KAE 609, NITD 609, Cipargamin for Malaria

Cipargamin, NITD 609

IUPAC Name: (3R,3'S)-5,7'-dichloro-6'-fluoro-3'-methylspiro[1H-indole-3,1'-2,3,4,9-tetrahydropyrido[3,4-b]indole]-2-one |

CAS Registry Number: 1193314-23-6
Synonyms: NITD609, NITD 609, NITD-609, GNF-609
KAE-609
NITD-609  

 390.238, C19 H14 Cl2 F N3 O

(1'R,3'S)-5,7'-Dichloro-6'-fluoro-3'-methyl-1,2,2',3',4',9'-hexahydrospiro[indole-3,1'-pyrido[3,4-b]indole]-2-one

(1R,3S)-5′,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,3′-indol]-2′(1′H)-one

Novartis Ag    innovator

CURRENTLY IN -PHASE2

NITD609 is an experimental synthetic antimalarial molecule belonging to the spiroindolone class.^[1][2] The compound was developed at the Novartis Institute for Tropical Diseases in Singapore, through a collaboration with the Genomics Institute of the Novartis Research Foundation (GNF), the Biomedical Primate Research Centre and the Swiss Tropical Institute. NITD609 is a novel, synthetic antimalarial molecule belonging to the spiroindolone class, awarded MMV Project of the Year 2009.

It is structurally related to GNF 493, a compound first identified as a potent inhibitor of Plasmodium falciparum growth in a high throughput phenotypic screen of natural products conducted at the Genomics Institute of the Novartis Research Foundation in San Diego, California in 2006. NITD609 was discovered by screening the Novartis library of 12,000 natural products and synthetic compounds to find compounds active against Plasmodium falciparum. The first screen turned up 275 compounds and the list was narrowed to 17 potential candidates.

KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analogue with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum.http://www.nejm.org/doi/full/10.1056/NEJMoa1315860

KAE609 shows promise as next generation treatment for malaria

http://www.novartis.com/newsroom/media-releases/en/2014/1843976.shtml

• KAE609 is the first antimalarial drug candidate with a novel mechanism of action to achieve positive clinical proof-of-concept in over 20 years
  
• KAE609 was tested in adult patients with uncomplicated malaria and showed a median parasite clearance time of 12 hours, including in patients with resistant infections[1]
  
• For more than a decade, Novartis has been a leader in the fight against malaria, setting the current gold standard for treatment and building one of the strongest malaria pipelines in the industry

KAE609 shows promise as next generation treatment for malaria

• KAE609 is the first antimalarial drug candidate with a novel mechanism of action to achieve positive clinical proof-of-concept in over 20 years
  
• KAE609 was tested in adult patients with uncomplicated malaria and showed a median parasite clearance time of 12 hours, including in patients with resistant infections[1]
  
• For more than a decade, Novartis has been a leader in the fight against malaria, setting the current gold standard for treatment and building one of the strongest malaria pipelines in the industry

The digital press release with multimedia content can be accessed here:

Basel, Switzerland, July 30, 2014 - Today, Novartis published clinical trial results in the New England Journal of Medicine showing that KAE609 (cipargamin), a novel and potent antimalarial drug candidate, cleared the parasite rapidly in Plasmodium falciparum (P. falciparum) and Plasmodium vivax (P. vivax) uncomplicated malaria patients[1]. Novartis currently has two drug candidates in development. Both KAE609 and KAF156 are new classes of anti-malarial compounds that treat malaria in different ways from current therapies, important to combat emerging drug resistance. Novartis has also identified PI4K as a new drug target with potential to prevent, block and treat malaria.

"Novartis is in the fight against malaria for the long term and we are committed to the continued research and development of new therapies to eventually eliminate the disease," said Joseph Jimenez, CEO of Novartis. "With two compounds and a new drug target currently under investigation, Novartis has one of the strongest malaria pipelines in the industry."

Malaria is a life-threatening disease primarily caused by parasites (P. falciparum and P. vivax) transmitted to people through the bites of infected Anopheles mosquitoes. Each year it kills more than 600,000 people, most of them African children[2].

"KAE609 is a potential game-changing therapy in the fight against malaria," said Thierry Diagana, Head of the Novartis Institute for Tropical Diseases (NITD), which aims to discover novel treatments and prevention methods for major tropical diseases. "Novartis has given KAE609 priority project status because of its unique potential of administering it as a single-dose combination therapy."

In June 2012, 21 patients infected by one of the two main malaria-causing parasite types took part in a proof-of-concept clinical study conducted in Bangkok and Mae Sot near the Thailand/Burma border where resistance to current therapies had been reported. Researchers saw rapid parasite clearance in adult patients (median of 12 hours)[2] with uncomplicated P. vivax or P. falciparum malaria infection including those with resistant parasites. No safety concerns were identified, however the study was too small for any safety conclusions.

"The growing menace of artemisinin resistance threatens our current antimalarial treatments, and therefore our attempts to control and eliminate falciparum malaria," said Nick White, Professor of Tropical Medicine at Mahidol University in Thailand and lead author of the NEJM article. "This is why we are so enthusiastic about KAE609; it is the first new antimalarial drug candidate with a completely novel mechanism of action to reach Phase 2 clinical development in over 20 years."

KAE609, the first compound in the spiroindolone class of treatment, works through a novel mechanism of action that involves inhibition of a P-type cation-transporter ATPase4 (PfATP4), which regulates sodium concentration in the parasite. Because KAE609 also appears to be effective against the sexual forms of the parasite, it could potentially help prevent disease transmission. The clinical trial was done in collaboration with the Wellcome Trust-Mahidol University - Oxford Tropical Medicine Research Programme. Research was supported by the Wellcome Trust, Singapore Economic Development Board, and Medicines for Malaria Venture.

KAE609 represents one of two new classes of antimalarial compounds that Novartis has discovered and published in the last four years.[3],[4] This drug candidate has shown potent in vitro activity against a broad range of parasites that have developed drug resistance against current therapies. KAE609 is currently being planned for Phase 2b trials.

References
[1] http://www.nejm.org/doi/full/10.1056/NEJMoa1315860
[2] World Health Organization, http://www.who.int/mediacentre/factsheets/fs094/en/
[3] Spiroindolones, a Potent Compound Class for the Treatment of Malaria, KAE609, Science, Sept. 2010
[4] Imaging of Plasmodium liver stages to drive next generation antimalarial drug discovery. Science Express, Nov. 17, 2011

http://www.ukmi.nhs.uk/applications/ndo/record_view_open.asp?newDrugID=6368

The current spiroindolone was optimized to address its metabolic liabilities leading to improved stability and exposure levels in animals. As a result, NITD609 is one of only a handful of molecules capable of completely curing mice infected withPlasmodium berghei (a model of blood-stage malaria).

Given its good physicochemical properties, promising pharmacokinetic and efficacy profile, the molecule was recently approved as a preclinical candidate and is now entering GLP toxicology studies with the aim of entering Phase I studies in humans in late 2010. If its safety and tolerability are acceptable, NITD609 would be the first antimalarial not belonging to either the artemisinin or peroxide class to go into a proof-of-concept study in malaria.

If NITD609 behaves similarly in people to the way it works in mice, it may be possible to develop it into a drug that could be taken just once - far easier than current standard treatments in which malaria drugs are taken between one and four times a day for up to seven days. NITD609 also has properties which could enable it to be manufactured in pill form and in large quantities. Further animal studies have been performed and researchers have begun human-stage trials.

NITD609
IUPAC name[hide] (1R,3S)-5’,7-Dichloro-6-fluoro-3-methyl-spiro[2,3,4,9-tetrahydropyrido[3,4-b]indole-1,3’-indoline]-2’-one
Identifiers
ChemSpider	24662493
Jmol-3D images	Image 1
Properties
Molecular formula	C19H14Cl2FN3O
Molar mass	390.24 g mol−1

Malaria is an old infectious disease caused by four protozoan parasites, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. These four parasites are typically transmitted by the bite of an infected female Anopheles mosquito. Malaria is a problem in many parts of the world, and over the last few decades the malaria burden has steadily increased. An estimated 1 to 3 million people die every year from malaria - mostly children under the age of 5. This increase in malaria mortality is due in part to the fact that Plasmodium falciparum, the deadliest malaria parasite, has acquired resistance against nearly all available antimalarial drugs, with the exception of the artemisinin derivatives.
Leishmaniasis is caused by one of more than twenty (20) varieties of parasitic protozoa that belong to the genus Leishmania, and is transmitted by the bite of female sandflies. Leishmaniasis is endemic in some 90 countries, including many tropical and sub-tropical areas.
There are four main forms of leishmaniasis. Visceral leishmaniasis, also called kala-azar, is the most serious form and is caused by the parasite Leishmania donovani. Patients who develop visceral leishmaniasis can die within months unless they receive treatment. The two main therapies for visceral leishmaniasis are the antimony derivatives sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantim®). Sodium stibogluconate has been used for about 70 years and resistance to this drug is a growing problem. In addition, the treatment is relatively long and painful, and can cause undesirable side effects. Human African Trypanosomiasis, also known as sleeping sickness, is a vector-bome parasitic disease. The parasites concerned are protozoa belonging to the Trypanosoma Genus. They are transmitted to humans by tsetse fly {Glossina Genus) bites which have acquired their infection from human beings or from animals harbouring the human pathogenic parasites.
Chagas disease (also called American trypanosomiasis) is another human parasitic disease that is endemic amongst poor populations on the American continent. The disease is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by blood-sucking insects. The human disease occurs in two stages: the acute stage, which occurs shortly after the infection, and the chronic stage, which can develop over many years. Chronic infections result in various neurological disorders, including dementia, damage to the heart muscle and sometimes dilation of the digestive tract, as well as weight loss. Untreated, the chronic disease is often fatal.
The drugs currently available for treating Chagas disease are nifurtimox and benznidazole. However, problems with these current therapies include their adverse side effects, the length of treatment, and the requirement for medical supervision during treatment. Furthermore, treatment is really only effective when given during the acute stage of the disease. Resistance to the two frontline drugs has already arisen. The antifungal agent amphotericin b has been proposed as a second-line drug, but this drug is costly and relatively toxic.


PAPER

Stereoselective Total Synthesis of KAE609 via Direct Catalytic Asymmetric Alkynylation to Ketimine

Hisashi Takada^†, Naoya Kumagai^*†, and Masakatsu Shibasaki^*†‡

^† Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

^‡ JST, ACT-C, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

Org. Lett., 2015, 17 (19), pp 4762–4765

DOI: 10.1021/acs.orglett.5b02300

Publication Date (Web): September 14, 2015

Copyright © 2015 American Chemical Society

*E-mail: nkumagai@bikaken.or.jp., *E-mail: mshibasa@bikaken.or.jp.

Abstract

A direct catalytic asymmetric alkynylation protocol is applied to provide the requisite enantioenriched propargylic α-tertiary amine, allowing for the stereoselective total synthesis of KAE609 (formerly NITD609 or cipargamin).

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http://pubs.acs.org/doi/abs/10.1021/acs.orglett.5b02300?journalCode=orlef7
http://pubs.acs.org/doi/suppl/10.1021/acs.orglett.5b02300/suppl_file/ol5b02300_si_001.pdf

 








PATENT
WO 2009/132921

In this process, the chiral amine is installed via an enzymatic resolution via deacylation of the acetamide 2. In addition to the wasteful resolution, other inefficiencies of this route include protection/deprotection (Ac/Boc, 2 to 4, and 5 to 6) and a three-step sequence to reduce the carboxylic acid to a methyl group (3 to 6).
Patent
US 2015/0045562

Improved Route to Cipargamin Employing Transaminase Reaction

For the transamination step, the enzyme ATA-256 was engineered by Codexis to accommodate the non-natural indole substrate 12. Since the substrate is not water-soluble, PEG 200 (approximately 20 vol %) is used as a cosolvent, an interesting selection given that DMSO or methanol are the most common cosolvents for enzymatic reactions. Isopropylamine is employed as the amine donor, a strategy that was adopted from the work of Merck and Codexis for the transamination of sitagliptin ketone.(2) During the transamination, which is a reversible reaction,i-PrNH₂ is converted to acetone, which can be readily removed by evaporation to drive the reaction to completion. The workup involves filtration to remove enzyme residues followed by pH swings in which the product is extracted into the aqueous layer under acidic conditions, then basified for extraction into the organic layer. Addition of (+)-camphorsulfonic acid (CSA) provides the amine 14 as the crystalline CSA salt. No details are provided on enantioselectivity for the transamination, and it is not clear if the (+)-CSA is required to upgrade the ee or whether this salt was selected based on physical properties and the ability to develop a scalable crystallization process.

The final step to generate the spiroindole involves a diastereoselective condensation of the chiral amine with 5-chloroisatin (7) under acidic conditions. The diastereoselectivity of this reaction is not provided, nor any ee or de data for the final product. The spiroindole is also isolated as a (+)-CSA salt, which is then converted to the crystalline free base hemihydrate as the final form of cipargamin.

Example 12: Process for Preparing a Compound of formula (IVA) ¹/z Hydrate

622.54 399.25
In a 750ml reactor with impeller stirrer 50g of compound (IVB) salt were dissolved in 300ml Ethanol (ALABD) and 100 ml deionised Water (WEM). The clear, yellowish sollution was heated to 58°C internal temperature. To the solution 85 g of a 10% aqueous sodium carbonate solution was added within 10 minutes. The clear solution was particle filtered into a second reaction vessel. Vessel and particle filter were each rinsed with 25 ml of a mixture of ethanohwater (3:1 v/v) in the second reaction vessel. The combined particle filtered solution is heated to 58°C internal temperature and 200ml water (WEM) were added dropwise within 15 minutes. Towards the end of the addition the solution gets turbid. The mixture is stirred for 10 minutes at 58°C internal temperature and is then cooled slowely to room temperature within 4hours 30 minutes forming a thick, well stirable white suspension. To the suspension 200 ml water are added and the mixture is stirred for additional 15hours 20 minutes at room temperature. The suspension is filtered and the filter cake is washed twice with 25 ml portions of a mixture of ethanohwater 9: 1 (v/v). The colourless crystals are dried at 60°C in vacuum yielding 26.23g (=91.2% yield). H NMR (400 MHz, DMSO-d₆)
0.70 (s, 1H), 10.52 (s, 1H), 7.44 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H),.26 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.83 - 4.00 (m,H), 3.13 (d, J = 6.0 Hz, 1H), 2.77 (dd, J = 15.1, 3.8 Hz, 1H), 2.38 (dd, J = 15.1, 10.5 Hz,H), 1.17 (d, J = 6.3 Hz, 3H).

Patent
http://www.google.com/patents/WO2009132921A1?cl=en

SCHEME G: Preparation of (lR,3S)-5',7-dichloro-6-fluoro-3-methyl-2,3,4,9- tetrahydrospiro[β-carboline-l,3'-indol-2'(l'iϊ)-one (35) and (lR,3S)-5'-chloro-6-fluoro-3- methyl-2,3,4,9-tetrahydrospiro[β-carboline-l,3'-indoI-2'(l'H0-one (36)

Step 1 : POCl₃ (2.43 mL, 26.53 mmol) was added dropwise to N, N-dimethylformamide (15.0 mL) at -20 °C and stirred below -5 ⁰C for one hour. A solution of 6-chloro-5-fluoroindole (3.0 g, 17.69 mmol) in dimethylformamide (5.0 mL) was added dropwise to the above reaction mixture at -20 °C. The salt-ice bath was removed and the reaction mixture was warmed to 35 ⁰C, After one hour, the reaction was poured onto ice and basified by solid sodium bicarbonate and extracted with ethyl acetate. The combined organic layer was washed with water and then concentrated to give 6-chloro-5-fluoro-1H-indole-3-carbaldehyde (3.4 g, 97 %) as a light brown solid. ¹H ΝMR (500 MHz, CDCl₃): δ 10.02 (s, 1 H), 8.10 (d, IH, J = 9.5 Hz), 7.87 (s, 1 H), 7.49 (d, IH, J= 5.5 Hz).
Step 2: The solution (0.2 M) of 6-chloro-5-fluoro-1H-indole-3-carbaldehyde (4.0 g, 20.24 mmol) in nitroethane (100 mL) was refluxed with ammonium acetate (1.32 g, 0.85 mmol) for 4 hours. The reaction mixture was concentrated under vacuum to remove nitroethane, diluted with ethylacetate and washed with brine. The organic layer was concentrated to give 6-chloro-5- fluoro-3-(2-nitro-propenyl)-1H-indole (5.0 g, 97 %) as a reddish orange solid. ¹H ΝMR (500 MHz, CDCl₃): δ 8.77 (s, IH), 8.32 (s, IH), 7.58 (d, IH, J= 2.5 Hz), 7.54 (d, IH, J = 9 Hz), 7.50 (d, IH, J= 5.9 Hz), 2.52 (s, 3H). Step 3: A solution of 6-chloro-5-fluoro-3-(2-nitro-propenyl)-1H-indole (5.0 g, 19.63 mmol) in tetrahydrofuran (10 mL) was added to the suspension of lithium aluminium hydride (2.92 g, 78.54 mmol) in tetrahydrofuran (20 mL) at 0 ⁰C and then refluxed for 3 hours. The reaction mixture was cooled to 0 °C, and quenched according to the Fischer method. The reaction mixture was filtered through celite and the filtrate concentrated to give 2-(6-chloro-5-fluoro-1H-indol-3- yl-1-methyl-ethylamine (4.7 g crude) as a viscous brown liquid. The residue was used without further purification. ¹H NMR (500 MHz, CDCl₃): δ 8.13 (s, IH), 7.37 (d, IH, 6.Hz), 7.32 (d, IH, J = 10 Hz), 7.08 (s, IH), 3.23-3.26 (m, IH), 2.77-2.81 (m, IH), 2.58-2.63 (m, IH), 1.15 (d, 3H, J= 6.5 Hz).
Step 4: A mixture of 2-(6-chloro-5-fluoro-1H-indol-3-yl-l-methyl-ethylamine (4.7 g, 20.73 mmol), 5-chloroisatin (3.76 g, 20.73 mmol) and p-toluenesulphonic acid (394 mg, 2.07 mmol) in ethanol (75 mL) was refluxed overnight. The reaction mixture was concentrated to remove ethanol, diluted with ethyl acetate and washed with saturated aqueous NaHCO₃. The organic layer was concentrated to give a brown residue, which was purified by silica gel chromatography (20 % ethyl acetate in hexane) to provide the corresponding racemate (4.5 g, 56 %) as a light yellow solid. The racemate was separated into its enantiomers by chiral chromatography to provide 35.
Compound 36 can be obtained in a similar fashion from 5-fluoroindole.
Alternatively 35 and 36 were be prepared in enantiomerically pure form by the following scheme.
SCHEME H: Alternative preparation of (lR,3S)-5',7-dichloro-6-fluoro-3-methyl-2,3,4,9- tetrahydrospiro[β-carboline-l,3'-indol-2'(1'H)-one (35)

Step 1 : To a solution of 6-chloro-5-fluoroindole (1.8 g, 10.8 mmol) and Ac₂O (10 niL) in AcOH (3OmL) was added L-serine (2.2 g, 20.9 mmol), the mixture was heated to 80 °C. After TLC indicated the reaction was complete, the mixture was cooled to 0 °C, neutralized to pH 11 , and washed with MTBE. The aqueous phase was acidified to pH 2 and extracted with EtOAc. The combined organic layers were washed with water and bπne, dπed with Na₂SO₄, filtered, and concentrated. The residue was purified with chromatography (Petroleum ether /EtOAc 1:1) to give 2-acetylamino-3-(6-chloro-5-fluoro-1H-mdol-3-yl)-propπonic acid as a light yellow solid (1.2 g, 37% yield).
Step 2: 2-Acetylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-proprionic acid (2.5g, 8.4mmol) was dissolved in aqueous NaOH (IN, 10 niL) and water added (70 mL). The mixture was heated to 37-38⁰C and neutralized with HCl (IN) to pΗ 7.3-7.8. L-Aminoacylase (0.5 g) was added to the mixture and allowed to stir for 2 days, maintaining 37-38⁰C and pΗ 7.3-7.8. The mixture was heated to 60 °C for another hour, concentrated to remove part of water, cooled and filtered. The filtrate was adjusted to pΗ 5.89 and filtered again. The filtrate was adjusted to pΗ 2.0 and extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether /EtOAc 1 : IEtOAc) to give R- 2-acetylamino-3-(6-chloro-5-fluoro-1H-mdol-3-yl)-propπonic acid as a light yellow solid (1.2 g, 48% yield). Step 3: R-2-acetylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-proprionic acid (1.2 g, 4.0 mmol) was dissolved in HCl (6N, 10 mL) and the mixture heated to reflux for 4 hours, and then concentrated to dryness. Toluene (50 mL) was added to the residue and concentrated to dryness to remove water and HCl. The residue was dried under vacuum and then dissolved in MeOH (20 mL). To the solution was added dropwise SOCl₂ (0.5 mL, 6.8 mmol) at 0 °C, and the mixture was stirred overnight. After removal of solvent, the residue was dissolved in THF/water (40/10 mL) and NaHCO₃ (1.0 g, 11.9 mmol) was added portionwise. Upon basifϊcation, BoC₂O (1.2 g, 5.5 mmol) added at 0 °C and allowed to stir at room temperature. After TLC indicated the reaction was finished, EtOAc was added and separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether /EtOAc: 5/1) to give R-2-tert-butoxycarbonylamino-3-(6-chloro-5-fluoro-l/-/-indol-3-yl)-proprionic acid methyl ester 460 g, 31% yield for 3 steps).
Step 4: To a solution of R-2-tert-butoxycarbonylamino-3-(6-chloro-5-fluoro-l//-indol-3-yl)- proprionic acid methyl ester (460mg, 1.2mmol) in dry ether (20 mL) was added portionwise LiAlH₄ (92 mg, 2.4 mmol) at 0 °C. The mixture was heated to reflux for 2 hours. After TLC indicated the reaction was finished, the mixture was cooled and carefully quenched with Na₂SO₄. The mixture was filtered and the filtrate was washed with saturated aqueous NH₄Cl and water, dried with Na₂SO₄, filtered, concentrated to give a crude product (400 mg), which was used without further purification.
Step 5: To a solution of the crude product (400 mg, 1.2mmol) and Et₃N (0.3 mL, 2.2 mmol) in CH₂Cl₂ (5 mL) was added MsCl (160 mg, 1.4 mmol) dropwise at 0 °C. The mixture was stirred for 2 hours at room temperature. After TLC indicated the reaction was completed, the mixture was washed with water and brine, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 5:1) to give methansulfonic acid (R)-2- ?ert-butoxycarbonylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-propyl ester as a light yellow solid (300 mg, 57% yield, 2 steps)
Step 6: To a solution of mesylate (300 mg, 0.7mmol) in dry ether (20 mL) was added portionwise LiAlH₄ (55 mg, 1.4 mmol) at 0 °C. The mixture was stirred at room temperature overnight. After TLC indicated the reaction was finished, the mixture was cooled and carefully quenched with Na₂SO₄. The mixture was filtered and the filtrate was washed with saturated aqueous NH₄Cl and water, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 10: 1) to give [(5)-2-(6-chloro-5-fluoro-1H-indol-3-yl)- 1 -methyl-ethyl] -carbamic acid tert-butyl ester as a light yellow solid (200 mg, 87% yield).
Step 7: A solution of [(S)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l-methyl-ethyl]-carbamic acid tert-butyl ester (200 mg, 0.6 mmol) in HCl/MeOH (10 mL) was stirred at room temperature. After TLC indicated the reaction was finished, the mixture was concentrated to remove the solvent. To the residue was added EtOAc (5OmL), and the mixture was neutralized with saturated NaHCO₃ to pH 8~9, and then extracted with EtOAc. The combined organic phases were dried with Na₂SO₄, filtered, concentrated to give a crude (S)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l- methyl-ethylamine which was used without further purification.
Step 8: To a solution of (5)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l-methyl-ethylamine (120 mg, 0.5 mmol) in EtOH (1OmL) was added 5-chloroisatin (90 mg, 0.5 mmol) and p-TsOΗ (8 mg, 0.04 mmol). The mixture was heated in a sealed tube at 110⁰C for 16 hours. After TLC indicated the reaction was finished, the mixture was cooled and concentrated. The residue was dissolved in EtOAc (2OmL) and washed with NaOH (IN) and brine, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 5:1) to give 36 (150mg, 64% yield over two steps).

Example 48 (15,3R)-5'-Chloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-l,3'-indol]-2'(l'_JH)-one
(35)

35
Compound 35 may be prepared according to Scheme F using the same or analogous synthetic techniques and/or substituting with alternative reagents.
(lS^RVS'-Chloro-S-methyl-l^^^-tetrahydrospirotβ-carboline-l.S'-indoll-l^l'ZO-one: ¹H NMR (300 MHz, DMSO-^₆): δ 10.45 (s, IH), 10.42 (s, IH), 7.43 (d, J= 7.5 Hz, IH), 7.31 (dd, J = 2.1, 8.4 Hz, IH), 7.16 (d, J = 7.2 Hz, IH), 7.05-7.02 (m, 2H), 7.00-6.96 (m, IH), 6.92 (d, J = 8.1 Hz, IH), 3.98-3.86 (m, IH), 2.78 (dd, J= 3.6, 14.9 Hz, IH), 2.41 (dd, J= 4.5, 25.5 Hz, IH), 1.18 (d, J= 6.3 Hz, 3H); MS (ESI) m/z 338.0 (M+H)⁺.
Chiral compounds such as 36 and 37 can be prepared according to Scheme G or H using the same or analogous synthetic techniques and/or substituting with alternative reagents. Example 49
(IR^^-S'.T-Dichloro-ό-fluoro-S-methyl-l^^^-tetrahydrospiroIβ-carboline-l^'-indol]- 2\VH)-one (36)

36
35: ¹H NMR (500 MHz, DMSO-Jd) δ 10.69 (s, IH), 10.51 (s, IH), 7.43 (d, J = 10.0 Hz, IH), 7.33 (dd, J= 8.4, 2.2 Hz, IH), 7.27 (d, J= 6.5 Hz, IH), 7.05 (d, J= 2.3, IH), 6.93 (d, J= 8.5 Hz, IH), 3.91 (m, IH), 3.13 (bd, J= 6.2 Hz, IH), 2.74 (dd, J= 15.0 , 3.0 Hz, IH), 2.35 (dd, J= 15.0, 10.3, IH), 1.15 (d, J= 6.0, 3H);
MS (ESI) m/z 392.0 (M+2H)⁺;
[α]²⁵ _D = + 255.4°
Example 50
(lS,3R)-5',7-Dichloro-6-fluoro-3-methyI-2,3,4,9-tetrahydrospiro[β-carboline-l,3'-indol]- 2'(l'H)-one (37)

37
(lS^^-S'^-Dichloro-o-fluoro-S-methyl^jS^^-tetrahydrospirojP-carboline-l-S'-indol]- 2'(l'H)-one: ¹H NMR (500 MHz, CDCl₃) δ 8.49 (s, IH), 7.54 (s, IH), 7.24 (d, J= 9.7 Hz, IH), 7.21 (dd, J = 8.6, 2.0 Hz, IH), 7.14 (d, J= 6.0 Hz, IH), 7.11 (d, J= 1.8, IH), 6.77 (d, J= 8.3 Hz, IH), 4.14 (m, IH), 2.89 (dd, J = 15.4, 3.7 Hz, IH), 2.49 (dd, J = 15.3, 10.5, IH), 1.68 (bs, IH), 1.29 (d, J= 6.4 Hz, 3H); MS (ESI) m/z 392.0 (M+2H)⁺; [α]²⁵_D -223.3°
PATENT
US 2011275613
http://www.google.com/patents/WO2013139987A1?cl=en

Prior art:
(1 'R, 3'S)-5, 7'-dichloro-6'-fIuoro-3'-methyl-2', 3',4', 9'-tetrahydrospiro[indoline-3, 1 - pyrido[3,4-b]indol]-2-one (eg. a compound of formula (IV), which comprises a spiroindolone moiety) and a 6-steps synthetic method for preparing, including known chiral amine intermediate compound (MA) are known (WO 2009/132921 ):

he present invention relates to processes for the preparation of spiroindolone compounds, such as (1'R,3'S)-5, 7'-dichloro-6'-fIuoro-3'-methyl-2',3',4',9'- tetrahydrospiro[indoline-3, 1 '-pyhdo[3.4-b]indol]-2-one.
(1 'R, 3'S)-5, 7'-dichloro-6'-fluoro-3'-methyl-2', 3',4 9'-tetrahydrospiro[indoline-3, 1 '- pyrido[3, 4-b]indol]-2-one is useful in the treatment and/or prevention of infections such as those caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Trypanosoma cruzi and parasites of the Leishmania genus such as, for example, Leishmania donovani., and it has the following structure:

(IVA)
(1 'R, 3'S)-5, 7'-dichloro-6'-fluoro-3'-methyl-2 3', 4', 9'-tetrahydrospiro[indoline-3, 1 - pyhdo[3, 4-b]indol]-2-one and a synthesis thereof are described in WO 2009/132921 Al in particular in Example 49 therein.

Example 10: Process for Conversion of Compound (IA) to Compound (IIA) in 30g Scale

458.97
152.48g /so-propylamine hydrochloride and 0.204g pyridoxalphosphate monohydrate were dissolved in 495ml water while stirring. To this yellow clear solution a solution of 30. Og ketone in 85ml poly ethylene glycol (average mol weight 200) within 15 minutes. Upon addition the ketone precipitates as fine particles which are evenly distributed in the reaction media. To the suspension 180ml triethanolamine buffer (0.1 mol/l, pH 7) were added and the pH was adjusted to 7 by additon of aqueous sodium hydroxide solution (1 mol/l). The reaction mixture is heated to 50°C and a solution of 1.62g transaminase SEQ ID NO: 134 dissolved in 162ml triethanolamine buffer (0 1 mol/l, pH 7) is added. The reaction mixture is continiously kept at pH 7 by addition of 1 mol/l aqueous sodium hydroxide solution. The reaction mixture is stirred 24h at 50°C and a stream of Nitrogen is blown over the surface of the reaction mixture to strip off formed acetone. The reaction mixture is then cooled to 25°C and filtered over a bed of cellulose flock. The pH of the filtrate is adjusted to «1 by addition of concentrated sulfuric acid. The acidified filtrated is extracted with 250 ml /so-Propyl acetate. The layers are separated and the pH of the aqueous phase is adjusted to ¾10 by additon of concentrated aqueous sodium hydroxide solution. The basified aqueous phase is extracted with /^'so-propyl acetate. The layers are seperated and the organic phase is washed with 100 ml water. The organic phase is concentrated by distillation to 2/3 of its origin volume. In a second reactor 33.98g (+)- camphor sulfonic acid is dissolved in 225 ml /^'so-propyl acetate upon refluxing and the concentrated organic phase is added within 10 minutes. After complete addition the formed thin suspension is cooled to 0°C within 2 hours and kept at 0°C for 15 hours. The precipitated amine-(+)-camphor sulfonate salt is filtered, washed with 70 ml /so-propyl acetate and dried at 40°C in vaccuum yielding 51.57g of colourless crystals (84.5% yield t.q.)
Analytical Data
IR:
v (crn ¹)=3296, 3061 , 2962, 2635, 2531 , 2078, 1741 , 1625, 1577, 1518, 1461 , 1415, 1392, 1375, 1324, 1302, 1280, 1256, 1226, 1 170, 1 126, 1096, 1041 , 988, 966, 937, 868, 834, 814, 790, 766, 746, 719, 669, 615.
LC-MS (ESI +):
Ammonium ion: m/z =227 ([M+H]), 268 ([M+H+CH₃CN]), 453 ([2M+H]).
Camphorsulfonate ion: m/z =250 ([M+NH₄]), 482 ([2M+NH4]).
LC-MS (ESI -):
Camphorsulfonate ion: m/z=231 ([M-H]), 463 ([2M-H]).
1H-NMR (DMSO-d6, 400 MHz):
1 1.22 (br. s., 1 H), 7.75 (br. s., 3H), 7.59 (d, J = 10.3 Hz, 1 H), 7.54 (d, J = 6.5 Hz, 1 H), 7.36 (d, J = 2.3 Hz, 1 H), 3.37 - 3.50 (m, 1 H), 2.98 (dd, J = 14.3, 5.8 Hz, 1 H), 2.91 (d, J = 14.8 Hz, 1 H), 2,81 (dd, J = 14.3, 8.0 Hz, 1 H), 2.63 - 2.74 (m, 1 H), 2.41 (d, J = 14.6 Hz, 1 H), 2.24 (dt, J = 18.3, 3.8 Hz, 1 H), 1 .94 (t, J = 4.4 Hz, 1 H), 1.86 (dt, J = 7.4, 3 6 Hz, 1 H), 1.80 (d, J = 18.1 Hz, H), 1.23 - 1 .35 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H), 1.05 (s, 3H), 0.74 (s, 3H)
Free Amine (obtained by evaporatig the iso-Propylacetate layer after extraction of the basified aqueous layer):
¹H NMR (400MHz, DMSO-d₆): 11 .04 (br. s., 1 H), 7.50 (d, J = 10.5 Hz, 1 H), 7.48 (d, J = 6.5 Hz, 1 H), 7.25 (s, 1 H), 3.03 (sxt, J = 6.3 Hz, 1 H), 2.61 (dd, J - 14.3, 6.5 Hz, 1 H), 2.57 (dd, J = 14.1 , 6.5 Hz, 1 H), 1.36 (br. s., 2H), 0.96 (d, J = 6.3 Hz, 3H)
Example 11: Process for Conversion of Compound (HA) to Compound (IVB)

3. solvent exchange to TP
13.62 g 5-chloroisatin is suspended in 35 ml /so-propanol and 2.3 g triethyl amine is added. The suspension is heated to reflux and a solution of 34.42g amine-(+)-camphor sulfonate salt dissolved in 300 ml /^'so-propanol is added within 50 minutes. The reaction mixture is stirred at reflux for 17 hours. The reaction mixture is cooled to 75°C and 17.4g (+)-camphorsulfonic acid are added to the reaction mixture. Approximately 300 ml /so- propanol are removed by vacuum distillation. Distilled off /so-propanol is replaced by iso- propyl acetate and vacuum distillation is continued. This is distillation is repeated a second time. To the distillation residue 19 ml ethanol and 265 ml ethyl acetate is added and the mixture is heated to reflux. The mixture is cooled in ramps to 0°C and kept at 0°C for 24 hours. The beige to off white crystals are filtered off, washed with 3 portions (each 25 ml) precooled (0°C) ethylacetate and dried in vacuum yielding 40.3 g beige to off white crystals. (86.3% yield t.q.)
IR:
v (crrr)= 3229, 3115, 3078, 3052, 2971 , 2890, 2841. 2772. 2722, 2675, 2605, 2434. 1741 , 1718, 1621 , 1606, 1483, 1460, 1408, 1391 , 1372, 1336, 1307, 1277, 1267, 1238, 1202, 1 184, 1 162, 1 149, 1 128, 1067, 1036, 987, 973, 939, 919, 896, 871 , 857, 843, 785, 771 , 756, 717, 690, 678, 613.
LC-MS (ESI +):
Ammonium ion: m/z =390 ([M+H]), 431 ([M+H+CH₃CN]) Camphorsulfonate ion: m/z =250 ([M+NH₄]), 482 ([2M+NH4])
LC-MS (ESI -):
Camphorsulfonate ion: m/z=231 ([M-H]), 463 ([2M-H])
1H NMR (DMSO-d₆, 600 MHz):
11.49 (s, 1 H), 1 1.23 (s, 1 H), 10.29 - 10.83 (m, 1 H), 9.78 - 10.31 (m, 1 H), 7.55 - 7.60 (m, 2H), 7.52 (s, 1 H), 7.40 (d, J = 6.2 Hz, H), 7.16 (d, J = 8.8 Hz, 1 H), 4.52 - 4.63 (m, 1 H). 3.20 (dd, J = 16.3, 4.2 Hz, 1 H), 2.96 (dd, J = 16.1 , 11.3 Hz, 1 H), 2.90 (d, J = 15.0 Hz, 1 H), 2.56 - 2.63 (m, 1 H), 2.39 (d, J = 14.6 Hz, 1 H), 2.21 (dt, J = 18.0, 3.8 Hz, 1 H), 1.89 - 1.93 (m, 1 H), 1.81 (ddd, J = 15.3, 7.8, 3.7 Hz, 1 H), 1.76 (d, J = 18.3 Hz, 1 H), 1 .53 (d, J = 6.6 Hz, 3H), 1.20 - 1.33 (m, 2H), 0.98 (s, 3H), 0.70 (s, 3H)
Example 12: Process for Preparing a Compound of formula (IVA) ¹/z Hydrate

mw622.54 .............................................................................mw399.25
In a 750ml reactor with impeller stirrer 50g of compound (IVB) salt were dissolved in 300ml Ethanol (ALABD) and 100 ml deionised Water (WEM). The clear, yellowish sollution was heated to 58°C internal temperature. To the solution 85 g of a 10% aqueous sodium carbonate solution was added within 10 minutes. The clear solution was particle filtered into a second reaction vessel. Vessel and particle filter were each rinsed with 25 ml of a mixture of ethanohwater (3:1 v/v) in the second reaction vessel. The combined particle filtered solution is heated to 58°C internal temperature and 200ml water (WEM) were added dropwise within 15 minutes. Towards the end of the addition the solution gets turbid.
The mixture is stirred for 10 minutes at 58°C internal temperature and is then cooled slowely to room temperature within 4hours 30 minutes forming a thick, well stirable white suspension. To the suspension 200 ml water are added and the mixture is stirred for additional 15hours 20 minutes at room temperature. The suspension is filtered and the filter cake is washed twice with 25 ml portions of a mixture of ethanohwater 9: 1 (v/v). The colourless crystals are dried at 60°C in vacuum yielding 26.23g (=91.2% yield). H NMR (400 MHz, DMSO-d₆)
0.70 (s, 1H), 10.52 (s, 1H), 7.44 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H),.26 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.83 - 4.00 (m,H), 3.13 (d, J = 6.0 Hz, 1H), 2.77 (dd, J = 15.1, 3.8 Hz, 1H), 2.38 (dd, J = 15.1, 10.5 Hz,H), 1.17 (d, J = 6.3 Hz, 3H).

PAPER

 Journal of Medicinal Chemistry, 2010 ,  vol. 53,   14  p. 5155 - 5164

http://pubs.acs.org/toc/jmcmar/53/14

(1R,3S)-5′,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,3′-indol]-2′(1′H)-one (19a)

¹H NMR (500 MHz, DMSO-d₆): δ 10.69 (s, 1H), 10.51 (s, 1H), 7.43 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.0, 2.2 Hz, 1H), 7.27 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.13 (bd, J = 6.2 Hz, 1H), 2.74 (dd, J = 15.0, 3.0 Hz, 1H), 2.35 (dd, J = 15.0, 10.3 Hz, 1H), 1.15 (d, J = 6.0 Hz, 3H). MS (ESI) m/z 392.0 (M + 2H)⁺; [α]_D²⁵ = +255.4° (c = 0.102 g/L, methanol).

CLIPS

http://www.google.com/patents/CN102432526A?cl=en&dq=%22cipargamin%22+OR+%22cipargamina%22+OR+%22cipargamine%22+OR+%22cipargaminum%22+OR+%22NITD609%22&source=uds

Z.Zhang, WO 2007 / 104714,2007).

[0008] (2) year 2008 Roche pharmaceutical company disclosed a spiro [oxindole - cyclohexenone] skeleton biomedicine, PCT International Application No. W02008 / 055812. It also announced the preparation of anti-cancer agents and antagonists of the application of the compound is used as the interaction with MDM2 (reference:. Liu, J.-J; Zhang, Z; (Hoffmann-LaRoche AG), PCT Int App 1. . W02008 / 055812, 2008), its structural formula is as follows:
[0009]

(3) Melchiorre research group abroad chiral amines and o-fluoro-3-benzyl benzoate as catalyst methylene-indole-2-one (3-benzylideneindolin-2-one, CAS Number: 3359-49- 7) with α, β - unsaturated ketone synthesis of chiral spiro [cyclohexane _1,3'- indole] _2,4 '- dione [s pir0 [cycl0hexane-l, 3' -indoline] - 2 ', 4-diones] compounds (see:.. Bencivenni, G; ffu, LY; Mazzanti, A .; Giannichi, B.; Pesciaioli, F; Song, Μ P.; Bartoli, G.; Melchiorre, P .... .Angew Chem Int Ed 2009,48,7200), the structure of the total formula is as follows:

(4) Gong Flow column team found to cyclohexanediamine derived Bronsted acid - a bifunctional catalyst Lewis base catalysis of 3-benzyl-methylene-indole-2-one and α, β- unsaturated 1,3 tandem reaction dicarbonyl compound (Nazarov reagent) can be obtained with high stereoselectivity chiral spiro [cyclohexane _1,3'- indol] -2 ', 4-dione [spiro [cyclohexane-l, 3 '-indoline] -2', 4-diones] compounds; and by this method successfully synthesized 7 Roche pharmaceutical companies to develop chiral anti-tumor agents (see: Q Wei, L -Z Gong, Org Lett 2010..... , 12, 1008.).
(5) Wang Lixin research group recently reported that primary amines derived from cinchona alkaloids and Bronsted acid as catalyst N- protected indolone compounds and double Michael addition reaction of diketene generate hand spiro [cyclohexane-1, 3'-indol] -2 ', 4-dione [spiro [cyclohexane-l, 3' -indoline] -2 ', 4-diones] type of tx ^ (: L. -L. Wang, L. Peng, J. -F. Bai, L. -N. Jia, X. -Y. Luo, QC Huang, L. -X. Wang, Chem. Commum. 2011,47, 5593.).

WO2009132921A1 *	Apr 1, 2009	Nov 5, 2009	Novartis Ag	Spiro-indole derivatives for the treatment of parasitic diseases
WO2010081053A2 *	Jan 8, 2010	Jul 15, 2010	Codexis, Inc.	Transaminase polypeptides
WO2012007548A1 *	Jul 14, 2011	Jan 19, 2012	Dsm Ip Assets B.V.	(r)-selective amination
AT507050A1 *	Title not available
EP0036741A2 *	Mar 17, 1981	Sep 30, 1981	THE PROCTER & GAMBLE COMPANY	Phosphine compounds, transition metal complexes thereof and use thereof as chiral hydrogenation catalysts
EP0120208A2 *	Jan 24, 1984	Oct 3, 1984	Degussa Aktiengesellschaft	Microbiologically produced L-phenylalanin-dehydrogenase, process for obtaining it and its use
EP0135846A2 *	Aug 31, 1984	Apr 3, 1985	Genetics Institute, Inc.	Production of L-amino acids by transamination
GB974895A *	Title not available
US3282959 *	Mar 21, 1962	Nov 1, 1966	Parke Davis & Co	7-chloro-alpha-methyltryptamine derivatives
US4073795 *	Jun 22, 1976	Feb 14, 1978	Hoffmann-La Roche Inc.	Synthesis of tryptophans

WO2005009370A2 *	Jul 22, 2004	Feb 3, 2005	Pharmacia Corp	Beta-carboline compounds and analogues thereof and their use as mitogen-activated protein kinase-activated protein kinase-2 inhibitors
EP0466548A1 *	Jun 27, 1991	Jan 15, 1992	Adir Et Compagnie	1,2,3,4,5,6-Hexahydroazepino[4,5-b]indole and 1,2,3,4-tetrahydro-beta-carbolines, processes for their preparation, and pharmaceutical compositions containing them

Рисунок из Science 2010, 329, 1175

Исследовательская группа Элизабет Винцелер (Elizabeth A. Winzeler) разработала новый препарат, первоначально проведя скрининг библиотеки, состоящей из 12000 соединений, а затем получив производные наиболее перспективных кандидатов. В результате долгой работы исследователи отобрали единственное соединение спироиндолоновой структуры, получившее регистрационный номер NITD609. В случае успешного прохождения экспертизы фармакологических и токсикологических свойств нового соединения исследователи надеются приступить к первой фазе его клинических испытаний уже в конце этого года.
Было обнаружено, что NITD609 быстро останавливает белковый синтез в организме возбудителя малярии, ингибируя ген аденозинтрифосфатазы, ответственной за транспорт катионов через мембрану клетки возбудителя. То, что механизм действия нового соединения отличается от механизма, характерного для других средств лечения малярии, объясняет причины успешного действия нового препарата в том числе и против штаммов малярии, выработавших резистентность.

 HPLC

Analyte quantization was performed byLC/MS/MS. Liquid chromatography was performed using an Agilent

1100 HPLC system(Santa Clara, CA), with the Agilent Zorbax XDB Phenyl (3.5μ, 4.6 x75 mm) column at

an oven temperature of 35 °C, coupled with a QTRAP4000 triple quadruple mass

spectrometer (Applied Biosystems, Foster City, CA). Instrumentcontrol and dataacquisition were performed using Applied Biosystems software Analyst 1.4.2. Themobile phases used were A: water:acetic acid (99.8:0.2, v/v) and B: acetonitrile:aceticacid (99.8:0.2, v/v), using a gradient, with flow rate of 1.0 mL/min, and run time of 5minutes. Under these conditions the retention time of9a

was 3.2 minutes. Compounddetection on the mass spectrometer was performed in electrospraypositive ionizationmode and utilized multiple reaction monitoring (MRM) for specificity (9atransitions338.3/295.1, 338.3/259.2) together with their optimized MS parameters. The lower limitof quantification for9awas 70 ng/mL.

Extraction and LCMS analysis of 20a.Plasma samples were extracted withacetonitrile:methanol-acetic acid (90:9.8:0.2 v/v) for the analyte and internal standard(17a) using a 3.6 to 1 extractant to plasma ratio. Analyte quantitation was performed by

LC/MS/MS. Liquid chromatography was performed using an Agilent1100 HPLC systemS7(Santa Clara, CA), with the Agilent Zorbax XDB-Phenyl (3.5μ, 4.6x75mm) column atan oven temperature of 45 °C coupled with a QTRAP 4000 triple quadruple massSpectrometer (Applied Biosystems, Foster City, CA). Instrumentcontrol and dataacquisition were performed using Applied Biosystems software Analyst 1.4.2. Themobile phases used were A: water:acetic acid (99.8:0.2, v/v) and B: methanol:acetic acid

(99.8:0.2, v/v), using gradient elution conditions with a flow rate of 1.0 mL/min and a runtime of 6 minutes

References

1.  "NITD 609". Medicines for Malaria Venture.
2.  Rottmann M, McNamara C, Yeung BK, Lee MC, Zou B, Russell B, Seitz P, Plouffe DM, Dharia NV, Tan J, Cohen SB, Spencer KR, González-Páez GE, Lakshminarayana SB, Goh A, Suwanarusk R, Jegla T, Schmitt EK, Beck HP, Brun R, Nosten F, Renia L, Dartois V, Keller TH, Fidock DA, Winzeler EA, Diagana TT (2010). "Spiroindolones, a potent compound class for the treatment of malaria". Science329 (5996): 1175–80. doi:10.1126/science.1193225. PMC 3050001. PMID 20813948.

Ang, S. H., Krastel, P., Leong, S. Y., Tan, L. J., Wong, W. L. J., Yeung, B. K., and Zou, B. Spiro-indole derivatives for the treatment of parasitic diseases. WO2009132921 A1, November 5, 2009.

Cipargamin

Names
IUPAC name (1R,3S)-5’,7-Dichloro-6-fluoro-3-methyl-spiro[2,3,4,9-tetrahydropyrido[3,4-b]indole-1,3’-indoline]-2’-one
Identifiers
CAS Number	1193314-23-6
ChemSpider	24662493
Jmol interactive 3D	Image
PubChem	44469321
InChI[show]
SMILES[show]
Properties
Chemical formula	C19H14Cl2FN3O
Molar mass	390.24 g·mol−1

////
C[C@H]1Cc2c3cc(c(cc3[nH]c2[C@]4(N1)c5cc(ccc5NC4=O)Cl)Cl)F

//////////
SEE.........https://newdrugapprovals.org/2014/12/27/nitd609/

[slideshare id=14159927&w=425&h=355&style=border:1px solid #CCC; border-width:1px; margin-bottom:5px; max-width: 100%;&sc=no]

Acs presentation march 2011 from fnesbitt

++++++++++++++++

PF 04995274, a 5-HT4Partial Agonist

1 Vote

PF-04995274,

(R)-4-((4-(((4-(Tetrahydrofuran-3-yloxy)-1,2-benzisoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol

4-(4-{4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidin-1-ylmethyl)-tetrahydro-pyran-4-ol

CAS  1331782-27-4
UNII: XI179PG9LV

MF C23-H32-N2-O6

MW 432.5138

a 5-HT4Partial Agonist

PHASE 1 Alzheimer’s type dementia.

Pfizer Inc. INNOVATOR

5-HT4 agonists have attracted attention for therapeutic value in the treatment of Alzheimer’s Disease (AD) and cognitive impairment.Acting to increase levels of acetylcholine and soluble APP alpha, 5-HT4 agonists have the potential to demonstrate both ameliorative and disease modifying effects

(R)-4-((4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidin-1-yl)methyl)tetrahydro-2/-/-pyran-4-ol and pharmaceutically acceptable salts thereof. This invention also is directed, in part, to a method for treating a 5-HT4 mediated disorder in a mammal. Such disorders include acute neurological and psychiatric disorders, stroke, cerebral ischemia, spinal cord trauma, head trauma, perinatal hypoxia, cardiac arrest, hypoglycemic neuronal damage, dementia, Alzheimer’s disease, Huntington’s Chorea, amyotrophic lateral sclerosis, ocular damage, retinopathy, cognitive disorders, idiopathic and drug- induced Parkinson’s disease, muscular spasms and disorders associated with muscular spasticity including tremors, depression, epilepsy, convulsions, migraine, urinary incontinence, substance tolerance, substance withdrawal, psychosis, schizophrenia, anxiety, mood disorders, trigeminal neuralgia, hearing loss, tinnitus, macular degeneration of the eye, gastroesophageal reflux disease, gastrointestinal disease, gastric motility disorder, non-ulcer dyspepsia, functional dyspepsia, irritable bowel syndrome, constipation, dyspepsia, esophagitis, gastroesophageral disease, nausea, emesis, brain edema, pain, tardive dyskinesia, sleep disorders, attention deficit/hyperactivity disorder, attention deficit disorder, disorders that comprise as a symptom a deficiency in attention and/or cognition, and conduct disorder

a(a) SOCl2, DMAP, acetone, DME, RT, 81%;

(b) DEAD, PPh3, THF, RT, 65%;

(c) K2CO3, MeOH, RT, 92%;

(d) K2CO3, water, MeOH, 50 °C, 76%;

(e) CDI, THF, 50 °C, 43%;

(f) DEAD, PPh3, THF, reflux, 51%;

(g) HCl, Et2O, RT, 81%;

(h) TEA, MeOH, reflux, 50%.

PAPER

Journal of Medicinal Chemistry (2012), 55(21), 9240-9254

http://pubs.acs.org/doi/abs/10.1021/jm300953p

The cognitive impairments observed in Alzheimer’s disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT4) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT4agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure–response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT4 partial agonist candidates 2d and 3 were chosen for clinical development.

PATENT

https://www.google.co.in/patents/WO2011101774A1?cl=en

(R)-4-((4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol , hereinafter referred to as “Compound X,” and having the following structure:

Compound X

Example 1 : Synthesis of iR)-4-ii4-i(4-itetrahvdrofuran-3-yloxy)benzord1isoxazol-3-yloxy)methyl)piperidin-1 -yl)methyl)tetrahvdro- 2 -pyran-4-ol

Methyl 2-fluoro-6-hydroxybenzoate (2): To a 20L jacketed reactor were charged 2-fluoro-6-hydroxybenzoic acid (Oakwood Products; 0.972 kg, 6.31 mol), methanol (7.60 L) and sulfuric acid (0.710 kg, 7.24 mol, 1 .15 eq). The jacket temperature was heated to 60°C and the reaction mixture was stirred for 45 h. The reaction mixture was concentrated under vacuum and approximately 7.5 L of methanol distillates were collected. The resulting thin oil was cooled to 20°C. Water (7.60 L) and ethyl acetate (7.60 L) were charged to the reactor, and the product extracted into the organic layer. The EtOAc solution was washed with a solution of sodium bicarbonate (1.52 Kg) in water (6.92 L) followed by a brine solution of sodium chloride (1.74 kg) in water (4.08 L). The resulting EtOAc solution was concentrated to dryness. A light orange oil was isolated; the oil slowly crystallized upon standing to give the title compound (2) (0.952 Kg, 5.60 mol, 89% yield). 1 H NMR (400 MHz, CDCI3) δ ppm 3.97 (s, 3H), 6.59 (ddd, J=10.9, 8.2,1 .2, 1 H), 6.76 (dt, J=8.2, 1 .1 , 1 H), 7.35 (td, J=8.6, 6.3, 1 H), 1 1.24 (s, 1 H); 13C NMR (400 MHz, CDCI3) δ ppm 52.65, 102.56 (d, J=13), 106.90 (d, J=23), 1 13.31 (d, J=3.1 ), 135.34 (d, J=1 1 .5), 161 .02, 163.31 (d, J=62.2), 169.87 (d, 3.8); MS 171.045 (m+1 ). 2-Fluoro-N,6-dihydroxybenzamide (3): To a 50L reactor was charged water (4.47 L) and hydroxylamine sulfate (6.430 kg, 39.17 mol), the mixture was stirred at 25°C. A solution of potassium carbonate (3.87 Kg, 27.98 mol) in water (5.05 L) was slowly added to the reaction mixture to form a thick white mixture that was stirred at 20°C. A solution of methyl 2-fluoro-6-hydroxybenzoate (2) (0.952 Kg, 5.60 mol) in methanol (9.52 L) was slowly added to the reactor resulting in mild off gassing. The reaction mixture was then heated to 35°C and stirred for 20 h. The reaction mixture was cooled to 15°C and stirred for 1 h. The mixture was filtered to remove inorganic material. The reactor was rinsed with methanol (2.86 L) and the tank rinse was used to wash the inorganic cake.

Analysis of the cake indicated that it contained product. To a 20L reactor was charged methanol (10 L) and the inorganic cake and the mixture was stirred at 25°C for 30 min. The mixture was filtered and the cake washed with methanol (3 L).

The combined filtrates were charged back into the reactor and concentrated under vacuum with the jacket temperature set at 40°C until approximately 10 L remained. The mixture was held at 25°C and cone. HCI (5.51 L) was added. The reactor was cooled to 15°C and stirred for 2 h. The white slurry was filtered and the resulting product cake was washed with water (4.76L), blown dry with nitrogen and then dried in a vacuum oven at 40°C for 12 h. The desired product (3) (747 g, 4.36 mol), was isolated in 78% yield. 1 H NMR (400 MHz, CD3OD) δ ppm 4.91 (s, 3H), 6.63 (ddd, J=10.9, 8.5, 0.8, 1 H), 6.72 (dt, J=8.2, 0.8, 1 H), 7.31 (td, J=8.2, 6.6, 1 H); MS 172.040 (m+1 ).

4-Fluorobenzo[d]isoxazol-3-ol (4): To a 20L jacketed reactor were charged tetrahydrofuran (2.23 L) and 1 ,1 ‘-carbonyldiimidazole (0.910 Kg, 5.64 mol). The resulting mixture was stirred at 20°C. Then a solution of 2-fluoro-N,6-dihydroxybenzamide (3) (744 g, 4.34 mol) in tetrahydrofuran (4.45 L) was slowly charged to the reactor maintaining the temperature below 30°C and stirred at 25°C for 30 min during which some off gassing was observed. The reaction mixture was heated to 60°C over 30 min and stirred for 6 h. The reactor was cooled to 20°C followed by the addition of 1 N aqueous hydrogen chloride (7.48L) over 15 min to adjust the pH to 1. The jacket temperature was set to 35°C and the reaction mixture concentrated under vacuum to remove approximately 6.68L of THF. The reactor was cooled to 15°C and stirred for 1 h. The resulting white slurry was filtered, the cake was washed with water (3.71 L) and dried in a vacuum oven at 40°C for 12 h. The desired product, (4) (597 g, 3.90 mol), was isolated in 90% yield. 1 H NMR (400 MHz, CD3OD) δ ppm 4.93 (b, 1 H), 6.95 (dd, J=10.1 , 8.6, 1 H), (d, J=8.6, 1 H), 7.52-7.57 (m, 1 H); LRMS 154.029 (m+1 ).

Tert-butyl 4-(tosyloxymethyl)piperidine-1-carboxylate (5): To a 20L jacketed reactor were charged dichloromethane (8 L), N-boc-4-piperdine methanol (0.982 Kg, 4.56 mol) and p-toluenesulfonyl chloride (0.970 Kg, 5.09 mol) and the resulting mixture was stirred at 20°C for 5 min. Triethylamine (0.94 Kg, 9.29 mol) was added to the reactor via an addition funnel and the resulting deep red solution was stirred at 25°C for 16 h. A solution of sodium carbonate (0.96 Kg, 9.06 mol) in water (7.04 L) was charged to the reaction mixture and stirred for 1 h at 20°C. The phases were split and the organic layer washed with brine (6 L) and concentrated at 40°C to a low stir volume. Dimethylacetamide (2 L) was charged to the reactor and concentration continued under full vacuum at 40°C for 1 h. The solution of tert-butyl 4-(tosyloxymethyl)piperidine-l -carboxylate (5) in dimethyl acetamide was held for further processing. Yield was assumed to be 100% with approximately

90% potency. A sample was pulled and concentrated to dryness for purity analysis. 1 H NMR (400 MHz, CDCI3) δ ppm 1 .02-1 .12 (m, 2H), 1.14 (s, 9H), 1 .59-1.64 (m, 2H), 1.75-1.87 (m, 1 H), 2.43 (s, 3H), 2.55-2.75 (m, 2H), 3.83 (d, J=6.7, 2H), 3.95-4.20 (b, 2H), 7.33 (d, 8.6, 2H), 7.76 (d, 8.2, 2H); 13C NMR (400 MHz, CDCI3) δ ppm 21 .64, 28.15, 28.39, 35.74, 73.97, 79.50, 126.99, 127.84, 129.86, 132.84, 144.84, 154.63; LRMS 739.329 (2m+1 ).

Tert-butyl 4-((4-fluorobenzo[d]isoxazol-3-yloxy)methyl)piperidine-1-carboxylate (6): To a 20L jacketed reactor were charged dimethylacetamide (4.28 L), tert-butyl 4-(tosyloxymethyl)piperidine-1 -carboxylate (5) (1.68 Kg, 4.56 mol), 4-fluorobenzo[d]isoxazol-3-ol (4) (540 g, 3.51 mol), and potassium carbonate (960 g, 6.98 mol) resulting in a thick beige slurry. The reaction mixture was heated to 50°C and stirred for 20 h and then cooled to 20°C, followed by the addition of water (7.5 L) and ethyl acetate (5.37 L). After mixing for 15 min, the phases were settled and split. The organic layer was washed with water (5.37 L), sending the aqueous wash to waste. The organic mixture was distilled under vacuum with a maximum jacket temperature of 40°C until approximately 5 L remained in the reactor. Methanol (2.68 L) was added and the resulting solution concentrated under vacuum to about 3 L of a yellow oil. Methanol (2.68 L) was charged to the reactor and the resulting solution was stirred at 25°C for 15 min. Water (0.54 L) was added over 15 min resulting in a white slurry. The mixture was cooled to 15°C, stirred for 1 h and then filtered. The filter cake was washed with a solution of water (0.54 L) in methanol (2.14 L), then air dried for 30 min, transferred to a vacuum oven and dried at 40°C for 12 h. The desired product, (6) (746 g, 2.13 mol), was isolated in 61 % yield. 1 H NMR (400 MHz, CDCI3) δ ppm 1.23-1 .37 (m, 2H), 1 .45 (s, 9H), 1 .78-1 .88 (m, 2H), 2.04-2.17 (m, 1 H), 2.67-2.83 (m, 2H), 4.02-4.26 (m, 2H), 4.28 (d, 6.6, 2H), 6.89 (dd, J=8.6, 7.5, 1 H), 7.21 (d, J=9, 1 H), (td, 8.6, 4.9); LRMS 351.171 (m+1 ).

(R)-Tert-butyl 4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidine-1-carboxylate (8): To a 20 L glass reactor with the jacket set to 20°C were charged (R)-tetrahydrofuran-3-ol (7) (297 g, 3.37 mol) and dimethylacetamide (5.1 L). 2.0 M sodium bis(trimethylsilyl)amide in THF (1.37 L, 2.74 mol) was slowly added via an addition funnel while maintaining a pot temperature less than 30°C. The resulting orange/red solution was stirred at 25°C for 30 min. Then, tert-butyl 4-((4-fluorobenzo[d]isoxazol-3-yloxy)methyl)piperidine-1 -carboxylate (6) (640.15 g, 1.83 mol) was charged and the reaction mixture was stirred at 25°C for 16 h. The reaction mixture was cooled to 20°C and water (6.4 L) was slowly added over 45 min maintaining a pot temperature of less than 35°C. Ethyl acetate (6 L) was added and the biphasic mixture was stirred for 15 min and then separated. The aqueous layer was back extracted with additional ethyl acetate (4 L). The combined organics were then washed with water (5 L) and a 20% brine solution (5 L). The organic mixture was concentrated under vacuum with the jacket temperature set to 40°C to approximately 3 L and held for further processing. Quantitative yield of the desired product, (8) (0.76 Kg, 1 .82 mol), in ethyl acetate was assumed. A sample was pulled and concentrated to dryness for purity analysis. 1 H NMR (400 MHz, CDCI3) δ ppm 1 .25-1.38 (m, 2H), 1 .44 (s, 9H), 1.76-1 .84 (m, 2H), 1 .89-1.97 (b, 1 H), 1 .99-2.12 (m, 1 H), 2.14-2.28 (m, 2H), 2.63-2.84 (m, 2H), 3.90-4.21 (m, 6H), 4.24 (d, J=6.3, 2H), 5.00-5.05 (m, 1 H), 6.48 (d, J=8.2, 1 H), 6.98 (d, J=8.6, 1 H), 7.37 (t, J=8.2, 1 H); LRMS 419.216 (m+1 ).

(R)-3-(Piperidin-4-ylmethoxy)-4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazole 4-methylbenzenesulfonate (9): To a 20L jacketed reactor charged ethyl acetate (6.1 L), (R)-tert-butyl 4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidine-1 -carboxylate (8) (0.76 kg, 1 .82 mol) and p-toluenesulfonic acid monohydrate (0.413 kg, 2.17 mol) and stirred at 20°C for 30 min. The reactor jacket was heated from 20 to 65°C over

1 h and then held at 65°C for 16 h. The reactor was cooled to 15°C over 1 h and granulated for 2 h. The resulting slurry was filtered, the cake was washed with EtOAc (3 L) and then air dried on the filter for 30 min. The cake was transferred to a vacuum oven and dried at 40°C for 12 h. The desired product, (9) (854 g, 1.74 mol), was isolated in 96% yield (two steps). 1 H NMR (400

MHz, CD3OD) δ ppm 1.54-1 .67 (m, 2H), 2.04-2.18 (m, 3H), 2.19-2.36 (m, 2H), 2.33 (s, 3H), 3.01 -3.12 (m, 2H), 3.41-3.50 (m, 2H), 3.86-4.01 (m, 4H), 4.26 (d, J=6.3, 2H), 4.90 (s, 2H), 5.14-5.19 (m, 1 H), 6.72 (d, J=8.2, 1 H), 7.02 (d, J=8.6, 1 H), 7.21 (d, J=7.8, 2H), 7.48 (t, J=8.6, 1 H), 7.70 (d, J=8.2, 2H); LRMS 319.165 (m+1 ).

(R)-4-((4-((4-(Tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (11): To a

20L jacketed reactor were charged water (7.5 L) and sodium carbonate (0.98 kg); the mixture was stirred at 20°C until all solids had dissolved. Then (R)-3-(piperidin-4-ylmethoxy)-4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazole 4-methylbenzenesulfonate (9) (750 g, 1 .53 mol) and ethyl acetate (6.0 L) were added to the reactor and stirred at 20°C for 30 min. The phases were split and the lower aqueous layer was back extracted twice with ethyl acetate (6.0 L and then 3.75 L). The organic layers were combined in the 20L reactor and washed twice with brine (3.0 L). The ethyl acetate solution was concentrated to under vacuum at 45°C to a low stir volume. Isopropyl alcohol (3.75 L) was added and concentration continued until 2 L remained in the reactor.

Additional isopropyl alcohol (2.75 L) was added and the mixture cooled to 25°C. To the reactor was charged 1 ,6-dioxaspiro[2.5]octane (10) (260 g, 2.29 mol) and the resulting solution heated to 50°C and stirred for 16 h. The reaction mixture was cooled to 30°C and water (15 L) was added over 60 min. Product crystallized from solution and the resulting slurry was cooled to 15°C over 1 h and then granulated for 4 h. The product was filtered and washed with water (3.75 L). The cake was blown dry with nitrogen for 30 min and then transferred to a vacuum oven and dried at 40°C for 12 h. The desired product, (11 ) (588 g, 1 .36 mol), was isolated in 89% yield.

1 H NMR (400 MHz, CDCI3) δ ppm 1 .41-1 .63 (m, 6H), 1.71 -1.81 (m, 2H), 1.81 -1.94 (m, 1 H), 2.17-2.26 (m, 2H), 2.33 (s, 2H), 2.4 (td, J=1 1.7, 2.3, 2H), 2.92 (d, J=1 1 .8, 2H), 3.46 (s, 1 H), 3.71-3.84 (m, 4H), 3.91 -4.10 (m, 4H), 4.24 (d, J=5.9, 2H), 5.03-5.08 (m, 1 H), 6.50 (d, J=8.2, 1 H), 7.00 (d, J=8.2, 1 H), 7.38 (t, J=8.2, 1 H);

13C NMR (400 MHz, CDCI3) δ ppm 29.1 1 , 33.10, 35.20, 36.92, 36.96, 56.15, 63.93, 67.14, 67.46, 68.27, 72.94, 74.06, 78.37, 103.17, 105.15, 131.71 , 152.71 , 166.02, 166.28;

LRMS 433.232 (m+1 ).

Example 2: Synthesis of iR)-4-ii4-i(4-itetrahvdrofuran-3-yloxy)benzord1isoxazol-3-yloxy)methyl)piperidin-1 -yl)methyl)tetrahvdro- 2H-pyran-4-ol

5-Hydroxy-2,2-dimethyl-benzo[1,3]dioxin-4-one: Thionyl chloride (83.8 g, 0.71 mol) was slowly added to a solution of 2,6-dihydroxy-benzoic acid (77 g, 0.5 mol), acetone (37.7 g, 0.65 mol) and DMAP (3.1 g, 0.025 mol) in dimethoxyethane (375 mL). The mixture was stirred at RT for 7 h. The residue obtained after concentration under reduced pressure was dissolved in ethyl

acetate and washed with water and aqueous saturated sodium bicarbonate solution. The organic layer was dried (Na2S04) and concentrated to afford 79 g desired product as a red solid (81 % yield). 1 H NMR (400 MHz, CDCI3) δ ppm 1 .68 (s, 6H), 6.37 (dd, J=8, 0.8, 11-1) 6.56 (dd, J=8, 0.8, 1 H), 7.34 (t, J=8, 1 H), 10.27( brs, 1 H).

2,2-Dimethyl-5-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[1,3]dioxin-4-one:

Diethyl azodicarboxylate (130.5 g, 0.75 mol) was added in a dropwise fashion to a mixture of 5-hydroxy-2,2-dimethyl-benzo[1 ,3]dioxin-4-one (100 g, 0.51 mol), triphenylphosphine (196.5 g, 0.75 mol), and (S)-tetrahydro-furan-3-ol (44 g, 0.5 mol) in 600 ml. of anhydrous THF. The resulting mixture was stirred at RT for 18 h. The solvent was removed under reduced pressure and the crude material was purified on a silica gel flash column, eluting with petroleum ether/ ethyl acetate (15:1 -> 3:1 ). 86 g (65% yield) of product was isolated as a colorless oil. 1 H NMR (400 MHz, CDCI3) δ ppm 1.67 (s, 6H), 2.30 (m, 2H), 4.2 (m, 4H) 4.97 (m, 1 H), 6.49 (d, J=8.4, 1 H) 6.51 (d, J=8.4, 1 H), 7.39 (t,

J=8.4, 1 H).

2-Hydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzoic acid methyl ester: Potassium carbonate (134.8 g, 0.98 mol) was added to a solution of 2,2-dimethyl-5-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[1 ,3]dioxin-4-one (86 g, 0.33 mol) in 1 L methanol. The mixture was stirred at RT for 2 h, then concentrated in vacuo. The residue was dissolved in ethyl acetate and washed with aqueous ammonium chloride solution. The organic layer was dried (Na2S04) and concentrated to afford 72 g of the product as a yellow solid (92% yield). 1 H NMR (400 MHz, CDCI3) δ ppm 2.20 (m, 2H), 3.99 (s, 3H), 4.80(m, 4H). 4.94 (m, 1 H), 6.31 (dd, J=8.4, 0.8, 1 H), 6.59 (dd, J=8.4, 0.8, 1 H), 7.30 (t, J=8.4, 1 H).

2,N-Dihydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzamide: Potassium carbonate (121 g. 0.867mmol) was added portionwise to a solution of hydroxylamine sulfate (120 g, 0.732 mol) in 360 ml. of water at 0°C. After stirring for 30 min, sodium sulfite (3.74 g, 0.029 mol) and a solution of 2-hydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzoic acid methyl ester (35 g, 0.146 mol) in 360 ml. of methanol were added and the mixture was stirred at 50°C for 30 h. Methanol was removed from the cooled reaction mixture under reduced pressure and the resulting aqueous layer was acidified with 2N HCI. The aqueous layer was extracted with ethyl acetate and the organic layer was dried (Na2S04) and concentrated to afford 25 g (76% yield ) of the product as a yellow solid. 1 H NMR (400 MHz, CDCI3) δ ppm 2.00 (m, 1 H), 2.15 (m, 1 H), 3.80 (m, 4H), 5.05 (m, 1 H), 6.48 (d, J=8, 1 H), 6.49 (d, J=8, 1 H), 7.19 (t, J=8, 1 H), 10.41 (brs, 1 H), 1 1.49 (brs, 1 H); LRMS m/z 239 (m+1 ).

4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-ol: A solution of 2, N-dihydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzamide (25 g, 0.105 mol) in 250 ml. of THF was heated to 50°C. Carbonyl diimidazole was added portionwise and the resulting mixture was stirred at 50°C for 14 h. After cooling to RT, 100 ml. of 2N HCI was added and the aqueous layer was extracted with ethyl acetate. The combined organic layers were then extracted three times with 10% aqueous potassium carbonate. The potassium carbonate aqueous extracts were washed with ethyl acetate and then acidified to pH 2 – 3 with 2N HCI. The acidified aqueous layer was extracted with ethyl acetate. The ethyl acetate extracts were washed with brine, dried (Na2S04) and concentrated to afford 20 g of product as a yellow solid (43% yield). 1 H NMR (400 MHz, CDCI3) δ ppm 2.20 (m, 2H), 3.89 (m, 1 H), 4.01 (m, 3H), 5.05 (m, 1 H), 6.48 (d, J=7.6, 1 H). 6.92 (d, J=7.6, 1 H), 7.37 (t, J=7.6, 1 H); LRMS m/z 222 (m+1 ).

4-{4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidine-1-carboxylic acid tert-butyl ester: Diethyl azodicarboxylate (15.6 g, 0.09 mol) was added to a mixture of 4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-ol (10 g, 0.045 mol), 4-hydroxymethyl-piperidine-1 -carboxylic acid tert-butyl ester (1 1.6 g, 0.054 mol) and triphenylphosphine (23.5 g, 0.09 mol) in 300 mL THF. After the addition was complete the mixture was heated at reflux for 18 h. After concentration in vacuo, the crude product was purified on a silica gel flash column, eluting with petroleum ether/ ethyl acetate (15:1 -» 5:1 ) to afford 22 g of the product as an oil (51 % yield). 1 H NMR (400 MHz, CDCI3) δ ppm 1.25 (m, 2H), 1.39 (s, 9H), 1.76 (m, 2H), 1.99 (m, 1 H). 2.15 (m, 2H), 2.70 (bt, J=1 1.6, 2H), 3.95 (m, 4H). 4.13 (m, 2H). 4.34 (d J=6.4, 2H), 4.98 (m, 1 H), 6.43 (d, J=8, 1 H), 6.93 (d, J=8, 1 H), 7.31 (t, J=8, 1 H).

3-(Piperidin-4-ylmethoxy)-4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazole: A 0°C solution of 4-{4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidine-1 -carboxylic acid tert-butyl ester in 500 mL ether was treated with a saturated solution of HCI (g) in 200 mL ether. After addition was complete, the mixture was warmed to RT and stirred for 16 h. The reaction mixture was filtered. The white solid was washed with ethyl acetate followed by ether and dried to yield 15 g (81 % yield) of the desired product as a white solid. 1 H NMR (400 MHz, CD3OD) 5 ppm 1 .51 – 1.69 (m, 2 H) 2.04 – 2.19 (m, 3 H) 2.22 – 2.37 (m, 2 H) 2.99 – 3.14 (m, 2 H) 3.40 – 3.51 (m, 2 H) 3.85 – 4.02 (m, 4 H) 4.25 – 4.31 (m, 2 H) 5.17 (td, J= >1^ , 1 .56 Hz, 1 H) 6.72 (d, J=8.00 Hz, 1 H) 7.01 (d, J=8.59 Hz, 1 H) 7.47 (t, J=8.20 Hz, 1 H); LRMS m/z 319 (m+1 ).

4-(4-{4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidin-1-ylmethyl)-tetrahydro-pyran-4-ol: 1 ,6-Dioxa-spiro[2.5]octane (Focus Synthesis; 9.7 g, 0.084 mol) and triethylamine (8.6 g, 0.084 mol) were added to a solution of 3-(piperidin-4-ylmethoxy)-4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazole (15 g, 0.042 mol) in 200 mL methanol. The resulting solution was heated at reflux for 18 h. The cooled mixture was concentrated and ethyl acetate and water were added to the residue. The layers were separated and the organic extracts were washed with brine, dried (Na2S04) and concentrated to provide 17 g crude product as a yellow oil. The crude material was purified by prep HPLC to afford 10 g of the desired product as a white solid. (50% yield).

1 H NMR (400 MHz, CDCI3) δ ppm 1.41 -1.63 (m, 6H), 1.71-1.81 (m, 2H), 1 .81 -1 .94 (m, 1 H), 2.17-2.26 (m, 2H), 2.33 (s, 2H), 2.4 (td, J=1 1 .7, 2.3, 2H), 2.92 (d, J=1 1.8, 2H), 3.46 (s, 1 H), 3.71-3.84 (m, 4H), 3.91-4.10 (m, 4H), 4.24 (d, J=5.9, 2H), 5.03-5.08 (m, 1 H), 6.50 (d, J=8.2, 1 H), 7.00 (d, J=8.2, 1 H), 7.38 (t, J=8.2, 1 H);

13C NMR (101 MHz, CDCI3) δ ppm 29.1 1 , 33.10, 35.20, 36.92, 36.96, 56.15, 63.93, 67.14, 67.46, 68.27, 72.94, 74.06, 78.37, 103.17, 105.15, 131.71 , 152.71 , 166.02, 166.28.

PAPER

Two Routes to 4-Fluorobenzisoxazol-3-one in the Synthesis of a 5-HT4Partial Agonist

Daniel W. Widlicka*†, John C. Murray†, Karen J. Coffman†, Chunguang Xiao‡, Michael A. Brodney†, Joseph P. Rainville†, and Brian Samas†

† Groton Laboratories, Worldwide Research & Development, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340,United States

‡ Porton Fine Chemical, 1 Fine Chemical Zone, Chongqing Chemical Industrial Park, Changshou, Chongqing 401221China

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00389

Publication Date (Web): February 2, 2016

Copyright © 2016 American Chemical Society

*E-mail: daniel.w.widlicka@pfizer.com.

http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00389

A potent 5-HT4 partial agonist, 1 (PF-04995274), targeted for the treatment of Alzheimer’s disease and cognitive impairment, has been prepared on a multi-kilogram scale. The initial synthetic route, that proceeded through a 4-substituted 3-hydroxybenzisoxazole core, gave an undesired benzoxazolinone through a Lossen-type rearrangement. Route scouting led to two new robust routes to the desired 4-substituted core. Process development led to the efficient assembly of the API on a pilot plant scale under process-friendly conditions with enhanced throughput. In addition, crystallization of a hemicitrate salt of the API with pharmaceutically beneficial properties was developed to enable progression of clinical studies.

REFERNCES

Noguchi, H.; Waizumi, N. Preparation of benzisoxazole derivatives for treatment of 5-HT4 mediated disorders. PCT Int. Appl. WO/2011/101774 A1, 20110825

////////PF-04995274, PF 04995274, PFIZER, Alzheimer’s type dementia, PHASE 1

c1cc2c(c(c1)O[C@@H]3CCOC3)c(no2)OCC4CCN(CC4)CC5(CCOCC5)O