Allisartan isoproxil

Allisartan isoproxil

CAS: 947331-05-7

553.01, C27 H29 Cl N6 O5

Shanghai Allist Pharmaceutical, Inc.

Allist Shanghai Pharmaceutical Co., Ltd.

2-butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)-1,1′-biphenyl-methyl]-imidazole-5-carboxylic acid, 1-[(isopropoxy)-carbonyloxy] methyl ester,

2-Butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-imidazole-5-carboxylic acid isopropoxycarbonyloxymethyl ester

2-butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)1,1′-biphenyl-methyl]imidazole-5-carboxylic acid, 1-[(isopropoxycarbonyl)oxy]methyl ester

Allisartan is an orally-available angiotensin AT1 antagonist in phase II clinical trials at Shanghai Allist Pharmaceutical for the treatment of mild to moderate essential hypertension.

Shanghai Allist Pharmaceutical PHASE 2 for Hypertension

The prior art discloses Arleigh medoxomil illiquid, low bulk density, electrostatic phenomena evident. Chinese patent discloses a CN200710094131.0 Alicante medoxomil polymorph and method of preparation. Allie medoxomil based crystal prepared by the method has high stability characteristics, but relatively small bulk density of the crystal clear after the electrostatic phenomenon and poor liquidity dried, crushed and used for easy dispensing generate dust, operating the site clean and labor protection inconvenience, on the other hand also for accurate weighing and packaging products inconvenience.

CN200710094021.4 and CN201110289695.6 disclose the preparation of Alicante medoxomil, the inventor repeated, the proceeds of crystal and Chinese patent CN200710094131.0 consistent disclosed.

Allisartan isoproxil

Angiotensin II AT-1 receptor antagonist

Essential hypertension

Amorphous form of allisartan isoproxil is claimed in WO 2015062498. Useful for treating hypertension. Shenzhen Salubris Pharmaceuticals, in collaboration with Allist, has developed and launched allisartan isoproxil. In October 2012, Shenzhen Salubris signed a strategic cooperation framework agreement with Allist Pharmaceutical for the production and marketing of allisartan isoproxil. Family members of the product case of allisartanWO2007095789, expire in the EU and in the US in 2026. For a prior filing see WO2009049495 (assigned to Allist Pharmaceuticals), claiming the crystalline form of allisartan and its method of preparation.

The compound of formula (I) is an Ang II receptor antagonist. Its chemical name is 2-butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)-1,1′-biphenyl-methyl]-imidazole-5-carb-oxylic acid, 1-[(isopropoxy)-carbonyloxy] methyl ester. Chinese Patent CN101024643A describes the structure, and its use as antihypertensive drugs.

As regards to the solid physical properties of the compound of formula (I), the patent document of CN101024643A discloses that it is a white solid, and its melting point is 134.5-136° C. However, CN101024643A dose not disclose the crystalline structure of the compound of formula (I).

CHINA

NEW PATENT

WO-2015062498

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015062498

2-butyl-4-chloro -1- [2 ‘- (1H- tetrazol-5-yl) -1,1′-biphenyl- – methyl] – imidazole-5-carboxylic acid, 1 – [(isopropoxy) – oxy] -, methyl ester, is a novel angiotensin Ⅱ receptor antagonist. China Patent CN200680000397.8 disclosed structural formula Alicante medoxomil compound. Allie medoxomil toxicity, blood pressure better than the same type of products (such as losartan), which by generating active metabolite (EXP3174) in vivo metabolism, and thus play its antihypertensive effect.

The prior art discloses Arleigh medoxomil illiquid, low bulk density, electrostatic phenomena evident. Chinese patent discloses a CN200710094131.0 Alicante medoxomil polymorph and method of preparation. Allie medoxomil based crystal prepared by the method has high stability characteristics, but relatively small bulk density of the crystal clear after the electrostatic phenomenon and poor liquidity dried, crushed and used for easy dispensing generate dust, operating the site clean and labor protection inconvenience, on the other hand also for accurate weighing and packaging products inconvenience.

CN200710094021.4 and CN201110289695.6 disclose the preparation of Alicante medoxomil, the inventor repeated, the proceeds of crystal and Chinese patent CN200710094131.0 consistent disclosed.

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PATENT

http://www.google.com/patents/CN103965171A?cl=en

Hypertension is a major disease threat to human health, looking for efficiency, low toxicity anti-hypertensive drugs can help relieve social pressures and family responsibilities, with good social and economic benefits.

 Angiotensin II (Ang II) is the renin – angiotensin – aldosterone system (RAAS) main vasoconstrictor hormone, which plays an important role in the pathobiology of many chronic diseases, particularly its the role of blood pressure regulation is particularly prominent, and therefore Ang II receptor is believed to be a good target for the development of anti-hypertensive drugs.

EP0253310 discloses a series of imidazole derivatives, DuPont declared and obtained by the study of losartan potassium-listed in 1994, was the first non-peptide Ang II receptor antagonist anti-hypertensive drugs. Thereafter, he listed a series of losartan antihypertensive drugs: candesartan cilexetil, valsartan, irbesartan, telmisartan and olmesartan medoxomil, etc. (EP0253310, W02005049587, GB2419592, EP1719766, US5196444) .

The losartan potassium in the body, the active metabolite EXP3174 has a stronger antihypertensive effect than losartan potassium, but EXP3174 polar molecular structure, is difficult to form passive absorption by diffusion through the cell membrane. US5298915 discloses five carboxyl ester group transformation EXP3174 is a series of derivatives, focusing on the compound HN-65021, and discloses hypotensive test results HN-65021 administered by the oral route, its hypotensive activity with chlorine Similar losartan potassium (BritishJouurnal ofClinical Pharmacology, 40,1995,591).

CN200680000397.8 _5_ discloses a class of imidazole carboxylic acid derivatives, namely Alicante medoxomil compound 8 has a good blood pressure lowering effect, the structure of formula I, the preparation method disclosed in this patent document follows the route A, losartan potassium by oxidation, the protecting group into an ester, deprotected to give a compound of formula I, the route step oxidation process of hydroxyl to carboxyl groups, will be reduced to very fine granular potassium permanganate, manganese dioxide, filtration This manganese mud time-consuming, inefficient, polluting; the second step conversion was about 70%, and post-processing cumbersome; byproducts and produced the first two steps more. This makes the high cost of the entire route, not suitable for the production of amplification.

CN200710094021.4 discloses another method for preparing the compounds of formula I, the following route B, the starting material by nucleophilic substitution, oxidation, an ester, a tetrazole ring to obtain a compound of formula I, the first step of the method nucleophilic substitution easy to generate an imidazole ring -3 para isomer impurities difficult to remove; the last step into the ring to use sodium azide, operating dangerous.

CN201210020174.5 disclosed a series of anti-hypertensive compound and preparation method, the following line C, the temperature control in the first step of its preparation O ~ 5 ° C, a mixed solution of acetone and water, with a 5% aqueous solution of sodium hypochlorite oxidation, yield 70%, the second step use of potassium permanganate, manganese dioxide will produce the same, and a yield of only 40%, the first two steps total yield of 28%, is very low, and the post-treatment methods are by column separation, the first two steps are used are organic and inorganic mixed solvent is not conducive to recovery, not suitable for scale-up.

Example 8 2-Butyl-4-chloro _1- [2 ‘- (1-tetrazol-5-yl biphenyl – methyl] imidazole

5-carboxylic acid, 1 – [(isopropoxy) carbonyl] -L-methoxy ester (Alicante medoxomil crude)

To a 20L reactor 9800ml of methanol, stirring was started, the rotational speed is added at 200r / min 1225.3g solid compound of formula II, and heated to reflux. The reaction 8-10h evacuation HPLC detection, the formula II compound residue <1.0% seen as a response endpoint. After reaching the end of the reaction the heating was stopped, continued stirring speed of 180r / min. About 3_4h fell 20_25 ° C, colorless transparent crystalline solid precipitated. The reaction mixture was cooled to continue to 15-20 ° C, to maintain 15-20 ° C with stirring 3h, the reaction mixture was filtered to give a pale yellow clear filtrate. The filtrate was concentrated under reduced pressure to move 20L flask, vacuum degree of 0.075MPa, 40_45 ° C methanol distilled off under until no distillate. 800ml of absolute ethanol was added, a vacuum degree of 0.075MPa, 40-45 ° C under distillation until no distillate.

900ml of absolute ethanol was added, heated to reflux. N-heptane was added slowly 1100ml, reflux 15min, to -10 ° c / h speed cooled to 15 ± 2 ° C, keep stirring 3h. Filtered under reduced pressure, ethanol / n-heptane = 1 mixture of filter cake was washed / 3, the back pressure dry vacuum filtration lh, was Allie medoxomil crude (800.lg, yield 93.8%).Purification was used directly in the next step without drying.

 Example 9 2-butyl-4-chloro-_1- [2 ‘- (1-tetrazol-5-yl biphenyl – methyl] imidazole-5-carboxylic acid, 1 – [(isopropylamino oxy) carbonyl] -L-methoxy ester (Alicante medoxomil)

850ml of absolute ethanol was added to the 3L reaction vessel was charged with crude Alicante medoxomil (800.lg, 1.45mol), heated to reflux. After completely dissolved clear, slow addition of n-heptane 1300ml, reflux 15min, to -10 ° C / h speed cooled to 10 ± 2 ° C, keep stirring 3h. Filtered under reduced pressure, ethanol / n-heptane = 1 mixture of filter cake was washed / 3, the back pressure dry vacuum filtration, the purified Alicante medoxomil (780.9g, 97.6% yield).

Example 10 2-butyl-4-chloro _1- [2 ‘- (1-tetrazol-5-yl biphenyl – methyl] imidazole

5-carboxylic acid, 1 – [(isopropoxy) carbonyl] -L-methoxy ester (Alicante medoxomil)

950ml of absolute ethanol was added to the 5L reaction vessel was charged with crude Alicante medoxomil (549.9g, 1.72mol), heated to reflux. After completely dissolved clear, slow addition of n-heptane 1200ml, reflux 15min, to -10 ° C / h speed cooled to 10 ± 2 ° C, keep stirring 3h. Filtered under reduced pressure, ethanol / n-heptane = cake was washed with a mixture of 1/3, and dried under reduced pressure after filtration to obtain a purified Alicante medoxomil (540.0g, 98.2% yield).

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PATENT

http://www.google.com/patents/US20090036505

Example 122-butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)1,1′-biphenyl-methyl]imidazole-5-carboxylic acid, 1-[(isopropoxycarbonyl)oxy]methyl ester (compound 8)

To a 100 ml of one-necked flask, 0.523 g of material, 0.124 g of potassium carbonate, 5 ml of N,N-dimethylacetamide were added in turn. The solution was stirred at room temperature for 20 minutes. Then 0.562 g of 1-chloromethyl isopropyl carbonate was added and the mixture was reacted at 45-50° C. for 16 hours. After the reaction was completed, the mixture solution was filtered, and 30 ml of water was added into the filtrate. The resulting mixture was extracted with 30 ml of ethyl acetate twice. The organic phase was dried and concentrated to give 1.724 g of oil, which was directly used in the next reaction without purification.

10 ml of dioxane and 5 ml of 4 mol/L HCl were added, and the resulting mixture was reacted at room temperature for 16 hours. The reaction was stopped and the solution was adjusted to pH 6-7 using aqueous sodium bicarbonate solution. The solution went turbid, and was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried, concentrated to give 0.436 g of 2-butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)1,1′-biphenyl-methyl]imidazole-5-carboxylic acid, 1-[(isopropoxycarbonyl)oxy]methyl ester.

In addition, the following reaction condition can be used to deprotect the protecting group. To 1.7 g of oily product, 5 ml absolute methanol was added and the mixture was heated slowly to reflux and stirred for 8 hours. When the insoluble solid disappeared totally, the mixture was discontinued to heating and cooled to 5° C. The white solid precipitated, and was separated by filtration, and the filter cake was washed with a small quantity of methanol. The combined filtrate was concentrated to dryness to give 2-butyl-4-chloro-1-[2′-(1H-tetrazol-5-yl)1,1′-biphenyl-methyl]imidazole-5-carboxylic acid, 1-[(isopropoxycarbonyl)oxy]methyl ester with the yield of 70%.

1H-NMR (CDCl3) δ H (ppm): 0.89 (t, 3H, J=14.6), 1.24 (d, 6H, J=6.3), 0.37 (m, 2H, J=22.1), 1.69 (m, 2H, J=30.5), 2.64 (t, 2H, J=15.5), 4.81 (m, 1H, J=12.4), 5.54 (s, 2H), 5.86 (s, 2H), 6.95-7.64 (8H), 8.08 (d, 1H, J=7.42)

ESI(+) m/z: 552.7

Mp: 134.5-136° C.

Shanghai Allist Pharmaceutical, Inc.

 

WO2005011646A2*

20 Jul 2004

10 Feb 2005

Nicoletta Almirante

Nitrooxy derivatives of losartan, valsatan, candesartan, telmisartan, eprosartan and olmesartan as angiotensin-ii receptor blockers for the treatment of cardiovascular diseases

US7943648*	Jun 5, 2009	May 17, 2011	Shanghai Allist Pharmaceutical Inc.	Salts of imidazole-5-carboxylic acid derivatives, a method for preparing same and pharmaceutical compositions comprising same
US8138214	Jul 2, 2009	Mar 20, 2012	Shanghai Allist Pharmaceutical, Inc.	Pharmaceutical composition
USRE44873	Jul 31, 2006	Apr 29, 2014	Salubris Asset Management Co., Ltd.	Imidazole-5-carboxylic acid derivatives, the preparation method therefor and the uses thereof

CITING PATENT	FILING DATE	PUBLICATION DATE	APPLICANT	TITLE
US8455526 *	6 Jun 2008	4 Jun 2013	Shanghai Allist Pharmaceuticals, Inc.	Therapeutic use of imidazole-5-carboxylic acid derivatives
US20100168193*	6 Jun 2008	1 Jul 2010	Shanghai Allist Pharmaceuticals, Inc.	Therapeutic use of imidazole-5-carboxylic acid derivatives
USRE44873	31 Jul 2006	29 Apr 2014	Salubris Asset Management Co., Ltd.	Imidazole-5-carboxylic acid derivatives, the preparation method therefor and the uses thereof

CN101024643A	20 Feb 2006	29 Aug 2007	上海艾力斯医药科技有限公司	Imidazo-5-carboxylic-acid derivatives, its preparing method and use
US5298519 *	24 Sep 1992	29 Mar 1994	Chemish Pharmazeutische Forschungsgesellschaft M.B.H.	Acylals of imidazole-5-carboxylic acid derivatives, and their use as angiotensin (II) inhibitors

……………….

Shanghai , CHINA

RG-1577, EVT 302, Sembragiline, RO-4602522

RG-1577, EVT 302, Sembragiline, RO-4602522

Hoffmann La Roche

CAS 676479-06-4, MW 342.36

• C19 H19 F N2 O3
• Acetamide, N-​[(3S)​-​1-​[4-​[(3-​fluorophenyl)​methoxy]​phenyl]​-​5-​oxo-​3-​pyrrolidinyl]​-

UNII-K3W9672PNJ

RG-1577, a selective and reversible monoamine oxidase B inhibitor, for treating AD (phase 2 clinical, as of May 2015).

Family members of the product case for RG-1577 (WO2004026825) hold protection in EU until 2023 and expire in US in 2024 with US154 extension. Follows on from WO2006097197, claiming a process for preparing RG-1577.

Alzheimer‘s Disease is a brain disease that slowly destroys memory and thinking skills, up to loss of the ability to carry out the simplest tasks. It is the most common cause of dementia among older people. Mild Alzheimer‘s Disease manifests itself in memory loss and small changes in other cognitive abilities, e.g getting lost, trouble handling money and managing daily tasks, having some mood and personality changes, etc.

In the stage of Moderate Alzheimer‘s Disease, the control of language, reasoning, sensory processing, and conscious thought are impacted. Memory loss and con usion grow worse, e.g patients have problems recognizing family and friends and become unable to learn new things, etc. hallucinations, delusions, and paranoia may occur. .Severe Alzheimer‘s Disease is the final stage. Patients cannot communicate anymore and are completely dependent.

N-[(3S)-l-[4-[(3-fluorophenyl)methoxy]phenyl]-5-oxo-pyrrolidin-3-yl]acetamide has previously been described in the art. 1 WO 2006/097197 2 and WO 2006/0972703 relate to methods for preparing enantiomerically pure 4-pyrrolidinophenylbenzyl ether derivatives.

The processes of the prior art hamper from several drawbacks (e.g. long reaction sequence, low overall yield also due to loss of half of the product in the classical resolution step, the need for a chromatographic purification to remove by-products formed in the Mitsunobu reaction) and are therefore less suitable for the preparation of N-[(3S)-l-[4-[(3-fluorophenyl) methoxy]phenyl]-5-oxo-pyrrolidin-3-yl]acetamide on large scale.

Most Recent Events

• 01 Aug 2014Roche completes a phase I trial in volunteers in USA (NCT02104648)
• 14 May 2014Roche completes enrolment in the MAyflOwer RoAD trial for Alzheimer’s disease (combination therapy, adjunctive treatment) in Australia, Canada, Czech Republic, France, Germany, Italy, Poland, South Korea, Spain, Sweden the United Kingdom and the USA (NCT01677754)
• 01 Apr 2014Roche initiates enrolment in a phase I trial in healthy volunteers in USA (NCT02104648)

http://www.evotec.com/uploads/media_library/10/2012-09_Evotec_Company_presentation_September_e.pdf

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WO2004026825

http://www.google.com/patents/WO2004026825A1?cl=en

………………….

WO2006097197

http://www.google.com/patents/WO2006097197A1?cl=en

……………………………………………..

PATENT

WO 2015063001

https://patentscope.wipo.int/search/ja/detail.jsf;jsessionid=82F2EFFC078602A9E3061C7CF658B36C.wapp2nA?docId=WO2015063001&recNum=37&office=&queryString=&prevFilter=%26fq%3DOF%3AWO%26fq%3DICF_M%3A%22C07D%22&sortOption=%E5%85%AC%E9%96%8B%E6%97%A5%EF%BC%88%E6%96%B0%E3%81%97%E3%81%84%E9%A0%86%EF%BC%89&maxRec=57119

Novel, crystalline polymorphic forms A and B of a pyrrolidone derivative ie RG-1577, useful for treating Alzheimer’s disease (AD). Roche and its Japanese subsidiary Chugai, under license from Evotec, which previously licensed the drug from Roche, are developing RG 1577

formula 1 via the following routes

In a certain embodiment, present invention relates to a synthesis of a compound of formula he following route A

1

In a certain embodiment, present invention relates to a synthesis of a compound of formula he following route B

In a certain embodiment, present invention relates to a crystalline polymorph of a compound of formula 1.

synthesize a compound of formula 1 from a compound of formula 7

compound of formula 6 to a compound of formula 7

In a certain embodiment, present invention relates to a process to synthesize a compound of formula 1 as described herein, further comprising reacting a compound of formula 6 via the intermediate 6a to a compound of formula 7

further comprising reacting a compound of formula 3 with a compound of formula 5 to a compound of formula 6

comprising reacting a compound of formula 2 to a compound of formula 3

2 3

In a certain embodiment, present invention relates to a process to synthesize a compound of formula 1 as described herein, further comprising reacting a compound of formula 10 to a compound of formula 6

eacting a compound of formula 9 with a compound of formula 5 to a compound of formula 10

In a certain embodiment, present invention relates to a process to synthesize a compound of formula 1 as described herein, further comprising reacting a compound of formula 8 to a compound of formula 9

(lS’)-N-[l-[4-(3-fluoro-benzyloxy)-phenyl]-5-oxo-pyrrolidin-3-yl-]acetamide (1)

To a suspension of chloride (7) (37.9 g, 100 mmol) in 2-methyltetrahydrofurane (600 ml) was added under vigorous stirring at 0°C 1.65 M potassium ie/t-butoxide in THF (75.5 ml, 125 mmol, ACROS) over 2.5 h. After additional stirring at 0°C for 1 h, the cold suspension was hydrolyzed with 0.1 M HCl (600 ml) and the reaction mixture was stirred at 30°C for 0.5 h. The organic layer was washed with water (300 ml), dried (Na2S04) and filtered. Removal of the solvent by rotary evaporation (50°C/>10 mbar) afforded 32.1 g crystalline residue, which was dissolved in 2-butanone (400 ml) at ca. 95°C and hot filtered. Crystallization, which was induced by seeding and cooling to room temperature and 0°C (4 h) afforded 25.4 g (74.2%) of the titled compound (1) as an off-white, crystalline powder,

Mp. 162-164°C (polymorph B).

Ee >99.8%, [cc]D20 = – 17.8 (DMF; c = 1).

1H NMR (400 MHz, DMSO- 6) δ ppm 1.82 (s, 3H), 2.34 (dd, J1=n. l, J2=3.9, 1H), 2.84 (dd, J/=17.1, J2=8.2, 1H), 3.55 (dd, J/=10.2, J2=3.2, 1H), 4.07 (dd, J/=10.2, J2=6.7, 1H), 4.32-4.41 (m, 1H), 5.13 (s, 2H), 7.02 & 7.55 (d, J=9.1, each 1H), 7.11-7.19 (m, 1H), 7.24-7.31 (m, 1H), 7.40-7.47 (m, 1H), 8.40 (d, J=6.4, 1H).

ESI-MS (m/z) 343 [M+H]+, 365 [M+Na]\. Anal.Calcd for Ci9H19FN203(342.37): Calcd. C, 66.66; H, 5.59; N, 8.18; F, 5.02; O, 14.02. Found C, 66.76; H, 5.48; N, 8.13; F, 5.03; O, 13.99.

Crystallized (1) form previous step (9.5 g, 0.028 mol) was dissolved in 2-butanone (290 mL) upon heating. The hot solution was filtered over charcoal. The solution was concentrated by removal of 2-butanone (200 mL) by distillation prior to seeded cooling crystallization. Filtration, washing with chilled 2-butanone and drying at 50°C/25 mbar/16h afforded 9.18 g (93.9% corrected yield) of the title compound (1) as a crystalline powder of polymorphic form B with an assay of 100.4 %(w/w) and a purity of 99.97 %(area) (by HPLC).

Alternatively, to a stirred suspension of hydroxyamide (6) (30.0 g, 0.083 mol) in toluene (500 ml) was added at 50°C within 45 minutes thionyl chloride (10.40 g, 0.087 mol) and the resulting mixture was stirred for 3h at 50°C. The mixture was then heated up to 92°C and subsequently stirred at this temperature for 15 h. The Suspension was then cooled to 50°C and toluene was removed by distillation under reduced pressure. The distillation residue was cooled to ambient temperature and treated with N-methylpyrrolidone (210 ml) to obtain an almost clear solution. This solution was then cooled to -10°C and subsequently treated at this temperature within 2h with a solution of potassium iert-butoxide (12.40 g, 0.111 mol) in THF (60 g). The resulting mixture was stirred for another 60 minutes at -10°C, then warmed up to room temperature within 60 minutes and subsequently stirred at room temperature for 6 h. The reaction mixture was quenched with water (150 g) and the pH was adjusted with acetic acid (approx. 1.8 g) to pH 7-8. The mixture was then heated to 30-45°C and THF and toluene were distilled off under reduced pressure (<200 mbar) to obtain a clear NMP/water mixture (400 ml). This mixture was heated to 45°C and 260 mg of seed crystals were added. Water (320 ml) was then added within 3 h whereby the product crystallized. The resulting suspension was cooled to room temperature within 3 h and subsequently stirred at this temperature for 2 h. Filtration and washing of the filter cake with a mixture of water (100 ml) and N-methylpyrrolidone (20 ml) and subsequently only with water (150 ml) afforded after drying (70°C/10 mbar/20 h) 26.2 g (92%) of the title compound (1) as a crystalline powder with an assay of 99.6 %(w/w) and a purity of 99.7 %(area) (by HPLC).

HPLC

Purity (HPLC): Column: XSelect Phenyl Hexyl x2, 150 x 4.6mm, 3.5um. Starting

Pressure: 226 bar; temp.: 50°C. Inj. vol.: 2.0 μΐ^ + wash. Flow: 1.0 ml/min. Det: 204 nm. A: Water + 5% ACN, 77-2% in 7 min., hold for 1 min.; B: 0.1% HCOOH, 18% isocratic; C: MeOH, 5-80% in 7 min., hold for 1 min. Sample prep.: 2 mg/ml ACN. Retention times: β-acid 5.93 min., diacid 6.18 min., cc-acid 6.89 min., diester 6.96 min.

ee determination(HPLC): Column: Chiralpak IA-3 100 x 4.6mm, 3um; 91 bar, 2ml/min; temp.: 30°C. Inj. vol.: 10.0 μL· Det.: 206 nm. A: n-heptane, 80%; B: EtOH, 20%. Sample prep.: 4 mg/ml EtOH. Retention times: D-enantiomer 2.21 min., L-enantiomer 2.71 min

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US 20050065204

EXAMPLE 11

Preparation of (S)-1-(4-Hydroxyphenyl)-5-oxo-pyrrolidine-3-carboxylic Acid

8.00 g Polyethyleneglycol 6000 was dissolved in 150 mL (100 mM) magnesium acetate buffer pH 6.0 under stirring, and the solution added to a stirred suspension of 10.00 g (42.51 mmol) (RS)-1-(4-hydroxyphenyl)-5-oxo-pyrrolidine-3-carboxylic acid methyl ester (99.7%) in 40 mL methylcyclohexane. The mixture was heated to 28° C. and the pH readjusted to 6.0 with 2 M NaOH. The reaction was started by adding 33.2 mg Candida cylindraceae cholesterase (16.88 kU/g), and the pH was maintained at 6.0 by the controlled addition of 1.0 M NaOH solution under stirring. After a total consumption of 20.35 mL (20.35 mmol) 1.0 M sodium hydroxide solution (after 17.1 h; 47.9% conversion) the reaction mixture was passed through a sintered glass filter. The filtrate spontaneously separated into an aqueous and an organic phase.The aqueous phase was washed with 2×200 mL ethyl acetate to remove uncleaved ester. The aqueous phase was set to pH 4.0 with 25% sulfuric acid and concentrated in vacuo to a volume of ca. 80 mL (bath 60° C.). The solution was cooled to 1° C. (formation of white precipitate/crystals) and the pH set to 1.5 with 25% sulfuric acid. The precipitate/crystals were stirred overnight at 1° C., filtered off on a sintered glass filter (washed with a minimum amount of water) and dried overnight on high vacuum (RT, 6×10−2 mbar) to give 4.32 g (19.53 mmol; 45.9%) (S)-1-(4-hydroxyphenyl)-5-oxo-pyrrolidine-3-carboxylic acid. Analysis: HPLC (area A226nm): 99.3%, 0.7% ester. 98.9%ee. The product contains 5.3% water (according to Karl Fischer determination) and 2.1% (w/w) PEG (according to NMR).

Company	Evotec AG
Description	Small molecule monoamine oxidase B (MAO-B) inhibitor
Molecular Target	Monoamine oxidase B (MAO-B)
Mechanism of Action	Monoamine oxidase B (MAO-B) inhibitor
Therapeutic Modality	Small molecule

Latest Stage of Development	Phase II
Standard Indication	Alzheimer’s disease (AD)
Indication Details	Treat Alzheimer’s disease (AD)
Regulatory Designation
Partner	Chugai Pharmaceutical Co. Ltd.; Roche

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Chūō, japan

A Chūō Line (Rapid) E233 series (right) and A Chūō-Sōbu Line E231 series (June 2007)

Chuo Dori street on a weekend afternoon

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Treating the flu?

They walked out together into the fine fall day, scuffling bright ragged leaves under their feet, turning their faces up to a generous sky really blue and spotless. At the first corner they waited for a funeral to pass, the mourners seated straight and firm as if proud in their sorrow. [...] “It seems to be a plague,” said Miranda, “something out of the Middle Ages. Did you ever see so many funerals, ever?”
— from “Pale Horse, Pale Rider” by Katherine Anne Porter (1939)

And I looked, and behold a pale horse: and his name that sat on him was Death, and Hell followed with him.
— Revelations 6.8 (King James Version)

In 1918-1919 between 50 and 100 million people worldwide died from the flu. The "Spanish Flu" spread to nearly every part of the world with amazing speed, helped perhaps by the thousands of soldiers returning from Europe after the end of World War I. There was little that could be done to help the sick, and often people who were healthy one day were dead the next. The Spanish Flu was remarkable at the time in that it primarily killed young healthy adults, whereas most often it is very young children and elderly people who die from infectious disease.

Oddly enough, after going through two successive waves of infection and mortality, the Spanish Flu pandemic disappeared almost abruptly. By the end of the 20th century, it was almost forgotten, and influenza had come to be regarded as one of the many childhood diseases that most people went through without much difficulty.

The situation today is quite different. Everyone is now highly sensitized to the threat of influenza. Stories about the so-called "Bird Flu" and now the "Swine Flu" have appeared regularly on television and in newspapers. Our society is more mobile than ever before, and we have seen examples of the rapid spread of diseases worldwide in recent years. Population is far more dense than it was in 1918, and diseases spread and mutate in crowded cities around the world far faster than ever before. People are deeply concerned about the possibility of a new influenza pandemic that could rival the Spanish Flu.

On the other hand, we also now know much more about how to prevent and how to treat illnesses like influenza. The best way to slow or stop the spread of influenza is through public health measures - simple things like frequent hand washing and avoiding contact with infected people. In addition, immunization is an important protective measure if a safe and effective vaccine can be developed.

But what about treating people who are already infected? Because influenza is a viral disease, antibiotics that can deal with bacterial infections will not work. The story of how drugs to treat serious cases of influenza were developed shows how structural biology, biochemistry and synthetic organic chemistry work hand-in-hand to produce new and useful chemical substances. It remains to be seen if they can help in the event of a pandemic outbreak, which many people think is a question of "when" rather than "if".

Treating the flu? Part 1: The Influenza Virus

Influenza is caused by RNA viruses of the family Orthomyxoviridae. These virions are roughly 80-120 microns in diameter. Their surfaces consist of a lipid bilayer derived from the membrane of the host cell, which is decorated by glycoproteins that project like spikes from the viral particle. About 80% of these spikes are hemagglutinin, a protein that facilitates binding the virion to a host cell. The remainder areneuraminidase, which is an enzyme that cleaves glycosidic linkages to the sugar neuraminic acid (also calledsialic acid). You have probably heard the different strains of the flu virus ("serotypes") referred to as "H1N1" or "H5N1". These names refer to the different subtypes of the two surface glycoproteins, differences that distinguish the serotypes immunogenically.

There are several outstanding web sites that will tell you much more about the influenza virus. There is no point in just repeating what they contain here, so if you want more information you can follow the links below. Otherwise, click here to move to the next part of the drug development story.

• Some really nice electron micrographs of real influenza virions that show the hemagglutinin and neuraminidase "spikes".
• Influenza 101 - the virology blog. This takes you through the structure of the virus, how it enters cells, reproduces and then leaves to infect other cells. Very good quality information and very easy reading.
• Influenza Virus from Kimball's Biology Pages
• Influenza viruses on Wikipedia.

Treating the flu? Part 2: Targets for therapy

A drug must act by binding to and modulating the activity of some target receptor or enzyme. Viruses do not present very many potential targets because they typically have only a few unique proteins coded in their genomes. Recall that viruses hi-jack the enzymes of the host cell to manufacture new virions.

The Influenza A genome consists of 8 strands of RNA:

1. The HA gene. It encodes the hemagglutinin.
2. The NA gene. It encodes the neuraminidase.
3. The NP gene encodes the nucleoprotein. Influenza A, B, and C viruses have different nucleoproteins.
4. The M gene encodes two proteins (using different reading frames of the RNA): a matrix protein M1 and an ion channel M2 spanning the lipid bilayer.
5. The NS gene encodes two different non-structural proteins that are found in the cytoplasm of the infected cell but not within the virion itself.
6. – 8. one RNA molecule (PA, PB1, PB2) for each of the 3 subunits of the RNA polymerase.

Drugs against Influenza A could potentially be developed to inhibit the activity of any of the products of the influenza genome, but in fact only drugs acting against the NA (neuraminidase) and the M2 (ion channel) proteins have been successfully developed to date.

The M2 inhibitors amantadine and rimantadine were the first effective drugs against influenza, but the M2 protein seems quite easy for the virus to modify so resistance rapidly develops against these drugs. The latest H1N1 virus that is causing pandemic concern is resistant to both amantadine and rimantadine. The drugs that are being used against current pandemic threat strains target the viral neuraminidase, and it is these that form the basis of our discussion on drug development.

Treating the flu? Part 3: Neuraminidase

This is only a very short description of this important enzyme. It assumes that you have some basic knowledge of what enzymes are and what they do. If you need more background information, your Biochemistry textbook or the Wikipedia article on enzymes are good places to start.

Recall that the surface of the influenza virion is covered with spikes of hemagglutinin and neuraminidase. Hemagglutinin is a protein that binds tightly to the sugar portions of various cell-surface glycoproteins by recognizing and binding the sugarsialic acid, which is also called N-acetyl neuraminic acid. Sialic acid is found at the terminus of the carbohydrate portions of many cell-surface glycoproteins and plays a key role in cell-cell and cell-virus binding. The human ABO blood-group antigens are examples of sialylated oligosaccharides that play an important role in medical biochemistry. Hemagglutinin permits the influenza virus to attach to a host cell during the initial infection, which in turn causes the viral RNA to enter the cell by endocytosis. This is a common mechanism for infection and we know that many viruses including HIV as well as parasites such as the Plasmodium that causes malaria attack host cells via their cell-surface carbohydrates. However, the tight grip of viral hemagglutinin on cell-surface sialic acid is a problem when new viral particles need to break away from the host cell.

The neuraminidase on the surface of the virion is necessary for new viral particles to break away from the host cell. Neuraminidase is a glycosidase (an enzyme that catalyzes the hydrolysis of glycosidic linkages) that specifically promotes the cleavage of sialic acid from glycoprotein saccharide chains. When the glycosidic linkage is cleaved by hydrolysis, the sialic acid falls off the cell surface. The viral particle is now no longer tethered to the host cell and can move off to infect other cells.

If the activity of neuraminidase is blocked, the new virions remain bound to the host cell and viral reproduction is prevented. You can view a Flash animation showing this concept here.

The structure of the influenza A neuraminidase N9 bound to an analogue of sialic acid has been determined by X-ray crystallography, and a simplified ribbon diagram is shown here. The amino acid chains are represented by the yellow ribbons, and the bound inhibitor as well as some key side chain groups are shown in ball-and-stick format. The broad arrows designate regions in which the amino acid chains form a "beta sheet" structure, with the arrow heads indicating the C-terminal end of the sheet. Cylindrical sections represent "random coil" regions of the amino acid sequence. Notice that there is essentially no helical structure in this enzyme. This image shows only one sub-unit of the biologically active form of the enzyme which is actually a tetramer of identical sub-units. The binding site of the enzyme does not vary from strain to strain. It consists of 18 amino acid residues of which 12 are in direct contact with the bound sialic acid analogue (and presumably with sialic acid in catalytically active situations). Four of these 12 are positively-charged arginines, while another 4 are negatively-charged glutamic and aspartic acid residues. The remainder are neutral (tyrosine, asparagine, isoleucine and tryptophan). If you visit the RCSB Protein Data Bank you can find X-ray structures of many neuraminidases - this one is indexed under the code "1nna". The details of the structure are discussed in the original paper by Bossart-Whitaker et al. cited below.

Mark von Itzstein and coworkers (then at the Monash University Victorian College of Pharmacy in Melbourne Australia and now at the Institute for Glycomics at Australia's Griffith University) studied the mechanism of sialic acid hydrolysis catalyzed by influenza A N9 neuraminidase. This enzyme is what is called a retaining glycosidase because if the starting glycoside has the α-configuration (as shown) then the product that is formed will also have the α-configuration. In common with many glycosidase enzymes, its active site features a pair of carboxyl residues (Asp 151 and Glu 277 in the N9 neuraminidase they studied) which play central roles in the enzyme's catalytic mechanism. The proposed mechanism is shown below.

There are two important transition states shown in this mechanism, the first for the actual cleavage of the C-O bond leading to loss of the ROH fragment and the second for the formation of a new C-OH bond. In the first transition state, notice how the enzyme assists the ionization of a water molecule, the transfer of its proton to the leaving OR group, and stabilizes the transient positive charge on the ring oxygen.

With knowledge of how the enzyme functioned, von Itzstein decided that a compound that looked like the carbohydrate in that key first transition state would be a good candidate for an anti-influenza drug that would function by preventing the release of viral particles from infected cells. Click here to go to the next stage in the story - synthesizing and testing a new compound.

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Wikipedia article about neuraminidase.

The story of how neuraminidase was identified as a target for anti-influenza drug development is briefly outlined by Graeme Laver, one of the key researchers in this field. You can read his March 2007 article in Education in Chemistry here.

Bossart-Whitaker, P.; Carson, M.; Babu, Y.S.; Smith, C.D.; Laver, W.G.; Air, G.M. J. Mol. Biol. 1993, 232, 1069–1083. (Link requires valid U of Manitoba Library ID).

von Itzstein, M. et al. Nature 1993, 363, 418-423. (Link requires valid U of Manitoba Library ID).

Treating the flu? Part 4: Developing Neuraminidase Inhibitors

Zanamivir (Relenza)

Note: this document should not be taken as any form of endorsement of the substances mentioned or as a recommendation for treatment.

With the information gained from structural and mechanistic studies on influenza A neuraminidase, von Itzstein and his team set out to devise and synthesize a stable molecule that looked sufficiently like the transition state to bind very tightly to the enzyme, thus inhibiting it. Recall that a transition state is not a stable isolable molecule, but it is possible to mimic the geometry of a proposed transition state with other chemical structures. These are called transition state analogues.

The proposed transition state for glycosidic bond cleavage in the mechanism previously outlined is shown here. Recall that for clarity the sugar structure has been simplified. It is evident that the reactive centre of the sugar ring is planar in this transition state. It is not possible to make a stable structure that has a double bond between position 2 and the ring oxygen similar to the partial double bond in the transition structure. Thus, von Itzstein et al. decided that a good inhibitor needed a double bond between positions 2 and 3 - that is, it should be a 2,3-dehydro derivative of sialic acid.

They also concluded that a strongly basic guanidino group should replace the hydroxyl at C-4 in the sialic acid structure. This would be positively charged at physiological pH and would bind strongly to a region of negative charge in the active site.

They synthesized and tested the structure shown in 1989 and found that it was indeed a potent and very selective inhibitor of influenza neuraminidase. Their synthetic route, published in the journal Carbohydrate Research in 1994, is shown below.

Although some of the reagents used in this synthesis may be unfamiliar, organic chemistry students should be able to recognize what is going on in each step. In the first step shown, the Lewis acid boron trifluoride etherate promotes an internal S_N2 reaction in which the carbonyl of the acetamide displaces the acetate ester to form the new ring. Notice the inversion of configuration at C4. This is then subjected to another S_N2 reaction in which the nucleophile is the azide anion N₃^-. The reagent is trimethylsilyl azide, which also provides mildly Lewis acidic activation for the displacement. Azide groups are excellent precursors for amines, and the reduction of the azide is easily carried out. You can see that some care must be taken here, since if the reaction is left too long the hydrogenation of the alkene will also occur. Simple alkaline hydrolysis removes the methyl ester and the acetate ester protecting groups, and then the amino group is converted into the desired guanidino function using formamidine sulfonic acid. This provided the desired neuraminidase inhibitor 4-deoxy-4-guanidino-2,3-dehydro-N-acetyl neuraminic acid, which ultimately has become the anti-influenza drug zanamivir (sold under the trade name Relenza by GlaxoSmithKline).

You can see how well zanamivir fits into the active site of influenza A neuraminidase from the X-ray crystal structure obtained by Zu et al. and indexed in theProtein Data Bank as 3b7e. This is an interesting structure because the enzyme is the neuraminidase from the A/Brevig Mission/1/1918 H1N1 strain, one of the viruses that caused the 1918 Spanish Flu. The genome of this virus was obtained from the frozen body of a woman who died in the Alaskan village of Brevig Missionin 1918. An interesting New York Times article describes the discovery of this virus (and incidentally the Johan Hultin who found the virus is no relation to Dr. Hultin!). It is another variation of the H1N1 strain that is at the centre of the 2009/2010 concern about Swine Flu.

The ribbon diagram has simplified the enzyme structure considerably - only those amino acids near the active site are shown, and only the most important ones that interact with zanamivir have their sidechains drawn. The drug molecule is shown in a space-filling representation in which oxygen is red, nitrogen is blue and carbon is white. Hydrogens are not shown. The diagram places the carboxylate group of zanamivir at the 6 o'clock position, while the guanidinium group is projecting backwards deep into the binding site. The hydroxylated sidechain is projecting forward at about 9 o'clock. The schematic drawing (based on a diagram from the book by Levy and Fugedi referenced below) shows the key contacts between the enzyme and the drug.

Numerous other synthetic routes to zanamivir have been published since the original synthesis shown here, and you can be very sure that the industrial synthesis isquite different. The problem with Zanamivir is that it cannot be administered orally. Because the guanidino group is strongly basic, if it were taken orally it would be protonated in the stomach. The resulting positively-charged structure could not be taken up from the gut. Zanamivir is usually administered by inhalation, but this is not as acceptable to many people as a pill would be, and does not give a particularly high level of bioavailability.

Given this problem with zanamivir, it is not surprising that others tried to find similar compounds to inhibit influenza neuraminidase that could be orally administered. Click here to find out about the second-generation drug oseltamivir (Tamiflu).

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von Itzstein, M.; Wu, W.-Y.; Jin, B. Carbohydrate Research 1994, 259, 301-305.

Taylor, N.R.; von Itzstein, M. J. Med. Chem. 1994, 37, 616–624.

Magano, J. Chem. Rev. 2009, in press. (You must have a valid U of Manitoba library ID to access the full-text article)

Xu, X.; Zhu, X.; Dwek, R.A.; Stevens, J.; Wilson, I.A. J.Virol. 2008, 82, 10493-10501.

Levy, D.; Fugedi, P. (Eds.) The Organic Chemistry of Sugars, CRC/Taylor & Francis: 2006.

Treating the flu? Part 4: Developing Neuraminidase Inhibitors

Other neuraminidase inhibitors

Research and development of new anti-influenza drugs has not stopped. The need for more effective drugs remains a powerful incentive for academic and industrial scientists, and there is of course a strong profit motive as well.

One compound that is now in clinical trials is peramivir, under development by BioCryst Pharmaceuticals. If you look at the structure of peramivir, you can see its family resemblance to other neuraminidase inhibitors. However, peramivir must be administered by injection because it has rather poor oral bioavailability. In fact, peramivir was initially developed by Johnson and Johnson but was abandoned because it was not orally active. Renewed interest in it as an injectable drug may be because only the most severe cases of influenza really need antiviral therapy, and such patients are likely already hospitallized.

Another new compound is CS-8958, from Japan's Daiichi Sankyo Co. Ltd. This compound is structurally very similar to zanamivir, differing only in the functionalization of the hydroxylated sidechain.

CS-8958 is a prodrug and not the active form. The octyl ester group is hydrolyzed in the liver, releasing the active neuraminidase inhibitor which only differs from zanamivir in having a methyl ether at the C7 position rather than a hydroxyl group. The main advantage of CS-8958 is that it is long-acting. Oseltamivir and zanamivir must be taken twice daily, but in a clinical study a single inhaled treatment with CS-8958 gave the same anti-influenza effect as twice-daily doses of oseltamivir over 5 days.