L reactor is charged with 9.1 Kg of neat benzylamine. The reactor is brought to 55° C. and a solution of intermediate (6) (8.2 Kg) in 1,2-dimethoxyethane (DME) (14.1 Kg) is added to the reactor while maintaining a temperature of 60° C.±5° C. After complete addition of this solution, the reaction is stirred at 60° C.±5° C. for several hours and monitored for completion by TLC or HPLC. The reaction is cooled to ambient temperature and volatiles (DME) are removed by rotary evaporation under vacuum. The residue is diluted with 11.7 Kg of 15% (v/v) ethyl acetate/hexanes solution and treated, while agitating, with 18.7 Kg of 20% (wt) aqueous potassium carbonate solution. A triphasic mixture is obtained upon settling. The bottom aqueous phase is removed and the middle phase is set aside. The upper organic phase is collected and held for combination with extracts from additional extractions. The isolated middle phase is extracted twice again with 11.7 Kg portions of 15% (v/v) ethyl acetate/hexanes solution, each time combining the extracts with original organic phase. The combined organic extracts are transferred into a rotary evaporator and solvent is removed under vacuum until an oily residue remains. The residue is then purified via large-scale preparative chromatography to afford purified intermediate (7) as an oil.

(3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8). A 40 L pressure vessel is charged with 0.6 Kg 50% wet, solid palladium on carbon (E101, 10 wt. %) under flow of nitrogen. A solution of 3.2 Kg intermediate (7) in 13.7 Kg of absolute ethanol is then charged to the reactor under nitrogen. The reactor is purged with nitrogen and is then pressurized with hydrogen at 45 psi. The reaction is then heated to 45° C. while maintaining a hydrogen pressure of 45 psi. The reaction is monitored by TLC or LC until complete. The reaction is cooled to ambient temperature, vented, and purged with nitrogen. The reactor contents are filtered through a bed of Celite and the solids are washed with 2.8 Kg of absolute ethanol. The filtrate is concentrated by rotary evaporation under vacuum until a waxy solid is obtained to afford intermediate (8): TLC R_f(Silica F₂₅₄, 70:30 v/v ethyl acetate-hexanes, KMnO₄stain)=0.12; ¹H NMR (300 MHz, CDCl₃) δ 5.31 (br s, 1H), 3.80-3.68 (m, 1H), 2.92 (d, J=11.4 Hz, 1H), 2.77 (AB quart, J_AB=12.0 Hz, Δν=50.2 Hz, 2H), 2.19 (t, J=10.7 Hz, 1H), 1.82-1.68 (m, 2H), 1.54 (br s, 1H), 1.43 (s, 9H), 1.25-1.15 (m, 1H), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 155.3, 78.9, 54.3, 50.8, 45.3, 37.9, 28.4, 27.1, 19.2; MS (ESI⁺) m/z 215 (M+H), 429 (2M+H).

B. Synthesis of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (19)

Intermediate (12): A reactor is charged with a solution of intermediate (11) (1.2 Kg, 7.7 mol, 1.0 eq) in anhydrous toluene (12 L) followed by ethylene glycol (1.8 L, 15.7 mol, 4.2 eq) and solid p-toluenesulfonic acid (120 g, 10 wt. %). The reaction mixture is stirred at ambient temperature for at least 30 minutes and then heated to reflux, collecting the water/toluene azeotrope in a Dean Stark type trap apparatus until the reaction is complete as determined by TLC analysis (15% EtOAc/Hexanes v/v). Upon completion, the reaction is cooled to ambient temperature and poured into an aqueous solution of sodium bicarbonate (6 L). The organic toluene phase was removed and washed with saturated sodium bicarbonate solution (6 L), distilled water (2×6 L), and saturated aqueous brine (6 L). The organic phase was removed and dried over MgSO₄, filtered, and evaporated under reduced pressure to afford intermediate (12) as an oil (1.3 Kg, 86%). The material is used without further purification in subsequent reaction steps.

Intermediate (13): A reactor is charged with a solution of intermediate (12) (1.2 Kg, 6.0 mol, 1.0 eq) in anhydrous tetrahydrofuran (12 L) and n-butyllithium (2.5M in hexanes, 2.6 L, 6.6 mol, 1.1 eq) is added at −40° C., while maintaining this temperature throughout the addition. The reaction is stirred for at least one hour at −40° C. and trimethylborate (0.9 L, 7.8 mol, 1.3 eq) is added to the mixture while maintaining the temperature at or below −40° C. The reaction mixture is stirred for at least one hour at −40° C. until complete as determined by TLC analysis (30% EtOAc/Hexanes v/v). The reaction is warmed slightly to −30° C. and acetic acid (3 L) is added slowly. Upon complete addition, water is added (0.5 L) to the reaction and the mixture is allowed to quickly warm to ambient temperature while stirring overnight. Organic solvent is removed from the reaction by distillation under reduced pressure at 45° C. To the reaction residue is added 3-4 volumes of water (6 L) and 30% hydrogen peroxide (0.7 L, 1.0 eq) slowly at ambient temperature with cooling provided to control the exotherm. The reaction is stirred for at least an hour at ambient temperature until complete as determined by TLC (15% EtOAc/Hexanes v/v). The reaction mixture is cooled to 0-5° C. and excess peroxide is quenched with the addition of 10% aqueous sodium bisulfite solution (2 L). The mixture is tested to ensure a negative peroxide result and the reaction is acidified by the addition of 6N HCl (aq) (1.2 L). The reaction is stirred until the hydrolysis reaction is complete as determined by TLC or NMR analysis. The resulting solids are collected by suction filtration to afford intermediate (13) as a yellow solid (1.0 Kg, 79%).

Intermediate (14): A reactor is charged with intermediate (13) (0.53 Kg, 3.0 mol, 1.0 eq) and dissolved in dry toluene (2.7 Kg, 3.1 L). To this solution is added dimethylsulfate (0.49 Kg, 3.9 mol, 1.30 eq) followed by solid potassium carbonate (0.58 Kg, 4.2 mol, 1.4 eq). The reaction mixture is heated to reflux and held for at least 1 hour until complete as determined by HPLC. During this time, vigorous gas evolution is observed. The reaction is then cooled to ambient temperature and diluted with distilled water (3.2 L) along with 30% NaOH (aq) (0.13 Kg, 0.33 eq). The aqueous phase is separated and the remaining toluene phase is extracted twice more with distilled water (3.2 L) combined with 30% NaOH (aq) (0.13 Kg, 0.33 eq), removing the aqueous phase each time. The organic upper phase is concentrated by distillation in vacuo (<100 mbar) at approximately 40° C. until a concentrated toluene solution is achieved. The resulting solution is cooled to ambient temperature, checked for quality and yield by HPLC, and carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (14) assumed, 0.56 Kg).

Intermediate (15a,b): A reactor is charged with 1.8 Kg (2.1 L) anhydrous toluene along with sodium hydride (0.26 Kg, 6.6 mol, 2.20 eq) as a 60 wt. % dispersion in mineral oil. To this mixture is added (0.85 Kg, 7.2 mol, 2.4 eq) diethylcarbonate as the reaction mixture is heated to 90° C. over 1 hour. A solution of intermediate (14) (˜1.0 eq) in toluene from the previous step is added to the reaction while maintaining a temperature of 90° C.±5° C. Gas evolution can be observed during this addition. After complete addition, the reaction is stirred for at least 30 minutes or until complete as determined by HPLC analysis. Upon completion, the mixture is cooled to ambient temperature and diluted with 10 wt. % aqueous sulfuric acid (3.8 Kg, 3.9 mol, 1.3 eq) with agitation. The phases are allowed to separate and the lower aqueous phase is removed. The remaining organic phase is concentrated in vacuo (<100 mbar) at approximately 40° C. until a concentrated toluene solution is achieved. The resulting solution is cooled to ambient temperature and carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (15a,b) assumed, 0.85 Kg).

Intermediate (16a,b; 17a,b): A reactor is charged with a solution of intermediate (15a,b) (0.85 Kg, ˜3.0 mol, ˜1.0 eq) in toluene from the previous step. To the reactor is then added dimethylformamide-dimethylacetal (0.54 Kg, 4.5 mol, 1.5 eq) and the resulting solution is heated to reflux temperature (˜95-105° C.). The lower boiling solvent (methanol from reaction) is allowed to distill off while the temperature is maintained at ≧90° C. Heating is continued for at least 1 hour or until complete as determined by HPLC analysis. Upon completion, the reaction containing the mixture of intermediate (16a,b), is cooled to ambient temperature and toluene (1.8 Kg, 2.1 L) along with cyclopropylamine (0.21 Kg, 3.6 mol, 1.2 eq) are added to the reaction. The reaction is stirred at ambient temperature for at least 30 minutes until complete as determined by HPLC. Upon completion, the reaction is diluted with 10 wt. % aqueous sulfuric acid (2.9 Kg, 3.0 mol, 1.0 eq) with agitation, and the phases are then allowed to separate. The aqueous phase is removed and the organic phase is concentrated under reduced pressure (<100 mbar) at approximately 40° C. by distillation. When the desired concentration is achieved, the solution is cooled to ambient temperature and the toluene solution containing the mixture of intermediate (17a,b) is carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (17a,b) assumed, ˜1.1 Kg).

Intermediate (18): A reactor is charged with a solution of the mixture of intermediate (17a,b) (˜4.7 Kg, ˜3.0 mol) at ambient temperature. To the reactor is added N,O-bis(trimethylsilyl)acetamide (0.61 Kg, 3.0 mol, 1.0 eq) and the reaction is heated to reflux temperature (˜105-115° C.) for at least 30 minutes or until complete as determined by HPLC analysis. If not complete, an additional amount of N,O-bis(trimethylsilyl)acetamide (0.18 Kg, 0.9 mol, 0.3 eq) is added to the reaction to achieve completion. Upon completion, the reaction is cooled to below 40° C. and organic solvent is removed under reduced pressure (<100 mbar) at approximately 40° C. by distillation until a precipitate is formed. The reaction is cooled to ambient temperature and the precipitated solids are isolated by suction filtration and washed with distilled water twice (1×1.8 L, 1×0.9 L). The solid is dried to afford intermediate (18) as a white solid (0.76 Kg, 82%). The material is used without further purification in the next reaction step.

Intermediate (19): A reactor is charged with solid intermediate (18) (0.76 Kg, ˜2.5 mol, ˜1.0 eq) at ambient temperature followed by ethanol (5.3 Kg, 6.8 L) and 32 wt. % aqueous hydrochloric acid (1.1 Kg, 10 mol). The reaction mixture is brought to reflux temperature (76-80° C.) during which time the mixture first becomes homogeneous and later becomes heterogeneous. The mixture is heated at reflux for at least 5 hours or until complete as determined by TLC analysis (15% EtOAc/Hexanes v/v). Upon completion, the reaction is cooled to 0° C.±5° C. and the precipitated solid is isolated by filtration and washed with distilled water (1.7 Kg) followed by ethanol (1.7 Kg). The isolated solid is dried to afford intermediate (19) as a white solid (0.65 Kg, ˜95%). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 14.58 (s, 1H), 8.9 (s, 1H), 8.25 (m, 1H), 7.35 (m, 1H), 4.35 (m, 1H), 4.08 (s, 3H), 1.3 (m, 2H), 1.1 (m, 2H) ¹⁹F NMR (CDCl₃+CFCl₃, 292 MHz) δ (ppm): −119. HPLC: 99.5% by area.

C. Synthesis of borone ester chelate of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (20)

A reactor is charged with boron oxide (2.0 Kg, 29 mol) followed by dilution with glacial acetic acid (8.1 L, 142 mol) and acetic anhydride (16.2 L, 171 mol). The resulting mixture is heated to reflux temperature for at least 2 hours. The reaction contents are cooled to 40° C. and the solid 7-fluoroquinolone acid intermediate (19) (14.2 Kg, 51 mol) is added to the reaction mixture. The mixture is again heated to reflux temperature for at least 6 hours. Reaction progress is monitored by HPLC and NMR. The mixture is cooled to approximately 90° C. and toluene (45 L) is added to the reaction. The reaction is further cooled to 50° C. and tert-butylmethyl ether (19 L) is added to the reaction mixture to bring about precipitation of the product. The mixture is then cooled to 20° C. and the solid product 19 is isolated by filtration. The isolated solids are then washed with tert-butylmethyl ether (26 L) prior to drying in a vacuum oven at 40° C. (50 torr). The product yield obtained for intermediate (20) in this reaction is 86.4%. Raman (cm⁻¹): 3084.7, 3022.3, 2930.8, 1709.2, 1620.8, 1548.5, 1468.0, 1397.7, 1368.3, 1338.5, 1201.5, 955.3, 653.9, 580.7, 552.8, 384.0, 305.8. NMR (CDCl₃, 300 MHz) δ (ppm): 9.22 (s, 1H), 8.38-8.33 (m, 1H), 7.54 (t, J=9.8 Hz, 1H), 4.38-4.35 (m, 1H), 4.13 (s, 3H), 2.04 (s, 6H), 1.42-1.38 (m, 2H), 1.34-1.29 (m, 2H). TLC (Whatman MKC18F Silica, 60 Å, 200 μm), Mobile Phase: 1:1 (v/v) CH₃CN:0.5N NaCl (aq), UV (254/366 nm) visualization; R_f=0.4-0.5.

D. Coupling of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (20) to (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8), and synthesis of malate salt of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (25)

A reactor is charged with solid intermediate (20) (4.4 Kg, 10.9 mol) followed by dilution with a solution of triethylamine (TEA) (2.1 L, 14.8 mol) and piperidine side chain intermediate (8) (2.1 Kg, 9.8 mol) in acetonitrile (33.5 L, 15.7 L/Kg) at room temperature. The resulting mixture is warmed to approximately 50° C. until reaction is judged complete. Reaction progress is monitored by HPLC or reverse phase TLC. When complete, the reaction is cooled to approximately 35° C. and reaction volume is reduced to approximately half by distillation of acetonitrile under vacuum between 0-400 torr. The reactor is then charged with 28.2 Kg of 3.0N NaOH (aq) solution and the temperature is raised to approximately 40° C. Distillation under vacuum is continued between 1-4 hours or until no further distillates are observed. The reaction is then cooled to room temperature and the hydrolysis reaction is monitored by HPLC or reverse phase TLC. Upon completion, the reaction mixture is neutralized to a pH of between 6-8 by adding ˜4-5 Kg of glacial acetic acid. The reactor is then charged with 12.7 Kg (9.6 L) of dichloromethane as an extraction solvent, the mixture is agitated, phases are allowed to separate, and the organic dichloromethane phase is removed. The extraction process is repeated two additional times using 12.7 Kg (9.6 L) of dichloromethane, collecting the lower, organic phase each time. The aqueous phase is discarded and the organic extracts are combined in a single reactor. The reactor contents are heated to 40° C. and the reaction volume is reduced to approximately one half by distillation. The reactor is then charged with 20.2 Kg 6.0N HCl (aq) solution, the temperature is adjusted to 35° C., and agitation is allowed for at least 12 hours to permit the Boc deprotection reaction to occur. The reaction is monitored by HPLC or reverse phase TLC. When complete, agitation is discontinued and the phases are allowed to separate. The lower, organic phase is removed and set aside. The reactor is then charged with 12.7 Kg (9.6 L) of dichloromethane as an extraction solvent, the mixture is agitated, phases are allowed to separate, and the organic dichloromethane phase is removed. The organic extracts are combined and discarded. The remaining aqueous phase is diluted with 18.3 Kg distilled water and the temperature is raised to approximately 50° C. Distillation under vacuum (100-400 torr) is performed to remove residual dichloromethane from the reaction. The pH of the reaction is then adjusted to between 7.8-8.1 using about 9.42 Kg of 3.0N NaOH (aq) solution while keeping the temperature of the reaction below 65° C. The reaction is cooled to 50° C. and the precipitated solids are aged for at least an hour prior to cooling the mixture to room temperature. The solids are isolated by suction filtration and washed twice with 5.2 Kg portions of distilled water. The solids are dried for at least 12 hours with suction and then for an additional 12 hours in a convection oven at 55° C. The yield achieved for intermediate (23) in this example is 3.2 Kg (79%). A reactor is charged with 3.2 Kg solid intermediate (23) and the solids are suspended in 25.6 Kg of 95% ethanol as solvent. To the reactor is then added 1.1 Kg of solid D,L-malic acid (24), and the mixture is heated to reflux temperature (˜80° C.). Distilled water (˜5.7 L) is added to the reaction until a complete solution is achieved and 0.2 Kg of activated charcoal is added. The reaction mixture is passed through a filter to achieve clarification, cooled to 45° C. and held for a period of at least 2 hours to allow crystallization to occur. The reaction mixture is further cooled to 5° C. and the suspended solids are isolated by suction filtration. The solids are then washed with 6.6 KG of 95% ethanol and dried for at least 4 hours with suction under vacuum. The solids are then further dried in a convection oven for at least 12 hours at 45° C. to afford 3.1 Kg of intermediate (24) (70%). NMR (D₂O, 300 MHz) δ (ppm): 8.54 (s, 1H), 7.37 (d, J=9.0 Hz, 1H), 7.05 (d, J=9.0 Hz, 1H), 4.23-4.18 (m, 1H), 4.10-3.89 (m, 1H), 3.66 (br s, 1H), 3.58 (s, 3H), 3.45 (d, J=9.0 Hz, 1H), 3.34 (d, J=9.3 Hz, 1H), 3.16 (d, J=12.9 Hz, 1H), 2.65 (dd, J=16.1, 4.1 Hz, 1H), 2.64-2.53 (m, 1H), 2.46 (dd, J=16.1, 8.0 Hz, 1H), 2.06 (br s, 1H), 1.87 (d, J=14.4 Hz, 1H), 1.58-1.45 (m, 1H), 1.15-0.95 (m, 2H), 0.91 (d, J=6.3 Hz, 3H); 0.85-0.78 (m, 2H). TLC (Whatman MKC18F Silica, 60 Å, 200 μm), Mobile Phase: 1:1 (v/v) CH₃CN:0.5N NaCl (aq), UV (254/366 nm) visualization. HPLC: Mobile Phase H₂O with 0.1% formic acid/Acetonitrile with 0.1% formic acid, gradient elution with 88% H₂O/formic acid to 20% H₂O/formic acid, Zorbax SB-C8 4.6 mm×150 mm column, Part No. 883975.906, 1.5 ml/min rate, 20 min run time, 292 nm, Detector Model G1314A, S/N JP72003849, Quat Pump Model G1311A, S/N US72102299, Auto Sampler Model G1313A, S/N DE14918139, Degasser Model G1322A, S/N JP73007229; approximate retention time for intermediate (19): 13.0 min; approximate retention time for intermediate (20): 11.6 min; approximate retention time for intermediate (21): 16.3 min; approximate retention time for intermediate (22): 18.2 min; approximate retention time for intermediate (23): 8.6 min; approximate retention time for compound (25): 8.6 min.

………………..

REF

A. ARJONA ET AL: “Nemonoxacin“, DRUGS OF THE FUTURE, vol. 34, no. 3, 1 January 2009 (2009-01-01), page 196, XP55014485, ISSN: 0377-8282, DOI: 10.1358/dof.2009.034.03.1350294


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3	*	ANONYMOUS: “TaiGen Biotechnology Initiates Phase II Trial Of Nemonoxacin For Treatment Of Adult Community Acquired Pneumonia (CAP)“, 20070108, [Online] 8 January 2007 (2007-01-08), page 1, XP007919910, Retrieved from the Internet: URL:http://www.taigenbiotech.com/news.html#11> [retrieved on 2011-12-12]
4	*	ANONYMOUS: “TaiGen Initiates Phase 1B Trial of a Novel Quinolone Antibiotic“, 20050618, 18 June 2005 (2005-06-18), pages 1-2, XP007919904,
5	*	See also references of WO2010002415A1

WO2007110834A2 *	Mar 26, 2007	Oct 4, 2007	Procter & Gamble	Malate salts, and polymorphs of (3s,5s)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid
WO2009023473A2 *	Aug 5, 2008	Feb 19, 2009	Chi-Hsin Richard King	Antimicrobial parenteral formulation
WO2010009014A2 *	Jul 10, 2009	Jan 21, 2010	Taigen Biotechnology Co., Ltd.

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US2010152452	6-18-2010	STEREOSELECTIVE SYNTHESIS OF PIPERIDINE DERIVATIVES
US2010041697	2-19-2010	PNEUMONIA TREATMENT
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US2009042932	2-13-2009	ANTIMICROBIAL PARENTERAL FORMULATION
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US8158798	Oct 27, 2008	Apr 17, 2012	Taigen Biotechnology Co., Ltd.	Coupling process for preparing quinolone intermediates
US8211909	Sep 8, 2008	Jul 3, 2012	Taigen Biotechnology Co., Ltd.	Treatment of antibiotic-resistant bacteria infection
WO2010002965A2 *	Jul 1, 2009	Jan 7, 2010	Taigen Biotechnology Co., Ltd.	Pneumonia treatmen

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1, nemonoxacin; 2, delafloxacin; 3, finafloxacin; 4, zabofloxacin; 5, JNJ-Q2; 6, DS-8587; 7, KPI-10; 8, ozenoxacin; 9, chinfloxacin; 10, ACH-702.

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