SEE  CHINESE ARTICLE ON  Recent Progress in the Synthesis of Tamiflu Zhang Tiancai, Lu Hui, Zhang Fu-Min, Cheng Jun, Liu Tianyang 2013, 33 (06), pp 1235-1243 Publication Date: 25 June 2013 (Reviews) DOI:10.6023/cjoc201303044

Oseltamivir total synthesis concerns the total synthesis of the antiinfluenza drug oseltamivirmarketed by Hoffmann-La Roche under the trade name Tamiflu. Its commercial production starts from the biomolecule shikimic acid harvested from Chinese star anise with a limited worldwide supply. Due to its limited supply, searches for alternative synthetic routes preferably not requiring shikimic acid are underway and to date several such routes have been published. Control of stereochemistry is important: the molecule has three stereocenters and the sought-after isomer is only 1 of 8 stereoisomers.

Commercial production

The current production method is based on the first scalable synthesis developed by Gilead Sciences ^[1] starting from naturally occurring quinic acid or shikimic acid. Due to lower yields and the extra steps required (because of the additional dehydration), the quinic acid route was dropped in favour of the one based on shikimic acid, which received further improvements by Hoffmann-La Roche.^[2][3] The current industrial synthesis is summarised below:

Karpf / Trussardi synthesis

The current production method includes two reaction steps with potentially hazardous azides. A reported azide-free Roche synthesis of tamiflu is summarised graphically below:^[4]

The synthesis commences from naturally available (−)-shikimic acid. The 3,4-pentylidene acetal mesylate is prepared in three steps: esterification with ethanol and thionyl chloride; ketalization with p-toluenesulfonic acid and 3-pentanone; and mesylation with triethylamine and methanesulfonyl chloride. Reductive opening of the ketal under modified Hunter conditions^[5] in dichloromethane yields an inseparable mixture of isomeric mesylates. The corresponding epoxide is formed under basic conditions withpotassium bicarbonate. Using the inexpensive Lewis acid magnesium bromide diethyl etherate (commonly prepared fresh by the addition of magnesium turnings to 1,2-dibromoethane in benzene:diethyl ether), the epoxide is opened with allyl amine to yield the corresponding 1,2-amino alcohol. The water-immiscible solvents methyl tert-butyl ether and acetonitrile are used to simplify the workup procedure, which involved stirring with 1 M aqueous ammonium sulfate. Reduction on palladium, promoted byethanolamine, followed by acidic workup yielded the deprotected 1,2-aminoalcohol. The aminoalcohol was converted directly to the corresponding allyl-diamine in an interesting cascade sequence that commences with the unselective imination of benzaldehyde with azeotropic water removal in methyl tert-butyl ether. Mesylation, followed by removal of the solid byproduct triethylamine hydrochloride, results in an intermediate that was poised to undergo aziridination upon transimination with another equivalent of allylamine. With the librated methanesulfonic acid, the aziridine opens cleanly to yield a diamine that immediately undergoes a second transimination. Acidic hydrolysis then removed the imine. Selective acylation with acetic anhydride (under buffered conditions, the 5-amino group is protonated owing to a considerable difference in pK_a, 4.2 vs 7.9, preventing acetylation) yields the desired N-acetylated product in crystalline form upon extractive workup. Finally, deallylation as above, yielded the freebase of oseltamivir, which was converted to the desired oseltamivir phosphate by treatment with phosphoric acid. The final product is obtained in high purity (99.7%) and an overall yield of 17-22% from (−)-shikimic acid. It is noted that the synthesis avoids the use of potentially explosive azide reagents and intermediates; however, the synthesis actually used by Roche uses azides. Roche has other routes to oseltamivir that do not involve the use of (−)-shikimic acid as a chiral pool starting material, such as a Diels-Alder route involving furan and ethyl acrylate or an isophthalic acid route, which involves catalytic hydrogenation and enzymatic desymmetrization.

Corey synthesis

In 2006 the group of E.J. Corey published a novel route bypassing shikimic acid starting from butadiene and acrylic acid.^[6] The inventors chose not to patent this procedure which is described below.

Butadiene 1 reacts in an asymmetric Diels-Alder reaction with the esterfication product of acrylic acid and 2,2,2-Trifluoroethanol 2 catalysed by the CBS catalyst. The ester 3 is converted into an amide in 4 by reaction with ammonia and the next step to lactam 5 is an iodolactamization with iodine initiated by trimethylsilyltriflate. The amide group is fitted with a BOC protective group by reaction with Boc anhydride in 6 and the iodine substituent is removed in an elimination reaction with DBU to the alkene 7. Bromine is introduced in 8 by an allylic bromination with NBS and the amide group is cleaved with ethanol and caesium carbonate accompanied by elimination of bromide to the diene ethyl ester 9. The newly formed double bond is functionalized with N-bromoacetamide 10 catalyzed with Tin(IV) bromide with complete control of stereochemistry. In the next step the bromine atom in 11 is displaced by the nitrogen atom in the amide group with the strong base KHMDS to the aziridine 12 which in turn is opened by reaction with 3-pentanol 13 to the ether 14. In the final step the BOC group is removed with phosphoric acid and the oseltamivir phosphate 15 is formed.

Shibasaki synthesis

Also in 2006 the group of Masakatsu Shibasaki of the University of Tokyo published a synthesis again bypassing shikimic acid.^[7][8]

Shibasaki Tamiflu synthesis Part I		Part II

An improved method published in 2007 starts with the enantioselective desymmetrization of aziridine 1 with trimethylsilyl azide (TMSN₃) and a chiral catalyst to the azide 2. Theamide group is protected as a BOC group with Boc anhydride and DMAP in 3 and iodolactamization with iodine and potassium carbonate first gives the unstable intermediate 4and then stable cyclic carbamate 5 after elimination of hydrogen iodide with DBU.

The amide group is reprotected as BOC 6 and the azide group converted to the amide 7 by reductive acylation with thioacetic acid and 2,6-lutidine. Caesium carbonateaccomplishes the hydrolysis of the carbamate group to the alcohol 8 which is subsequently oxidized to ketone 9 with Dess-Martin periodinane. Cyanophosphorylation withdiethyl phosphorocyanidate (DEPC) modifies the ketone group to the cyanophosphate 10 paving the way for an intramolecular allylic rearrangement to unstable β-allylphosphate 11 (toluene, sealed tube) which is hydrolyzed to alcohol 12 with ammonium chloride. This hydroxyl group has the wrong stereochemistry and is therefore inverted in a Mitsunobu reaction with p-nitrobenzoic acid followed by hydrolysis of the p-nitrobenzoate to 13.

A second Mitsunobu reaction then forms the aziridine 14 available for ring-opening reaction with 3-pentanol catalyzed by boron trifluoride to ether 15. In the final step the BOC group is removed (HCl) and phosphoric acid added to objective 16.

Fukuyama synthesis

An approach published in 2007 ^[9] like Corey’s starts by an asymmetric Diels-Alder reaction this time with starting materials pyridine and acrolein.

Fukuyama Tamiflu synthesis Part I		Part II

Pyridine (1) is reduced with sodium borohydride in presence of benzyl chloroformate to the Cbz protected dihydropyridine 2. The asymmetric Diels-Alder reaction with acrolein3 is carried out with the McMillan catalyst to the aldehyde 4 as the endo isomer which is oxidized to the carboxylic acid 5 with sodium chlorite, Monopotassium phosphate and 2-methyl-2-butene. Addition of bromine gives halolactonization product 6 and after replacement of the Cbz protective group by a BOC protective group in 7 (hydrogenolysis in the presence of Di-tert-butyl dicarbonate) a carbonyl group is introduced in intermediate 8 by catalytic ruthenium(IV) oxide and sacrificial catalyst sodium periodate. Addition ofammonia cleaves the ester group to form amide 9 the alcohol group of which is mesylated to compound 10. In the next step iodobenzene diacetate is added, converting the amide in a Hofmann rearrangement to the allyl carbamate 12 after capturing the intermediate isocyanate with allyl alcohol 11. On addition of sodium ethoxide in ethanol three reactions take place simultaneously: cleavage of the amide to form new an ethyl ester group, displacement of the mesyl group by newly formed BOC protected amine to anaziridine group and an elimination reaction forming the alkene group in 13 with liberation of HBr. In the final two steps the aziridine ring is opened by 3-pentanol 14 and boron trifluoride to aminoether 15 with the BOC group replaced by an acyl group and on removal of the other amine protecting group (Pd/C, Ph₃P, and 1,3-dimethylbarbituric acid in ethanol) and addition of phosphoric acid oseltamivir 16 is obtained.

Trost synthesis

In 2008 the group of Barry M. Trost of Stanford University published the shortest synthetic route to date.^[10]

1. Rohloff John C., Kent Kenneth M., Postich Michael J., Becker Mark W., Chapman Harlan H., Kelly Daphne E., Lew Willard, Louie Michael S., McGee Lawrence R. et al. (1998). “Practical Total Synthesis of the Anti-Influenza Drug GS-4104”. J. Org. Chem. 63 (13): 4545–4550. doi:10.1021/jo980330q.
2.  Federspiel M., Fischer R., Hennig M., Mair H.-J., Oberhauser T., Rimmler G., Albiez T., Bruhin J., Estermann H. et al. (1999). “Industrial Synthesis of the Key Precursor in the Synthesis of the Anti-Influenza Drug Oseltamivir Phosphate (Ro 64-0796/002, GS-4104-02) Ethyl (3R,4S,5S)-4,5-epoxy-3-(1-ethyl-propoxy)-cyclohex-1-ene-1-carboxylate”. Org. Process Res. Dev. 3: 266–274. doi:10.1021/op9900176.
3.  Abrecht S., Federspiel M. C., Estermann H., Fischer R., Karpf M., Mair H.-J., Oberhauser T., Rimmler G., Trussardi R. et al.. “The Synthetic-Technical Development of Oseltamivir Phosphate Tamiflu™: A Race against Time Chimia”. 2007; 61: 93–99. doi:10.2533/chimia.2007.93.
4.  New, Azide-Free Transformation of Epoxides into 1,2-Diamino Compounds: Synthesis of the Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate (Tamiflu) Martin Karpf and René Trussardi J. Org. Chem.; 2001; 66(6) pp 2044 – 2051; (Article) doi:10.1021/jo005702l PMID 11300898.
5.  Birgit Bartels and Roger Hunter (1993). “A selectivity study of activated ketal reduction with borane dimethyl sulfide”. J. Org. Chem. 58 (24): 6756. doi:10.1021/jo00076a041.
6.  A Short Enantioselective Pathway for the Synthesis of the Anti-Influenza Neuramidase Inhibitor Oseltamivir from 1,3-Butadiene and Acrylic Acid Ying-Yeung Yeung, Sungwoo Hong, and E. J. Corey J. Am. Chem. Soc.; 2006; 128(19) pp 6310 – 6311; (Communication) doi:10.1021/ja0616433
7.  De Novo Synthesis of Tamiflu via a Catalytic Asymmetric Ring-Opening of meso-Aziridines with TMSN3 Yuhei Fukuta, Tsuyoshi Mita, Nobuhisa Fukuda, Motomu Kanai, and Masakatsu Shibasaki J. Am. Chem. Soc.; 2006; 128(19) pp 6312 – 6313; doi:10.1021/ja061696k
8.  Second Generation Catalytic Asymmetric Synthesis of Tamiflu: Allylic Substitution Route Tsuyoshi Mita, Nobuhisa Fukuda, Francesc X. Roca, Motomu Kanai, and Masakatsu Shibasaki Org. Lett.; 2007; 9(2) pp 259 – 262; (Letter) doi:10.1021/ol062663c
9. A Practical Synthesis of (-)-Oseltamivir Nobuhiro Satoh, Takahiro Akiba, Satoshi Yokoshima, Tohru Fukuyama Angew. Chem. Int. Ed. 2007, 46, 5734 –5736doi:10.1002/anie.200701754
10.  A Concise Synthesis of (−)-Oseltamivir Barry M.Trost, Ting Zhang Angew. Chem. Int. Ed. 2008, 47, 1-4 doi:10.1002/anie.200800282

Jiangsu Chengxin Pharmaceutical Co., Ltd with total area 60,000m2 and total invested amount of RMB 300 million, is a high-tech joint-stock enterprise established in 2012, located in Binjiang Pharm-Chem Industry Park, Qidong, the outstanding cultural city well known as the Rivers and Seas Pearl in Jiangsu Province, close to Chongming Island with merely one separated river. It takes around 1 hour by car from the manufacturing site to Shanghai, the Yangtze River Delta economic metropoli……More

http://www.cxpharma.com/en/about.asp?id=8

Dear sir,

We are just a manufacturer and very strong in the following intermediates:

Intermediates for Capecitabine:

2′,3′-Di-O-acetyl-5′-deoxy-5-fluorocytidine (CAS: 161599-46-8)

1,2,3-Triacetyl-5-deoxy-D-ribose (CAS: 62211-93-2)

Methyl-5-deoxy-2,3-O-isopropylidene-beta-D-ribofuranoside (CAS: 23202-81-5)

We have the dedicated workshop for Capecitabine intermediate. Our capacity is more than 20MT per month. Our company is complied with the requirement from EU GMP, US FDA and Chinese GMP. Just for your information.

If you are interested in any of above, please let me know.

Looking forward to your early reply.

Best regards,

Runya Wang(Ms) / Sales Department 

Add:     No.338 Shanghai Road, Binjiang Pharm-Chem

Industry Park, Qidong, Jiangsu, P.R.China 226221

Tel:       0086-513-86029596

Fax:      0086-513-86105399

Email:   runya.wang@cxpharma.com

Web:     www.cxpharma.com

http://www.cxpharma.com/en/home.asp   in english

We’ve all dreamt of popping a pill to help us safely lose weight, or at least eat that chocolate cake without guilt. But alas, even though the Food and Drug Administration has approved two new diet drugs in recent months, that dream probably isn’t any closer to reality.

In the current issue of the BMJ (formerly the British Medical Journal), Sidney Wolfe, founder of the advocacy group Public Citizen, slams the FDA for approving the drugs – lorcaserin (US brand name Belviq) and topiramate (called Qsymia). The FDA’s European counterpart rejected both of them because of heart risks that turned up during preliminary trials.

read all this at

http://commonhealth.wbur.org/2013/08/diet-pill-dilemma-why-is-fda-approving-drugs-when-europe-isnt

efavirenz

• Sustiva (USA, Bristol-Myers Squibb)
• Stocrin (EU, MSD)
• Aspen Efavirenz (Sub-Saharan Africa, Aspen Pharmacare)

Generic brands India:

• E.F (McNeil & Argus)
• Efavir (Cipla)
• Efcure (Emcure Pharmaceuticals)
• Efferven (Ranbaxy Laboratories)
• Estiva (Hetero)
• Evirenz (Alkem Laboratories)
• Viranz (Aurobindo Pharma)

Zhejiang Huahai Pharma received CFDA approval to produce efavirenz, an oral non-nucleoside reverse transcriptase inhibitor (NNRTI) used to control the symptoms of AIDS. Huahai is the first China drugmaker approved to make the drug. Huahai produced efavirenz API for Merck, which marketed the drug under the name Stocrin

read at

http://www.sinocast.com/readbeatarticle.do?id=99634

Efavirenz (EFV, brand names Sustiva, Stocrin, Efavir etc.) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and is used as part of highly active antiretroviral therapy(HAART) for the treatment of a human immunodeficiency virus (HIV) type 1.

For HIV infection that has not previously been treated, the United States Department of Health and Human Services Panel on Antiretroviral Guidelines currently recommends the use of efavirenz in combination with tenofovir/emtricitabine (Truvada) as one of the preferred NNRTI-based regimens in adults and adolescents.

Efavirenz is also used in combination with other antiretroviral agents as part of an expanded postexposure prophylaxis regimen to reduce the risk of HIV infection in people exposed to a significant risk (e.g. needlestick injuries, certain types of unprotected sex etc.).

The usual adult dose is 600 mg once a day. It is usually taken on an empty stomach at bedtime to reduce neurological and psychiatric adverse effects.

Efavirenz was combined with the popular HIV medication Truvada, which consists oftenofovir and emtricitabine, all of which are reverse transcriptase inhibitors. This combination of three medications approved by the U.S. Food and Drug Administration(FDA) in July 2006 under the brand name Atripla, provides HAART in a single tablet taken once a day. It results in a simplified drug regimen for many patients.

History

Efavirenz was approved by the FDA on September 21, 1998, making it the 14th approved antiretroviral drug.

•  Efavirenz is a non-nucleoside reverse trancriptase inhibitor being studied clinically for use in the treatment of HIV infections and AIDS.
• Efavirenz chemically known as (-) 6-Chloro-4-cyclopropylethynyl-4-trifluoromethyl- 1 , 4- dihydro-2H-3, 1-benzoxa zin-2-one, is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI).A number of compounds are effective in the treatment of the human immunodeficiency virus (HIV) which is the retrovirus that causes progressive destruction of the human immune system. Effective treatment through inhibition of HIV reverse transcriptase is known for non- nucleoside based inhibitors. Benzoxazinones have been found to be useful non-nucleoside based inhibitors of HIV reverse transcriptase.(-) β-chloro^-cyclopropylethynyM-trifluoromethyl-l ,4-dihydro-2H-3,l -ben zoxazin-2-one (Efavirenz) is efficacious against HIV reverse transcriptase resistance. Due to the importance of (-)6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-l,4-dihydro-2H-3,l-ben zoxazin-2- one, economical and efficient synthetic processes for its production needs to be developed.

  The product patent US5519021. discloses the preparation of Efavirenz, in Example-6, column-29, involving cyclisation of racemic mixture of 2-(2-amino-5-chlorophenyl)-4- cyclopropyl-l,l,l-trifluoro-3-butyn-2-ol using l ,l ‘-carbonyldiimidazole as carbonyl delivering agent to give racemic Efavirenz. Further, resolution of the racemic Efavirenz is carried out using (-) camphanic acid chloride to yield optically pure Efavirenz. However, research article published in the Drugs of the future, 1998, 23(2), 133-141 discloses process for manufacture of optically pure Efavirenz. The process involves cyclisation of racemic 2-(2-amino-5-chlorophenyl)-4-cyclopropyl-l, 1, l-trifluoro-3-butyn-2- ol using 1, 1-carbonyldiimidazole as carbonyl delivering agent to give racemic Efavirenz and further resolution by (-) camphanic acid chloride.

  Similarly research article published in Synthesis 2000, No. 4, 479-495 discloses stereoselective synthesis of Efavirenz (95%yield, 99.5%ee), as shown below

  Even though many prior art processes report method for the preparation of Efavirenz, each process has some limitations with respect to yield, purity, plant feasibility etc. Hence in view of the commercial importance of Efavirenz there remains need for an improved process.

• US 6 028 237 discloses a process for the manufacture of optically pure Efavirenz.
• The synthesis of efavirenz and structurally similar reverse transcriptase inhibitors are disclosed in US Patents 5,519,021, 5,663,169, 5,665,720 and the corresponding PCT International Patent Application WO 95/20389, which published on August 3, 1995. Additionally, the asymmetric synthesis of an enantiomeric benzoxazinone by a highly enantioselective acetylide addition and cyclization sequence has been described by Thompson, et al., Tetrahedron Letters 1995, 36, 8937-8940, as well as the PCT publication, WO 96/37457, which published on November 28, 1996.
• Additionally, several applications have been filed which disclose various aspects of the synthesis of(-)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one including: 1) a process for making the chiral alcohol, U.S.S.N. 60/035,462, filed 14 January 1997; 2) the chiral additive, U.S.S.N. 60/034,926, filed 10 January 1997; 3) the cyclization reaction, U.S.S.N. 60/037,059, filed 12 February 1997; and the anti-solvent crystallization procedure, U.S.S.N. 60/037,385 filed 5 February 1997 and U.S.S.N. 60/042,807 filed 8 April 1997.

Efavirenz has been obtained by two related ways: 1) The acylation of 4-chloroaniline (I) with pivaloyl chloride (II) by means of Na2CO3 in toluene gives the expected anilide (III), which is acylated with ethyl trifluoroacetate by means of butyllithium in THF yielding, after hydrolysis with HCl, 2′-amino-5′-chloro-2,2,2-trifluoroacetophenone (IV). The benzylation of (IV) with 4-methoxybenzyl chloride (V) in basic alumina affords the protected acetophenone (VI), which is regioselectively condensed with cyclopropylacetylene (VII) [obtained by cyclization of 5-chloro-1-pentyne (VIII) by means of butyllithium in cyclohexane] by means of butyllithium in THF in the presence of (1R,2S)-1-phenyl-2-(1-pyrrolidinyl)-1-propanol (IX) giving the (S)-isomer of the tertiary alcohol (X) exclusively. The cyclization of (X) with phosgene and triethylamine or K2CO3 in toluene/THF yields the benzoxazinone (XI), which is finally deprotected with ceric ammonium nitrate in acetonitrile/water. 2) The condensation of 2′-amino-5′-chloro-2,2,2-trifluoroacetophenone (IV) with cyclopropylacetylene (VIII) by means of butyllithium or ethylmagnesium bromide in THF gives (?-2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol (XII). The cyclization of (XII) with carbonyldiimidazole (XIII) in hot THF yields the racemic benzoxazinone (XIV). Compound (XIV) is submitted to optical resolution by condensation with (S)-(-)-camphanoyl chloride by means of dimethylaminopyridine (DMAP) in dichloromethane to give the acyl derivative (XVI) as a diastereomeric mixture that is resolved by crystallization and finally decomposed with HCl in ethanol or butanol.

The China Food and Drug Administration (CFDA) has accepted Sihuan Pharmaceutical’s application for clinical trial approval for its Pirotinib, a Category 1.1 innovative oncology drug developed by the company’s drug R&D team.

By developing Pirotinib, Sihuan Pharma has demonstrated its capability for the oncology products market. The company holds the largest cardio-cerebral vascular (CCV) drug franchise in China’s prescription market.

The new drug is a second generation (pan-HER) inhibitor intended to treat patients with lung and breast cancer.http://www.pharmaceutical-technology.com/news/newssihuan-pharmas-clinical-study-application-oncology-drug-pirotinib-accepted-cfda?WT.mc_id=DN_News

Bristol-Myers Squibb licensed exclusive China rights to market Coniel, a calcium channel blocker treatment for hypertension and angina pectoris, from Kyowa Hakko Kirin Co. BMS said the transaction, its first China-specific in-licensing deal, demonstrated the company’s long-term commitment to China. Previously, Kyowa Hakko Kirin handled China marketing of the product itself. 

O5-methyl O3-[(3R)-1-(phenylmethyl)piperidin-3-yl] 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate

Benidipine (INN), also known as Benidipinum or benidipine hydrochloride, is a dihydropyridine calcium channel blocker for the treatment of high blood pressure (hypertension).

Dosing

Benidipine is dosed as 2–4 mg once daily.^[1]

Benidipine is sold as Coniel by Kyowa Hakko Kogyo.

Benidipine is only licensed for use in Japan and selected Southeast Asian countries, where it is sold as 4 mg tablets.

Also known as: 105979-17-7, NCGC00185768-01, Benidipene, AC1LCVDP, SureCN24516, CTK8E8626, AKOS015895389, H007

Benidipine hydrochloride, A calcium channel protein inhibitor
Alternative Name: KW 3049 Chemical Name: (4R)-rel-1,4-Dihydro-2,6-dimethyl-4​-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 3-methyl 5-[(3R)-1-(phenylmethyl)-3-piperidinyl] ester hydrochloride

Xuezhikang

Xuezhikang, the extract of red yeast rice, has been widely used as a Chinese traditional medicine for the therapy of patients with cardiovascular diseases. It contains natural Lovastatin and its homologues, as well as unsaturated fatty acids, flavonoids, plant sterols and other biologically active substances

The product is a world-recognized blood lipid regulator, which is made by extracting from “specially-made red yeast rice”. It combines modern high-tech biotechnology with traditional Chinese medicine, which can safely and effectively regulate blood lipids in a comprehensive way with proven curative effects and reliable safety.

Pharmacological Effects: the product can reduce blood cholesterol, triglycerides, low density lipoprotein cholesterol, improve high density lipoprotein cholesterol, inhibit atherosclerotic plaque formation, and protect vascular endothelial cells; and inhibit lipid deposition in the liver. The large-scale evidence-based research has proven that long-term use of XUEZHIKANG can greatly reduce the risk of CHD occurrence and decrease the mortality. XUEZHIKANG is the only Chinese medicine with blood lipids regulating function which is listed into the National Basic Medicine List.

Beijing Peking University WBL Biotech (WPU) has developed and launched Xuezhikang, a capsule formulation of Monascus purpureus-fermented rice, for the oral treatment of hyperlipidemia and cardiovascular disease

CLINICAL TRIAL, NCT01327014  PHASE 2

The data had shown that Xuezhikang significantly reduced the level of low density lipoprotein cholesterin (LDL-C) in patients in a similar manner to statins and increased the level of the beneficial high density lipoprotein cholesterin (HDL-C). It had a good safety profile with no significant liver enzyme abnormal events observed. Besides regulation of dyslipidemia, the drug also signifcantly reduced cardiovascular events and general mortality rate of patients

NCT01686451 PHASE 4

Both XueZhiKang and Statins are cholesterol-lowering medications that are often prescribed for individuals with high cholesterol and who are at risk for cardiovascular disease (CVD). Several studies, including one randomized, double-blind, placebo-controlled clinical trial, have suggested that the use of statins is more frequently associated with fatigue. And XueZhiKang may be not. The purpose of this study is to compare the effect of these two medications on fatigue in persons who are at moderate to low CVD risk based on the risk estimation system in ESC(European Society of Cardiology)/ESA(European Atherosclerosis Society) guidelines (2011) for the management of dyslipidemias.

Those of you with high cholesterol will be happy to learn that there are some legitimate options to your statin pills. Many people cannot tolerate the extremely popular statin pills, especially from side effects of muscle aches. But there’s now some very strong evidence that herbal medicines, including red yeast rice, can be at least as effective as a statin, and without the side effects. Too good to be true? Maybe not…

Red yeast rice is a bright reddish purple fermented rice, which acquires its colour from being cultivated with the mold Monascus purpureus. Red yeast rice is known as Zhi Tai when in powdered form but is called Xue Zhi Kang in alcohol extract form. This has been used in China for many centuries for many reasons, but researchers have been very interested in its effectiveness in lowering cholesterol and preventing heart disease (similar benefits from statins). It seems that the main active ingredient is indeed the natural form of a common statin, lovastatin — but researchers feel that other ingredients inside may add more protective effects. There is an official patented Chinese TCM formulation, called Xue Zhi Kang (xue2 zhi1 kang2 jiao nang 血脂康 胶囊), which has the equivalent of 10mg of lovastatin. The ScienceDaily website has a nice 2008 review of a well-designed study, printed in American Journal of Cardiology, which followed 5,000 persons after their first heart attack, and divided them into two groups taking either xuezhikang or placebo. After 5 years:

Frequencies of the primary end point were 10.4% in the placebo group and 5.7% in the XZK-treated group, with absolute and relative decreases of 4.7% and 45%, respectively. Treatment with XZK also significantly decreased CV and total mortality by 30% and 33%, the need for coronary revascularization by 1/3, and lowered total and low-density lipoprotein cholesterol and triglycerides, but raised high-density lipoprotein cholesterol levels. In conclusion, long-term therapy with XZK significantly decreased the recurrence of coronary events and the occurrence of new CV events and deaths, improved lipoprotein regulation, and was safe and well tolerated.

This is impressive data, and the study design is very well done, which means the evidence is quite strong. One co-author, Dr Capuzzi, has a nice summary:

“It’s very exciting because this is a natural product and had very few adverse side effects including no abnormal blood changes,” said Capuzzi. “People in the Far East have been taking Chinese red yeast rice as food for thousands of years, but no one has ever studied it clinically in a double-blind manner with a purified product against a placebo group until now and we are pleased with the results. However, people in the United States should know that the commercially available over-the-counter supplement found in your average health food store is not what was studied here. Those over-the-counter supplements are not regulated, so exact amounts of active ingredient are unknown and their efficacy has not been studied yet.”

In another randomized trial study, printed last year in the Annals of Internal Medicine, patients who had previously failed treatment of statins due to side effects were given 1800mg of red yeast rice twice a day versus placebo. The red yeast rice group had a significant improvement in cholesterol numbers — with no major reports of severe muscle aches they previously had on the statins.

There are other studies that also show similar benefits. In fact, the evidence is so strong that it is classified as Grade A evidence: “Strong scientific evidence for use”. This is the highest grade that any therapy can get. There are a number of good reviews of red yeast rice in Western literature, including from Medscape; the Mayo Clinic; WebMD; MedlinePlus; and NCCAM. There’s also more informal information from the TCM blog Qi Spot. You can find more scholarly information in the 2008 review from Chinese Medical Journal.

http://www.hindawi.com/journals/ecam/2012/636547/

(U.S. patent #6,046,022), ethanol extract of red yeast rice, with a total monacolins content of approx. 0.8%.

1  Heber D et al. Cholesterol-lowering effects of a proprietary Chinese red-yeast-rice dietary supplement. American Journal of Clinical Nutrition 1999;69(2): 231-236
2) SoRelle R. Appeals court says Food and Drug Administration can regulate cholestin. Circulation 200;102 (7): E9012?E9013.
3) Li, C et al. Monascus purpureus-fermented rice (red yeast rice): a natural food product that lowers blood cholesterol in animal models of hypercholesterolemia. Nutrition Research 1998;18 (1): 71-81
4) Becker DJ et al. Red yeast rice for dyslipidemia in statin-intolerant patients: a randomized trial.Ann Intern Med. 2009 Jun 16;150(12):830-9, W147-9
5) Lu Z et al.Effect of Xuezhikang, an extract from red yeast Chinese rice, on coronary events in a Chinese population with previous myocardial infarction. Am J Cardiol. 2008 Jun 15;101(12):1689-93.

Hypochol is the same product. Xuezhikang is the brand name marketed in China. Hypochol, is manufactured by a Singapore comapany who have a joint venture agreement with Peking University who perfected the processing and quality control of the Red Yeast Rice Extract Product. You can order directly from: http://www.hypocol.com/wbm.html
or thru their New Zealand distributor (very good service) at: http://www.hypocol.co.nz/

Dried grain red yeast rice

In addition to its culinary use, red yeast rice is also used in Chinese herbology and traditional Chinese medicine. Its use has been documented as far back as the Tang Dynasty in China in 800 AD. It is taken internally to invigorate the body, aid in digestion, and revitalize the blood. A more complete description is in the traditional Chinese pharmacopoeia, Ben Cao Gang Mu-Dan Shi Bu Yi, from the Ming Dynasty (1378–1644).

What other names is Red Yeast known by?

Arroz de Levadura Roja, Cholestin, Hong Qu, Koji Rouge, Levure de Riz Rouge, Monascus, Monascus purpureus, Monascus Purpureus Went, Red Rice, Red Rice Yeast, Red Yeast Rice, Red Yeast Rice Extract, Riz Rouge, Xue Zhi Kang, XueZhiKang, XZK, Zhibituo, Zhi Tai.

What is Red Yeast?

Red yeast is the product of rice fermented with Monascus purpureus yeast. Red yeast supplements are different from red yeast rice sold in Chinese grocery stores. People use red yeast as medicine.

Possibly Effective for…

• High cholesterol.
• High cholesterol and triglyceride levels caused by human immunodeficiency virus (HIV) disease (AIDS).

Insufficient Evidence to Rate Effectiveness for…

• Indigestion, diarrhea, improving blood circulation, spleen and stomach problems, and other conditions.

In the late 1970s, researchers in the United States and Japan were isolating lovastatin from Aspergillus and monacolins fromMonascus, respectively, the latter being the same fungus used to make red yeast rice but cultured under carefully controlled conditions. Chemical analysis soon showed that lovastatin and monacolin K are identical. The article “The origin of statins” summarizes how the two isolations, documentations and patent applications were just months apart.^[5] Lovastatin became the patented, prescription drug Mevacor for Merck & Co. Red yeast rice went on to become a contentious non-prescription dietary supplement in the United States and other countries.

Lovastatin and other prescription “statin” drugs inhibit cholesterol synthesis by blocking action of the enzyme HMG-CoA reductase. As a consequence, circulating total cholesterol and LDL-cholesterol are lowered. In a meta-analysis of 91 randomized clinical trial of ≥12 weeks duration, totaling 68,485 participants, LDL-cholesterol was lowered by 24-49% depending on the statin. Different strains ofMonascus fungus will produce different amounts of monacolins. The ‘Went’ strain of Monascus purpureus (purpureus = dark red in Latin), when properly fermented and processed, will yield a dried red yeast rice powder that is approximately 0.4% monacolins, of which roughly half will be monacolin K (identical to lovastatin). Monacolin content of a red yeast rice product is described in a 2008 clinical trial report.

• Website about medicinal use of Monascus purpureus
• Medicinal use of Red yeast rice
• Fermentiertes Rotes Reismehl (in German)

Harbin Gloria to Commercialize Constipation Drug in China

– See more at: http://www.chinabiotoday.com/articles/20150512_1#sthash.YbauZ6qM.dpuf

http://www.chinabiotoday.com/articles/20150512_1

AMITIZA (lubiprostone)

Harbin Gloria Pharmaceuticals Co., Ltd. engages in the research, development, production, and sale of pharmaceutical products primarily in the People’s Republic of China. The company offers orthopedic medicines, antineoplastic products, medical-nutrition products, rheumatology drugs, digestive and respiratory system medicines, cardiovascular medicines, liver disease medications, gynecology medications, and antibiotics. It also provides circulatory system, pediatrics, uropoiesis and reproduction, immune regulation, and other products. Harbin Gloria Pharmaceuticals Co., Ltd. was founded in 2000 and is based in Harbin, the People’s Republic of China.

MORINIDAZOLE

1- [3- (4-morpholinyl) -2-hydroxypropyl] -2-methyl-5- nitro -1H- imidazole

CAS 92478-27-8

Jiangsu Hansoh Pharmaceutical Co., Ltd.

Morinidazole was approved by China Food and Drug Administration (CFDA) on February 24, 2014. It was developed and marketed as a step Lingda ® by Hansoh Pharmaceutical.

Morinidazole is a nitroimidazoles antibiotic indicated for the treatment of bacterial infections including appendicitis and pelvic inflammatory disease (PID) caused by anaerobic bacteria.

PATENT

WO2006058457A1.

http://www.google.com/patents/WO2006058457A1?cl=en

……………………….

PATENT
CN1981764A.

https://www.google.com/patents/CN1981764A?cl=en

1- (2,3-epoxypropoxy yl) -2-methyl-5-nitro-imidazole (10g), morpholino (10g), 100ml of acetonitrile under reflux for 2 hours, vacuum recovery of acetonitrile, water was added 100ml, heating to the whole solution, filtered hot, let cool, filtering, washing and drying to obtain an off-white solid (11g).

Proton nuclear magnetic resonance data: 1HNMR (CD3Cl) δ2.39 ~ 2.73 (6H, m) δ2.61 (3H, s) δ3.71 ~ 3.81 (4H, m) δ4.10 ~ 4.17 (2H, m) δ4 .63 ~ 4.66 (1H, m) δ8.00 (1H, s)

CN 102199147

http://www.google.com/patents/CN102199147A?cl=en

CN 1605586

https://www.google.com/patents/CN1605586A?cl=en

Example 7 Preparation of α- (morpholino-1-yl) methyl-2-methyl-5-nitroimidazole-1-ethanol according to Example 4 the same manner as in Preparation α- (morpholino-1-yl) methyl-2-methyl-5-nitroimidazole-1-ethanol, except for using morpholine instead of 4-hydroxypiperidine, prepared by the present invention Compound 7. Proton nuclear magnetic resonance data: 1HNMR (CD3Cl) δ2.39 ~ 2.73 (6H, m) δ2.61 (3H, s) δ3.71 ~ 3.81 (4H, m) δ4.10 ~ 4.17 (2H, m) δ4

NMR PREDICT

1H NMR  PREDICT

13C NMR PREDICT

COSY

China Generic Drugmakers Poaching Indian Execs

Written by Richard Daverman, PhD, Executive Editor, Greg B. Scott.

In the competition between China and India pharmas, China’s generic drug industry leads in the supply of APIs to global drugmakers, but India supplies more finished generic drugs to the world’s marketplace. That may be changing. According to press reports,

China drugmakers have begun hiring experienced Indian pharma execs, offering them two to three times their present salaries.

The China companies are willing to pay at these levels because the Indian professionals have two skills the Chinese want: drug formulation experience and English.

China’s drugmakers want help as they target the western world’s lucrative generic drug market.

More details…. http://www.chinabiotoday.com/articles/20150903

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Icaritin;  118525-40-9; AC1NSXIV; UNII-UFE666UELY;

3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromen-4-one

3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromen-4-one

The roots of Epimedium brevicornu Maxim

Beijing Shenogen Granted Fast Track Status for Novel Cancer Drug

Written by Richard Daverman, PhD, Executive Editor, Greg B. Scott.

Beijing Shenogen Biomedical announced that Icaritin, a China Class I cancer drug, was granted Fast Track Review status after the company filed its New Drug Approval submission to the Beijing Food & Drug Administration. Icaritin is an oral traditional Chinese medicine, derived from barrenwort, which targets the estrogen receptor α36. Shenogen has conducted clinical trials of Icaritin in patients with liver cancer, though it expects the drug will also prove effective in breast cancer and other estrogen-related cancers as well. More details…. http://www.chinabiotoday.com/articles/20150917

Antiproliferative agent (IC₅₀ values are 8,13 and 18 μM for K562, CML-CP and CML-BC cells respectively). Inhibits H/R-induced PTK activation. Induces G(2)/M cell cycle arrest and mitochondrial transmembrane potential drop. Modulates MAPK/ERK/JNK and JAK2/STAT3 /AKT signaling. Inhibits PPAR-g. Modulates differentiation. Inhibits cytochrome P450 in vivo. Orally active.

Cardiovascular function improvement, hormone regulation and antitumor activity.
2. The anti-MM activity of Icaritin was mainly mediated by inhibiting IL-6/JAK2/STAT3 signaling.
3. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosis.
4. The Icaritin at low concentration (4 or 8 μmol/L) can promote rat chondrocyte proliferation and inhibit cell apoptosis, while the effect of Icaritin on rat chondrocyte at high concentration was reversed.
5. Icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.
6. Icaritin dose-dependently inhibits ENKL cell proliferation and induces apoptosis and cell cycle arrest at G2/M phase. Additionally, Icaritin upregulates Bax, downregulates Bcl-2 and pBad, and activates caspase-3 and caspase-9.

What is Epimedium ?

Herba epimedii (Epimedium, also called bishop’s hat, horny goat weed or yin yang huo), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. It is a genus of about 60 flowering herbs, cultivated as a ground cover plant and an aphrodisiac. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Most of them bloom in the early spring, and the leaves of some species change colors in the fall, while other species retain their leaves year round.

Figure 1 Epimedium

Epimedium Raw Material

The herbs we used to extract icariin is one species of Epimedium, which name is Epimedium brevicornum Maxim. This kind of epimedium only can be abundantly found in Gansu province of China. And because of the growth habit of this kind of herb, which only grows under trees, it can’t to be planted, only can harvest the wild one.

This wild epimedium contains quite a bit of active components, depending on its long growth time and rich nutrient. Usually the content of the icariin is not lower than 1%.

Below photo is the herb specimen which we use. Picking in the epimedium full-bloom stage. And the medicinal value of the herb is the best at this time. The herb we select contains roots, stems, leaves and flowers. And we extract with the whole herb.

Figure 2 Epimedium for extract

Epimedium Extract

Epimedium extract is a herbal supplement claimed to be beneficial for the treatment of sexual problems such as impotence. It is believed to contain a number of active components, including plant compounds that may have antioxidant activity and estrogen-like compounds. The major components of Epimedium brevicornum are icariin, epimedium B and epimedium C. It is reported to have anti-inflammatory, anti-proliferative, and anti-tumor effects. It is also reported to have potential effects on the management of erectile dysfunction.

Figure 3 HPLC spectrum of icariin

Our specification available is Icariin HPLC 50%- 98%. Below please see the the information for reference:

      Figure 4 Epimedium Extract(Icariin)

Derivatives

The plant extracts of epimedium traditionally used for male impotence, and the individual compounds is icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. The E. brevicornum extract and its active principle icariin were active. To improve its inhibitory activity, some derivatives ware subjected to various structural modifications, which include icaritin, icariside II and 3,7-bis(2-hydroxyethyl) icaritin. There have some scientific papers report that the improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make such compounds a promising candidate for further development. We hope that our new products can help you to find more commercial opportunity.

In this way, we can introduce those products as below, and we can also provide more details about the products according to your demand. The 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin is as below.

Figure 4 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin

Main Function of Epimedium Extract 

horny goat weed; epimedium; Icariin; penis medicine;epimedium p.e;epimedium brevicornum; shorthorned epimedium herb; Icariins; Icaritin; 3,7-Bis(2-Hydroxyethyl)Icaritin; icariin 60%; icariin 98%; epimedium graepimedium; icarisides II;epimedium sagittatum;epimedium leaf; barrenwort.powder extract

Epimedium has been used to treat male erectile dysfunction in Traditional Chinese Medicine for many centuries. The main functions of Epimedium brevicornum in ancient Chinese books focused on the nourishment of kidney viscera and reinforcement of ‘yang’, resulting in the restoration of erectile function in males.

Epimedium contains chemicals which might help increase blood flow and improve sexual function. It also contains phytoestrogens, chemicals that act somewhat like the female hormone estrogen that might reduce bone loss in postmenopausal women.

Figure 5 some products from epimedium extract

………..

PAPER

The novel total synthesis of icaritin (1), naturally occurring with important bioactive 8-prenylflavonoid, was performed via a reaction sequence of 8 steps including Baker-Venkataraman reaction, chemoselective benzyl or methoxymethyl protection, dimethyldioxirane (DMDO) oxidation, O-prenylation, Claisen rearrangement and deprotection, starting from 2,4,6-trihydroxyacetophenone and 4-hydroxybenzoic acid in overall yields of 23%. The key step was Claisen rearrangement under microwave irradiation. MS, ¹H and ¹³C NMR techniques have been used to confirm the structures of all synthetic compounds. – See more at: http://www.eurekaselect.com/124334/article

…….

PAPER

Synthesis of icariin from kaempferol through regioselective methylation and para-Claisen–Cope rearrangement

Qinggang Mei^1,2, Chun Wang¹, Zhigang Zhao³, Weicheng Yuan² and Guolin Zhang¹

¹Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
²Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China
³College of Chemistry and Environmental Protection Engineering, Southwest University for Nationalities, Chengdu 610041, China…http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-11-135

http://www.beilstein-journals.org/bjoc/content/supplementary/1860-5397-11-135-S1.pdf

Scheme 1: Reagents and conditions: (a) Ac₂O, pyridine, 94%; (b) BnBr, KI, K₂CO₃, acetone, 85%; (c) Me₂SO₄, K₂CO₃, acetone, MeOH, 82%; (d) MOMCl, N,N-diisopropylethylamine (DIPEA), CH₂Cl₂, 93%; (e) 3,3-dimethylallyl bromide, 18-crown-6, K₂CO₃, acetone, 86%; (f) Eu(fod)₃, NaHCO₃, PhCl, 85 °C, 61%; (g) MeOH, 3 M HCl (aq), reflux, 95%; (h) Pd/C, 1,4-cyclohexadiene, MeOH, 84%.

Scheme 2: Decomposition of 8.

Scheme 3: Claisen rearrangement of flavonol 8.

Scheme 4: Reagents and conditions: (a) 15, DMF/CHCl₃, Ag₂CO₃, molecular sieves (4 Å, powder); (b) 16, CH₂Cl₂, Ag₂O, molecular sieves (4 Å powder), 31% for 2 steps; (c) NH₃ (g), MeOH, 94%; (d) NH₃ (g), MeOH, 63% for 2 steps.

3 Nguyen, V.-S.; Shi, L.; Li, Y.; Wang, Q.-A. Lett. Org. Chem. 2014, 11, 677–681.

4. Dell’Agli, M.; Galli, G. V.; Dal Cero, E.; Belluti, F.; Matera, R.; Zironi, E.; Pagliuca, G.; Bosisio, E. J. Nat. Prod. 2008, 71, 1513–1517.

 1H NMR

13C NMR

HMBC

NOESY

………….

The present invention relates to compositions comprising icariside I, and to a novel, one step method of preparing such compositions, comprising converting specific prenylated flavonol glycosides such as epimedium A, epimedium B, epimedium C, icariin, and their corresponding acetate derivatives contained in an Epimedium plant extract to a single compound, namely icariside I shown below as compound I, which was surprisingly discovered to be a strong PDE-5 inhibitor.

This invention further comprises compositions enriched for anhydroicaritin, and to methods of preparing such compositions. One method of this invention for preparing compositions enriched for anhydroicaritin comprises a one-step method of converting prenylated flavonol glycosides, specifically the sagittatoside compounds A, B, and C, and the corresponding acetate derivatives, present in Epimedium plant extracts to a single compound, namely anhydroicaritin shown below as compound II, which was also discovered to be a strong PDE-5 inhibitor.

http://www.google.com/patents/US6399579

EXAMPLES Example 1 Acid Hydrolysis of a 50% EtOH Extract and Purification by Reversed Phase ChromatographyWhole Epimedium grandiflorum leaves were extracted with a 1:1 mixture of ethanol and water at 55° C. The resulting extract (referred to as a “50% EtOH extract”) was filtered and the filtrate concentrated at 40-50° C. under vacuum and then dried under vacuum at 60° C. to a dry solid. The dried extract (131 g) containing approximately 5.8 g of total PFG’s was placed in a 2 liter round bottom flask and 1 L of 90% ethanol was added. The mixture was heated to reflux to help dissolve the solids. Concentrated sulfuric acid (28 mL) was added. The mixture refluxed for 2 hr, cooled to room temperature, and 900 mL of water added with stirring. Next the mixture was filtered using vacuum to remove insoluble sulfate salts and other solids and loaded on a 2.5×56 cm (275 mL) column packed with 250-600 micron divinylbenzene cross-linked polystyrene resin (Mitsubishi Chemical). The column was washed with 2 column volumes (CVs) of 60% ethanol and the icariside I was eluted with 2 CVs of 95% ethanol. The product pool was air-dried producing 11.3 g of brown solids. HPLC analysis (FIG. 5) showed that the solids contained 18% icariside I (peak 15.27 min) and 12% anhydroicaritin (peak 25.15 min). The recovery of the icariside I in the product pool was 87% of the amount present in the hydrolyzate.

Example 2 Purification of a Hydrolyzate by Liquid/liquid ExtractionThe ethanolic hydrolyzate (25 mL) prepared in Example 1 was mixed with 62.5 mL of de-ionized water and the pH was adjusted to 7.0 using 50% (w/w) sodium hydroxide solution. The resulting mixture was extracted with three 25 mL portions of ethyl acetate and the combined ethyl acetate extracts were back extracted with 150 mL of water. The ethyl acetate layers were combined, dried, and assayed for icariside I. HPLC analysis (FIG. 6) showed that the dried EtOAc fractions contained 22% icariside I (peak 15.29 min) and 11% anhydroicaritin (peak 25.27 min), and icariside I recovery into the ethyl acetate was 97% of the amount present in the hydrolyzate. The partition coefficient for icariside I between ethyl acetate and water was found to be 16, indicating that the icariside I has a high affinity for ethyl acetate over water.

Example 3 Acid Hydrolysis of a 50% EtOH Extract and Purification by PrecipitationThe dried extract (204 g) described in Example 1 was mixed with 1 L of 90% EtOH and then heated to reflux to help dissolve the solids. Sulfuric acid (25 ML) was added slowly with swirling. The mixture was refluxed 90 minutes and immediately chilled to stop the reaction. After cooling to room temperature, the mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials, and the cake was washed with about 350 mL of 90% ethanol. The resulting ethanolic hydrolyzate (1.34 L) contained 4.1 g of icariside I.

The ethanolic hydrolyzate prepared above (1.32 L) was placed in a 10 L container and 40 g of 50% (w/w) sodium hydroxide solution was added followed by 20 mL of phosphoric acid. Next 3.3 L of deionized water was added with stirring. The pH of this mixture was 2.4. Sodium hydroxide solution (50% w/w ) was added until the pH was 8.25. The mixture was heated to 65° C. to assist with the coagulation of the precipitate. The mixture was cooled to room temperature and stirred for 0.5 hr at room temperature before filtering through a cellulose filter using vacuum. The resulting brown solids were washed with 715 mL of 10% ethanol and dried either under vacuum at room temperature or in air at 55° C. to yield brown solids. HPLC analysis (FIG. 7) showed the solids contained 20% icariside I (peak 15.27 min) and 10% anhydroicaritin. Recovery of icariside I using this precipitation procedure was 94% of the amount present in the hydrolyzate.

Example 4 Acid Hydrolysis of a Water Extract and Purification by PrecipitationGround Epimedium grandiflorum leaves (0.40 kg) were mixed with 5 L water in a 10 L round bottom flask. The flask was placed on a rotary evaporator for two hours at a rotation speed of 120 rpm and a water bath temperature of 90° C. The extract was filtered under reduced pressure through cellulose paper. The resulting filtrate (3.2 L) was evaporated using the rotary evaporator to a volume of 100 mL and dried under vacuum at 50° C.

The dark brown solids prepared above (40.4 g) were mixed with 200 mL of 90% ethanol and 6.0 mL of sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with 15 mL of 90% ethanol. The resulting ethanolic hydrolyzate (215 mL) contained 0.53 g of icariside I.

The hydrolyzate prepared above (50 mL) was transferred to a 250 mL beaker and 2.5 mL of 50% (w/w) sodium hydroxide solution was added with stirring to adjust the pH of the solution to pH 9, followed by 1.5 mL of concentrated phosphoric acid. Deionized water (125 mL) was added, and the mixture was adjusted to pH 8.2 using 1.5 mL of 50% sodium hydroxide solution. The mixture was heated to 65° C. to assist with coagulation of the precipitate and cooled to room temperature. The mixture was allowed to sit undisturbed at room temperature for 30 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 25 mL of de-ionized water and dried under vacuum at room temperature or in air at 80° C. to produce olive-green solids. HPLC analysis (FIG. 8) showed the solids contained 60% icariside I (peak 15.33 min) and 2.4% anhydroicaritin (peak 25.40 min). Recovery of icariside I using this precipitation procedure was 92% of the amount present in the hydrolyzate.

Example 5 Enzymatic Hydrolysis of Icariside Ia) The substrate was a partially purified icariside I product with 20% icariside I and 11% anhydroicaritin. About 50 mg was dissolved in 10 mL of ethanol, and water or buffer was added until the mixture became cloudy (about 20% ethanol). The following dry enzymes were added to separate samples: α-amylase, α-glucosidase, β-amylase, β-glucosidase, hesperidinase, lactase, and pectinase. The samples were incubated overnight at 40 ° C. and analyzed by HPLC. The results were only semi-quantitative due to the difficulty in dissolving the anhydroicaritin that precipitated from the samples. However, several of the chromatograms did show a definite reduction in icariside I and increase in the ratio of anhydroicaritin to icariside I. The best results were obtained using hesperidinase, lactase, β-glucosidase and pectinase.

A larger scale experiment was done using hesperidinase in order to isolate pure anhydroicaritin for characterization. Pure icariside I (20 mg )was dissolved in 10 mL of ethanol and 50 mL of water and 200 mg of hesperidinase enzyme was added and the mixture was incubated for 24 hr at 40 ° C. Crude anhydroicaritin was collected via filtration and purified on a 2.5×30 cm semi-prep C-18 HPLC column using a gradient of 50:50 (MeCN/H₂O) to 80:20 (MeCN/H₂O) in 20 min. The pure anhydroicaritin was analyzed by LC/MS and proton NMR.

b) Enzymatic Hydrolysis of PFG’s: The purified PFG solids (55.3%, purified by reversed-phase chromatography of a 50% EtOH extract) were subjected to enzymatic hydrolysis with the same enzymes and conditions described in part (a). Hesperidinase, lactase, β-glucosidase and pectinase appeared to convert the mixture of PFG’s to a mixture of sagittatosides, but no icariside I or anhydroicaritin were observed. This indicated that these enzymes were specific for the 7-β-glucosyl group and did not hydrolyze the 3-position sugar(s).

Example 6 Preparation of a High Anhydroicaritin-containing ProductA high sagittatosides Epimedium sagittatum extract containing 24.7% total sagittatosides (assayed as icariin) and 8.1% icariin and other expected prenylated flavonol glycosides was obtained from China. A 50 g portion of this extract was mixed with 250 mL of 90% ethanol and 7.5 mL of concentrated sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes, then allowed to cool to room temperature. The hydrolyzed mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 20 mL of 90% ethanol. The resulting filtered ethanolic hydrolyzate (305 mL) contained 3.75 g of anhydroicaritin and 2.50 g of icariside I.

The filtered hydrolyzate prepared above (200 mL) was transferred to a 1000 mL container and 8.0 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 4.0 mL of phosphoric acid. De-ionized water (500 mL) was then added. This mixture was adjusted to pH 4.9 using 50% sodium hydroxide solution. The mixture was allowed to sit undisturbed at room temperature for 24 hours prior to decanting off the liquid. The resulting solids were macerated using de-ionized water and filtered under reduced pressure through cellulose paper. The resulting dark brown solids (11.9 g) were washed with de-ionized water and dried in air overnight. The dark brown solids contained 20% anhydroicaritin and 12% icariside I and an anhydroicaritin/icariside I ratio of 1.66. The recovery of anhydroicaritin in the precipitation procedure was 94% from the hydrolyzate.

Example 7 Recrystallization of Icariside IIcariside 1 (30 mg) obtained by a method described in Example 1 was dissolved in a minimum of hot tetrahydrofuran (THF). Hot methanol (approximately 10 mL) was then added. The hot THF/MeOH solution was filtered through a PTFE filter into a vial and allowed to evaporate at room temperature to about 5 mL, whereupon crystals began to form, and then placed in a 4° C. refrigerator for 24 hours. The crystals were filtered and washed with cold methanol and dried in a vacuum. Icariside I (21 mg) was isolated as yellow crystals and had a chromatographic purity of 97.4%.

Example 8 Large Scale Acid Hydrolysis of an Epimedium extractAn 800 g portion of an Epimedium sagittatum powder extract obtained from China containing about 13% total prenylflavonol glycosides as icariin was mixed with 4.0 L of 90% ethanol and 120 mL of sulfuric acid in a 10 L round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 200 mL of 90% ethanol. The resulting ethanolic hydrolyzate (4.0 L) contained 33.7 g of icariside I.

The ethanolic hydrolyzate prepared above was transferred to a 34 L container and 200 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 120 mL of phosphoric acid. De-ionized water (10 L) was then added. This mixture was adjusted to pH 8.2 using 120 mL of 50% sodium hydroxide solution. The mixture was stirred for 10 minutes and allowed to sit undisturbed at room temperature for 60 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 750 mL of de-ionized water and dried under vacuum at 50° C. or in air at 80° C. The olive-green solids contained 44.6% icariside I. Recovery of icariside I in the precipitation procedure was 96% from the hydrolyzate.

Example 9 Large Scale Purification of an Epimedium Extract Containing Prenylflavonoid GlycosidesA 3.7 kg portion of an Epimedium sagittatum powdered extract obtained from China containing approximately 10% total prenylflavonol glycosides (PFG’s) assayed as icariin was stirred with 35 L of 85/15 acetone/water (v/v) in a 50 L mixing tank. The mixture was stirred vigorously for 30 minutes and allowed to sit for 5 minutes. The acetone extract layer (36 L) was decanted from the tank and contained 362 g of PFG’s. Recovery of the PFG’s in this extraction procedure was 96%.

A portion (about 500 mL) of the acetone extract was dried under reduced pressure at 50° C. or less, providing 16.1 g of brown solids which were analyzed to contain 28.6% total PFG’s when assayed as icariin.

TABLE 1
			PDE-5
			IC50
Entry	Sample description	% PFG’s	(μg/mL)
1	Vat extraction of Epimedium leaves,	8.0	5.78
	refluxing for 17 hours with methanol
2	Extract prepared by extracting Epimedium	7.2	4.24
	leaves with 50% ethanol
3	Extract prepared by extracting Epimedium	10.2	12.50
	leaves with 90% ethanol
4	Extract prepared by extracting Epimedium	16.30	5.27
	leaves with 50% EtOH and then purifying
	the extract (after removal of EtOH) by
	liq/liq extraction with butanol. Sample
	tested was the butanol fraction.
5	Extract prepared by extracting Epimedium	19.3	3.97
	leaves with 50% EtOH and purifying by
	liquid/liquid extraction. Sample tested was
	the aqueous fraction of the liq/liq extraction.
6	Purification of a 90% ethanol extract on	65.60	1.87
	a HP-20 reversed phase column

TABLE 2
			PDE-5
		%	IC50
Entry	Sample description	icarside I	(μg/mL)
7	Crude hydrolyzate composition obtained	2.1	24.30
	from a 50% EtOH extract of Epimedium
	leaves
8	Crude hydrolyzate composition obtained	5.3	9.39
	from a 90% EtOH extract of Epimedium
	leaves
9	Icariside I fraction obtained from	21.4	1.50
	purifying hydrolyzate Sample No. 7 on a
	SP-70 reversed-phase column and
	eluting icariside I with alcohol
10	Pure (recrystallized) icariside I	100	0.33
11	Pure anhydroicaritin	0	1.50
12	icariside I hydrate	0	21.50
13	sildenafil	0	0.031

• Liang DL & Zheng SL Effects of icaritin on cytochrome P450 enzymes in rats. Pharmazie 69:301-5 (2014).Read more (PubMed: 24791596) »
• Guo Y  et al. An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells. Eur J Pharmacol 658:114-22 (2011). Read more (PubMed: 21376032) »
• Zhu Jf  et al. Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings. PLoS One 6:e23720 (2011). Read more (PubMed: 21887305) »
• The roots of Epimedium brevicornu Maxim

/////////Beijing Shenogen,  Granted Fast Track Status,  Novel Cancer Drug, Icaritin, New Drug Approval submission,  Beijing Food & Drug Administration, oral traditional Chinese medicine, barrenwort

Lefucoxib (乐福昔布)

5-(3,4-dimethyl-phenyl)-1-methanesulfonyl-3-trifluoromethol-pyrazole

1 [4- (methylsulfonyl) phenyl] -3-trifluoromethyl-5- (3,4-dimethylphenyl) – pyrazole

CAS 849048-84-6

IND FILED

Prostaglandin G/H Synthase 2 (PTGS2; COX-2) Inhibitors

A COX-2 inhibitor potentially for the treatment of rheumatoid arthritis.

cyclooxygenase-2 (COX-2) inhibitor

National Center of Biomedical Analysis

冉允章, 梅世昌

梅世昌, 冉允章

CHINA FLAG

PATENT

CN 1468854

http://www.google.com/patents/CN1468854A?cl=en

Example 1

1 [4- (methylsulfonyl) phenyl] -3-trifluoromethyl-5- (3,4-dimethylphenyl) – pyrazole (I1)

1- (3,4- two toluene-yl) -4,4,4-trifluoro-methyl – D-1,3-dione (IV1) of sodium metal was weighed 2.3g (0.1mol) was added 50ml of anhydrous toluene to prepare a sodium sand. After cooling, ethanol was added dropwise 12ml, and then heated at 60 ℃, complete reaction of sodium metal. After cooling to room temperature, was added 3,4-dimethylphenyl ethanone 23.8g (0.1mol) and trifluoroacetic ethyl acetate 20ml (0.2mol), reacted at 100 ℃ 5 hours. Toluene was distilled off under reduced pressure, a 10% aqueous hydrochloric acid was added, the pH was adjusted to 2-3, extracted with ethyl acetate, washed with water, dried over anhydrous MgSO4, ethyl acetate was distilled off under reduced pressure. Then under reduced pressure, distillation, collecting fractions 105-107 ℃ / 0.7mmHg, was 14.6g, 60% yield.

1- [4- (methylsulfonyl) phenyl] -3-trifluoromethyl-5- (3,4-dimethylphenyl) – pyrazole (I1) take the above-prepared substituted (IV1) 2.38g (0.01mol ), 15ml of ethanol, then added p-methanesulfonyl phenyl hydrazine salt alkoxide 2.3g (0.01ml). Was refluxed for 15 hours. Place the refrigerator overnight, the crystals were collected by filtration, recrystallized from ethanol, mp 129-31 ℃, to give 3.1 g.

Elemental analysis: C19H17F3N2O2S Calculated: C, 57.86; H, 4.34; N, 7.10 Found: C, 57.97; H, 4.29; N, 7.20MS (m / z): 395 (M + 1)

References

Cheng, Feixiong, Edited by Lee, Philip W, From Handbook of Metabolic Pathways of Xenobiotics (2014), 4, 1655-1656

Bi, X.; Meng, Z.; Chen, H.; Zhu, X.; Dou, G.
In vivo and in vitro metabolism of lefucoxib in rats, J Pharm Biomed Anal. 2008 Sep 10;48(1):134-9. doi: 10.1016/j.jpba.2008.04.024. Epub 2008 Apr 30.

Bi, X.; Meng, Z.; Dou, G.   Determination of lefucoxib in rat plasma, urine, and feces by high-performance liquid chromatography with fluorescence detection: Application in pharmacokinetic studies
J Chromatogr B Anal Technol Biomed Life Sci 2007, 850(1-2): 199

Talanta (2011), 85(1), 8-27

Jiefangjun Yaoxue Xuebao (2009), 25(6), 496-498.

Yaowu Fenxi Zazhi (2006), 26(9), 1222-1224.

Zhongguo Yaolixue Yu Dulixue Zazhi (2007), 21(2), 147-151.

China PLA General Hospital

.

//////////c1c(ccc(c1C)C)c2n(nc(c2)C(F)(F)F)c3ccc(cc3)S(=O)(=O)C

CC1=C(C=C(C=C1)C2=CC(=NN2C3=CC=C(C=C3)S(=O)(=O)C)C(F)(F)F)C

Chidamide (Epidaza)

CS055; HBI-8000

CAS   743438-44-0  CORRECT

Molecular Weight, 390.41

• Benzamide, N-(2-amino-4-fluorophenyl)-4-[[[1-oxo-3-(3-pyridinyl)-2-propenyl]amino]methyl]-
• N-(2-Amino-4-fluorophenyl)-4-[[[1-oxo-3-(3-pyridinyl)-2-propen-1-yl]amino]methyl]benzamide
• CS 055
• Chidamide
• Epidaza

 

 

ERROR IN STRUCTURE

FLUORO IN WRONG POSITION

CAS Registry Number: 743420-02-2

CN103833626A

As described for Example 2 according to the patent ZL03139760.3 obtained chidamide poor purity (about 95%). LC / MS analysis results shown in Figure 1, show that the product contains N- (2- amino-5-fluorophenyl) -4- (N- (3- pyridin-acryloyl group of 4.7% of the structure shown in formula II) aminomethyl) benzamide. 1H NMR analysis of the results shown in Figure 2, show that the product contains 1.80% of tetrahydrofuran, far beyond the technical requirements for people with drug registration International Conference on Harmonization (ICH, International Conference of Harmonizition) provided 0.072% residual solvent limits. Therefore, the solid

Body not for pharmaceutical manufacturing.

WRONG COMPD….https://www.google.com/patents/CN103833626A?cl=en

Chidamide (Epidaza) is an HDAC inhibitor (HDI) developed wholly in China.^[1] It was originally known as HBI-8000.^[2]

It is a benzamide HDI) and inhibits Class I HDAC1, HDAC2, HDAC3, as well as Class IIb HDAC10.^[3]

It is approved by the Chinese FDA for relapsed or refractory peripheral T-cell lymphoma (PTCL), and having orphan drug status in Japan.^[2]

As of April 2015 it is only approved in China.^[1]

It shows potential in treating pancreatic cancer.^[4][5][6]

Is NOT approved for the treatment of pancreatic cancer.

Chidamide drug administration and clinical milestone

November 2005: China declared IND

November 2006: eligible for Phase I clinical documents of approval

November 2006: completion of the International Patent Licensing, China entered the international fray original new drug development

May 2008: completed Phase I clinical, showing international mechanism similar drugs have the potential to become the best

February 2009: eligible lymphoma indications II / III of this document

March 2009: Start of the Phase II clinical trial for the NDA to ①CTCL goal of clinical trials and ②PTCL

March 2009: IND by the FDA application is eligible to start Phase I clinical in the United States

July 2009: eligible for non-small cell lung cancer, breast cancer and prostate cancer clinical documents of approval

December 2010: of PTCL by a conventional phase II directly into Phase II clinical trial registered drug trial center and by recognition

March 2011: combination chemotherapy for non-small cell lung cancer clinical trials enter phase Ib

September 2012: of PTCL indication test deadline

December 2012: of PTCL clinical summary will be held

January 2013: Chidamide declare China NDA

December 2014: the State Food and Drug Administration (CFDA) approved the listing

Chidamide overview, location and clinical significance

Chidamide (Epidaza), a class I HDAC inhibitor, was discovered and developed by ChipScreen and approved by the CFDA in December 2014 for the treatment of recurrent of refractory peripheral T-cell lymphoma. Chidamide, also known as CS055 and HBI- 8000, is an orally bioavailable benzamide type inhibitor of HDAC isoenzymes class I , as well as class IIb 10, with potential antineoplastic activity. It selectively binds to and inhibits HDAC, leading to an increase in acetylation levels of histone protein H3.

Chidamide, the English called Chidamide, by the Shenzhen-core biotechnology limited liability company independent design and synthesis of a novel anti-cancer drugs with new chemical structures and global intellectual property, and its chemical name N- (2-amino-_4_ fluorophenyl) -4_ (N- (3- topiramate Li acryloyl) aminomethyl) benzamide, its chemical structure of the structural formula I

The patent ZL03139760.3 and said US7,244,751, Chidamide have histone deacetylase inhibitory activity can be used to treat the differentiation and proliferation-related diseases such as cancer and psoriasis, especially for leukemia and solid tumors with excellent results.

 Patent No. ZL03139760.3 and US7,244,751 discloses a method for preparing chidamide, but did not specify whether the resulting product is a crystalline material, nor did the presence or absence of the compound polymorphism. In the above patent, the activity of the compound for evaluation is not conducted in a solid state and, therefore, does not disclose any description about characteristics of the crystal.

CLIP

Chiamide synthesis: US7244751B2

Procedure:

Step a: To a suspension of 0.33 g (2.01 mmol) of N,N’-carbonyldiimidazole in tetrahydrofunan (10 ml) is added drop-wise a solution of 0.30 g (2.01 mmol) of 3-pyridineacrylic acid at 0 °C. Then, the mixture is stirred at room temperature for 3 hours and added drop-wise to a separately prepared 2.0 ml (2.00 mmol) of 1N aqueous sodium hydroxide solution including 0.30 g (2.00 mmol) of 4-aminomethylbenzoic acid, followed by stirring at room temperature for 8 hours. The reaction mixture is evaporated under vacuum. To the residue is added a saturated solution of sodium chloride (2 ml), then the mixture is neutralized with concentrated hydrochloric acid to pH 5. The deposited white solid is collected by filtration, washed with ice-water, and then dried to give 4-[N-(Pyridin-3-ylacryloyl)aminomethyl]benzoic acid (0.46 g, 82%). HRMS calcd for C16H14N2O3: 282.2988. Found: 282.2990. MA calcd for: C16H14N2O3: C, 68.07%; H, 5.00%; N, 9.92%. Found: C, 68.21%; H, 5.03%; N, 9.90%.

Step b: To a suspension of 0.29 g (1.78 mmol) of N,N’-carbonyldiimidazole in tetrahydrofunan (15 ml) is added 0.50 g (1.78 mmol) of 4-[N-(Pyridin-3-ylacryloyl)aminomethyl]benzoic acid, followed by stirring at 45 °C. for 1 hour. After cooling, the reaction mixture is added to a separately prepared tetrahydrofiman (10 ml) solution including 0.28 g (2.22 mmol) of 4-fluoro-1,2-phenylenediamine and 0.20 g (1.78 mmol) of trifluoroacetic acid at room temperature. After reaction at room temperature for 24 hours, the deposited white solid is collected by filtration, washed with tetrahydrofunan, and then dried to give N-(2-amino-4-fluorophenyl)-4-[N-(Pyridin-3-ylacryloyl)aminomethyl]benzamide (0.40 g, 57%). 1H NMR (300 MHz, DMSO-d6): dppm: 4.49 (2H, d), 4.84 (2H, br.s), 6.60 (1H, t), 6.80 (2H, m),696 (1H, t), 7.18 (1H, d), 7.42 (2H, d), 7.52 (1H, d), 7.95 (2H, d), 8.02 (1H, d), 8.56 (1H, d), 8.72 (1H, br. t), 8.78 (1H, s), 9.60 (1H, br.s). IR (KBr) cm1: 3310, 1655, 1631, 1524, 1305, 750. HRMS calcd for C22H19N4O2F: 390.4170. Found: 390.4172. MA calcd for C22H19N4O2F: C, 67.68%; H, 4.40%; N, 14.35%. Found: C, 67.52%; H, 4.38%; N, 14.42%.

http://www.google.co.in/patents/US7244751

EXAMPLE 1

Preparation of 4-[N-(Pyridin-3-ylacryloyl)aminomethyl]benzoic acid

To a suspension of 0.33 g (2.01 mmol) of N,N′-carbonyldiimidazole in tetrahydrofunan (10 ml) is added drop-wise a solution of 0.30 g (2.01 mmol) of 3-pyridineacrylic acid at 0° C. Then, the mixture is stirred at room temperature for 3 hours and added drop-wise to a separately prepared 2.0 ml (2.00 mmol) of 1N aqueous sodium hydroxide solution including 0.30 g (2.00 mmol) of 4-aminomethylbenzoic acid, followed by stirring at room temperature for 8 hours. The reaction mixture is evaporated under vacuum. To the residue is added a saturated solution of sodium chloride (2 ml), then the mixture is neutralized with concentrated hydrochloric acid to pH 5. The deposited white solid is collected by filtration, washed with ice-water, and then dried to give the title compound (0.46 g, 82%). HRMS calcd for C₁₆H₁₄N₂O₃: 282.2988. Found: 282.2990. MA calcd for: C₁₆H₁₄N₂O₃: C, 68.07%; H, 5.00%; N, 9.92%. Found: C, 68.21%; H, 5.03%; N, 9.90%.EXAMPLE 2

Preparation of N-(2-amino-4-fluorophenyl)-4-[N-(Pyridn-3-ylacryloyl)aminomethyl]benzamide

To a suspension of 0.29 g (1.78 mmol) of N,N′-carbonyldiimidazole in tetrahydrofunan (15 ml) is added 0.50 g (1.78 mmol) of 4-[N-(Pyridn-3-ylacryloyl)aminomethyl]benzoic acid, followed by stirring at 45° C. for 1 hour. After cooling, the reaction mixture is added to a separately prepared tetrahydrofiman (10 ml) solution including 0.28 g (2.22 mmol) of 4-fluoro-1,2-phenylenediamine and 0.20 g (1.78 mmol) of trifluoroacetic acid at room temperature. After reaction at room temperature for 24 hours, the deposited white solid is collected by filtration, washed with tetrahydrofunan, and then dried to give the title compound (0.40 g, 57%). ¹H NMR (300 MHz, DMSO-d₆): δppm: 4.49 (2H, d), 4.84 (2H, br.s), 6.60 (1H, t), 6.80 (2H, m),696 (1H, t), 7.18 (1H, d), 7.42 (2H, d), 7.52 (1H, d), 7.95 (2H, d), 8.02 (1H, d), 8.56 (1H, d), 8.72 (1H, br. t), 8.78 (1H, s), 9.60 (1H, br.s). IR (KBr) cm¹: 3310, 1655, 1631, 1524, 1305, 750. HRMS calcd for C₂₂H₁₉N₄O₂F: 390.4170. Found: 390.4172. MA calcd for C₂₂H₁₉N₄O₂F: C, 67.68%; H, 4.40%; N, 14.35%. Found: C, 67.52%; H, 4.38%; N, 14.42%.EXAMPLE 3

Preparation of 4-[N-cinnamoylaminomethyl]benzoic acid

To a suspension of 0.33 g (2.01 mmol) of N,N′-carbonyldiimidazole in tetrahydrofunan (10 ml) is added drop-wise a solution of 0.30 g (2.01 mmol) of cinnamic acid at 0° C. Then, the mixture is stirred at room temperature for 3 hours and added drop-wise to a separately prepared 2.0 ml (2.00 mmol) of 1N aqueous sodium hydroxide solution including 0.30 g (2.00 mmol) of 4-aminomethylbenzoic acid, followed by stirring at room temperature for 8 hours. The reaction mixture is evaporated under vacuum. To the residue is added a saturated solution of sodium chloride (2 ml), then the mixture is neutralized with concentrated hydrochloric acid to pH 7. The deposited white solid is collected by filtration, washed with ice-water, and then dried to give the title compound (0.51 g, 91%). HRMS calcd for C₁₇H₁₅NO₃: 281.3242. Found: 281.3240. MA calcd for C₁₇H₁₅NO₃: C, 72.58%; H, 5.38%; N, 4.98. Found: C, 72.42%; H, 5.37%; N, 4.98%.

EXAMPLE 4

Preparation of N-(2-amino-4-fluorophenyl)-4-[N-cinnamoylaminomethyl]benzamide

To a suspension of 0.29 g (1.78 mmol) of N,N′-carbonyldiimidazole in tetrahydrofunan (15 ml) is added 0.50 g (1.78 mmol) of 4-[N-cinnamoylaminomethyl]benzoic acid, followed by stirring at 45° C. for 1 hour. After cooling, the reaction mixture is added to a separately prepared tetrahydrofunan (10 ml) solution including 0.28 g (2.22 mmol) of 4-fluoro-1,2-phenylenediamine and 0.20 g (1.78 mmol) of trifluoroacetic acid at room temperature. After reaction at room temperature for 16 hours, the deposited white solid is collected by filtration, washed with tetrahydrofunan, and then dried to give the title compound (0.45 g, 64%). ¹H NMR (300 MHz, DMSO-d₆): δppm: 4.42 (2H, d), 4.92 (2H, br.s), 6.62 (1H, t), 6.78 (2H, m), 7.01 (1H, t), 7.32 (5H, m), 7.54 (5H, m), 8.76 (1H, br.t), 9.58 (1H, br.s). IR (KBr) cm⁻¹: 3306, 1618, 1517, 1308, 745. HRMS calcd for C₂₃H₂₀N₃O₂F: 389.4292. Found: 389.4294. MA calcd for C₂₃H₂₀N₃O₂F: C, 70.94%; H, 5.18%; N, 10.79%. Found: C, 70.72%; H, 5.18%; N, 10.88%.

PATENT

https://www.google.com/patents/US20150299126

• FIG. 2 is the ¹H NMR spectrum of the solid prepared according to Example 2 of patent ZL 03139760.3;

NMR, MS ETC CLICK TO VIEW

C-NMR

CLIP

Chidamide (Epidaza), a class I HDAC inhibitor, was discovered and developed by ChipScreen and approved by the CFDA in December 2014 for the treatment of recurrent of refractory peripheral T-cell lymphoma. Chidamide, also known as CS055 and HBI- 8000, is an orally bioavailable benzamide type inhibitor of HDAC isoenzymes class I 1–3, as well as class IIb 10, with potential antineoplastic activity. It selectively binds to and inhibits HDAC, leading to an increase in acetylation levels of histone protein H3.74

This agent also inhibits the expression of signaling kinases in the PI3K/ Akt and MAPK/Ras pathways and may result in cell cycle arrest and the induction of tumor cell apoptosis.75

Currently, phases I and II clinical trials are underway for the treatment of non-small cell lung cancer and for the treatment of breast cancer, respectively.76 The scalable synthetic approach to chidamide very closely follows the discovery route,77–79 and is described in Scheme 10. The sequence began with the condensation of commercial nicotinaldehyde (52) and malonic acid (53) in a mixture of pyridine and piperidine. Next, activation of acid 54 with N,N0-carbonyldiimidazole (CDI) and subsequent reaction with 4-aminomethyl benzoic acid (55) under basic conditions afforded amide 56 in 82% yield.

Finally, activation of 56 with CDI prior to treatment with 4-fluorobenzene- 1,2-diamine (57) and subsequent treatment with TFA and THF yielded chidamide (VIII) in 38% overall yield from 52. However, no publication reported that mono-N-Boc-protected bis-aniline was used to approach Chidamide.

74. Ning, Z. Q.; Li, Z. B.; Newman, M. J.; Shan, S.; Wang, X. H.; Pan, D. S.; Zhang, J.;
Dong, M.; Du, X.; Lu, X. P. Cancer Chemother. Pharmacol. 2012, 69, 901.
75. Liu, L.; Chen, B.; Qin, S.; Li, S.; He, X.; Qiu, S.; Zhao, W.; Zhao, H. Biochem.
Biophys. Res. Commun. 2010, 392, 190.
76. Gong, K.; Xie, J.; Yi, H.; Li, W. Bio. Chem. J. 2012, 443, 735.
77. Lu, X. P.; Li, Z. B.; Xie, A. H.; Shi, L. M.; Li, B. Y.; Ning, Z. Q.; Shan, S.; Deng, T.;
Hu, W. M. US Patent 2004224991A1, 2004.
78. Lu, X. P.; Li, Z. B.; Xie, A. H.; Shi, L. M.; Li, B. Y.; Ning, Z. Q.; Shan, S.; Deng, T.;
Hu, W. M. CN Patent 1513839A, 2003.
79. Yin, Z. H.; Wu, Z. W.; Lan, Y. K.; Liao, C. Z.; Shan, S.; Li, Z. L.; Ning, Z. Q.; Lu, X.
P.; Li, Z. B. Chin. J. New Drugs 2004, 13, 536.

see  CN 105457038

CN 1513839

WRONG COMPD

WO2004071400

Example 2. Preparation of
N-(2-amino-5-fluorophenyl)-4-[N-(Pyridn-3-ylacryloyl)aminomethyl]benzamide

To a suspension of 0.29 g (1.78 mmol) of N, N’-carbonyldiimidazole in tetrahydrofunan (15 ml) is added 0.50 g (1.78 mmol) of 4-[N-(Pyridn-3-ylacryloyl)aminomethyl]benzoic acid, followed by stirring at 45°C for 1 hour. After cooling, the reaction mixture is added to a separately prepared tetrahydrofunan (10 ml) solution including 0.28 g (2.22 mmol) of 4-fluoro-1,2-phenylenediamine and 0.20 g (1.78 mmol) of trifluoroacetic acid at room temperature. After reaction at room temperature for 24 hours, the deposited white solid is collected by filtration, washed with tetrahydrofunan, and then dried to give the title compound (0.40 g, 57%). 1H NMR (300 MHz, DMSO-d₆): δppm: 4.49 (2H, d), 4.84 (2H, br.s), 6.60 (IH, t), 6.80 (2H, m), 6.96 (IH, t), 7.18 (IH, d), 7.42 (2H, d), 7.52 (IH, d), 7.95 (2H, d), 8.02 (IH, d), 8.56 (IH, d), 8.72 (IH, br. t), 8.78 (IH, s), 9.60 (IH, br.s). IR (KBr) cm^“1: 3310, 1655, 1631, 1524, 1305, 750. HRMS calcd for C₂₂Hι₉N₄O₂F: 390.4170. Found: 390.4172. MA calcd for C₂₂Hι₉N₄O₂F: C, 67.68%; H, 4.40%; N, 14.35. Found: C, 67.52%; H, 4.38%; N, 14.42%.

Photo taken on May 22, 2015 shows a box of Chidamide in Shenzhen, south China’s Guangdong Province. Chidamide is the world’s first oral HDAC inhibitor …

Xian-Ping Lu left his research job at a drug maker in the U.S. to co-found a biotech company in his native China. PHOTO: SHENZHEN CHIPSCREEN BIOSCIENCES

HONG KONG— Xian-Ping Lu left his job as director of research at drug maker Galderma R&D in Princeton, N.J., to co-found a biotech company to develop new medicines in his native China.

It took more than 14 years but the bet could be paying off. In February, Shenzhen Chipscreen Biosciences’ first therapy, a medication for a rare type of lymph-node cancer, hit the market in China.

The willingness of veterans like Dr. Lu and others to leave multinational drug companies for Chinese startups reflects a growing optimism in the industry here. The goal, encouraged by the government, is to move the Chinese drug industry beyond generic medicines and drugs based on ones developed in the West.

Chipscreen’s drug, called chidamide, or Epidaza, was developed from start to finish in China. The medicine is the first of its kind approved for sale in China, and just the fourth in a new class globally. Dr. Lu estimates the research cost of chidamide was about $70 million, or about one-tenth what it would have cost to develop in the U.S.

“They are a good example of the potential for innovation in China,” said Angus Cole, director at Monitor Deloitte and pharmaceuticals and biotechnology lead in China.

China’s spending on pharmaceuticals is expected to top $107 billion in 2015, up from $26 billion in 2007, according to Deloitte China. It will become the world’s second-largest drug market, after the U.S., by 2020, according to an analysis published last year in the Journal of Pharmaceutical Policy and Practice.

China has on-the-ground infrastructure labs, a critical mass of leading scientists and interested investors, according to Franck Le Deu, head of consultancy McKinsey & Co.’s pharmaceuticals and medical-products practice in China. “There’re all the elements for the recipe for potential in China,” he said.

But there are obstacles to an industry where companies want big payoffs for a decade or more of work and tremendous costs it takes to develop a drug.

While the protection of intellectual property has improved, China’s cumbersome rules for drug approval and a government effort to cut health-care costs, particularly spending on drugs, could hurt the Chinese drug companies’ efforts, said Mr. Cole of Deloitte.

“Will you start to see success? Of course you will,” said Mr. Cole. However, “I’ve yet to see convincing or compelling evidence that it’s imminent.”

To date, many of the Chinese companies that are flourishing in the life sciences are contract research organizations that help carry out clinical trials, as well as providers of related services.

Some companies, like Shanghai-based Hua Medicine, are buying the rights to develop new compounds in China from multinational drug companies, what some experts consider more akin to an intermediate step to innovation.

Late last year, Hua Medicine completed an early-stage human clinical trial of a diabetes drug in China and in March filed an application to the Food and Drug Administration to develop it in the U.S. as well. The company has raised $45 million in venture funding to date.

Li Chen, who left an 18-year career at Roche Holding AG as head of research and development in China to help start Hua Medicine, said the company’s goal is to “create a game-changer of drug discovery.”

At Chipscreen Biosciences, Dr. Lu and his co-founders set up the company in 2001 in Shenzhen, a city that was quickly growing into a technology and research hub, just over the border from Hong Kong. They created a lab of 10 scientists to use a new analytic technique known as “chemical genomics” to examine the relationships between molecular structures of the existing and failed drugs, how they act on different targets in the body and what genes were being activated or repressed. Now they have more than 60 scientists.

By better predicting how chemicals would act on the body before entering human testing, they hoped they would be more likely get a drug to market.

“How can a small company compete with a multinational?” said Dr. Lu. “The only thing we can compete with is the scientific brain.”

The biggest challenges for the company have been financing and the Chinese regulatory system, said Dr. Lu. The company has raised a total of 300 million yuan ($48 million) over five rounds of venture funding, said Dr. Lu. Chipscreen also receives grant money from the Chinese government.

The company filed its application for approval of chidamide to the Chinese Food and Drug Administration, or CFDA, in early 2013. It had to wait nearly two years for approval, receiving the OK only in December.

Chidamide now is on the market in China for 26,500 yuan ($4,275) a month, a price far lower than patients in the U.S. pay for some of the newest cancer medicines but much more than the typical Chinese patient pays for drugs. Dr. Lu said the price reflects a balance between affordability for patients and return for shareholders. Some investors wanted to price the drug higher.

PAPER

Discovery of an orally active subtype-selective HDAC inhibitor, chidamide, as an epigenetic modulator for cancer treatment

De-Si Pan,^a   Qian-Jiao Yang,^a   Xin Fu,^a   Song Shan,^a  Jing-Zhong Zhu,^a   Kun Zhang,^a   Zhi-Bin Li,^a   Zhi-Qiang Ning^a and  Xian-Ping Lu*^a  

Corresponding authors

^aShenzhen Chipscreen Biosciences Ltd., BIO-Incubator, Suit 2-601, Shenzhen Hi-Tech Industrial Park, Shenzhen, P. R. China
E-mail: xplu@chipscreen.com

Med. Chem. Commun., 2014,5, 1789-1796

DOI: 10.1039/C4MD00350K, http://pubs.rsc.org/en/content/articlelanding/2014/md/c4md00350k#!divAbstract

Tumorigenesis is maintained through a complex interplay of multiple cellular biological processes and is regulated to some extent by epigenetic control of gene expression. Targeting one signaling pathway or biological function in cancer treatment often results in compensatory modulation of others, such as off-target drivers of cell survival. As a result, overall survival of cancer patients is still far from satisfactory. Epigenetic-modulating agents can concurrently target multiple aberrant or compensatory signaling pathways found in cancer cells. However, existing epigenetic-modulating agents in cancer treatment have not yet fully translated into survival benefits beyond hematological tumors. In this article, we present a historical rationale for use of chidamide (CS055/Epidaza), an orally active and subtype-selective histone deacetylase (HDAC) inhibitor of the benzamide chemical class. This compound was discovered and successfully developed as mono-therapy for relapsed and refractory peripheral T cell lymphoma (PTCL) in China. We discuss the evidence supporting chidamide as a durable epigenetic modulator that allows cellular reprogramming with little cytotoxicity in cancer treatments.

Chidamide

Systematic (IUPAC) name
N-(2-Amino-5-fluorophenyl)-4-[[[1-oxo-3-(3-pyridinyl)-2-propen-1-yl]amino]methyl]-benzamide
Clinical data
Trade names	Epidaza
Identifiers
CAS Number	743420-02-2
PubChem	CID 9800555
ChemSpider	7976319
UNII	87CIC980Y0
Chemical data
Formula	C22H19FN4O2
Molar mass	390.4 g/mol

References

/////////Chidamide, Epidaza, CS055,  HBI-8000, orally active subtype-selective HDAC inhibitor, epigenetic modulator,  cancer treatment, CFDA, CHINA, CANCER

Fc3ccc(NC(=O)c1ccc(cc1)CNC(=O)/C=C/c2cccnc2)c(N)c3

.CF3COOH

HAO 472

PHASE 1 CHINA

PRoject Name: HAO472 treatment Phase I clinical trial in relapsed / refractory AML,  M2b type of AML

The main purpose: to determine HAO472 treatment of relapsed / refractory C the maximum tolerated dose (MTD). Secondary objectives: 1) evaluation of drug safety and tolerability; 2) study HAO472 in pharmacokinetic characteristics of the human body; 3) the effectiveness of HAO472 treatment of relapsed / refractory M2b type of AML.

Introduction Test

Sponsor Name:

Jiangsu Hengrui Medicine Co., Ltd. 1/
2 Ruijin Hospital, Shanghai Jiaotong University School of Medicine /
3 Jiangsu Hengrui Medicine Co., Ltd. /
4 Shanghai Hengrui Medicine Co., Ltd. /

Natural products have historically been, and continue to be, an invaluable source for the discovery of various therapeutic agents. Oridonin, a natural diterpenoid widely applied in traditional Chinese medicines, exhibits a broad range of biological effects including anticancer and anti-inflammatory activities. To further improve its potency, aqueous solubility and bioavailability, the oridonin template serves as an exciting platform for drug discovery to yield better candidates with unique targets and enhanced drug properties. A number of oridonin derivatives (e.g. HAO472) have been designed and synthesized, and have contributed to substantial progress in the identification of new agents and relevant molecular mechanistic studies toward the treatment of human cancers and other diseases. This review summarizes the recent advances in medicinal chemistry on the explorations of novel oridonin analogues as potential anticancer therapeutics, and provides a detailed discussion of future directions for the development and progression of this class of molecules into the clinic.

Highlights

Oridonin displays significant anticancer activities via multi-signaling pathways.

Recent advances in medicinal chemistry of oridonin-like compounds are presented.

The article summarizes the SAR and mechanism studies of relevant drug candidates.

The milestones and future direction of oridonin-based drug discovery are discussed.

European Journal of Medicinal Chemistry

Volume 122, 21 October 2016, Pages 102–117

////////Natural product, Oridonin, Diterpenoids, Anticancer agents, Drug discovery, Chemical biology, AML, HAO 472, relapsed / refractory AML. Jiangsu Hengrui Medicine Co., Ltd, PHASE1, LEUKEMIA

C[C@H](N)C(=O)O[C@]15OC[C@@]2([C@H](O)CCC(C)(C)[C@@H]2[C@H]1O)[C@H]3CC[C@@H]4C(=C)C(=O)[C@@]35C4O

TL-487

Molecular Weight, 507.58, MF C₃₀ H₂₉ N₅ O₃

Teligene Inc(2E)-N-[3-Cyano-7-ethoxy-4-[(4-phenoxyphenyl)amino]-6-quinolinyl]-4-(dimethylamino)-2-butenamide

(E)-N-(3-cyano-7-ethoxy-4-((4-phenoxyphenyl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide

Maleate in anhydrous or monohydrate CAS, 2326561-36-6, AND 2326561-38-8 form are BTK and HER-2 kinase inhibitor useful for treating cancer

Useful for treating breast cancer, ovary cancer and colon cancer. are BTK and HER-2 kinase inhibitor useful for treating cancer.

Anticancer protein kinase inhibitor

The compound was originally claimed in WO2013152135 , and may provide the structure of TL-487 , a small molecule inhibitor to HERs, being investigated by Teligene for the treatment of breast cancer; in July 2016, the company intended to develop the product as a class 1.1 chemical drug in China.

PATENT

US 20150057312

PATENT

WO2013152135

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2013152135&tab=PCTDESCRIPTION&queryString=%28ET%2Fkinase%29+&recNum=8&maxRec=4574

PATENT

WO-2019096327

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019096327&redirectedID=true

Novel crystalline maleate salt of (E)-N-(3-cyano-7-ethoxy-4-((4-phenoxyphenyl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide (first disclosed in WO2013152135) and its hydrates (monohydrate) and anhydrates, process for its preparation, composition comprising it and its use for treating cancers such as breast cancer, ovary cancer, colon cancer, prostate cancer, kidney cancer, bladder cancer, stomach cancer, lung cancer, mantle cell lymphoma and multiple myeloma are claimed. The compound is disclosed to be an irreversible inhibitor to BTK and Her-2 (also known as Erb-2 or neu).

///////////////TL-487, PRECLINICAL, CHINA, breast cancer, ovary cancer, olon cancer,  BTK, HER-2 kinase inhibitor,

CN(C)C\C=C\C(=O)Nc3cc4c(Nc2ccc(Oc1ccccc1)cc2)c(cnc4cc3OCC)C#N

CS-3001

BB 7, VX 033

Molecular Weight, 478.37

C₁₇ H₁₈ Br F₂ N₃ O₂ S₂

CStone Pharmaceuticals Co Ltd, JUNE 2018 IND FILED CHINA

URAT1 inhibitor – useful for treating hyperuricemia and gout.

The compound was originally claimed in WO2017202291 , covering thiophene derivative URAT1 inhibitors, useful for treating hyperuricemia and gouty arthritis, assigned to Medshine Discovery Inc , but naming the inventors.and has been reported in some instances to be a URAT1 modulator. In June 2018, an IND application was filed in

SYN

PATENT

WO 2017202291

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017202291

WO-2019101058

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019101058&tab=FULLTEXT&maxRec=1000

Novel crystalline forms of URAT1 inhibitor (designated as Forms A and B) are claimed. The compounds are disclosed to be useful for treating hyperuricemia and gouty arthritis.

Novel crystalline forms of a URAT1 inhibitor, designated as Forms A and B, and their preparation.

For example, remove 2.0 mL of phosphoric acid into 2000 mL of water, sonicate for 10 minutes, mix, and let cool to room temperature as mobile phase A.

////////////CS-3001, BB 7, VX 033, CHINA, PRECLINICAL, CStone Pharmaceuticals, URAT1 inhibitor,  hyperuricemia, gout

O=C(O)C(C)(C)Sc4nnc(Br)n4c2sc(c1CC(F)(F)CCc12)C3CC3

HS-10340

CAS 2156639-66-4

CAS 2307670-65-9

Jiangsu Hansoh Pharmaceutical Group Co Ltd

Being investigated by Jiangsu Hansoh, Shanghai Hansoh Biomedical and Changzhou Hengbang Pharmaceutical ; in June 2018, the product was being developed as a class 1 chemical drug in China.

Useful for treating liver cancer, gastric cancer and prostate cancer.

Use for treating cancers, liver cancer, gastric cancer, prostate cancer, skin cancer, ovary cancer, lung cancer, breast cancer, colon cancer, glioma and rhabdomyosarcoma

At present, some FGFR inhibitors have entered the clinical research stage as anti-tumor drugs, but these are mainly inhibitors of FGFR1, 2 and 3, and the inhibition of FGFR4 activity is weak, and the inhibition of FGFR1-3 has hyperphosphatemia. Such as target related side effects. Highly selective inhibitor of FGFR4 can effectively treat cancer diseases caused by abnormal FGFR4 signaling pathway, and can avoid the side effects of hyperphosphatemia caused by FGFR1-3 inhibition. Highly selective small molecule inhibitors against FGFR4 in tumor targeted therapy The field has significant application prospects.

SYN

PATENT

WO2017198149

where it is claimed to be an FGFR-4 inhibitor for treating liver and prostate cancers, assigned to Jiangsu Hansoh Pharmaceutical Group Co Ltd and Shanghai Hansoh Biomedical Co Ltd .

PATENT

WO2019085860

[0003]

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019085860

Example 1: Preparation of a compound of formula (I)

[0048]

First step 4-(((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)amino)butane Preparation of 1-propanol

[0049]

[0050]

2-(Dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-carbaldehyde (1.0 g, 4.2 mmol), 4-aminobutyl at room temperature l-ol (0.45g, 5.1mmol) was dissolved in DCE (15mL), stirred for 2 hours, followed by addition of NaBH (OAc) _{. 3} (1.35 g of, 6.4 mmol), stirred at room temperature overnight. The reaction was treated with CH ₂ CI ₂ was diluted (100 mL), the organic phase was washed with water (10mL) and saturated brine (15mL), and dried over anhydrous sodium sulfate, and concentrated by column chromatography to give compound 4 – (((2- ( Dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)amino)butan-1-ol (0.9 g, 69%) .

[0051]

^{. 1} H NMR (400 MHz, CDCl3 _{. 3} ) [delta] 7.13 (S, IH), 5.17 (S, IH), 4.84 (S, IH), 3.73 (S, 2H), 3.66-3.49 (m, 2H), 3.42 ( s, 6H), 3.40-3.36 (m, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.68-2.56 (m, 2H), 1.95-1.81 (m, 2H), 1.74-1.55 (m, 4H);

[0052]

MS m/z (ESI): 310.2 [M+H] ⁺ .

[0053]

The second step is 3-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)-1,3- Preparation of oxazepine-2 ketone

[0054]

[0055]

4-(((2-(Dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)amino) in an ice water bath Butan-1-ol (0.6 g, 1.94 mmol) was dissolved in DCE (15 mL), then bis(trichloromethyl) carbonate (0.22 g, 0.76 mmol) was added and triethylamine (0.78 g, 7.76) was slowly added dropwise. Methyl) and then stirred at room temperature for 3 hours. The reaction temperature was raised to 80 ° C, and the reaction was carried out at 80 ° C for 6 hours. After the reaction was cooled to room temperature, it was diluted with CH ₂ Cl ₂ (100 mL), and the organic phase was washed sequentially with water (10 mL) and brine (15 mL) Drying with sodium sulfate, concentration and column chromatography to give the compound 3-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl) )methyl)-1,3-oxazepin-2-one (0.37 g, 57%).

[0056]

MS m/z (ESI): 336.2 [M+H] ⁺ .

[0057]

The third step is phenyl 7-(dimethoxymethyl)-6-((2-carbonyl-1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1, Preparation of 8-naphthyridin-1(2H)-carboxylate

[0058]

[0059]

3-((2-(Dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)-1,3-oxan -2-one (670mg, 2mmol), diphenyl carbonate (643mg, 3mmol) mixing in of THF (15 mL), N ₂ in an atmosphere, cooled to -78 deg.] C, was added dropwise LiHMDS in THF (4mL, 4mmol) was Naturally, it was allowed to react to room temperature overnight. After adding saturated aqueous NH ₄ Cl (100 mL), ethyl acetate (100 mL×2), EtOAc. Methyl)-6-((3-carbonylmorpholino)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate (432 mg, 47%) .

[0060]

^{. 1} H NMR (400 MHz, CDCl3 _{. 3} ) [delta] 7.56 (S, IH), 7.38 (m, 2H), 7.21 (m, 3H), 5.22 (S, IH), 4.77 (S, 2H), 4.16 (m, 2H), 3.95 (m, 2H), 3.39 (s, 6H), 3.25 (m, 2H), 2.84 (t, J = 6.5 Hz, 2H), 1.87 (m, 2H), 1.64 (m, 4H);

[0061]

MS m/z (ESI): 456.2 [M+H] ⁺ .

[0062]

The fourth step: (R)-N-(5-cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-(dimethoxymethyl) -6-((2-carbonyl-1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxamide synthesis

[0063]

[0064]

(R)-6-Amino-4-((1-methoxypropan-2-yl)amino) nicotinenitrile (30 mg, 0.14 mmol), phenyl 7-(dimethoxymethyl)-6- ( (2-carbonyl-1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate (60 mg, 0.13 Methyl acetate was dissolved in THF (5 mL), cooled to -78 ° C under N ₂atmosphere, and a solution of THF (0.3 mL, 0.3 mmol) of LiHMDS was added dropwise to the reaction mixture. After adding a saturated aqueous solution of NH ₄ Cl (50 mL), EtOAc (EtOAc) (5-Cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-(dimethoxymethyl)-6-((2-carbonyl-1) 3-oxoheptyl-3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxamide (65 mg, 86%).

[0065]

1H NMR (400MHz, CDCl3) δ 13.70 (s, 1H), 8.18 (s, 1H), 7.60 (s, 2H), 5.41 (s, 1H), 5.12 (d, J = 7.8 Hz, 1H), 4.73 (s, 2H), 4.20-4.11 (m, 2H), 4.06-3.99 (m, 2H), 3.93 (s, 1H), 3.52-3.48 (m, 7H), 3.46-3.42 (m, 1H), 3.39 (s, 3H), 3.26-3.21 (m, 2H), 2.83 (t, J = 6.2 Hz, 2H), 2.03-1.95 (m, 2H), 1.91-1.83 (m, 2H), 1.67-1.62 (m , 2H), 1.31 (d, J = 6.6 Hz, 3H);

[0066]

MS m/z (ESI): 568.3 [M+H] ⁺ .

[0067]

Step 5: (R)-N-(5-Cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-formyl-6-((2) Synthesis of -carbonyl-1,3-oxoheptyl-3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxamide

[0068]

[0069]

(R)-N-(5-Cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-(dimethoxymethyl)-6-( (2-carbonyl-1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxamide (65 mg, 0.12 mmol) Dissolved in THF/water (volume ratio: 11/4, 4.5 mL), concentrated HCl (0.45 mL, 5.4 mmol), and allowed to react at room temperature for 2 h. Saturated NaHC03 _{. 3} solution (50mL), (50mL × 2 ) and extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by column chromatography to give the title compound (R) -N- ( 5-cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-formyl-6-((2-carbonyl-1,3-oxazepine) 3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1 (2H)-carboxamide (30 mg, 51%).

[0070]

^{. 1} H NMR (400 MHz, CDCl3 _{. 3} ) [delta] 13.57 (S, IH), 10.26 (S, IH), 8.17 (S, IH), 7.71 (S, IH), 7.63 (S, IH), 5.27 (S, 1H), 4.95 (s, 2H), 4.19-4.12 (m, 2H), 4.11-4.04 (m, 2H), 3.94 (s, 1H), 3.52 (m, 1H), 3.48-3.37 (m, 4H) , 3.33 – 3.28 (m, 2H), 2.93 (t, J = 6.3 Hz, 2H), 2.04 (m, 2H), 1.93-1.85 (m, 2H), 1.73 (m, 2H), 1.39-1.28 (m , 3H);

[0071]

MS m/z (ESI): 522.2 [M+H] ⁺ .

PATENT

WO-2019085927

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019085927&tab=FULLTEXT

Novel crystalline salt (such as hydrochloride, sulfate, methane sulfonate, mesylate, besylate, ethanesulfonate, oxalate, maleate, p-toluenesulfonate) forms of FGFR4 inhibitor, particularly N-[5-cyano-4-[[(1R)-2-methoxy-1-methyl-ethyl]amino]-2-pyridyl]-7-formyl-6-[(2-oxo-1,3-oxazepan-3-yl)methyl]-3,4-dihydro-2H-1,8-naphthyridine-1-carboxamide (designated as Forms I- IX), compositions comprising them and their use as an FGFR4 inhibitor for the treatment of cancer such as liver cancer, gastric cancer, prostate cancer, skin cancer, ovarian cancer, lung cancer, breast cancer, colon cancer and glioma or rhabdomyosarcoma are claimed.

///////////HS-10340 , HS 10340 , HS10340, CANCER, Jiangsu Hansoh, Shanghai Hansoh Biomedical,  Changzhou Hengbang, CHINA,  liver cancer, gastric cancer, prostate cancer, skin cancer, ovary cancer, lung cancer, breast cancer, colon cancer, glioma,  rhabdomyosarcoma

C[C@H](COC)Nc1cc(ncc1C#N)NC(=O)N4CCCc3cc(CN2CCCCOC2=O)c(C=O)nc34

CCS(=O)(=O)O.C[C@H](COC)Nc1cc(ncc1C#N)NC(=O)N4CCCc3cc(CN2CCCCOC2=O)c(C=O)nc34

Fluazolepali

CAS  2170504-09-1

Fluzoparib; SHR-3162, (HS10160)

• HS 10160
• SHR 3162

An orally available inhibitor of poly(ADP-ribose) polymerase 1 and 2 (PARP-1/2) for treatment of solid tumors (Jiangsu Hengrui Medicine Co. Ltd., Lianyungang, China)

Fluazolepali, developed by Hengrui and Howson, is intended for the treatment of recurrent ovarian cancer, triple-negative breast cancer, advanced gastric cancer and other advanced solid tumors. Currently, the drug has been introduced into China for recurrent ovarian cancer. Clinical stage.

In February 2019, a randomized, double-blind, controlled, multicenter, phase III clinical study (CTR20190294) of flazopril capsule versus placebo for maintenance of recurrent ovarian cancer was initiated in China and was sponsored by Hengrui Medicine.

Jiangsu Hansoh Pharmaceutical , in collaboration with  Jiangsu Hengrui Medicine , is developing an oral capsule formulation of fluazolepali (fluzoparib; SHR-3162), a small molecule inhibitor to PARP-1 and PARP-2, for the treatment of solid tumors including epithelial ovarian, fallopian tube or primary peritoneal, breast and gastric cancer.

• Originator Jiangsu Hengrui Medicine Co.
• Class Antineoplastics
• Mechanism of Action Poly(ADP-ribose) polymerase 1 inhibitors; Poly(ADP-ribose) polymerase 2 inhibitors

• Phase II Ovarian cancer

• Phase I Breast cancer; Fallopian tube cancer; Gastric cancer; Peritoneal cancer; Solid tumours

• 09 Jul 2019 Jiangsu HengRui Medicine initiates a phase I trial in Solid tumors in China (NCT04013048) [14C]-Fluzoparib
• 01 Jul 2019 Jiangsu HengRui Medicine plans a phase I drug-drug interaction trial (In volunteers) in China (PO) (NCT04011124)
• 12 Jun 2019 Jiangsu HengRui Medicine completes a phase I trial in Gastric cancer (Combination therapy, Recurrent, Metastatic disease, Second-line therapy or greater, Late-stage disease) in China (PO) (NCT03026881)

Fluzoparib (SHR 3162) is a selective poly [ADP-ribose] polymerase 1 (PARP1) and poly [ADP-ribose] polymerase 2 inhibitor (PARP2), being developed by Jiangsu HengRui Medicine, for the treatment of cancer. PARP enzymes play a vital role in repair of DNA damage and maintaining genomic stability. Fluzoparib inhibits PARP enzymes and induces DNA-double strands breaks, G₂/M arrest and apoptosis in homologous recombination repair (HR)-deficient cells. Clinical development for ovarian cancer, breast cancer, fallopian tube cancer, peritoneal cancer, gastric cancer and solid tumours is underway in China and Australia.

An orally available inhibitor of poly (ADP-ribose) polymerase (PARP) types 1 and 2, with potential antineoplastic activity. Upon oral administration, fluzoparib inhibits PARP 1 and 2 activity, which inhibits PARP-mediated repair of damaged DNA via the base excision repair (BER) pathway, enhances the accumulation of DNA strand breaks, promotes genomic instability, and leads to an induction of apoptosis. The PARP family of proteins catalyze post-translational ADP-ribosylation of nuclear proteins, which then transduce signals to recruit other proteins to repair damaged DNA. PARP inhibition may enhance the cytotoxicity of DNA-damaging agents and may reverse tumor cell chemoresistance and radioresistance. Check for active clinical trials using this agent. (NCI Thesaurus)

PATENT

WO-2019137358

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019137358&tab=FULLTEXT&_cid=P20-JYI5A2-54836-1

Process for preparing heterocyclic compounds (presumed to be fluazolepali ) and its intermediates as PARP inhibitors useful for treating cancer.

PATENT

WO2019109938

claiming synergistic combination comprising PARP inhibitor fluazolepali and apatinib mesylate .

PATENT

WO 2018005818

WO 2018129553

WO 2018129559

WO 2018208968

WO 2018213732

WO 2018191277

WO 2018201096

WO 2018085469

WO 2018085468

WO 2019090227

WO 2019133697

WO 2019067978

WO 2019071123

WO 2019090141

///////////Fluazolepali, Jiangsu Hansoh Pharmaceutical,  Jiangsu Hengrui Medicine, fluzoparib,  SHR-3162, 氟唑帕利 , Phase II,  Ovarian cancer, HS10160, CHINA, HS 10160

https://med.sina.com/article_detail_103_2_64751.html

SY-008

CAS 1878218-66-6

FREE FORM 1480443-32-0

SGLT1 inhibitor (type 2 diabetes),

β-D-Glucopyranoside, 4-[[4-[(1E)-4-(2,9-diazaspiro[5.5]undec-2-yl)-1-buten-1-yl]-2-methylphenyl]methyl]-5-(1-methylethyl)-1H-pyrazol-3-yl, acetate (1:1)

acetic acid;(2S,3R,4S,5S,6R)-2-[[4-[[4-[(E)-4-(2,9-diazaspiro[5.5]undecan-2-yl)but-1-enyl]-2-methylphenyl]methyl]-5-propan-2-yl-1H-pyrazol-3-yl]oxy]-6-(hydroxymethyl)oxane-3,4,5-triol

4-{4-[(1E)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-1-en-1-yl]-2-methylbenzyl}-5-(propan-2-yl)-1H-pyrazol-3-yl beta-D-glucopyranoside acetate

MF H₅₀ N₄ O₆ . C₂ H₄ O₂

MW 58.8 g/mol,C₃₅H₅₄N₄O₈

Originator Eli Lilly

• Developer Eli Lilly; Yabao Pharmaceutical Group
• Class Antihyperglycaemics; Small molecules
• Mechanism of Action Sodium-glucose transporter 1 inhibitors

• Phase I Diabetes mellitus

• 28 Aug 2018 No recent reports of development identified for phase-I development in Diabetes-mellitus in Singapore (PO)
• 24 Jun 2018 Biomarkers information updated
• 12 Mar 2018 Phase-I clinical trials in Diabetes mellitus (In volunteers) in China (PO) (NCT03462589)

• Eli Lilly is developing SY 008, a sodium glucose transporter 1 (SGLT1) inhibitor, for the treatment of diabetes mellitus. The approach of inhibiting SGLT1 could be promising because it acts independently of the beta cell and could be effective in both early and advanced stages of diabetes. Reducing both glucose and insulin may improve the metabolic state and potentially the health of beta cells, without causing weight gain or hypoglycaemia. Clinical development is underway in Singapore and China.

  As at August 2018, no recent reports of development had been identified for phase-I development in Diabetes-mellitus in Singapore (PO).

Suzhou Yabao , under license from  Eli Lilly , is developing SY-008 , an SGLT1 inhibitor, for the potential oral capsule treatment of type 2 diabetes in China. By April 2019, a phase Ia trial was completed

PATENT

WO 2013169546

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2013169546&recNum=43&docAn=US2013039164&queryString=EN_ALL:nmr%20AND%20PA:(ELI%20LILLY%20AND%20COMPANY)%20&maxRec=4416

The present invention is in the field of treatment of diabetes and other diseases and disorders associated with hyperglycemia. Diabetes is a group of diseases that is characterized by high levels of blood glucose. It affects approximately 25 million people in the United States and is also the 7^th leading cause of death in U.S. according to the 201 1 National Diabetes Fact Sheet (U.S. Department of Health and Human Services, Centers for Disease Control and Prevention). Sodium-coupled glucose cotransporters (SGLT’s) are one of the transporters known to be responsible for the absorption of carbohydrates, such as glucose. More specifically, SGLTl is responsible for transport of glucose across the brush border membrane of the small intestine. Inhibition of SGLTl may result in reduced absorption of glucose in the small intestine, thus providing a useful approach to treating diabetes.

U.S. Patent No. 7,655,632 discloses certain pyrazole derivatives with human SGLTl inhibitory activity which are further disclosed as useful for the prevention or treatment of a disease associated with hyperglycemia, such as diabetes. In addition, WO 201 1/039338 discloses certain pyrazole derivatives with SGLT1/SGLT2 inhibitor activity which are further disclosed as being useful for treatment of bone diseases, such as osteoporosis.

There is a need for alternative drugs and treatment for diabetes. The present invention provides certain novel inhibitors of SGLTl which may be suitable for the treatment of diabetes.

Accordingly, the present invention provides a compound of Formula II:

Preparation 1

Synthesis of (4-bromo-2-methyl-phenyl)methanol.

Scheme 1, step A: Add borane-tetrahydrofuran complex (0.2 mol, 200 mL, 1.0 M solution) to a solution of 4-bromo-2-methylbenzoic acid (39 g, 0.18 mol) in

tetrahydrofuran (200 mL). After 18 hours at room temperature, remove the solvent under the reduced pressure to give a solid. Purify by flash chromatography to yield the title compound as a white solid (32.9 g, 0.16 mol). 1H NMR (CDCI3): δ 1.55 (s, 1H), 2.28 (s, 3H), 4.61 (s, 2H), 7.18-7.29 (m, 3H).

Alternative synthesis of (4-bromo-2-methyl-phenyl)methanol.

Borane-dimethyl sulfide complex (2M in THF; 1 16 mL, 0.232 mol) is added slowly to a solution of 4-bromo-2-methylbenzoic acid (24.3 g, 0.1 13 mol) in anhydrous tetrahydrofuran (THF, 146 mL) at 3 °C. After stirring cold for 10 min the cooling bath is removed and the reaction is allowed to warm slowly to ambient temperature. After 1 hour, the solution is cooled to 5°C, and water (100 mL) is added slowly. Ethyl acetate (100 mL) is added and the phases are separated. The organic layer is washed with saturated aqueous NaHC0₃ solution (200 mL) and dried over Na₂S0₄. Filtration and concentration under reduced pressure gives a residue which is purified by filtration through a short pad of silica eluting with 15% ethyl acetate/iso-hexane to give the title compound (20.7 g, 91.2% yield). MS (m/z): 183/185 (M+l-18).

Preparation 2

Synthesis of 4-bromo- l-2-methyl-benzene.

Scheme 1, step B: Add thionyl chloride (14.31 mL, 0.2 mol,) to a solution of (4-bromo-2-methyl-phenyl)methanol (32.9 g, 0.16 mol) in dichloromethane (200 mL) and

-Cl-

dimethylformamide (0.025 mol, 2.0 mL) at 0°C. After 1 hour at room temperature pour the mixture into ice-water (100 g), extract with dichloromethane (300 mL), wash extract with 5% aq. sodium bicarbonate (30 mL) and brine (200 mL), dry over sodium sulfate, and concentrate under reduced pressure to give the crude title compound as a white solid (35.0 g, 0.16 mol). The material is used for the next step of reaction without further purification. ^XH NMR (CDC1₃): δ 2.38 (s, 3H), 4.52 (s, 2H), 7.13-7.35 (m, 3H).

Alternative synthesis of 4-bromo- 1 -chloromethyl-2-methyl-benzene. Methanesulfonyl chloride (6.83 mL, 88.3 mmol) is added slowly to a solution of (4-bromo-2-methyl-phenyl)methanol (16.14 g, 80.27 mmol) and triethylamine (16.78 mL; 120.4 mmol) in dichloromethane (80.7 mL) cooled in ice/water. The mixture is allowed to slowly warm to ambient temperature and is stirred for 16 hours. Further

methanesulfonyl chloride (1.24 mL; 16.1 mmol) is added and the mixture is stirred at ambient temperature for 2 hours. Water (80mL) is added and the phases are separated. The organic layer is washed with hydrochloric acid (IN; 80 mL) then saturated aqueous sodium hydrogen carbonate solution (80 mL), then water (80 mL), and is dried over Na₂S0_4. Filtration and concentration under reduced pressure gives a residue which is purified by flash chromatography (eluting with hexane) to give the title compound (14.2 g; 80.5% yield). ^XH NMR (300.1 1 MHz, CDC1₃): δ 7.36-7.30 (m, 2H), 7.18 (d, J= 8.1 Hz, 1H), 4.55 (s, 2H), 2.41 (s, 3H).

Preparation 3

Synthesis of 4-[(4-bromo-2-methyl-phenyl)methyl]-5-isopropyl-lH-pyrazol-3-ol.

Scheme 1, step C: Add sodium hydride (8.29 g, 0.21 mol, 60% dispersion in oil) to a solution of methyl 4-methyl-3-oxovalerate (27.1 mL, 0.19 mol) in tetrahydrofuran at 0°C. After 30 min at room temperature, add a solution of 4-bromo- l-chloromethyl-2-methyl-benzene (35.0 g, 0.16 mol) in tetrahydrofuran (50 mL). Heat the resulting mixture at 70 °C overnight (18 hours). Add 1.0 M HC1 (20 mL) to quench the reaction.

Extract with ethyl acetate (200 mL), wash extract with water (200 rnL) and brine (200 mL), dry over a₂S04, filter and concentrate under reduced pressure. Dissolve the resulting residue in toluene (200 mL) and add hydrazine monohydrate (23.3 mL, 0.48 mol). Heat the mixture at 120 °C for 2 hours with a Dean-Stark apparatus to remove water. Cool and remove the solvent under the reduced pressure, dissolve the residue with dichloromethane (50 mL) and methanol (50 mL). Pour this solution slowly to a beaker with water (250 mL). Collect the resulting precipitated product by vacuum filtration. Dry in vacuo in an oven overnight at 40 °C to yield the title compound as a solid (48.0 g, 0.16 mol). MS (m/z): 311.0 (M+l), 309.0 (M-l).

Alternative synthesis of 4-r(4-bromo-2-methyl-phenyl)methyl1-5-isopropyl- !H-pyrazol- 3-oL

A solution of 4-bromo- 1 -chloromethyl-2-methyl-benzene (13.16 g, 59.95 mmoles) in acetonitrile (65.8 mL) is prepared. Potassium carbonate (24.86 g, 179.9 mmol), potassium iodide (1 1.94 g, 71.94 mmol) and methyl 4-methyl-3-oxo valerate (8.96 mL; 62.95 mmol) are added. The resulting mixture is stirred at ambient temperature for 20 hours. Hydrochloric acid (2N) is added to give pH 3. The solution is extracted with ethyl acetate (100 ml), the organic phase is washed with brine (100 ml) and dried over Na₂S0₄. The mixture is filtered and concentrated under reduced pressure. The residue is dissolved in toluene (65.8 mL) and hydrazine monohydrate (13.7 mL, 0.180 mol) is added. The resulting mixture is heated to reflux and water is removed using a Dean and Stark apparatus. After 3 hours the mixture is cooled to 90 °C and additional hydrazine monohydrate (13.7 mL; 0.180 mol) is added and the mixture is heated to reflux for 1 hour. The mixture is cooled and concentrated under reduced pressure. The resulting solid is triturated with water (200 mL), filtered and dried in a vacuum oven over P2O5 at 60°C. The solid is triturated in iso-hexane (200 mL) and filtered to give the title compound (14.3 g; 77.1% yield). MS (m/z): 309/31 1 (M+l).

Preparation 4

Synthesis of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra- O-benzoyl-beta-D-glucopyranoside.

Scheme 1, step D: To a 1L flask, add 4-[(4-bromo-2-methyl-phenyl)methyl]-5-isopropyl-lH-pyrazol-3-ol (20 g, 64.7 mmol), alpha-D-glucopyranosyl bromide tetrabenzoate (50 g, 76 mmol), benzyltributylammonium chloride (6 g, 19.4 mmol), dichloromethane (500 mL), potassium carbonate (44.7 g, 323 mmol) and water (100 mL). Stir the reaction mixture overnight at room temperature. Extract with dichloromethane (500mL). Wash extract with water (300 mL) and brine (500 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by flash chromatography to yield the title compound (37 g, 64 mmol). MS (ml 2): 889.2 (M+l), 887.2 (M-l).

Preparation 5

Synthesis of 4- {4-[( lis)-4-hydroxybut- 1 -en- 1 -yl]-2-methylbenzyl} -5-(propan-2-yl)- 1H- pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside.

Scheme 1, step E: Add 3-buten-l-ol (0.58 mL, 6.8 mmol) to a solution of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside (3 g, 3.4 mmol) in acetonitrile (30 mL) and triethylamine (20 mL). Degas the solution with nitrogen over 10 minutes. Add tri-o-tolylphosphine (205 mg, 0.67 mmol) and palladium acetate (76 mg, 0.34 mmol). Reflux at 90 °C for 2 hours. Cool to room temperature and concentrate to remove the solvent under the reduced pressure. Purify the residue by flash chromatography to yield the title compound (2.1 g, 2.4 mmol). MS (m/z): 878.4 (M+l).

Preparation 6

Synthesis of 4-{4-[(l£)-4-oxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH- pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside.

Scheme 1, step F: Add 3,3,3-triacetoxy-3-iodophthalide (134 mg, 0.96 mmol) to a solution of 4-{4-[(l£)-4-hydroxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside (280 mg, 0.32 mmol) and sodium bicarbonate (133.8 mg, 1.6 mmol) in dichloromethane (20 mL) at 0 °C. After 15 minutes at room temperature, quench the reaction with saturated aqueous sodium thiosulfate (10 mL). Extract with dichloromethane (30 mL). Wash extract with water (30 mL) and brine (40 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (270 mg, 0.31 mmol). MS (m/z): 876.5 (M+l), 874.5 (M-l).

Preparation 7

Synthesis of tert-butyl 2- {(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-benzoyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9- diazaspiro[5.5]undecane-9-carboxylate.

Scheme 1, step G: Add sodium triacetoxyborohydride (98 mg, 0.46 mmol) to a solution of 4- {4-[(lis)-4-oxybut- 1 -en-1 -yl]-2-methylbenzyl} -5-(propan-2-yl)- lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside (270 mg, 0.31 mmol) and tert-butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (179 mg, 0.62 mmol) in 1,2-dichloroethane (5 mL). After 30 minutes at room temperature, quench the reaction with saturated aqueous sodium bicarbonate (10 mL). Extract with dichloromethane (30 mL). Wash extract with water (30 mL) and brine (40 mL), dry organic phase over sodium sulfate, filter and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (275 mg, 0.25 mmol).

MS (m/z): 1115.6 (M+1).

Preparation 8

Synthesis of 4-{4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en-l-yl]-2- methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D- glucopyranoside dihydrochloride.

Scheme 1, step H: Add hydrogen chloride (4.0 M solution in 1,4-dioxane, 0.6 mL, 2.4 mmol) to a solution of tert-butyl 2-{(3is)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-benzoyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (275 mg, 0.25 mmol) in dichloromethane (5 mL). After overnight (18 hours) at room temperature, concentrate to remove the solvent under reduced pressure to yield the title compound as a solid (258 mg, 0.24 mmol). MS (m/z): 1015.6 (M+l).

Example 1

Synthesis of 4-{4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en-l-yl]-2- methylbenzyl} -5-(propan-2-yl)- lH-pyrazol-3-yl beta-D-glucopyranoside.

Scheme 1, step I: Add sodium hydroxide (0.5 mL, 0.5 mmol, 1.0 M solution) to a solution of 4-{4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside dihydrochloride (258 mg, 0.24 mmol) in methanol (2 mL). After 2 hours at 40 °C, concentrate to remove the solvent under reduced pressure to give a residue, which is purified by preparative HPLC method: high pH, 25% B for 4 min, 25-40 B % for 4 min @ 85 mL/min using a 30 x 75 mm, 5 um C18XBridge ODB column, solvent A – 1¾0 w NH₄HCO₃ @ pH 10, solvent B – MeCN to yield the title compound as a solid (46 mg, 0.08 mmol). MS (m/z): 598.8 (M+l), 596.8 (M-l).

 Preparation 9

Synthesis of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra- O-acetyl-beta-D-glucopyranoside.

Scheme 2, step A: To a 1 L flask, add 4-[(4-bromo-2-methyl-phenyl)methyl]-5-isopropyl-lH-pyrazol-3-ol (24 g, 77.6 mmol), 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide (50.4 g, 116 mmol), benzyltributylammomum chloride (5 g, 15.5 mmol), dichloromethane (250 mL), potassium carbonate (32 g, 323 mmol) and water (120 mL). Stir the reaction mixture overnight at room temperature. Extract with dichloromethane (450 mL). Wash extract with water (300 mL) and brine (500 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (36.5 g, 57 mmol). MS (m/z): 638.5 (M+l), 636.5 (M-l).

Alternative synthesis of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl

2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside.

Reagents 4-[(4-bromo-2-methyl-phenyl)methyl]-5-isopropyl-lH-pyrazol-3-ol (24.0 g, 77.6 mmol), 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide (50.4 g, 116 mmol), benzyltributylammonium chloride (4.94 g, 15.52 mmol), potassium carbonate

(32.18 g, 232.9 mmol), dichloromethane (250 mL) and water (120 mL) are combined and the mixture is stirred at ambient temperature for 18 hours. The mixture is partitioned between dichloromethane (250 mL) and water (250 mL). The organic phase is washed with brine (250 mL), dried over Na₂S0₄, filtered, and concentrated under reduced pressure. The resulting residue is purified by flash chromatography (eluting with 10% ethyl acetate in dichloromethane to 70% ethyl acetate in dichloromethane) to give the title compound (36.5 g, 74% yield). MS (m/z): 639/641 (M+l).

Preparation 10

Synthesis of 4- {4-[( lis)-4-hydroxybut- 1 -en- 1 -yl]-2-methylbenzyl} -5-(propan-2-yl)- 1H- pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside.

Scheme 2, step B: Add 3-buten-l-ol (6.1 mL, 70 mmol) to a solution of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside (15 g, 23.5 mmol) in acetonitrile (200 mL) and triethylamine (50 mL). Degas the solution with nitrogen over 10 minutes. Add tri-o-tolylphosphine (1.43 g, 4.7 mmol) and palladium acetate (526 mg, 2.35 mmol). After refluxing at 90 °C for 2 hours, cool, and concentrate to remove the solvent under the reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (7.5 g, 11.9 mmol). MS (m/z): 631.2 (M+l), 629.2 (M-l).

Preparation 11

Synthesis of 4-{4-[(l£)-4-oxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH- pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside.

Scheme 2, step C: Add 3,3,3-triacetoxy-3-iodophthalide (2.1g, 4.76 mmol) to a solution of 4-{4-[(l£)-4-hydroxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside ( 1.5 g, 2.38 mmol) and sodium bicarbonate (2 g, 23.8 mmol) in dichloromethane (50 mL) at 0 °C. After 15 minutes at room temperature, quench the reaction with saturated aqueous sodium thiosulfate (10 mL). Extract with dichloromethane (30 mL), wash extract with water (30 mL) and brine (40 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (0.95 g, 1.51 mmol). MS (m/z): 628.8(M+1), 626.8 (M-l).

Preparation 12

Synthesis of tert-butyl 2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0- acetyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9- diazaspiro[5.5]undecane-9-carboxylate.

Scheme 2, Step D: Add sodium triacetoxyborohydride (303 mg, 1.4 mmol) to a solution of 4- {4-[(lis)-4-oxybut- 1 -en-1 -yl]-2-methylbenzyl} -5-(propan-2-yl)- lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside (600 mg, 0.95 mmol) and tert-butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (333 mg, 1.2 mmol) in 1,2-dichloroethane (30 mL). After 30 minutes at room temperature, quench the reaction with saturated aqueous sodium bicarbonate (15 mL). Extract with dichloromethane (60 mL). Wash extract with water (30 mL) and brine (60 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (500 mg, 0.58 mmol).

MS (m/z): 866.8, 867.8 (M+l), 864.8, 865.8 (M-l).

Preparation 13

Synthesis oftert-butyl 2-{(3E)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-acetyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,8- diazaspiro[4.5]decane-8-carboxylate.

The title compound is prepared essentially by the method of Preparation 12. S (m/z): 852.8, 853.6 (M+l), 850.8, 851.6 (M-l).

Preparation 14

Synthesis oftert-butyl 9-{(3E)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-acetyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-3,9- diazaspiro[5.5]undecane-3-carboxylate.

The title compound is prepared essentially by the method of Preparation 12. S (m/z): 866.8, 867.6 (M+l), 864.8, 865.6 (M-l).

Preparation 15

Synthesis of 4-{4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en-l-yl]-2- methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D- glucopyranoside dihydrochloride.

Scheme 2, step E: Add hydrogen chloride (4.0 M solution in 1,4-dioxane, 1.5 mL, 5.8 mmol) to a solution of tert-butyl 2-{(3£)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-acetyl-beta-D-glucopyranosyl)oxy]- lH-pyrazol-4-yl} methyl)phenyl]but-3 -en- 1 -yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (500 mg, 0.58 mmol) in dichloromethane (20 mL). After 2 hours at room temperature, concentrate to remove the solvent under reduced pressure to yield the title compound as a solid (480 mg, 0.57 mmol).

MS (m/z): 767.4 (M+l).

Preparation 16

Synthesis of 4-{4-[(lE)-4-(2,8-diazaspiro[4.5]dec-2-yl)but-l-en-l-yl]-2-methylbenzyl}-5- (propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside

dihydrochloride.

The title compound is prepared essentially by the method of Preparation 15. MS (m/z): 752.8, 753.8 (M+1), 750.8 (M-1).

First alternative synthesis of Example 1

First alternative synthesis of 4-{4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en- 2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside.

Scheme 2, step F: Add methanol (5 mL), triethylamine (3 mL), and water (3 mL) to 4-{4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside dihydrochloride (480 mg, 0.24 mmol). After 18 hours (overnight) at room temperature, concentrate to dryness under reduced pressure. Purify the resulting residue by preparative HPLC method: high pH, 25% B for 4 min, 25-40 B % for 4 min @ 85 mL/min using a 30 x 75 mm, 5 urn C18XBridge ODB column, solvent A – H₂0 w NH₄HCO₃ @ pH 10, solvent B – MeCN to yield the title compound as a solid (50 mg, 0.08 mmol).

MS (m/z): 598.8 (M+1), 596.8 (M-1). 1H MR (400.31 MHz, CD3OD): δ 7.11 (d, J=1.3

Hz, 1H), 7.04 (dd, J=1.3,8.0 Hz, 1H), 6.87 (d, J= 8.0 Hz, 1H), 6.36 (d, J= 15.8 Hz, 1H), 6.16 (dt, J= 15.8, 6.3 Hz, 1H), 5.02 (m, 1H), 3.81 (d, J= 11.7 Hz, 1H), 3.72 (d, J= 16.8 Hz, 1H), 3.68 (d, J= 16.8 Hz, 1H) , 3.64 (m, 1H), 3.37-3.29 (m, 4H), 2.79 (m, 1H), 2.72 (t, J= 5.8 Hz, 4H), 2.44-2.33 (m, 6H), 2.30 (s, 3H), 2.26 ( broad s, 2H), 1.59 (m, 2H), 1.50 (m, 2H), 1.43 (m, 2H), 1.36 (m, 2H), 1.1 1 (d, J= 7.0 Hz, 3H), 1.10 (d, J= 7.0 Hz, 3H).

Example 2

Synthesis of 4- {4-[(lE)-4-(2,8-diazaspiro[4.5]dec-2-yl)but-l-en-l-yl]-2-methylbi

(propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside.

O H

The title compound is prepared essentially by the method of the first alternative synthesis of Example 1. MS (m/z): 584.7 (M+l), 582.8 (M-l).

Example 3

Synthesis of 4- {4-[( 1 E)-4-(3 ,9-diazaspiro[5.5]undec-3 -yl)but- 1 -en- 1 -yl]-2- methylbenzyl} -5-(propan-2-yl)- lH-pyrazol-3-yl beta-D-glucopyranoside.

The title compound is prepared essentially by first treating the compound of Prearation 14 with HC1 as discussed in Preparation 15 then treating the resulting hydrochloride salt with triethyl amine as discussed in the first alternative synthesis of Example 1. MS (m/z): 598.8, 599.8 (M+l), 596.8, 597.8 (M-l).

Example 1 Preparation 17

Synthesis of tert-butyl 4-but-3- nyl-4,9-diazaspiro[5.5]undecane-9-carboxylate.

Scheme 3, step A: Cesium carbonate (46.66 g, 143.21 mmol) is added to a suspension of tert-butyl 4,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (16.66 g, 57.28 mmoles) in acetonitrile (167 mL). The mixture is stirred for 10 minutes at ambient temperature then 4-bromobutyne (6.45 mL, 68.74 mmol) is added. The reaction is heated to reflux and stirred for 18 hours. The mixture is cooled and concentrated under reduced pressure. The residue is partitioned between water (200 mL) and ethyl acetate (150 mL). The phases are separated and the aqueous layer is extracted with ethyl acetate (100 mL). The combined organic layers are washed with water (200 mL), then brine (150 mL), dried over MgSC^, filtered, and concentrated under reduced pressure to give the title compound (17.2 g, 98% yield). iH MR (300.11 MHz, CDC1₃): δ 3.43-3.31 (m, 4H),

2.53-2.48 (m, 2H), 2.37-2.29 (m, 4H), 2.20 (s, 2H), 1.94 (t, J= 2.6 Hz, 1H), 1.44 (s, 17H).

Preparation 18

Synthesis of tert-butyl 4-[(£)-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)but-3-enyl]- 4,9-diazaspiro[5.5]undecane-9-carboxylate.

Scheme 3, step B: Triethylamine (5.62 mmoles; 0.783 mL), 4,4,5, 5-tetramethyl-1,3,2-dioxaborolane (8.56 mL, 59.0 mmol) and zirconocene chloride (1.45 g, 5.62 mmoles) are added to tert-butyl 4-but-3-ynyl-4,9-diazaspiro[5.5]undecane-9-carboxylate (17.21 g, 56.16 mmoles). The resulting mixture is heated to 65 °C for 3.5 hours. The mixture is cooled and dissolved in dichloromethane (150 mL). The resulting solution is passed through a ~4cm thick pad of silica gel, eluting with dichloromethane (2 x 200 mL). The filtrate is concentrated under reduced pressure to give the title compound (21.2 g, 87% yield), !H NMR (300.1 1 MHz, CDC1₃): δ 6.65-6.55 (m, 1H), 5.49-5.43 (m, 1H),

3.42-3.29 (m, 4H), 2.40-2.27 (m, 6H), 2.25-2.08 (m, 2H), 1.70 – 1.13 (m, 29H).

Preparation 19

Synthesis of tert-butyl 2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-beta-D- glucopyranosyl)oxy]- lH-pyrazol-4-yl} methyl)phenyl]but-3 -en- 1 -yl} -2,9- diazaspiro[5.5]undecane-9-carboxylate.

Scheme 3, step C: A solution of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside (20 g, 31.3 mmol), tert-butyl 4-[(£)-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)but-3-enyl]-4,9-diazaspiro[5.5]undecane-9-carboxylate (16.3 g, 37.5 mmol) and potassium carbonate (12.97 g, 93.82 mmol) in tetrahydrofuran (200 mL) and water (40 mL) is degassed for 15 min by bubbling nitrogen gas through it. Pd(OAc)2 (140 mg, 625 μιηοΐ) and 2-dicyclohexylphosphino-2′,4′,6′-tri-i-propyl-l, r-biphenyl (0.596 g, 1.25 mmol) are added and the reaction is heated to reflux for 16 h. The solution is cooled to ambient temperature and methanol (200 mL) is added. After 30 minutes the solvent is removed under reduced pressure. The mixture is partitioned between ethyl acetate (500 mL) and brine (500 ml) adding aqueous MgS0₄ (1M; 500 ml) to aid the phase separation. The layers are separated and the organic layer is dried over MgS0₄ and filtered through a 10 cm pad of silica gel, eluting with ethyl acetate (-1.5 L). The filtrate is discarded and the silica pad is flushed with 5% MeOH in THF (2 L). The methanolic filtrate is concentrated under reduced pressure to give the title compound (20. lg, 92%).

MS (m/z): 699 (M+l).

Second alternative Synthesis of Example 1

Second alternative synthesis of 4- {4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but-l-en-l- yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside.

Scheme 3, step D: Trifluoroacetic acid (32.2 mL; 0.426 mol) is added to a solution of tert-butyl 2- {(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (14.87 g; 21.28 mmol) in dichloromethane (149 mL) cooled in iced water. The solution is allowed to warm to room temperature. After 30 minutes, the mixture is slowly added to ammonia in MeOH (2M; 300 mL), applying cooling as necessary to maintain a constant temperature. The solution is stirred at room temperature for 15 min. The mixture is concentrated under reduced pressure and the residue is purified using SCX-2 resin. The basic filtrate is concentrated under reduced pressure and the residue is triturated/sonicated in ethyl acetate, filtered and dried. The resulting solid is dissolved in MeOH (200ml) and concentrated in vacuo. This is repeated several times give the title compound (12.22 g, yield 96%). MS (m/z): 599 (M+l). [a]_D²⁰ = -12 ° (C=0.2, MeOH).

PATENT

WO 2015069541

https://patents.google.com/patent/WO2015069541A1

4-{4-[(1 E)-4-(2,9-DIAZASPIRO[5.5]UNDEC-2-YL)BUT-1 -EN-1

-YL]-2-METHYLBENZYL}-5-(PROPAN-2-YL)-1 H-PYRAZOL-3-YL

BETA-D- GLUCOPYRANOSIDE ACETATE

The present invention relates to a novel SGLT1 inhibitor which is an acetate salt of a pyrazole compound, to pharmaceutical compositions comprising the compound, to methods of using the compound to treat physiological disorders, and to intermediates and processes useful in the synthesis of the compound.

The present invention is in the field of treatment of diabetes and other diseases and disorders associated with hyperglycemia. Diabetes is a group of diseases that is characterized by high levels of blood glucose. It affects approximately 25 million people in the United States and is also the 7^th leading cause of death in U.S. according to the 2011 National Diabetes Fact Sheet (U.S. Department of Health and Human Services, Centers for Disease Control and Prevention). Sodium-coupled glucose cotransporters (SGLT’s) are one of the transporters known to be responsible for the absorption of carbohydrates, such as glucose. More specifically, SGLT1 is responsible for transport of glucose across the brush border membrane of the small intestine. Inhibition of SGLT1 may result in reduced absorption of glucose in the small intestine, thus providing a useful approach to treating diabetes.

U.S. Patent No. 7,655,632 discloses certain pyrazole derivatives with human SGLT1 inhibitory activity which are further disclosed as useful for the prevention or treatment of a disease associated with hyperglycemia, such as diabetes. In addition, WO 2011/039338 discloses certain pyrazole derivatives with SGLT1/SGLT2 inhibitor activity which are further disclosed as being useful for treatment of bone diseases, such as osteoporosis.

There is a need for alternative drugs and treatment for diabetes. The present invention provides an acetate salt of a pyrazole compound, which is an SGLT1 inhibitor, and as such, may be suitable for the treatment of certain disorders, such as diabetes. Accordingly, the present invention provides a compound of Formula I:

or hydrate thereof.

Preparation 1

(4-bromo-2-methyl-phenyl)methanol

Scheme 1, step A: Add borane-tetrahydrofuran complex (0.2 mol, 200 mL, 1.0 M solution) to a solution of 4-bromo-2-methylbenzoic acid (39 g, 0.18 mol) in

tetrahydrofuran (200 mL). After 18 hours at room temperature, remove the solvent under the reduced pressure to give a solid. Purify by flash chromatography to yield the title compound as a white solid (32.9 g, 0.16 mol). !H NMR (CDCI3): δ 1.55 (s, 1H), 2.28 (s, 3H), 4.61 (s, 2H), 7.18-7.29 (m, 3H).

Alternative synthesis of (4-bromo-2-methyl-phenyl)mefhanol.

Borane-dimethyl sulfide complex (2M in THF; 116 mL, 0.232 mol) is added slowly to a solution of 4-bromo-2-methylbenzoic acid (24.3 g, 0.113 mol) in anhydrous tetrahydrofuran (THF, 146 mL) at 3 °C. After stirring cold for 10 min the cooling bath is removed and the reaction is allowed to warm slowly to ambient temperature. After 1 hour, the solution is cooled to 5°C, and water (100 mL) is added slowly. Ethyl acetate (100 mL) is added and the phases are separated. The organic layer is washed with saturated aqueous NaHC0₃ solution (200 mL) and dried over Na₂S0₄. Filtration and concentration under reduced pressure gives a residue which is purified by filtration through a short pad of silica eluting with 15% ethyl acetate/iso-hexane to give the title compound (20.7 g, 91.2% yield). MS (m/z): 183/185 (M+l-18).

Preparation 2

4-bromo- 1 -chloromethyl -2 -methyl -benzene

Scheme 1, step B: Add thionyl chloride (14.31 mL, 0.2 mol,) to a solution of (4- bromo-2 -methyl -phenyl)methanol (32.9 g, 0.16 mol) in dichloromethane (200 mL) and dimethylformamide (0.025 mol, 2.0 mL) at 0°C. After 1 hour at room temperature pour the mixture into ice-water (100 g), extract with dichloromethane (300 mL), wash extract with 5% aq. sodium bicarbonate (30 mL) and brine (200 mL), dry over sodium sulfate, and concentrate under reduced pressure to give the crude title compound as a white solid (35.0 g, 0.16 mol). The material is used for the next step of reaction without further purification. ^!H NMR (CDC1₃): δ 2.38 (s, 3H), 4.52 (s, 2H), 7.13-7.35 (m, 3H).

Alternative synthesis of 4-bromo-l-chloromethyl-2-methyl -benzene. Methanesulfonyl chloride (6.83 mL, 88.3 mmol) is added slowly to a solution of (4-bromo-2-methyl-phenyl)methanol (16.14 g, 80.27 mmol) and triethylamine (16.78 mL; 120.4 mmol) in dichloromethane (80.7 mL) cooled in ice/water. The mixture is allowed to slowly warm to ambient temperature and is stirred for 16 hours. Further

methanesulfonyl chloride (1.24 mL; 16.1 mmol) is added and the mixture is stirred at ambient temperature for 2 hours. Water (80mL) is added and the phases are separated. The organic layer is washed with hydrochloric acid (IN; 80 mL) then saturated aqueous sodium hydrogen carbonate solution (80 mL), then water (80 mL), and is dried over Na₂S0₄. Filtration and concentration under reduced pressure gives a residue which is purified by flash chromatography (eluting with hexane) to give the title compound (14.2 g; 80.5% yield). ^!H NMR (300.11 MHz, CDC1₃): δ 7.36-7.30 (m, 2H), 7.18 (d, J= 8.1 Hz, 1H), 4.55 (s, 2H), 2.41 (s, 3H).

Preparation 3

4- [(4-bromo-2-methyl-phenyl)methyl] -5 -isopropyl- lH-pyrazol-3 -ol

Scheme 1, step C: Add sodium hydride (8.29 g, 0.21 mol, 60% dispersion in oil) to a solution of methyl 4-methyl-3-oxovalerate (27.1 mL, 0.19 mol) in tetrahydrofuran at 0°C. After 30 min at room temperature, add a solution of 4-bromo-l-chloromethyl-2- methyl-benzene (35.0 g, 0.16 mol) in tetrahydrofuran (50 mL). Heat the resulting mixture at 70 °C overnight (18 hours). Add 1.0 M HC1 (20 mL) to quench the reaction. Extract with ethyl acetate (200 mL), wash extract with water (200 mL) and brine (200 mL), dry over Na₂S0₄, filter and concentrate under reduced pressure. Dissolve the resulting residue in toluene (200 mL) and add hydrazine monohydrate (23.3 mL, 0.48 mol). Heat the mixture at 120 °C for 2 hours with a Dean-Stark apparatus to remove water. Cool and remove the solvent under the reduced pressure, dissolve the residue with dichloromethane (50 mL) and methanol (50 mL). Pour this solution slowly to a beaker with water (250 mL). Collect the resulting precipitated product by vacuum filtration. Dry in vacuo in an oven overnight at 40 °C to yield the title compound as a solid (48.0 g, 0.16 mol). MS (m/z): 311.0 (M+l), 309.0 (M-l). Alternative synthesis of 4-[(4-bromo-2-methyl-phenyl)methyl] -5 -isopropyl- lH-pyrazol-

3-ol.

A solution of 4-bromo-l-chloromethyl-2-methyl-benzene (13.16 g, 59.95 mmoles) in acetonitrile (65.8 mL) is prepared. Potassium carbonate (24.86 g, 179.9 mmol), potassium iodide (11.94 g, 71.94 mmol) and methyl 4-methyl-3-oxovalerate (8.96 mL; 62.95 mmol) are added. The resulting mixture is stirred at ambient temperature for 20 hours. Hydrochloric acid (2N) is added to give pH 3. The solution is extracted with ethyl acetate (100 ml), the organic phase is washed with brine (100 ml) and dried over Na₂S0₄. The mixture is filtered and concentrated under reduced pressure. The residue is dissolved in toluene (65.8 mL) and hydrazine monohydrate (13.7 mL, 0.180 mol) is added. The resulting mixture is heated to reflux and water is removed using a Dean and Stark apparatus. After 3 hours the mixture is cooled to 90 °C and additional hydrazine monohydrate (13.7 mL; 0.180 mol) is added and the mixture is heated to reflux for 1 hour. The mixture is cooled and concentrated under reduced pressure. The resulting solid is triturated with water (200 mL), filtered and dried in a vacuum oven over P₂Os at 60°C. The solid is triturated in iso-hexane (200 mL) and filtered to give the title compound (14.3 g; 77.1% yield). MS (m/z): 309/311 (M+l).

Preparation 4

4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl- beta-D-glucopyranoside

Scheme 1, step D: To a 1L flask, add 4-[(4-bromo-2-methyl-phenyl)methyl]-5- isopropyl-lH-pyrazol-3-ol (20 g, 64.7 mmol), alpha-D-glucopyranosyl bromide tetrabenzoate (50 g, 76 mmol), benzyltributylammonium chloride (6 g, 19.4 mmol), dichloromethane (500 mL), potassium carbonate (44.7 g, 323 mmol) and water (100 mL). Stir the reaction mixture overnight at room temperature. Extract with dichloromethane (500mL). Wash extract with water (300 mL) and brine (500 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by flash chromatography to yield the title compound (37 g, 64 mmol). MS (m/z): 889.2 (M+l), 887.2 (M-l).

Preparation 5

4- {4- [(lis)-4-hydroxybut- 1 -en- 1 -yl] -2-methylbenzyl } -5 -(propan-2-yl)- lH-pyrazol-3-yl

2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside

Scheme 1, step E: Add 3-buten-l-ol (0.58 mL, 6.8 mmol) to a solution of 4-(4- bromo-2-methylbenzyl)-5 -(propan-2-yl)- lH-pyrazol-3 -yl 2,3 ,4,6-tetra-O-benzoyl-beta-D- glucopyranoside (3 g, 3.4 mmol) in acetonitrile (30 mL) and triethylamine (20 mL). Degas the solution with nitrogen over 10 minutes. Add tri-o-tolylphosphine (205 mg, 0.67 mmol) and palladium acetate (76 mg, 0.34 mmol). Reflux at 90 °C for 2 hours. Cool to room temperature and concentrate to remove the solvent under the reduced pressure. Purify the residue by flash chromatography to yield the title compound (2.1 g, 2.4 mmol). MS (m/z): 878.4 (M+l).

Preparation 6

4-{4-[(l£)-4-oxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl

2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside

Scheme 1, step F: Add 3,3,3-triacetoxy-3-iodophthalide (134 mg, 0.96 mmol) to a solution of 4-{4-[(l£)-4-hydroxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH- pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside (280 mg, 0.32 mmol) and sodium bicarbonate (133.8 mg, 1.6 mmol) in dichloromethane (20 mL) at 0 °C. After 15 minutes at room temperature, quench the reaction with saturated aqueous sodium thiosulfate (10 mL). Extract with dichloromethane (30 mL). Wash extract with water (30 mL) and brine (40 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (270 mg, 0.31 mmol). MS (m/z): 876.5 (M+l), 874.5 (M-l).

Preparation 7

tert-butyl 2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-benzoyl-beta-D- glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl} -2,9- diazaspiro[5.5]undecane-9-carboxylate

Scheme 1, step G: Add sodium triacetoxyborohydride (98 mg, 0.46 mmol) to a solution of 4-{4-[(l£)-4-oxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol- 3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside (270 mg, 0.31 mmol) and tert-butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (179 mg, 0.62 mmol) in 1,2- dichloroethane (5 mL). After 30 minutes at room temperature, quench the reaction with saturated aqueous sodium bicarbonate (10 mL). Extract with dichloromethane (30 mL). Wash extract with water (30 mL) and brine (40 mL), dry organic phase over sodium sulfate, filter and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (275 mg, 0.25 mmol).

MS (m/z): 1115.6 (M+l).

Preparation 8

4- {4- [( l£)-4-(2,9-diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl} -5-(propan- 2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside dihydrochloride

Scheme 1, step H: Add hydrogen chloride (4.0 M solution in 1,4-dioxane, 0.6 mL, 2.4 mmol) to a solution of tert-butyl 2-{(3£)-4-[3-methyl-4-({5-(propan-2-yl)-3- [(2,3,4,6-tetra-0-benzoyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4- yl}methyl)phenyl]but-3-en-l-yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (275 mg, 0.25 mmol) in dichloromethane (5 mL). After overnight (18 hours) at room temperature, concentrate to remove the solvent under reduced pressure to yield the title compound as a solid (258 mg, 0.24 mmol). MS (m/z): 1015.6 (M+l).

Preparation 9

4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl- beta-D-glucopyranoside.

Scheme 2, step A: To a 1 L flask, add 4-[(4-bromo-2-methyl-phenyl)mefhyl]-5- isopropyl-lH-pyrazol-3-ol (24 g, 77.6 mmol), 2,3,4,6-tetra-O-acetyl-alpha-D- glucopyranosyl bromide (50.4 g, 116 mmol), benzyltributylammonium chloride (5 g, 15.5 mmol), dichloromethane (250 mL), potassium carbonate (32 g, 323 mmol) and water (120 mL). Stir the reaction mixture overnight at room temperature. Extract with dichloromethane (450 mL). Wash extract with water (300 mL) and brine (500 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (36.5 g, 57 mmol). MS (m/z): 638.5 (M+l), 636.5 (M-l).

Alternative synthesis of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)-lH-pyrazol-3-yl

2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside.

Reagents 4-[(4-bromo-2-methyl-phenyl)methyl]-5-isopropyl-lH-pyrazol-3-ol (24.0 g, 77.6 mmol), 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide (50.4 g, 116 mmol), benzyltributylammonium chloride (4.94 g, 15.52 mmol), potassium carbonate (32.18 g, 232.9 mmol), dichloromethane (250 mL) and water (120 mL) are combined and the mixture is stirred at ambient temperature for 18 hours. The mixture is partitioned between dichloromethane (250 mL) and water (250 mL). The organic phase is washed with brine (250 mL), dried over Na₂S0₄, filtered, and concentrated under reduced pressure. The resulting residue is purified by flash chromatography (eluting with 10% ethyl acetate in dichloromethane to 70% ethyl acetate in dichloromethane) to give the title compound (36.5 g, 74% yield). MS (m/z): 639/641 (M+l). Preparation 10

4- {4- [(lis)-4-hydroxybut- 1 -en- 1 -yl] -2-methylbenzyl } -5 -(propan-2-yl)- lH-pyrazol-3-yl

2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside

Scheme 2, step B: Add 3-buten-l-ol (6.1 mL, 70 mmol) to a solution of 4-(4- bromo-2-methylbenzyl)-5 -(propan-2-yl)- 1 H-pyrazol-3 -yl 2,3 ,4,6-tetra-O-acetyl-beta-D- glucopyranoside (15 g, 23.5 mmol) in acetonitrile (200 mL) and triethylamine (50 mL). Degas the solution with nitrogen over 10 minutes. Add tri-o-tolylphosphine (1.43 g, 4.7 mmol) and palladium acetate (526 mg, 2.35 mmol). After refluxing at 90 °C for 2 hours, cool, and concentrate to remove the solvent under the reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (7.5 g, 11.9 mmol) MS (m/z): 631.2 (M+l), 629.2 (M-l).

Preparation 11

4-{4-[(l£)-4-oxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol-3-yl

2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside

Scheme 2, step C: Add 3,3,3-triacetoxy-3-iodophthalide (2.1g, 4.76 mmol) to a solution of 4-{4-[(l£)-4-hydroxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH- pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside ( 1.5 g, 2.38 mmol) and sodium bicarbonate (2 g, 23.8 mmol) in dichloromethane (50 mL) at 0 °C. After 15 minutes at room temperature, quench the reaction with saturated aqueous sodium thiosulfate (10 mL). Extract with dichloromethane (30 mL), wash extract with water (30 mL) and brine (40 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (0.95 g, 1.51 mmol). MS (m/z): 628.8(M+1), 626.8 (M-l).

Preparation 12a

tert-butyl 2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-acetyl-beta-D- glucopyranosyl)oxy] -lH-pyrazol-4-yl}methyl)phenyl]but-3-en- 1 -yl} -2,9- diazaspiro[5.5]undecane-9-carboxylate

Scheme 2, Step D: Add sodium triacetoxyborohydride (303 mg, 1.4 mmol) to a solution of 4-{4-[(l£)-4-oxybut-l-en-l-yl]-2-methylbenzyl}-5-(propan-2-yl)-lH-pyrazol- 3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside (600 mg, 0.95 mmol) and tert-butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (333 mg, 1.2 mmol) in 1,2- dichloroethane (30 mL). After 30 minutes at room temperature, quench the reaction with saturated aqueous sodium bicarbonate (15 mL). Extract with dichloromethane (60 mL). Wash extract with water (30 mL) and brine (60 mL). Dry organic phase over sodium sulfate, filter, and concentrate under reduced pressure. Purify the resulting residue by flash chromatography to yield the title compound (500 mg, 0.58 mmol).

MS (m/z): 866.8, 867.8 (M+l), 864.8, 865.8 (M-l).

Preparation 13

4- {4- [( l£)-4-(2,9-diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl} -5-(propan- 2-yl)- lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside dihydrochloride

Scheme 2, step E: Add hydrogen chloride (4.0 M solution in 1,4-dioxane, 1.5 mL, 5.8 mmol) to a solution of tert-butyl 2-{(3£)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6- tetra-0-acetyl-beta-D-glucopyranosyl)oxy] – lH-pyrazol-4-yl} methyl)phenyl]but-3 -en- 1 – yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (500 mg, 0.58 mmol) in dichloromethane (20 mL). After 2 hours at room temperature, concentrate to remove the solvent under reduced pressure to yield the title compound as a solid (480 mg, 0.57 mmol).

MS (m/z): 767.4 (M+l).

Scheme 3

Preparation 14

tert-butyl 4-but-3-ynyl-4,9-diazas iro[5.5]undecane-9-carboxylate

Scheme 3, step A: Cesium carbonate (46.66 g, 143.21 mmol) is added to a suspension of tert-butyl 4,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (16.66 g, 57.28 mmoles) in acetonitrile (167 mL). The mixture is stirred for 10 minutes at ambient temperature then 4-bromobutyne (6.45 mL, 68.74 mmol) is added. The reaction is heated to reflux and stirred for 18 hours. The mixture is cooled and concentrated under reduced pressure. The residue is partitioned between water (200 mL) and ethyl acetate (150 mL). The phases are separated and the aqueous layer is extracted with ethyl acetate (100 mL). The combined organic layers are washed with water (200 mL), then brine (150 mL), dried over MgS0₄, filtered, and concentrated under reduced pressure to give the title compound (17.2 g, 98% yield). ^lH NMR (300.11 MHz, CDC1₃): δ 3.43-3.31 (m, 4H), 2.53-2.48 (m, 2H), 2.37-2.29 (m, 4H), 2.20 (s, 2H), 1.94 (t, J= 2.6 Hz, 1H), 1.44 (s, 17H).

Preparation 15

tert-butyl 4-[(£)-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)but-3-enyl]-4,9- diazaspiro[5.5]undecane-9-carboxylate

Scheme 3, step B: Triethylamine (5.62 mmoles; 0.783 mL), 4,4,5,5-tetramethyl- 1,3,2-dioxaborolane (8.56 mL, 59.0 mmol) and zirconocene chloride (1.45 g, 5.62 mmoles) are added to tert-butyl 4-but-3-ynyl-4,9-diazaspiro[5.5]undecane-9-carboxylate (17.21 g, 56.16 mmoles). The resulting mixture is heated to 65 °C for 3.5 hours. The mixture is cooled and dissolved in dichloromethane (150 mL). The resulting solution is passed through a ~4cm thick pad of silica gel, eluting with dichloromethane (2 x 200 mL). The filtrate is concentrated under reduced pressure to give the title compound (21.2 g, 87% yield). 1H NMR (300.11 MHz, CDCI3): δ 6.65-6.55 (m, 1H), 5.49-5.43 (m, 1H), 3.42-3.29 (m, 4H), 2.40-2.27 (m, 6H), 2.25-2.08 (m, 2H), 1.70 – 1.13 (m, 29H).

Preparation 16

tert-butyl 2-{(3£’)-4-[3-methyl-4-({5-(propan-2-yl)-3-beta-D-glucopyranosyl)oxy]-lH- pyrazol-4-yl} methyl)phenyl]but-3 -en- 1 -yl} -2,9-diazaspiro [5.5]undecane-9-carboxylate

Scheme 3, step C: A solution of 4-(4-bromo-2-methylbenzyl)-5-(propan-2-yl)- lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside (20 g, 31.3 mmol), tert- butyl 4-[(£)-4-(4,4,5 ,5 -tetramethyl- 1 ,3,2-dioxaborolan-2-yl)but-3 -enyl] -4,9- diazaspiro[5.5]undecane-9-carboxylate (16.3 g, 37.5 mmol) and potassium carbonate (12.97 g, 93.82 mmol) in tetrahydrofuran (200 mL) and water (40 mL) is degassed for 15 min by bubbling nitrogen gas through it. Pd(OAc)₂ (140 mg, 625 μιηοΐ) and 2- dicyclohexylphosphino-2′,4′,6′-tri-i -propyl- Ι, -biphenyl (0.596 g, 1.25 mmol) are added and the reaction is heated to reflux for 16 h. The solution is cooled to ambient temperature and methanol (200 mL) is added. After 30 minutes the solvent is removed under reduced pressure. The mixture is partitioned between ethyl acetate (500 mL) and brine (500 ml) adding aqueous MgS0₄ (1M; 500 ml) to aid the phase separation. The layers are separated and the organic layer is dried over MgS0₄ and filtered through a 10 cm pad of silica gel, eluting with ethyl acetate (-1.5 L). The filtrate is discarded and the silica pad is flushed with 5% MeOH in THF (2 L). The methanolic filtrate is concentrated under reduced pressure to give the title compound (20. lg, 92%).

MS (m/z): 699 (M+l).

Preparation 17

tert-butyl 4- [(E)-4- [4- [(3 -hydroxy-5-isopropyl- 1 H-pyrazol-4-yl)methyl] -3 -methyl- phenyl]but-3-enyl]-4,9-diazaspiro[5.5]undecane-9-carboxylate

Scheme 4, step A: Add tert-butyl 4-[(£)-4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)but-3-enyl]-4,9-diazaspiro[5.5]undecane-9-carboxylate (35.8 kg, 82.4 mol) in methanol (130 L) to a solution of (4-[(4-bromo-2-methyl-phenyl)methyl]-5- isopropyl-lH-pyrazol-3-ol (23.9 kg, 77.3 mol) in methanol (440 L) at room temperature. Add water (590 L) and tripotassium phosphate (100 kg, 471.7 mol) and place the reaction under nitrogen atmosphere. To the stirring solution, add a suspension of

tris(dibenzylideneacetone) dipalladium (1.42 kg, 1.55 mol) and di-tert- butylmethylphosphonium tetrafluoroborate (775 g, 3.12 mol) in methanol (15 L). The resulting mixture is heated at 75 °C for 2 hours. Cool the mixture and filter over diatomaceous earth. Rinse the the filter cake with methanol (60 L), and concentrate the filtrate under reduced pressure. Add ethyl acetate (300 L), separate the layers, and wash the organic layer with 15% brine (3 x 120 L). Concentrate the organic layer under reduced pressure, add ethyl acetate (300 L), and stir the mixture for 18 to 20 hours. Add heptane (300 L), cool the mixture to 10 °C, and stir the mixture for an additional 18 to 20 hours. Collect the resulting solids by filtration, rinse the cake with ethyl acetate/heptane (2:3, 2 x 90 L), and dry under vacuum at 40°C to give the title compound (29.3 kg, 70.6% yield) as a white solid. ^lH NMR (400 MHz, CD₃OD): δ 7.14 (s, 1H), 7.07 (d, J= 8.0 Hz, 1H), 6.92 (d, J= 7.6 Hz, 1H), 6.39 (d, J= 16.0 Hz, 1H), 6.25-6.12 (m, 1H), 3.63 (s, 2H), 3.45-3.38 (bs, 3H), 3.34 (s, 3 H), 3.33 (s, 3H), 2.85-2.75 (m, 1H), 2.49-2.40 (m, 5 H), 2.33 (s, 3H), 1.68-1.62 (m, 2H), 1.60-1.36 (m, 15H), 1.11 (s, 3H), 1.10 (s, 3H).

Preparation 12b

Alterternative preparation of tert-butyl 2-{(3£)-4-[3-methyl-4-({5-(propan-2-yl)-3- [(2,3,4,6-tetra-0-acetyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but- 3-en-l-yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate.

Scheme 4, step B: Combine tert-butyl 4-[(E)-4-[4-[(3-hydroxy-5-isopropyl-lH- pyrazol-4-yl)methyl] -3-methyl-phenyl]but-3 -enyl] -4,9-diazaspiro [5.5]undecane-9- carboxylate (17.83 kg, 33.2 moles), acetonitrile (180 L), and benzyltributylammonium chloride (1.52 kg, 4.87 moles) at room temperature. Slowly add potassium carbonate (27.6 kg, 199.7 moles) and stir the mixture for 2 hours. Add 2,3,4,6-tetra-O-acetyl-alpha- D-glucopyranosyl bromide (24.9 kg, 60.55 mol), warm the reaction mixture to 30°C and stir for 18 hours. Concentrate the mixture under reduced pressure and add ethyl acetate (180 L), followed by water (90 L). Separate the layers, wash the organic phase with 15% brine (3 x 90 L), concentrate the mixture, and purify using column chromatography over silica gel (63 kg, ethyl acetate/heptanes as eluent (1 :2→1 :0)) to provide the title compound (19.8 kg, 94% purity, 68.8% yield) as a yellow foam, !H NMR (400 MHz, CDC1₃): δ 7.13 (s, 1H), 7.03 (d, J= 8.0 Hz, 1H), 6.78 (d, J= 8.0 Hz, 1H), 6.36 (d, J= 16.0,

1H), 6.25-6.13 (m, 1H), 5.64 (d, J= 8.0 Hz, 1H), 5.45-5.25 (m, 2H), 5.13-4.95 (m, 2H), 4.84-4.76 (m, 1H), 4.25-4.13 (m, 2H), 4.10-4.00 (m, 2H), 3.90-3.86 (m, 1H), 3.58-3.50 (m, 2H), 3.40-3.22 (m, 4H), 2.89-2.79 (m, 1H), 2.10-1.90 (m, 18 H), 1.82 (s, 3H), 1.62- 0.82 (m, 22H).

Preparation 18

2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-[(2,3,4,6-tetra-0-acetyl-beta-D- glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl} -2,9- diazaspiro[5.5]undecane

Scheme 4, step C: Combine tert-butyl 2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)- 3-[(2,3,4,6-tetra-0-acetyl-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4- yl}methyl)phenyl]but-3-en-l-yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (19.6 kg, 22.6 moles) with dichloromethane (120 L) and cool to 0°C. Slowly add trifluoroacetic acid (34.6 L, 51.6 kg, 452 moles) and stir for 9 hours. Quench the reaction with ice water (80 L), and add ammonium hydroxide (85-90 L) to adjust the reaction mixture to pH (8- 9). Add dichloromethane (120 L), warm the reaction mixture to room temperature, and separate the layers. Wash the organic layer with water (75 L), brine, and concentrate under reduced pressure to provide the title compound (16.2 kg, 95.0% purity, 93% yield) as a yellow solid. ^lH NMR (400 MHz, CDC1₃): δ 7.08 (s, IH), 6.99 (d, J= 8.0 Hz, IH),

6.76 (d, J= 7.6 Hz, IH), 6.38 (d, J=15.6 Hz, IH), 6.00-5.83 (m, IH), 5.31 (d, J= 7.6 Hz, IH), 5.25-5.13 (m, 4H), 4.32 (dd, J= 12.8, 9.2 Hz, IH), 4.14 (d, J= 11.2 Hz, IH), 3.90 (d, J= 10.0 Hz, IH), 3.75-3.50 (m, 3H), 3.30-3.00 (m, 5 H), 2.85-2.75 (m, IH), 2.70-2.48 (m, 3H), 2.25 (s, IH), 2.13-1.63 (m, 19H), 1.32-1.21 (m, IH), 1.14 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.10 (s, 3H).

Example 1

Hydrated crystalline 4- {4-[(l£)-4-(2,9-diazaspiro[5.5]undec-2-yl)but- 1 -en- 1 -yl]-2- methylbenzyl} -5-(propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside acetate

First alternative preparation of 4-{4-[(l£’)-4-(2.9-diazaspiro[5.5]undec-2-yl)but-l-en-l- yl]-2-methylbenzyl| -5-(propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside (free base).

Scheme 1, step I: Add sodium hydroxide (0.5 mL, 0.5 mmol, 1.0 M solution) to a solution of 4- {4-[( l£)-4-(2,9-diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl} – 5-(propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranoside dihydrochloride (258 mg, 0.24 mmol) in methanol (2 mL). After 2 hours at 40°C, concentrate to remove the solvent under reduced pressure to give a residue, which is purified by preparative HPLC method: high pH, 25% B for 4 min, 25-40 B % for 4 min @ 85 mL/min using a 30 x 75 mm, 5 μιη C18XBridge ODB column, solvent A – H.0 with NH₄HCO₃ @ pH 10, solvent B – MeCN to yield the title compound (free base) as a solid (46 mg, 0.08 mmol). MS (m/z): 598.8 (M+l), 596.8 (M-l).

Second alternative preparation of 4-{4-r(l-£’)-4-(2.9-diazaspiror5.51undec-2-yl)but-l-en- 1 -yl] -2-methylbenzyl I -5 -(propan-2-yl)- lH-pyrazol-3 -yl beta-D-glucopyranoside (free base^“).

Scheme 2, step F: Add methanol (5 mL), triethylamine (3 mL), and water (3 mL) to 4- {4-[( l_JE)-4-(2,9-diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl } -5 – (propan-2-yl)-lH-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside dihydrochloride (480 mg, 0.24 mmol). After 18 hours (overnight) at room temperature, concentrate to dryness under reduced pressure. Purify the resulting residue by preparative HPLC method: high pH, 25% B for 4 min, 25-40 B % for 4 min @ 85 mL/min using a 30 x 75 mm, 5 μιη C18XBridge ODB column, solvent A – H₂0 with NH₄HCO₃ @ pH 10, solvent B – MeCN to yield the title compound (free base) as a solid (50 mg, 0.08 mmol).

MS (m/z): 598.8 (M+l), 596.8 (M-l). 1H NMR (400.31 MHz, CD3OD): δ 7.11 (d, J=1.3

Hz, 1H), 7.04 (dd, J=l .3,8.0 Hz, 1H), 6.87 (d, J= 8.0 Hz, 1H), 6.36 (d, J= 15.8 Hz, 1H), 6.16 (dt, J= 15.8, 6.3 Hz, 1H), 5.02 (m, 1H), 3.81 (d, J= 11.7 Hz, 1H), 3.72 (d, J= 16.8 Hz, 1H), 3.68 (d, J= 16.8 Hz, 1H) , 3.64 (m, 1H), 3.37-3.29 (m, 4H), 2.79 (m, 1H), 2.72 (t, J= 5.8 Hz, 4H), 2.44-2.33 (m, 6H), 2.30 (s, 3H), 2.26 ( broad s, 2H), 1.59 (m, 2H), 1.50 (m, 2H), 1.43 (m, 2H), 1.36 (m, 2H), 1.11 (d, J= 7.0 Hz, 3H), 1.10 (d, J= 7.0 Hz, 3H).

Third alternative preparation of 4-{4-[(l£^,)-4-(2,9-diazaspiro[5.51undec-2-yl)but-l-en-l- yll-2-methylbenzyl|-5-(propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside.

Scheme 3, step D: Trifluoroacetic acid (32.2 mL; 0.426 mol) is added to a solution of tert-butyl 2-{(3_JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-beta-D- glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9- diazaspiro[5.5]undecane-9-carboxylate (14.87 g; 21.28 mmol) in dichloromethane (149 mL) cooled in iced water. The solution is allowed to warm to room temperature. After 30 minutes, the mixture is slowly added to ammonia in MeOH (2M; 300 mL), applying cooling as necessary to maintain a constant temperature. The solution is stirred at room temperature for 15 min. The mixture is concentrated under reduced pressure and the residue is purified using SCX-2 resin. The basic filtrate is concentrated under reduced pressure and the residue is triturated/sonicated in ethyl acetate, filtered and dried. The resulting solid is dissolved in MeOH (200mL) and concentrated in vacuo. This is repeated several times to give the title compound (free base) (12.22 g, yield 96%). MS (m/z): 599 (M+l); [a]_D ²0 = -12 ° (C=0.2, MeOH).

Preparation of final title compound, hydrated crystalline 4-{4-|YlE)-4-(2.9- diazaspiro [5.5^“|undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl I -5-(propan-2-vD- 1 H-pyrazol-3 – yl beta-D-glucopyranoside acetate.

4- {4- [(1 E)-4-(2,9-diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl } -5 – (propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside (902 mg) is placed in a round bottom flask (100 mL) and treated with wet ethyl acetate (18 mL). [Note – wet ethyl acetate is prepared by mixing ethyl acetate (100 mL) and dionized water (100 mL). After mixing, the layers are allowed to separate, and the top wet ethyl acetate layer is removed for use. Acetic acid is a hydrolysis product of ethyl acetate and is present in wet ethyl acetate.] The compound dissolves, although not completely as wet ethyl acetate is added. After several minutes, a white precipitate forms. An additional amount of wet ethyl acetate (2 mL) is added to dissolve remaining compound. The solution is allowed to stir uncovered overnight at room temperature during which time the solvent partially evaporates. The remaining solvent from the product slurry is removed under vacuum, and the resulting solid is dried under a stream of nitrogen to provide the final title compound as a crystalline solid. A small amount of amorphous material is identified in the product by solid-state NMR. This crystalline final title compound may be used as seed crystals to prepare additional crystalline final title compound.

Alternative preparation of final title compound, hvdrated crystalline 4-{4-[(lE)-4-(2.,9- diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl I -5-(propan-2-yl)- 1 H-pyrazol-3 – yl beta-D-glucopyranoside acetate.

Under a nitrogen atmosphere combine of 4-{4-[(lE)-4-(2,9-diazaspiro[5.5]undec- 2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl} -5-(propan-2-yl)- 1 H-pyrazol-3-yl 2,3,4,6-tetra-O- acetyl-beta-D-glucopyranoside (2.1 kg, 2.74 mol), methanol (4.4 L), tetrahydrofuran (4.2 L), and water (210 mL). Add potassium carbonate (460 g, 3.33 moles) and stir for four to six hours, then filter the reaction mixture to remove the solids. Concentrate the filtrate under reduced pressure, then add ethanol (9.0 L) followed by acetic acid (237 mL, 4.13 mol) and stir at room temperature for one hour. To the stirring solution add wet ethyl acetate (10 L, containing approx. 3 w/w% water) slowly over five hours, followed by water (500 mL). Stir the suspension for twelve hours and add wet ethyl acetate (4.95 L, containing approx. 3 w/w% water) over a period of eight hours. Stir the suspension for twelve hours and add additional wet ethyl acetate (11.5 L, containing approx. 3 w/w% water) slowly over sixteen hours. Stir the suspension for twelve hours, collect the solids by filtration and rinse the solids with wet ethyl acetate (3.3 L, containing approx. 3 w/w% water). Dry in an oven under reduced pressure below 30°C to give the title compound as an off-white crystalline solid (1.55 kg, 2.35 mol, 96.7% purity, 72.4 w/w% potency, 68.0% yield based on potency). HRMS (m/z): 599.3798 (M+l).

PATENT

CN105705509

https://patentscope.wipo.int/search/en/detail.jsf?docId=CN175101669&tab=PCTDESCRIPTION

The present invention is in the field of treatment of diabetes and other diseases and conditions associated with hyperglycemia. Diabetes is a group of diseases characterized by high blood sugar levels. It affects approximately 25 million people in the United States, and according to the 2011 National Diabetes Bulletin, it is also the seventh leading cause of death in the United States (US Department of Health and Human Resources Services, Centers for Disease Control and Prevention). Sodium-coupled glucose cotransporters (SGLT’s) are one of the transporters known to be responsible for the uptake of carbohydrates such as glucose. More specifically, SGLT1 is responsible for transporting glucose across the brush border membrane of the small intestine. Inhibition of SGLT1 can result in a decrease in glucose absorption in the small intestine, thus providing a useful method of treating diabetes.

Alternative medicines and treatments for diabetes are needed. The present invention provides an acetate salt of a pyrazole compound which is an SGLT1 inhibitor, and thus it is suitable for treating certain conditions such as diabetes.

U.S. Patent No. 7,655,632 discloses certain pyrazole derivatives having human SGLT1 inhibitory activity, which are also disclosed for use in the prevention or treatment of diseases associated with hyperglycemia, such as diabetes. Moreover, WO 2011/039338 discloses certain pyrazole derivatives having SGLT1/SGLT2 inhibitor activity, which are also disclosed for use in the treatment of bone diseases such as osteoporosis.

PATENT

WO-2019141209

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019141209&tab=FULLTEXT&_cid=P10-JYNZF2-05384-1

Process for preparing pyranoglucose-substituted pyrazole compound, used as a pharmaceutical intermediate in SGLT inhibitor for treating diabetes.

/////////////SY-008 , SY 008 , SY008, ELI LILY, PHASE 1, GLT1 inhibitor, type 2 diabetes, Yabao Pharmaceutical, CHINA, DIABETES

CC(=O)O.Cc5cc(\C=C\CCN2CCCC1(CCNCC1)C2)ccc5Cc3c(nnc3C(C)C)O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O

Cc5cc(\C=C\CCN2CCCC1(CCNCC1)C2)ccc5Cc3c(nnc3C(C)C)O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4
O

CK-101, RX-518

CAS 1660963-42-7

N-[3-[2-[[2,3-Difluoro-4-[4-(2-hydroxyethyl)piperazin-1-yl]phenyl]amino]quinazolin-8-yl]phenyl]acrylamide

N-(3-(2-((2,3-Difluoro-4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)quinazolin-8-yl)phenyl)acrylamide

EGFR-IN-3

UNII-708TLB8J3Y

708TLB8J3Y

AK543910

Suzhou NeuPharma (Originator)
Checkpoint Therapeutics

Non-Small Cell Lung Cancer Therapy
Solid Tumors Therapy

PHASE 2 Checkpoint Therapeutics, Cancer, lung (non-small cell) (NSCLC), solid tumour

RX518(CK-101) is an orally available third-generation and selective inhibitor of certain epidermal growth factor receptor (EGFR) activating mutations, including the resistance mutation T790M, and the L858R and exon 19 deletion (del 19) mutations, with potential antineoplastic activity.

In August 2019, Suzhou Neupharma and its licensee Checkpoint Therapeutics are developing CK-101 (phase II clinical trial), a novel third-generation, covalent, EGFR inhibitor, as a capsule formulation, for the treatment of cancers including NSCLC and other advanced solid tumors. In September 2017, the FDA granted Orphan Drug designation to this compound, for the treatment of EGFR mutation-positive NSCLC; in January 2018, the capsule was being developed as a class 1 chemical drug in China.

CK-101 (RX-518), a small-molecule inhibitor of epidermal growth factor receptor (EGFR), is in early clinical development at Checkpoint Therapeutics and Suzhou NeuPharma for the potential treatment of EGFR-mutated non-small cell lung cancer (NSCLC) and other advanced solid malignancies.

In 2015, Suzhou NeuPharma granted a global development and commercialization license to its EGFR inhibitor program, excluding certain Asian countries, to Coronado Biosciences (now Fortress Biotech). Subsequently, Coronado assigned the newly acquired program to its subsidiary Checkpoint Therapeutics.

In 2017, the product was granted orphan drug designation in the U.S. for the treatment of EGFR mutation-positive NSCLC.

There are at least 400 enzymes identified as protein kinases. These enzymes catalyze the phosphorylation of target protein substrates. The phosphorylation is usually a transfer reaction of a phosphate group from ATP to the protein substrate. The specific structure in the target substrate to which the phosphate is transferred is a tyrosine, serine or threonine residue. Since these amino acid residues are the target structures for the phosphoryl transfer, these protein kinase enzymes are commonly referred to as tyrosine kinases or serine/threonine kinases.

[0003] The phosphorylation reactions, and counteracting phosphatase reactions, at the tyrosine, serine and threonine residues are involved in countless cellular processes that underlie responses to diverse intracellular signals (typically mediated through cellular receptors), regulation of cellular functions, and activation or deactivation of cellular processes. A cascade of protein kinases often participate in intracellular signal transduction and are necessary for the realization of these cellular processes. Because of their ubiquity in these processes, the protein kinases can be found as an integral part of the plasma membrane or as cytoplasmic enzymes or localized in the nucleus, often as components of enzyme complexes. In many instances, these protein kinases are an essential element of enzyme and structural protein complexes that determine where and when a cellular process occurs within a cell.

[0004] The identification of effective small compounds which specifically inhibit signal transduction and cellular proliferation by modulating the activity of tyrosine and serine/threonine kinases to regulate and modulate abnormal or inappropriate cell proliferation, differentiation, or metabolism is therefore desirable. In particular, the identification of compounds that specifically inhibit the function of a kinase which is essential for processes leading to cancer would be beneficial.

[0005] While such compounds are often initially evaluated for their activity when dissolved in solution, solid state characteristics such as polymorphism are also important. Polymorphic forms of a drug substance, such as a kinase inhibitor, can have different physical properties, including melting point, apparent solubility, dissolution rate, optical and mechanical properties, vapor pressure, and density. These properties can have a direct effect on the ability to process or manufacture a drug substance and the drug product. Moreover, differences in these properties

can and often lead to different pharmacokinetics profiles for different polymorphic forms of a drug. Therefore, polymorphism is often an important factor under regulatory review of the ‘sameness’ of drug products from various manufacturers. For example, polymorphism has been evaluated in many multi-million dollar and even multi-billion dollar drugs, such as warfarin sodium, famotidine, and ranitidine. Polymorphism can affect the quality, safety, and/or efficacy of a drug product, such as a kinase inhibitor. Thus, there still remains a need for polymorphs of kinase inhibitors. The present disclosure addresses this need and provides related advantages as well.

PATENT

WO2015027222

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015027222

PATENT

WO-2019157225

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019157225&tab=PCTDESCRIPTION&_cid=P10-JZNKMN-12945-1

Crystalline form II-VIII of the compound presumed to be CK-101 (first disclosed in WO2015027222 ), for treating a disorder mediated by epidermal growth factor receptor (EGFR) eg cancer.

SCHEME A

Scheme B

General Procedures

Example 1: Preparation of the compound of Formula I (N-(3-(2-((2,3-difluoro-4-(4-(2-hydroxyethyl)piperazin-l-yl)phenyl)amino)quinazolin-8-yl)phenyl)acrylamide)

[0253] To a solution of l,2,3-trifluoro-4-nitrobenzene (2.5 g, 14 mmol, 1.0 eq.) in DMF (20 mL) was added K₂C0₃ (3.8 g, 28 mmol, 2.0 eq.) followed by 2-(piperazin-l-yl)ethanol (1.8 g, 14 mmol, 1.0 eq.) at 0 °C and the mixture was stirred at r.t. overnight. The mixture was poured into ice-water (200 mL), filtered and dried in vacuo to afford 2-(4-(2,3-difluoro-4-nitrophenyl)piperazin-l-yl)ethanol (2.7 g, 67.5%).

[0254] To a solution of 2-(4-(2,3-difluoro-4-nitrophenyl)piperazin-l-yl)ethanol (2.7 g, 9.0 mmol) in MeOH (30 mL) was added Pd/C (270 mg) and the resulting mixture was stirred at r.t.

overnight. The Pd/C was removed by filtration and the filtrate was concentrated to afford 2-(4-(4-amino-2,3-difluorophenyl)piperazin-l-yl)ethanol (2.39 g, 99% yield) as off-white solid.

[0255] To a solution of 8-bromo-2-chloroquinazoline (15.4 g, 63.6 mmol, 1 eq. ) and (3-aminophenyl)boronic acid (8.7 g, 63.6 mmol, 1 eq.) in dioxane/H₂0 (200 mL/20 mL) was added Na₂C0₃ (13.5 g, 127.2 mmol, 2 eq.), followed by Pd(dppf)Cl₂ (2.6 g, 3.2 mmol, 0.05 eq.) under N₂, then the mixture was stirred at 80 °C for 12 h. Then the solution was cooled to r.t.,

concentrated and the residue was purified via column chromatography (PE/EA=3 :2, v/v) to afford 3-(2-chloroquinazolin-8-yl)aniline as yellow solid (8.7 g, 53.7% yield).

[0256] To a solution of 3-(2-chloroquinazolin-8-yl)aniline (8.7 g, 34 mmol, 1 eq.) in DCM ( 200 mL ) cooled in ice-bath was added TEA (9.5 mL, 68 mmol, 2 eq. ), followed by acryloyl chloride (4.1 mL, 51 mmol, 1.5 eq.) dropwise. The resulting mixture was stirred at r.t. for 1 h, then washed with brine, dried over anhydrous N₂S0₄ concentrated and the residue was purified via column chromatography (PE/EA=l : 1, v:v) to afford N-(3-(2-chloroquinazolin-8-yl)phenyl)acryl amide as yellow solid(6.6 g, 65% yield).

[0257] To a suspension of 2-(4-(4-amino-2,3-difluorophenyl)piperazin-l-yl)ethanol (83 mg,

0.32 mmol, 1 eq.) and N-(3-(2-chloroquinazolin-8-yl)phenyl)acrylamide (100 mg, 0.32 mmol, 1 eq.) in n-BuOH (5 mL) was added TFA (68 mg, 0.64 mmol, 2 eq.) and the resulting mixture was stirred at 90 °C overnight. The mixture was concentrated, diluted with DCM (20 mL) , washed with Na₂C0₃ solution (20 mL), dried over anhydrous Na₂S0₄, concentrated and the residue was purified via column chromatography (MeOH/DCM=l/30, v:v) to afford N-(3-(2-((2,3-difluoro-4-(4-(2-hydroxyethyl)piperazin-l-yl)phenyl)amino)quinazolin-8-yl)phenyl)acrylamide as a yellow solid(l6.3 mg, 9.5% yield). LRMS (M+H⁺) m/z calculated 531.2, found 531.2. 1H NMR

(CD₃OD, 400 MHz) d 9.21 (s, 1 H), 7.19-8.01 (m, 10 H), 8.90 (s, 1 H), 6.41-6.49 (m, 3 H), 5.86 (m, 1 H), 3.98-4.01 (m, 3 H), 3.70-3.76 (m, 3 H), 3.40-3.49 (m, 2 H), 3.37-3.39 (m, 4 H), 3.18 (m, 2H).

Example 2. Preparation of Form I of the compound of Formula I

[0258] Crude compound of Formula I (~30 g, 75% of weight based assay) was dissolved in ethyl acetate (3 L) at 55-65 °C under nitrogen. The resulting solution was filtered via silica gel pad and washed with ethyl acetate (3 L><2) at 55-65 °C. The filtrate was concentrated via vacuum at 30-40 °C to ~2.4 L. The mixture was heated up to 75-85 °C and maintained about 1 hour.

Then cooled down to 50-60 °C and maintained about 2 hours. The heat-cooling operation was repeated again and the mixture was then cooled down to 20-30 °C and stirred for 3 hours. The resulting mixture was filtered and washed with ethyl acetate (60 mL><2). The wet cake was dried via vacuum at 30-40 °C to get (about 16 g) of the purified Form I of the compound of Formula I.

Example 3. Preparation of Form III of the compound of Formula I

[0259] The compound of Formula I (2 g) was dissolved in EtOH (40 mL) at 75-85 °C under nitrogen. n-Heptane (40 mL) was added dropwise into reaction at 75-85 °C. The mixture was stirred at 75-85 °C for 1 hour. Then cooled down to 50-60 °C and maintained about 2 hours. The heat-cooling operation was repeated again and continued to cool the mixture down to 20-30 °C and stirred for 3 hours. The resulting mixture was filtered and washed with EtOH/n-Heptane (1/1, 5 mL><2). The wet cake was dried via vacuum at 30-40 °C to get the purified Form III of the compound of Formula I (1.7 g).

Example 4. Preparation of Form IV of the compound of Formula I The crude compound of Formula I (15 g) was dissolved in ethyl acetate (600 mL) at 75-85 °C under nitrogen and treated with anhydrous Na₂S0₄, activated carbon, silica metal scavenger for 1 hour. The resulting mixture was filtered via neutral Al₂0₃ and washed with ethyl acetate (300 mL><2) at 75-85 °C. The filtrate was concentrated under vacuum at 30-40 °C and swapped with DCM (150 mL). n-Heptane (75 mL) was added into this DCM solution at 35-45 °C, and then the mixture was cooled down to 20-30 °C slowly. The resulting mixture was filtered and washed with DCM/n-Heptane (2/1, 10 mL><3). The wet cake was dried via vacuum at 35-40 °C to get the purified Form IV of the compound of Formula I (9.6 g).

Example 5. Preparation of Form V of the compound of Formula I

[0260] Polymorph Form III of the compound of Formula I was dried in oven at 80 °C for 2 days to obtain the polymorph Form V.

Example 6. Preparation of Form VI of the compound of Formula I

[0261] The compound of Formula I (1 g) was dissolved in IPA (20 mL) at 75-85 °C under nitrogen. n-Heptane (20 mL) was added dropwise into reaction at 75-85 °C. The mixture was stirred at 45-55 °C for 16 hours. Then heated up to 75-85 °C and maintained about 0.5 hour.

Then cooled down to 45-55 °C for 0.5 hour and continued to cool the mixture down to 20-30 °C and stirred for 3 hours. Filtered and washed with IPA/n-Heptane (1/1, 3 mL><2). The wet cake was dried via vacuum at 75-80 °C for 2 hours to get the purified Form VI of the compound of Formula I.

Example 7. Preparation of Form VIII of the compound of Formula I

[0262] The polymorph Form VI of the compound of Formula I was dried in oven at 80 °C for 2 days to obtain the polymorph Form VIII.

Example 8. X-ray powder diffraction (XRD)

[0263] X-ray powder diffraction (XRD) patterns were obtained on a Bruker D8 Advance. A CuK source (=1.54056 angstrom) operating minimally at 40 kV and 40 mA scans each sample between 4 and 40 degrees 2-theta. The step size is 0.05°C and scan speed is 0.5 second per step.

Example 9. Thermogravimetric Analyses (TGA)

[0264] Thermogravimetric analyses were carried out on a TA Instrument TGA unit (Model TGA 500). Samples were heated in platinum pans from ambient to 300 °C at 10 °C/min with a nitrogen purge of 60mL/min (sample purge) and 40mL/min (balance purge). The TGA temperature was calibrated with nickel standard, MP=354.4 °C. The weight calibration was performed with manufacturer-supplied standards and verified against sodium citrate dihydrate desolvation.

Example 10. Differential scanning calorimetry (DSC)

[0265] Differential scanning calorimetry analyses were carried out on a TA Instrument DSC unit (Model DSC 1000 or 2000). Samples were heated in non-hermetic aluminum pans from ambient to 300 °C at 10 °C/min with a nitrogen purge of 50mL/min. The DSC temperature was calibrated with indium standard, onset of l56-l58°C, enthalpy of 25-29J/g.

Example 11. Hygroscopicity (DVS)

[0266] The moisture sorption profile was generated at 25°C using a DVS Moisture Balance Flow System (Model Advantage) with the following conditions: sample size approximately 5 to 10 mg, drying 25°C for 60 minutes, adsorption range 0% to 95% RH, desorption range 95% to 0% RH, and step interval 5%. The equilibrium criterion was <0.01% weight change in 5 minutes for a maximum of 120 minutes.

Example 12: Microscopy

[0267] Microscopy was performed using a Leica DMLP polarized light microscope equipped with 2.5X, 10X and 20X objectives and a digital camera to capture images showing particle shape, size, and crystallinity. Crossed polars were used to show birefringence and crystal habit for the samples dispersed in immersion oil.

Example 13: HPLC

[0256] HPLCs were preformed using the following instrument and/or conditions.

///////////////CK-101 , CK 101 , CK101 , phase II , Suzhou Neupharma, Checkpoint Therapeutics ,  Orphan Drug designation, EGFR mutation-positive NSCLC, NSCLC, CANCER, SOLID TUMOUR,  China, RX-518, AK543910

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