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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK LIFE SCIENCES LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 30 PLUS year tenure till date June 2021, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 90 Lakh plus views on dozen plus blogs, 233 countries, 7 continents, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 33 lakh plus views on New Drug Approvals Blog in 233 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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AUR 101


AUR 101

AUR101-201

ANTIINNFLAMATORY

AUR-101, a ROR gamma inverse agonist for autoimmune disorders like psoriasis

AUR-101 is an ROR-gammaT inverse agonist in phase II clinical development at Aurigene for the treatment of patients with moderate-to-severe chronic plaque-type psoriasis.

  • DrugsAUR 101 (Primary)
  • IndicationsPlaque psoriasis
  • FocusAdverse reactions; First in man
  • AcronymsINDUS
  • SponsorsAurigene Discovery Technologies
  • OriginatorAurigene Discovery Technologies
  • ClassAntipsoriatics; Small molecules
  • Mechanism of ActionNuclear receptor subfamily 1 group F member 3 inverse agonists
  • Phase IIPsoriasis
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AURIGENE ANNOUNCES FIRST PATIENT DOSED WITH AUR101 IN PHASE II STUDY IN PATIENTS WITH MODERATE TO SEVERE PSORIASIS

https://www.aurigene.com/aurigene-announces-first-patient-dosed-with-aur101-in-phase-ii-study-in-patients-with-moderate-to-severe-psoriasis/

PRESS RELEASE

Aurigene Announces First Patient Dosed with AUR101 in Phase II Study in Patients with Moderate to Severe Psoriasis

Bangalore, February 17, 2020 — Aurigene, a development stage biotechnology company, today announced dose administration for the first patient in INDUS-2, a Phase II double blind placebo-controlled three-arm study of AUR101 in patients with moderate to severe psoriasis. AUR101 is an oral small molecule inverse agonist of RORγ and has shown desirable pharmacodynamic modulation of IL-17 and acceptable safety in a completed Phase I human study conducted in Australia.

“The initiation of this Phase II study under a US FDA IND represents a significant milestone for Aurigene, as it marks the first program which Aurigene has led from the bench side to the clinic all by itself,” said Murali Ramachandra, PhD, Chief Executive Officer of Aurigene. “We look forward to producing important clinical data by the end of 2020 to guide our future development plans and demonstrating Aurigene’s unique expertise in conducting Proof-of-Concept studies in a quality and fast-paced manner.”

About AUR101-201 and the Phase II Study of AUR101 in Patients with Moderate to Severe Psoriasis

The purpose of the Phase II multi-center, blinded, placebo-controlled, three-arm study is to evaluate the clinical activity of AUR101 in patients with moderate to severe psoriasis. In two of the arms, AUR101 will be administered twice daily, at 400 mg PO BID and 600 mg PO BID, for 12 weeks. Patients in the third arm will receive matched blinded placebo in a double dummy fashion. The trial is listed at clinicaltrials.gov with identifier NCT04207801.

About Aurigene

Aurigene is a development stage biotech company engaged in discovery and clinical development of novel and best-in-class therapies to treat cancer and inflammatory diseases and a wholly owned subsidiary of Dr. Reddy’s Laboratories Ltd. (BSE: 500124, NSE: DRREDDY,NYSE: RDY). Aurigene is focused on precision- oncology, oral immune checkpoint inhibitors, and the Th-17 pathway. Aurigene currently has several programs from its pipeline in clinical development. Aurigene has also submitted an IND to DCGI, India for a Phase IIb/III trial of CA-170, a dual inhibitor of PD-L1 and VISTA, in non-squamous NSCLC. Additionally, Aurigene has multiple compounds at different stages of pre-clinical development. Aurigene has partnered with many large and mid-pharma companies in the United States and Europe and has 15 programs currently in clinical development. For more information, please visit Aurigene’s website at https://www.aurigene.com/.

CLIP

Signalling of multiple interleukin (IL)-17 family cytokines via IL-17 receptor A drives psoriasis-related inflammatory pathways

https://onlinelibrary.wiley.com/doi/10.1111/bjd.20090

M.A.X. Tollenaere,J. Hebsgaard,D.A. Ewald,P. Lovato,S. Garcet,X. Li,S.D. Pilger,M.L. Tiirikainen,M. Bertelsen,J.G. Krueger,H. Norsgaard,First published: 01 April 2021 https://doi.org/10.1111/bjd.20090Citations: 2Funding sources LEO Pharma A/S funded this study.Conflicts of interest M.A.X.T., J.H., D.A.E., P.L., S.D.P., M.L.T., M.B. and H.N. are employees of LEO Pharma. J.G.K. received grants paid to his institution from Novartis, Pfizer, Amgen, Lilly, Boehringer, Innovaderm, BMS, Janssen, AbbVie, Paraxel, LEO Pharma, Vitae, Akros, Regeneron, Allergan, Novan, Biogen MA, Sienna, UCB, Celgene, Botanix, Incyte, Avillion and Exicure; and personal fees from Novartis, Pfizer, Amgen, Lilly, Boehringer, Biogen Idec, AbbVie, LEO Pharma, Escalier, Valeant, Aurigene, Allergan, Asana, UCB, Sienna, Celgene, Nimbus, Menlo, Aristea, Sanofi, Sun Pharma, Almirall, Arena and BMS.Data Availability Statement The gene array dataset described in this publication has been deposited in NCBI’s Gene Expression Omnibus and is accessible through GEO Series accession number GSE158448 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158448).

CLOP

https://www.drugdiscoverychemistry.com/Anti-Inflammatories/16

10:35 Small Molecule Inhibitors of RORgamma and IRAK4 for the Treatment of Autoimmune Disorders

Susanta_SamajdarSusanta Samajdar, Ph.D., Director, Medicinal Chemistry, Aurigene Discovery Technologies Limited

Although biologics such as anti-TNFα antibody are fairly successful in the treatment of autoimmune disorders, there is significant unmet need due to heterogeneity in diseases and lack of response to established therapies in some patients. While biologics typically target one cytokine signaling pathway, small molecule therapeutics directed towards intracellular target(s) can interfere in the signaling from multiple cytokines potentially leading to improved response. Development of small molecule oral inhibitors of IRAK4 and RORgamma to target TLR/IL-R and Th17 pathway respectively will be discussed.

PATENT

2448/CHE/2015 15.05.2015 IN

PATENT

PATENT

This application claims the benefit of Indian provisional application number 5641/CHE/2013 filed on 06th December 2013 which hereby incorporated by reference.

PATENT

  • KOTRABASAIAH UJJINAMATADA, Ravi
  • PANDIT, Chetan

 2049005-13-0

2-​Quinolinecarboxamide​, 6-​(2,​6-​dimethyl-​4-​pyrimidinyl)​-​N-​[[4-​(ethylsulfonyl)​phenyl]​methyl]​-​5,​6,​7,​8-​tetrahydro-​6-​methyl-​5-​oxo-​, (6S)​-

Molecular Weight492.59, C26 H28 N4 O4 S

EXAMPLE

PATENT

CLIP

https://www.sciencedirect.com/science/article/abs/pii/S0223523419301011

2013239366 CA 170

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///////////////////////AUR 101, AURIGENE, ROR, IL-17, PHASE 2, CDSCO, Ravi Ujjinamatada, KOTRABASAIAH UJJINAMATADA Ravi, PANDIT Chetan, AUR101-201, plaque-type psoriasis

IMG_3542.jpg
Ravi Ujjinamatada

Ravi Ujjinamatada

VX 148


img

VX 148

297730-05-3

Name: VX-148
CAS#: 297730-05-3
Chemical Formula: C23H25N5O4
Exact Mass: 435.19065
Molecular Weight: 435.48
Elemental Analysis: C, 63.44; H, 5.79; N, 16.08; O, 14.70

Molecular Weight435.48
FormulaC23H25N5O4
CAS No.297730-05-3 (VX 148);
Chemical NameCarbamic acid, N-[(1S)-1-[3-[[[(4-cyano-3-methoxyphenyl)amino]carbonyl]amino]phenyl]ethyl]-, (1R)-1-(cyanomethyl)propyl ester
  • OriginatorVertex Pharmaceuticals
  • ClassAntipsoriatics
  • Mechanism of ActionInosine monophosphate dehydrogenase inhibitors
  • DiscontinuedPsoriasis; Transplant rejection; Viral infections
  • 13 Nov 2003Interim data from a media release have been added to the adverse events and Skin Disorders therapeutic trials sections
  • 23 May 2003Vertex Pharmaceuticals has completed enrolment in a phase IIa trial for Psoriasis in Iceland
  • 24 Dec 2002Phase-II clinical trials in Psoriasis in Iceland (unspecified route)

VX-148 is a second-generation, orally administered inhibitor of inosine monophosphate dehydrogenase (IMPDH). The IMPDH enzyme plays a key role in regulating immune response and proliferation of specific cell types, including lymphocytes. VX-148 is a developed for the treatment of autoimmune diseases.

Investigated for use/treatment in autoimmune diseases, psoriasis and psoriatic disorders, and viral infection.

VX-148 is a novel, uncompetitive IMPDH inhibitor with a K(i) value of 6 nM against IMPDH type II enzyme. VX-148 is slightly more potent than mycophenolic acid and VX-497 in inhibiting the proliferation of mitogen-stimulated primary human lymphocytes (IC(50) value of ~80 nM). The inhibitory activity of VX-148 is alleviated in the presence of exogenous guanosine. VX-148 does not inhibit proliferation of nonlymphoid cell types such as fibroblasts, indicating selectivity for inhibition of IMPDH activity. VX-148 is orally bioavailable in rats and mice; oral administration of VX-148 inhibits primary antibody response in mice in a dose-dependent manner with an ED(50) value of 38 mg/kg b.i.d. VX-148 significantly prolongs skin graft survival at 100 mg/kg b.i.d. in mice.

SYN

WO 0056331

The intermediate carbamate (V) has been obtained as follows. The reaction of 4-bromo-3-methoxynitrobenzene (I) with CuCN in NMP at 150 C gives 2-methoxy-4-nitrobenzonitrile (II), which is reduced with H2 over Pd/C in ethyl acetate to yield 4-amino-2-methoxybenzonitrile (III). Finally, this compound is condensed with phenyl carbamate (IV) by means of NaHCO3 in ethyl acetate to afford the desired carbamate intermediate (V).

SYN

The reduction of 3-nitroacetophenone (VI) by means of NaBH4 in ethanol gives 1-(3-nitrophenyl)ethanol (VII), which is treated with DPPA and DBU in hot toluene to yield the azido derivative (VIII). The reduction of (VIII) with PPh3 in THF/water affords 1-(3-nitrophenyl)ethylamine (IX) as a racemic mixture that is submitted to optical resolution with L-(+)-tartaric acid to provide the desired (S)-isomer (X). The reduction of the nitro group of (X) by means of H2 over Pd/C in methanol gives 1(S)-(3-aminophenyl)ethylamine (XI), which is condensed with 2(R)-hydroxypentanenitrile (XII) and CDI to yield the carbamate (XIII). Finally, this compound is condensed with intermediate carbamate (V) by means of TEA in hot ethyl acetate to afford the target urea.

  1. Jain J, Almquist SJ, Heiser AD, Shlyakhter D, Leon E, Memmott C, Moody CS, Nimmesgern E, Decker C: Characterization of pharmacological efficacy of VX-148, a new, potent immunosuppressive inosine 5′-monophosphate dehydrogenase inhibitor. J Pharmacol Exp Ther. 2002 Sep;302(3):1272-7. [Article]

////////////VX 148, phase 2

O=C(O[C@H](CC)CC#N)N[C@H](C1=CC=CC(NC(NC2=CC=C(C#N)C(OC)=C2)=O)=C1)C

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VX- ? (3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2- oxo-pyrrolidin-3-yl ] propanamide)


Schembl22118316.png

VX- ?

CAS  2446817-72-5

HYDRATE 2446818-26-2

Acetic acid, 1-​methylethyl ester 2446818-27-3

C21 H20 F N3 O3, 381.4

1H-Indole-3-propanamide, 2-(4-fluorophenyl)-N-[(3S,4R)-4-hydroxy-2-oxo-3-pyrrolidinyl]-

3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2- oxo-pyrrolidin-3-yl ] propanamide

use in treating focal segmental glomerulosclerosis (FSGS) and/or non-diabetic kidney disease (NDKD).

front page image
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PATENT

WO/2021/158666

SOLID FORMS OF APOL1 INHIBITOR AND METHODS OF USING SAME

Compound I is disclosed as Compound 87 in U.S. Provisional Application No.62/780,667 filed on December 17, 2018, U.S. Application No. 16/717,099 filed onDecember 17, 2019, and PCT International Application No. PCT/US2019/066746 filed on December 17, 2019, the entire contents of each of which are incorporated herein by reference.

Compound I, which can be employed in the treatment of diseases mediated by APOLl, such as FSGS and NDKD

Example 1. Synthesis of Compound

Preparation of Compound I and Forms Thereof

Compound I Compound I /– PrOAc solvate Form A

n-pentanol/

n-heptane

Compound I

Form B

Step 1. Synthesis of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid (C101)

[00156] To a mixture of C104 (100.0 g, 1.0 equiv) and phenyl hydrazine hydrochloride (72.2 g, 1.05 eqiv) was charged AcOH (800 mL, 8 vol). The mixture was agitated and heated to 85 °C for 16 hours. The batch was cooled to 22 °C. A vacuum was applied and the batch distill at <70 °C to ~3 total volumes. The batch was cooled to 19- 25 °C. The reactor was charged with iPrOAc (800 mL, 8 vol) and then charged with water (800 mL, 8 vol). The internal temperature was adjusted to 20 – 25 °C and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and the phases allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. 1 N HC1 (500 mL, 5 vol) was charged to the reactor. The internal temperature was adjusted to 20 – 25 °C, and the

biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. The reactor was charged with 1 N HC1 (500 mL, 5 vol). The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. Water (500 mL, 5 vol) was charged to the reactor. The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. Water (500 mL, 5 vol) was charged to the reactor. The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. The organic phase was distilled under vacuum at <75 °C to 3 total volumes. The reactor was charged with toluene (1000 mL, 10 vol). The organic phase was distilled under vacuum at <75 °C to 5 total volumes. The reactor was charged with toluene (1000 mL, 10 vol). The organic phase was distilled under vacuum at <75 °C to 5 total volumes. The resulting slurry was heated to an internal temperature of 85 °C until complete dissolution of solids was achieved. The mixture was allowed to stir for 0.5 h at 85 °C and then cooled to an internal temperature of 19 – 25 °C over 5 h. The mixture was allowed to stir at 25 °C for no less than 2 h. The slurry was filtered. The filter cake was washed with toluene (1 x 2 vol (200 mL) and 1 x 1.5 vol (150 mL)). The solids were dried under vacuum with nitrogen bleed at 60 °C to afford product C101 (95.03 g, 70%).

Step 2. Synthesis of Compound I

[00157] A mixture of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid C101 (50 g, 1.0 equiv), S2 hydrochloride (28.3 g, 1.05 equiv), and CDMT (34.1 g, 1.1 equiv) was charged with 2-MeTHF (200 mL, 4 vol) and DMF (50 mL, 1 vol) and the mixture was agitated. The internal temperature adjusted to <13 °C. The reactor was charged with NMM (64.5 g, 3.5 equiv) over 1 h, while maintaining internal temperature <20 °C. The internal temperature was adjusted to 25 °C and the batch was stirred at that temperature for 14 h. The batch was cooled to 10 °C and charged with water (250 mL, 5 vol) while keeping the internal temperature <20 °C. The batch was then warmed to 20 – 25 °C. Stirring was stopped, and the phases allowed to separate for 10 min. The lower aqueous phase was removed. The aqueous layer was back extracted with 2-MeTHF (2 x 200 mL, 2 x 4 vol) at

20 – 25 °C. The combined organic phases were washed with 1 N HC1 (500 mL, 10 vol) at 20 – 25 °C by mixing for 10 min and settling for 10 min. The lower aqueous phase was removed. The organic phases were washed with 0.25 N HC1 (2 x 250 mL, 2 x 5 vol) at 20 – 25 °C by mixing for 10 min and settling for 10 min for each wash. Lower aqueous phases were removed after each wash. The organic phase was washed with water (250 mL, 5 vol) at 20 – 25 °C by mixing for 10 min and settling for 10 min. The reactor was charged with 20 wt % Nuchar RGC® and stirred for 4 h. The reaction mixture was filtered through a pad of celite®. The reactor and celite® pad were rinsed with 2-MeTHF. The combined organics were distilled under vacuum at <50 °C to 5 total volumes. The reactor was charged with iPrOAc (500 mL, 10 vol). The organic phase was distilled under vacuum at <50 °C to 5 total volumes. The mixture was charged with additional iPrOAc (400 mL, 8 vol) and distillation under vacuum was repeated. The mixture was charged with additional iPrOAc (250 mL, 5 vol), heated to an internal temperature of 75 °C and stirred for 5 h. The slurry was cooled to 25 °C, over 5 h and stirred for no less than 12 h. The slurry was filtered and the filter cake washed with iPrOAc (2 x 50 mL, 2 x 1 vol). The solids were dried under vacuum with nitrogen bleed at 55 – 60 °C to afford Compound I as an iPrOAc solvate (60.38 g including 9.9% w/w iPrOAc, 80.8% yield).

Recrystallization to Form A of Compound I

[00158] Compound I as an iPrOAc solvate (17.16 g after correction for iPrOAc content, 1.0 equiv) was charged to a reactor. A mixture of IP A (77 mL, 4.5 vol) and water (137 mL, 8 vol) were charged to the reactor. The slurry was heated to an internal temperature of 75 °C. The batch was cooled to an internal temperature of 25 °C over 10 h and then stirred at 25 °C for at least 12 h. The slurry was filtered. The filter cake was washed with 36/64 IP A/water (2 x 52 mL, 2 x 3 vol). The solids were dried under vacuum with nitrogen bleed at 60 °C to afford Compound I as a neat, crystalline form (Form A, 15.35 g, 89%).

[00159] The X-ray powder diffractogram of Compound I Form A (FIG. 50) was acquired at room temperature using a PANalytical Empyrean diffractometer equipped with PIXcel ID detector. The peaks are listed in Table A below.

Table A. XRPD of Form A of Compound I

|

I

PATENT

  • WO2020131807

Alternative Preparation I of Compound 87 (Indole preparation route C)

Step 1. Synthesis of 2-(4-fluorophenyl)-lH-indole (C98)

[00401] To a stirred suspension of indole (5 g, 42.7 mmol) and (4- fluorophenyl)boronic acid (8.96 g, 64.0 mmol) in AcOH (200 mL) was

added Pd(OAc)2.Trimer (1.44 g, 6.4 mmol) and the mixture stirred at room temperature for 16 h under 02-balloon pressure. Then the reaction mixture was filtered through a Celite® pad, washed with EtOAc (500 mL). The filtrates were washed with water, sat. NaHC03 solution, brine solution, then dried over Na2S04 and concentrated under reduced pressure. Purification by silica gel chromatography (Gradient: 0-10 % EtOAc in heptane) yielded the product afforded 2-(4-fluorophenyl)-lH-indole (5.5 g, 61 %). ‘H NMR (300 MHz, DMSO-de) 5 11.51 (s, 1H), 7.9 (t, J = 5.4 Hz, 2H), 7.52 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.30 (t, J = 8.7 Hz, 2H), 7.09 (t, J = 12 Hz, 1H), 6.99 (t, J = 7.5 Hz, 1H), 6.86 (s, 1H). LCMS m/z 212.4 [M+H]+.

Step 2. Synthesis of methyl (E)-3-[2-(4-fluorophenyl)-lH-indol-3-yl]prop-2-enoate (C99)

[00402] 2-(4-fluorophenyl)-lH-indole (1.0 g, 4.76 mmol) and methyl 3,3-dimethoxypropanoate (0.81 mL, 5.7 mmol) were suspended in dichloromethane (15 mL). Trifluoroacetic acid (2.00 mL, 26 mmol) was added rapidly via syringe, resulting in a clear brown solution. The reaction mixture was heated to 40 °C for three hours. The reaction was diluted with dichloromethane (15 mL) to give an amber solution which was washed with saturated aqueous NaHCCh (25 mL) to yield a bright yellow/light amber biphasic mixture. The phases were separated and the organic layer was washed with saturated NaHCCh (30 mL), then dried (MgSCh) and filtered. The mixture was concentrated under a nitrogen stream overnight. The crude product was obtained as a yellow powder. The product was dissolved in minimum 2-MeTHF and pentane added until the suspension became lightly cloudy. The suspension was allowed to stand overnight, and the precipitate was filtered off. The filter cake was washed with heptane (2 x 15 mL), and dried in vacuo at 40 °C to afford the product as a yellow powder. Methyl (E)-3-[2-(4-fluorophenyl)-lH-indol-3-yl]prop-2-enoate (1.30 g, 86 %). ¾ NMR (300 MHz, Chloroform -if) d 8.41 (s, 1H), 8.01 – 7.95 (m, 1H), 7.92 (d, J = 16.0 Hz,

1H), 7.58 – 7.50 (m, 2H), 7.46 – 7.41 (m, 1H), 7.33 – 7.27 (m, 2H), 7.22 (t, J = 8.6 Hz, 2H), 6.59 (d, J = 16.0 Hz, 1H), 3.79 (s, 3H). LCMS m/z 295.97 [M+H]+.

Step 3. Synthesis of methyl 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoate (CIOO)

[00403] To a solution of methyl (E)-3-[2-(4-fluorophenyl)-lH-indol-3-yl]prop-2-enoate (7 g, 0.02 mol) in EtOAc (350 mL) was added Palladium on carbon (4 g, 10 %w/w, 0.004 mol) and stirred at room temperature for 2 h under an atmosphere of H2 (bladder pressure). The reaction mixture was filtered through a pad of Celite® and washed with EtOAc (400 mL). The filtrates was concentrated to afford methyl 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoate (7.1 g, 100 %). 1H MR (300 MHz, DMSO-<fc) 5 11.2 (s, 1H), 7.65 (q, J = 5.4 Hz, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 9.0 Hz, 3H), 7.10 (t, J = 8.1 Hz, 1H), 7.02 (t, J = 7.8 Hz, 1H), 3.53 (s, 3H), 3.10 (t, J = 15.9 Hz, 2H), 2.63 (t, J = 15.9 Hz, 2H). LCMS m/z 298.21 [M+H]+. The product was used directly in the subsequent step without further purification.

Step 4. Synthesis of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid (C101)

[00404] To stirred solution of methyl 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoate (14.4 g, 0.05mol) in THF (300 mL), MeOH (300 mL) and H2O (250 mL) was cooled to -10°C. LiOH.H20 (10.1 g, 0.24 mol) was slowly added in a portion-wise manner. The reaction mixture was allowed to stir at room temperature for 16 h. The mixture was

evaporated and ice cold water (200 mL) was added, pH was adjusted to pH- 2 with 1M HC1 (400 mL, Cold solution). The mixture was stirred for 10 minutes, filtered and dried to afford 3-[2-(4-fhiorophenyl)-lH-indol-3-yl]propanoic acid (12.9 g, 94 %). ‘H NMR (400 MHz, DMSCMJ) 5 12.11 (s, 1H), 11.18 (s, 1H), 7.65 (q, J = 5.2 Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.36 (t, J = 8.8 Hz, 3H), 7.10 (t, J = 8 Hz, 1H), 7.01 (t, J = 8 Hz, 1H), 3.06 (t, J = 16.4 Hz, 2H), 2.55 (t, J = 16 Hz, 2H). LCMS m/z 284.21 [M+H]+.

Step 5. Synthesis of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2- oxo-pyrrolidin-3-yl ] propanamide (87)

[00405] A mixture of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid C101 (40 g, 120.0 mmol) and (3S,4R)-3-amino-4-hydroxy-pyrrolidin-2-one (Hydrochloride salt) S2 (23.8 g, 156.0 mmol) in DMF (270 mL) was stirred at room temperature for 5 minutes. CDMT (27.2 g, 154.9 mmol) and NMM (53 mL, 482.1 mmol) were added and the mixture was stirred at room temperature for 2 h. The mixture was poured into water (140 mL) and then stirred for 1 h at room temperature, then filtered and washing the solids with water (50 mL). The solids were dissolved in 1 : 1 IP A/water (-400 mL, until all solids dissolved) with heating (reflux) and stirring. The mixture was allowed to cool slowly to room temperature overnight. The mixture was cooled to 0 oC and stirred to break up crystals for filtration. The crystals were then filtered off, rinsed with cold 1 : 1 IP A/water to afford a tan solid (45 g). The solid was dissolved in IPA (200 mL) and heated to 80 °C to dissolve the solid. Activated charcoal (10 g) was added and the mixture was heated with stirring for 30 minutes. The mixture was filtered through Celite ® and solvent removed under reduced pressure. A mixture of 40:60 IP A/water (350 mL) was added to the solid and the mixture was heated until all solids dissolved. The mixture was cooled to room temperature over 5 h. Solids precipitated within the mixture. The mixture was then cooled to 0 °C and stirred for 1 h. The solids were filtered off and air dried on funnel for 1 h, then in a vacuum at 55 °C overnight to afford the product. 3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide (36.6 g, 79 %). ¾ NMR (300 MHz, Methanol-i¾) d 7.63 (ddt, J= 8.6, 5.1, 2.7 Hz, 3H), 7.35 (dt, J= 8.1, 1.0 Hz, 1H), 7.25 – 7.16 (m, 2H), 7.11 (ddd, J= 8.1, 7.0, 1.3 Hz, 1H), 7.03 (ddd, J = 8.0, 7.0, 1.2 Hz, 1H), 4.34 (td, J= 7.6, 6.8 Hz, 1H), 4.22 (d, J= 7.7 Hz, 1H), 3.55 (dd, J= 9.9, 7.5 Hz, 1H), 3.26 – 3.18 (m, 2H), 3.10 (dd, J= 9.9, 6.8 Hz, 1H), 2.69 – 2.59 (m, 2H). LCMS m/z 382.05 [M+H]+. The

product contained 0.23 % IPA by weight by NMR (1439 ppm IPA by residual solvent analysis). Purity is 99.5 % by (qNMR).

Alternative Preparation II of Compound 87 ( Indole Preparation route D)

Step 1. Synthesis of 5-(4-fluorophenyl)-5-oxo-pentanoic acid (Cl 04)

[00406] To a stirred suspension of AlCb(13.9 g, 0.10 mol) in dichloromethane (50 mL) was added a solution of tetrahydropyran-2,6-dione (5.93 g, 0.05

mol) in dichloromethane (100 mL) at 0 °C over a period of 15 minutes and stirred for 30 min. Then to the reaction mixture was added fluorobenzene (5 g, 0.05 mol) at 0 °C over a period of 15 min, gradually allowed to room temperature and stirred for 16 h. Then the reaction mixture was added to ice water (50 mL) under stirring. The resulting solid was filtered to afford a light yellow solid. The solid was diluted with 3 % NaOH solution (50 mL) and dichloromethane (50 mL). The aqueous layer was separated and acidified with IN HC1 at 0 °C. The mixture was then extracted with EtOAc (100 mL), dried over Na2SC>4, and concentrated under reduced pressure. The solid was then washed with pentane and dried to afford 5-(4-fluorophenyl)-5-oxo-pentanoic acid as an off white solid. (6 g, 53 %). ¾ NMR (300 MHz, DMSO-^) d 12.07 (s, 1H), 8.06 (d, J = 6 Hz, 1H), 8.02 (d, J = 5.4 Hz, 1H), 7.36 (t, J = 8.7 Hz, 2H), 3.06 (t, J = 12 Hz,

2H), 2.31 (t, J = 7.2 Hz, 2H), 1.86-1.78 (m, 2H). LCMS m/z 211.18 [M+H]+.

Step 2. Synthesis of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid (Cl 01) [00407] Phenylhydrazine (Hydrochloride salt) (375.7 g, 2.6 mol) was combined with the 5-(4-fluorophenyl)-5-oxo-pentanoic acid (507.7 g, 2.4 mol) in a 12 L three-necked round-bottomed flask equipped with an overhead stirrer, temperature probe, and reflux condenser. AcOH (5 L) was added. The stirring was initiated and ZnCk (605 g, 4.44 mol) was added. The white suspension rapidly thickened after a few minutes (due to formation of the hydrazine intermediate). Approx. 500 mL of extra AcOH was added to aid stirring. The reaction was then heated to 100 °C for three hours. The reaction was cooled to room temperature and poured into water (approx. 6 L). The mixture was extracted with EtOAc (approx 8 L). The extract was washed with water, dried

(MgS04), filtered, and evaporated in vacuo to afford a golden yellow solid. The solid was triturated with approx. 4 L of 10 % EtOAc/DCM and filtered. The filter cake was washed with 50 % dichloromethane/heptane (approx 1 L). The filter cake was dissolved in 40 % EtOAc/dichloromethane (approx. 2L) and filtered over a plug of silica gel. The plug was eluted with 40 % EtOAc/ dichloromethane until the product had been eluted (checked by TLC (25 % EtOAc/ dichloromethane)). The filtrate was evaporated in vacuo to afford 382.6 g of an off-white solid (Crop 1). All filtrates were combined and evaporated in vacuo. The remaining solid was dissolved in 10 %

EtOAc/dichloromethane (approx. 1 L) and chromatographed on a 3 kg silica gel cartridge on the ISCO Torrent (isocratic gradient of 10 % EtOAc/dichloromethane). Product fractions were combined and evaporated in vacuo to afford a yellow solid that was slurried with dichloromethane, cooled under a stream of nitrogen, and filtered. The filter cake was washed with 50 % dichloromethane/heptane and dried in vacuo to afford 244.2 g of product (Crop 2). Altogether, both crops afforded 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid (626.8 g, 93 %). ¾ NMR (300 MHz, DMSO-i/e) d 12.15 (s, 1H), 11.20 (s, 1H), 7.74 – 7.62 (m, 2H), 7.57 (d, J = 7.8 Hz, 1H), 7.47 – 7.28 (m, 3H), 7.11 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.02 (ddd, J = 7.9, 7.0, 1.1 Hz, 1H), 3.17 – 2.85 (m, 2H), 2.61 – 2.52 (m, 2H) ppm. 19F NMR (282 MHz, DMSO-i/e) d -114.53 ppm. LCMS m/z 284.15 [M+H]+.

Step 3. Synthesis of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2- oxo-pyrrolidin-3-yl ] propanamide (87)

[00408] A 3-L three neck RBF under nitrogen was equipped with a 150 mL addition funnel and thermocouple, then loaded with 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid (77.2 g, 228.6 mmol), (3S,4R)-3-amino-4-hydroxy-pyrrolidin-2-one

(Hydrochloride salt) (36.6 g, 239.9 mmol) and CDMT (44.2 g, 251.7 mmol). DMF (320 mL) was added and the orange slurry was cooled to -5 °C (acetone/brine/dry ice). NMM (88 mL, 800.4 mmol) was added via a funnel over 75 minutes to keep the internal temp <0 °C. The slurry was stirred at between -10 and 0 °C for 1 hour, then allowed to warm to ambient temperature progressively over 2 hours. Additional reagents were added (10 % of the initial quantities), and the mixture was stirred overnight at ambient temperature. Water (850 mL) was added over 60 minutes, maintaining the internal temperature at <25 °C (ice bath). This slow water addition allows for complete dissolution of any visible salt before precipitation of the product. The resulting thick slurry was stirred at ambient temperature overnight. The solid was recovered by filtration and washed with water (3 x 500 mL). The solid was dried under a stream of air at ambient temperature, then purified by crystallization.

Crystallization of 3- [2-( 4-fluorophenyl)-lH-indol-3-yl ]-N-[ ( 3S, 4R)-4-hydroxy-2-oxo- pyrrolidin-3-yl ] propanamide (87)

[00409] Under nitrogen atmosphere, a 2-L, 3 -neck flask equipped with addition funnel and thermocouple was charged with a light brown suspension of the crude 3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yljpropanamide (89.5 g) in IPA (225 mL, 2.5 vol). The slurry was heated to 50 °C and water (675 mL, 7.5 vol) was added until near-complete dissolution of solid was observed. The temperature was adjusted to 70 °C-to achieve full dissolution, yielding a clear amber solution. After 30 minutes, the heat source was removed and the mixture was cooled to ambient temperature over the weekend, stirring gently while maintaining the nitrogen atmosphere. The solid was recovered by filtration, washed with IPA:H20 = 1 :2 (2 x 300 mL, 2 x 3.3 vol) dried under a stream of air overnight to afford the product. 3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide (84.8 g, 92 %). ¾ NMR (300 MHz, DMSO-^) d 11.19 (s, 1H), 8.23 (d, J= 7.5 Hz, 1H), 7.77 (s, 1H), 7.72 – 7.63 (m, 2H), 7.60 (d, J= 7.8 Hz, 1H), 7.41 -7.31 (m, 3H), 7.12 (ddd, J= 8.1, 7.0, 1.2 Hz, 1H), 7.03 (ddd, J= 8.0, 7.0, 1.1 Hz, 1H), 5.49 (d, J= 5.0 Hz, 1H), 4.20 – 4.06 (m, 2H), 3.38 (s, 1H), 3.11 – 3.00 (m, 2H), 2.92 (dd, J= 9.4, 6.6 Hz, 1H). LCMS m/z 382.15 [M+H]+.

Crystallization of 3- [2-( 4-fluorophenyl)-lH-indol-3-yl J-N-[ ( 3S, 4R)-4-hydroxy-2-oxo- pyrrolidin-3-yl ] propanamide (87)

[00410] A 2-L, 3-neck flask equipped with addition funnel and thermocouple was charged with a light brown suspension of the crude 3-[2-(4-fluorophenyl)-lH-indol-3- yl]-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide in IPA (225 mL, 1 vol). The slurry was heated to 50 °C and water (675 mL, 3 vol) was added until near- complete dissolution of solid observed (mL). Temperature was increased to 70 °C under nitrogen (full dissolution, yielding a clear amber solution). After 30 minutes, the heat was removed and the mixture cooled to ambient temperature over the weekend, stirring gently under nitrogen atmosphere. The solid was recovered by filtration and washed with IPAiLLO = 1 :2 (2 x 300 mL).The solid was dried under a stream of air overnight to afford the product. 3-[2-(4-fluorophenyl)-lH-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxo- pyrrolidin-3-yl]propanamide (84.8 g, 92 %). ¾ NMR (300 MHz, DMSO-i/e) d 11.19 (s, 1H), 8.23 (d, J= 7.5 Hz, 1H), 7.77 (s, 1H), 7.72 – 7.63 (m, 2H), 7.60 (d, J= 7.8 Hz,

1H), 7.41 – 7.31 (m, 3H), 7.12 (ddd, J= 8.1, 7.0, 1.2 Hz, 1H), 7.03 (ddd, 7= 8.0, 7.0,

1.1 Hz, 1H), 5.49 (d, J= 5.0 Hz, 1H), 4.20 – 4.06 (m, 2H), 3.38 (s, 1H), 3.11 – 3.00 (m, 2H), 2.92 (dd, J= 9.4, 6.6 Hz, 1H). LCMS m/z 382.15 [M+H]+.

Large Scale Preparation of Compound 87

/- PrOAc solvate Form A

Step 1. Synthesis of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid (C101)

[00411] To a mixture of C104 (100.0 g, 1.0 equiv) and phenyl hydrazine hydrochloride (72.2 g, 1.05 eqiv) was charged AcOH (800 mL, 8 vol). The mixture was agitated and heated to 85 °C for 16 hours. The batch was cooled to 22 °C. A vacuum was applied and the batch distill at <70°C to ~3 total volumes. The batch was cooled to 19- 25 °C. The reactor was charged with iPrOAc (800 mL, 8 vol) and then charged with water (800 mL, 8 vol). The internal temperature was adjusted to 20 – 25 °C and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and the phases allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. 1 N HC1 (500 mL, 5 vol) was charged to the reactor. The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. The reactor was charged with 1 N HC1 (500 mL, 5 vol). The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h.

Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. Water (500 mL, 5 vol) was charged to the reactor.

The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. Water (500 mL, 5 vol) was charged to the reactor. The internal temperature was adjusted to 20 – 25 °C, and the biphasic mixture was stirred for no less than 0.5 h. Stirring was stopped and phases were allowed to separate for no less than 0.5 h. The lower aqueous layer was removed. The organic phase was distilled under vacuum at <75 °C to 3 total volumes. The reactor was charged with toluene (1000 mL, 10 vol). The organic phase was distilled under vacuum at <75 °C to 5 total volumes. The reactor was charged with toluene (1000 mL, 10 vol). The organic phase was distilled under vacuum at <75 °C to 5 total volumes. The resulting slurry was heated to an internal temperature of 85 °C until complete dissolution of solids was achieved. The mixture was allowed to stir for 0.5 h at 85 °C and then cooled to an internal temperature of 19 – 25 °C over 5 h. The mixture was allowed to stir at 25 °C for no less than 2 h. The slurry was filtered. The filter cake was washed with toluene (1 x 2 vol (200 mL) and 1 x 1.5 vol (150 mL)). The solids were dried under vacuum with nitrogen bleed at 60 °C to afford product C101 (95.03 g, 70%).

Purification of Compound 87 by Recrystallization to Form A

[00412] Compound 87 as an iPrOAc solvate (17.16 g after correction for iPrOAc content, 1.0 equiv) was charged to a reactor. A mixture of IP A (77 mL, 4.5 vol) and water (137 mL, 8 vol) were charged to the reactor. The slurry was heated to an internal temperature of 75 °C. The batch was cooled to an internal temperature of 25 °C over 10 h and then stirred at 25 °C for at least 12 h. The slurry was filtered. The filter cake was washed with 36/64 IP A/water (2 x 52 mL, 2 x 3 vol). The solids were dried under vacuum with nitrogen bleed at 60 °C to afford Compound 87 as a neat, crystalline form (Form A, 15.35 g, 89%).

Synthetic Procedure

[00413] A mixture of 3-[2-(4-fluorophenyl)-lH-indol-3-yl]propanoic acid C101 (50 g, 1.0 equiv), S2 hydrochloride (28.3 g, 1.05 equiv), and CDMT (34.1 g, 1.1 equiv) was charged with 2-MeTHF (200 mL, 4 vol) and DMF (50 mL, 1 vol) and the mixture was agitated. The internal temperature adjusted to <13 °C. The reactor was charged with NMM (64.5 g, 3.5 equiv) over 1 h, while maintaining internal temperature <20 °C. The internal temperature was adjusted to 25 °C and the batch was stirred at that temperature for 14 h. The batch was cooled to 10 °C and charged with water (250 mL, 5 vol) while keeping the internal temperature <20 °C. The batch was then warmed to 20 – 25 °C. Stirring was stopped, and the phases allowed to separate for 10 min. The lower aqueous phase was removed. The aqueous layer was back extracted with 2-MeTHF (2 x 200 mL, 2 x 4 vol) at 20 – 25 °C. The combined organic phases were washed with 1 N HC1 (500 mL, 10 vol) at 20 – 25 °C by mixing for 10 min and settling for 10 min. The lower aqueous phase was removed. The organic phases were washed with 0.25 N HC1 (2 x 250 mL, 2 x 5 vol) at 20 – 25 °C by mixing for 10 min and settling for 10 min for each wash. Lower aqueous phases were removed after each wash. The organic phase was washed with water (250 mL, 5 vol) at 20 – 25 °C by mixing for 10 min and settling for 10 min. The reactor was charged with 20 wt % Nuchar RGC® and stirred for 4 h. The reaction mixture was filtered through a pad of celite®. The reactor and celite® pad were rinsed with 2-MeTHF. The combined organics were distilled under vacuum at <50 °C to 5 total volumes. The reactor was charged with iPrOAc (500 mL, 10 vol). The organic phase was distilled under vacuum at <50 °C to 5 total volumes. The mixture was charged with additional iPrOAc (400 mL, 8 vol) and distillation under vacuum was repeated. The mixture was charged with additional iPrOAc (250 mL, 5 vol), heated to an internal

temperature of 75 °C and stirred for 5 h. The slurry was cooled to 25 °C, over 5 h and stirred for no less than 12 h. The slurry was filtered and the filter cake washed with iPrOAc (2 x 50 mL, 2 x 1 vol). The solids were dried under vacuum with nitrogen bleed at 55 – 60 °C to afford Compound 87 as an iPrOAc solvate (60.38 g including 9.9% w/w iPrOAc, 80.8% yield).

Form A of Compound 87

[00414] Compound 87 hydrate form was converted to the dehydrated, neat crystalline form (Form A) after drying.

Hydrate Form A of Compound 87

[00415] A mixture of IP A (4.5 vol) and water (8 vol) was added to compound 87

(iPrOAc solvate containing ~2.5 – 11 wt% iPrOAc, 1.0 equiv). The slurry was heated to an internal temperature of 75 °C and filtered hot. The filtrate was cooled to 25 °C for at least 12 h. The slurry was filtered. The filter cake was washed with 36/64 IP A/water (2 x 3 vol). The solids were dried under vacuum with nitrogen bleed at 55 – 60 °C. The product was isolated as Hydrate form.

IPAC Solvate of Compound 87:

[00416] The large scale synthesis described above provided an iPrOAc solvate containing ~2.5 – 11 wt% iPrOAc after drying.

Amorphous Form of Compound 87

[00417] ~lg of compound 87 was dissolved in 22mL of acetone. The solution was evaporated using a Genevac. The resulted solid was dried at 60C under vacuum overnight. The dried solid was amorphous form.

Publication Number TitlePriority Date Grant Date
WO-2020131807-A1Inhibitors of apol1 and methods of using same2018-12-17 
US-2020377479-A1Inhibitors of apol1 and methods of using same2018-12-17

///////////

O=C(N[C@@H]1C(=O)NC[C@H]1O)CCc1c2ccccc2[NH]c1c1ccc(F)cc1

SIMILAR

https://d4crq6wjnrm5a.cloudfront.net/drugs/720/5842720.png?Expires=1629119288&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9kNGNycTZ3am5ybTVhLmNsb3VkZnJvbnQubmV0L2RydWdzLzcyMC81ODQyNzIwLnBuZyIsIkNvbmRpdGlvbiI6eyJEYXRlTGVzc1RoYW4iOnsiQVdTOkVwb2NoVGltZSI6MTYyOTExOTI4OH19fV19&Signature=cF-TptDVLQjX2ZetNPD5u1xkA-2MNWfoDI-idPuhS-blf-hpPJxOxXvstTNlxr0CfZBAGZwTR0LgoB5iSQzJJyu2NJXiXipepG0~Svx6zY6NdmxVK37PO7nzv61f9zTO-vjTUW4g0oiXzENMdRkJsansf2XgskWiwa-9piD0gV02R9jO2E9mmjtLygU5JlbJsfui91rsPYVHkW7qJQLVliePDWNXO4ykZpeGwy0N2UXxfphEgm3WsBDE1TomCJDgMZBY37ewn3Bk83lH2DBBb~EhC80sRaJr4mEcOkbdVI3hWISDfz-14L-A2tY0JQ8JOdpth31dNVYZIQZcsI-qZA__&Key-Pair-Id=APKAJYXZOHSJHO6RX3UQ

predicted

VX 147

cas 2446816-88-0 predicted

O=C(N[C@@H]1C(=O)NC[C@H]1O)CCc1c2cc(F)cc(F)c2[NH]c1c1ccc(F)cc1

  • OriginatorVertex Pharmaceuticals
  • ClassSmall molecules; Urologics
  • Mechanism of ActionApolipoprotein L1 inhibitors
  • Orphan Drug StatusNo
  • New Molecular EntityYes

Highest Development Phases

  • Phase IIFocal segmental glomerulosclerosis
  • Phase IKidney disorders

Most Recent Events

  • 14 Apr 2020Phase-II clinical trials in Focal segmental glomerulosclerosis in USA (PO) (EudraCT2020-000185-42) (NCT04340362)
  • 31 Dec 2019Vertex Pharmaceuticals completes phase I clinical trial in Focal segmental glomerulosclerosis and Kidney disorders (In volunteers) in USA (PO)
  • 05 Aug 2019Vertex Pharmaceuticals plans a phase II proof-of-concept trial for focal segmental glomerulosclerosis in 2020
NCT Number  ICMJENCT04340362
Other Study ID Numbers  ICMJEVX19-147-101
2020-000185-42 ( EudraCT Number )

MIRDAMETINIB


img
2D chemical structure of 391210-10-9

MIRDAMETINIB

391210-10-9
Chemical Formula: C16H14F3IN2O4
Molecular Weight: 482.19

PD0325901; PD 0325901; PD-325901; mirdametinib

IUPAC/Chemical Name: (R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide

SpringWorks Therapeutics (a spin out of Pfizer ) is developing mirdametinib, a second-generation, non-ATP competitive, allosteric MEK1 and MEK2 inhibitor derived from CI-1040, for treating type 1 neurofibromatosis (NF1) and advanced solid tumors. In June 2021, a phase I/II trial was initiated in patients with low grade glioma.

  • OriginatorPfizer
  • DeveloperAstraZeneca; BeiGene; BIOENSIS; Pfizer; SpringWorks Therapeutics; St. Jude Childrens Research Hospital; University of Oxford
  • ClassAniline compounds; Anti-inflammatories; Antineoplastics; Benzamides; Immunotherapies; Small molecules
  • Mechanism of ActionMAP kinase kinase 1 inhibitors; MAP kinase kinase 2 inhibitors
  • Orphan Drug StatusYes – Neurofibromatosis 1
  • Phase IINeurofibromatosis 1
  • Phase I/IIGlioma
  • Phase ISolid tumours
  • PreclinicalChronic obstructive pulmonary disease
  • No development reportedCervical cancer
  • DiscontinuedBreast cancer; Cancer; Colorectal cancer; Malignant melanoma; Non-small cell lung cancer
  • 22 Jul 2021SpringWorks Therapeutics receives patent allowance for mirdametinib from the US Patent and Trademark Office for the treatment of Neurofibromatosis type 1-associated plexiform neurofibromas
  • 16 Jun 2021SpringWorks Therapeutics and St. Jude Children’s Research Hospital agree to develop mirdametinib in USA for glioma
  • 15 Jun 2021Efficacy and safety data from the phase IIb RENEU trial for Neurofibromatosis type 1-associated plexiform neurofibromas released by SpringWorks Therapeutics

PATENT

US-11066358

On July 20, 2021, SpringWorks Therapeutics announced that the United States Patent and Trademark Office (USPTO) has issued US11066358 , directed to mirdametinib , the Company’s product candidate in development for several oncology indications, including as a monotherapy for patients with neurofibromatosis type 1-associated plexiform neurofibromas (NF1-PN) and was assigned to Warner-Lambert Company (a subsidiary of Pfizer ).This patent was granted on July 20, 2021, and expires on Feb 17, 2041. Novel crystalline forms of mirdametinib and compositions comprising them are claimed.

N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (“mirdametinib”, or “PD-0325901”) is a small molecule drug which has been designed to inhibit mitogen-activated protein kinase kinase 1 (“MEK1”) and mitogen-activated protein kinase kinase 2 (“MEK2”). MEK1 and MEK2 are proteins that play key roles in the mitogen-activated protein kinase (“MAPK”) signaling pathway. The MAPK pathway is critical for cell survival and proliferation, and overactivation of this pathway has been shown to lead to tumor development and growth. Mirdametinib is a highly potent and specific allosteric non-ATP-competitive inhibitor of MEK1 and MEK2. By virtue of its mechanism of action, mirdametinib leads to significantly inhibited phosphorylation of the extracellular regulated MAP kinases ERK1 and ERK2, thereby leading to impaired growth of tumor cells both in vitro and in vivo. In addition, evidence indicates that inflammatory cytokine-induced increases in MEK/ERK activity contribute to the inflammation, pain, and tissue destruction associated with rheumatoid arthritis and other inflammatory diseases.
      Crystal forms of N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide have been described previously. WO2002/006213 describes crystalline Forms I and II. U.S. Pat. No. 7,060,856 (“the ‘856 patent”) describes a method of producing Form IV. The ‘856 patent indicates that the material produced by this method was greater than 90% Form IV (The ‘856 patent, Example 1). The ‘856 patent also states that the differential scanning calorimetry (“DSC”) of the material produced shows an onset of melting at 110° C., as well as a small peak with an onset at 117° C., consistent with the material being a mixture of two forms.
      WO 2006/134469 (“the ‘469 PCT publication”) also describes a method of synthesizing N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide. The ‘469 PCT publication reports the method yields a product conforming to the polymorphic Form IV disclosed in U.S. patent application Ser. No. 10/969,681 which issued as the ‘856 patent.
      Compositions containing more than one polymorphic form are generally undesirable because of the potential of interconversion of one polymorphic form to another. Polymorphic interconversion can lead to differences in the effective dose or physical properties affecting processability of a drug, caused by differences in solubility or bioavailability. Thus, there is a need for a composition containing essentially pure Form IV of N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, for use in treatment of a tumor, a cancer, or a Rasopathy disorder.

Example 1: Production of Essentially Pure Form IV

Lab Scale Production of Essentially Pure Form IV

      2 kg PD-0325901 has been prepared using the below convergent synthesis scheme starting from commercially available 2,3,4-Trifluorobenzoic Acid (TFBA), 2-Fluoro-4-Iodoaniline (FIA) and chiral S-Glycerol Acetonide (SGA)

 (MOL) (CDX)
 (MOL) (CDX)
Step 1: Preparation of “Side Chain”, PD-0337792
      All reactions were performed in toluene other than otherwise stated. Triflic anhydride gave the best yield.

[TABLE-US-00002]TABLE 1 Coupling Agents for Step 1Entry   No.Coupling AgentYieldNotes 1Mesyl Chloridedid not   react 2Benzyl chloride27Had to heat 70° C.   for 166 hr34-fluorobenzensulfonylchloride27Ran 93 hrs. at 70° C.44-chlorobenzensulfonylchloride35Complete after 68 hrs.   50° C.5Tosyl Chloride36Had to heat to 70° C.   for 164 hrs6Benzyl chloride52study solvent effects:   DMF, DMSO, NMP –   all similar DMSO   fastest all complete   after 110 hrs., heated   to 70° C. after 66 hrs.7Triflic anhydride91Cooled to −74° C. 
      Recognizing that triflate gave the highest yield, the possibility of eliminating the cryogenic conditions was investigated, set possibly due to stability concerns of the “methanesulfonate” intermediate. The following experiments suggest no significant yield loss for experiments run at −20° C.
[TABLE-US-00003]TABLE 2 Yield of Coupling ReactionExperimentalHold time afterYield (Alcohol toDescription*TFMSA addn.IPGAP) 1.07 equiv. NHP15min.85%1.07 equiv. NHP2hours86%1.77 equiv. NHP2hours72%1.07 equiv. NHP (reverse1hours91%addition) * 2 g (1 eq.) SGA in 16 ml toluene was treated with triflic anhydride, trifluoromethanesulfonic acid (TFMSA) (4.2 g, 1.002 equiv.) at −20° C. and then stirred for a prescribed time prior to solid N-hydroxyphthalimide (NIP) addition or transfer to a flask containing solid NHP.
      The data presented above suggest no detrimental effect was observed after prolonged stirring of the “trifluoromethane sulfonate” intermediate prior to the N-hydroxyphthalimide addition. Reverse addition of intermediate mixture to solid NHP appears to give the highest yield.
      An additional advantage of the triflate usage was easy removal of the Et 3N triflate salts side product simply by water wash. This resulted in highly pure N-hydroxyphthalimide-protected alcohol, IPGAP (PD-0333760) in Toluene, which can be isolated as crystals or carried through to the final deprotection reaction.
      Both aqueous and anhydrous ammonia base were examined as deprotecting agents. The results were both successful. The phthalimide side product was simply filtered out from solution of product (PD-0337792) in toluene when anhydrous ammonia was used. Similarly, it was filtered out from the solution after performing azeotropic water removal from toluene when aqueous ammonia (28% solution) was used. Anhydrous ammonia however, requires the reaction to be performed at high-pressure containment. Experiments conducted by sparging the ammonia gas gave acceptable yields; however, they required large volumes and use of a cryogenic condenser (to avoid gas from escaping the reactor headspace).
[TABLE-US-00004]TABLE 3 Yields for base deprotection ReagentYield*   Methyl hydrazine85-95% Anhydrous NH(sparged)78-90% Anhydrous NH(50 psi)80-92% Aqueous NH390-97%   *from PD-0333760

Step 2: Fluoride Displacement

      Examination of the reaction in an automated reactor reveals that the reaction is essentially dosed-controlled after the initiation period. Increasing the amount of lithium amide and increased agitation rate appear to shorten the induction time. The addition of water was shown to prolong the induction time. However, it is not clear whether it is due to lithium hydroxide formation.
      Induction time is increased when 0.1 equivalent H 2O was added. The trend was reversed however when 0.1 equivalent lithium hydroxide was added. Induction times were decreased upon increasing lithium amide equivalents and agitation.

 (MOL) (CDX)
      CDI-assisted coupling of PD-0315209 acid and sidechain reagent followed by the acid (with aqueous HCl) hydrolysis consistently yielded good results in the laboratory. The development focus of this step was to ensure that impurity levels are within the specification limit. The known impurities in the final isolated diol product are excess PD-0315209 acid, dimeric impurities and chiral impurities. The chiral impurities are controlled by limiting the R-enantiomer in the starting s-glycerol acetonide. Elevated levels of dimeric impurity (d) has been known to cause difficulties in the polymorph transformation step. The dimeric impurity is formed initially by the reaction of imidazole (CDI-activated acid) in the presence of excess acid PD-0315209 forming dimer (a) and possibly (b) which are then carried through in the subsequent IPGA coupling and acid hydrolysis steps forming dimer (c) and (d), respectively. Impurity d is referred to as PF-00191189.

 (MOL) (CDX)
 (MOL) (CDX)
      The reaction can be easily carried out in the laboratory either by charging both solids, FIPFA and CDI, followed by solvent (acetonitrile) or charging solids CDI into a slurry of FIPFA in acetonitrile. None of the solids is initially soluble in acetonitrile. The acid activation reaction was fast (almost instantaneous), forming highly soluble imidazolide product that turned the slurry into a clear homogenous solution while CO gas evolution occurs.
      Lab experiments generally resulted in impurity levels under 3%, which can be completely removed by the subsequent recrystallization from a 3-5% ethanol-toluene system. An additional recrystallization was performed in the few instances where the impurity level was above 0.3%. Table 4 shows selected results of lab experiments where elevated levels of impurities were observed and how they were removed in the subsequent recrystallization. The crude PD-0325901 was obtained using the acetonitrile/toluene system and the purified product was recrystallized from a 5% ethanol/toluene system. Entries no. 4 and 5 used additional solvent to ensure impurity removal with entry 5 requiring two recrystallizations in order to achieve a level of “ND” in the polymorph transformation. The 8-10 ml/g crude crystallization volume was chosen to limit product loss while maintaining a filterable slurry and ensuring removal of impurities.
[TABLE-US-00005]TABLE 4 Purification of PD-0325901  Tot.     Imp. In  Final Tot.isolated Tot. Imp.assay (after Imp. InCrude PurifiedpolymorphEntryreactionPD-RecrystallizationPD-trans-Nomixture0325901Vol (ml/g crude)0325901formation) 1 2.4%ND8ND99.8%210.5% 2%8ND99.6%3   6% 1%8ND99.4%4  10%3.2%15ND98.6%5  20%12%130.6%98.4%* 
      A scale up procedure that would give tolerable levels of impurities prior to the polymorph transformation (<0.3%), without losing too much product in the recrystallization was developed considering the solid CDI addition rate. Fast addition is preferred to minimize impurity formation; however, the addition needs to be performed at a rate that ensures safely venting of the evolved CO 2.
      A half portion of solid CDI was initially added to the PD-0325901 acid, followed by solvent addition. The remaining CDI was added then through a hopper in less than 30 minutes to ensure that the impurity levels were below 3%.

Pilot Plant Preparation of Essentially Pure Form IV

Step 1: Preparation of “Side Chain”, PD-0337792

      14.4 kg alcohol (chemical purity 99.4%, optical purity 99.6% enantiomeric excess) was converted to 97.5 kg 9.7% w/w PD-0337792 (IPGA) solution in toluene (overall yield ˜60%). The triflate activation was performed in the 200 L reactor by maintaining temperatures under −20° C. during triflic anhydride addition. The resulting activated alcohol was then transferred to a 400 L reactor containing solid N-hydroxypthalimide (NHP) and the reaction was allowed to occur at ambient temperature to completion. The final base de-protection was performed by adding aqueous ammonia (˜28% soln, 5 equiv., 34 kg). After reaction completion, water was removed by distillation from toluene, and the resulting solid side product was filtered out to yield the product solution.

Step 2: Preparation of PD-0315209

      The process yielded 21.4 kg (99.4% w/w assay), which is 80% of theoretical from starting materials 2,3,4-trifluorobenzoic acid (12 kg, 1 eq.) and 2-fluoro-4-iodoaniline (16.4 kg, 1.02 eq.) with lithium amide base (5 kg, 3.2 eq.). The reaction was initiated by adding 5% of total solution of TFBA and FIA into lithium amide slurry at 50° C. This reaction demonstrated a minimal initiation period of ˜10 minutes, which was observed by color change and slight exotherm. The remaining TFBA/FIA solution in THE was slowly added through a pressure can in an hour while maintaining the reaction temperatures within 45-55° C. There was no appreciable pressure rise (due to ammonia gas release) observed during the entire operation.

Step 3: Preparation of PD-0325901

      A modification was made to the CDI charging to mitigate potential gas generation. Two equal portions of CDI were added into solid FIPFA before and after solvent addition (through a shot loader). The timing between the two solid CDI additions (4.6 kg each) should not exceed 30 minutes. Then two intermediate filter cakes were dissolved with ethanol. The excess ethanol was distilled and replaced with toluene to approximately 5% v/v ethanol prior to PD-0325901 recrystallization. Lab studies suggested that the crystallization from toluene and acetonitrile and recrystallization from ethanol in toluene would not be able to reduce impurities which is essential for the polymorph transformation. The presence of a dimeric impurity (PF-00191189) at a level greater than 0.2% has been known to result in the formation of undesired polymorph.

 (MOL) (CDX)
      The crude crystallization from the final reaction mixture reduced dimeric impurity PF-00191189 to approximately 1.9% and the subsequent recrystallization further reduced it to approximately 0.4%. As a consequence, undesired polymorphs were produced. The DSC patterns indicated two different melting points ˜80° C. (low melt Form II) and ˜117° C. (Form I). Also during the processing, the solids crystallized at a much lower temperature than expected (actual ˜10° C., expected ˜40° C.). It is suspected that the unsuccessful recrystallization is due to a change in the solvent composition as a result of incomplete drying of the crude. Drying of the crude wet cake prior to ethanol dissolution was stopped after about 36 hours when the crude product was ˜28 kg (26 kg theoretical).

Polymorph Transformation

      Approximately 7.4 kg of PD-0325901 (mixed polymorphs) from the final EtOH/Water crystallization and precipitated materials from the earlier EtOH/Toluene filtrate were taken forward to the polymorph transformation. Both crops were separately dried in the filter until constant weights and each was dissolved in EtOH. The combined EtOH solution was analyzed by HPLC and resulted in an estimated amount of 16.4 kg PD-0325901. The recrystallization was started after removing EtOH via vacuum distillation and adjusting the solvent composition to about 5% EtOH in Toluene at 65° C. (i.e., EtOH is added dropwise at 65° C. until complete solids dissolution).
      A slow 4-hour cooling ramp to 5° C. followed by 12 h stirring was performed to ensure satisfactory results. The resulting slurry was filtered and again it was completely dried in the filter until constant weight (approximately 3 days). The purified solid showed 99.8% pure PD-0325901 with not detected level of dimeric impurity PF-00191189.
      The dried solid (15.4 kg) was re-dissolved in exactly 4 volumes of EtOH (62 L) off of the filter, transferred to the reactor and precipitated by a slow (˜3 h) water addition (308 L) at 30-35° C., cooled to 20° C. and stirred for 12 h. The DSC analysis of a slurry sample taken at 2 h shows the solids to be completely Form IV (desired polymorph).
      21.4 kg PD-0315209, 9.7 kg CDI (1.05 equiv.), 91 kg solution of 9.7% PD-0337792 in Toluene (1.1 equiv.) were used and resulted in 12.74 kg of PD-0325901 (assay 99.4%, 100% Form IV, Yield 48%).

PATENT

WO2006134469 , claiming methods of preparing MEK inhibitor, assigned to Warner-Lambert Co .

https://patents.google.com/patent/WO2006134469A1/enThe compound Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide represented by formula 1

Figure imgf000002_0001

i is a highly specific non-ATP-competitive inhibitor of MEK1 and MEK2. The compound of formula ± (Compound I) is also known as the compound PD 0325901. Compound I is disclosed in WO 02/06213; WO 04/045617; WO 2005/040098; EP 1262176; U.S. Patent Application Pub. No. 2003/0055095 A1 ; U.S. Patent Application Pub. No. 2004/0054172 A1; U.S. Patent Application Pub. No. 2004/0147478 A1 ; and U.S. Patent Application No. 10/969,681, the disclosures of which are incorporated herein by reference in their entireties.Numerous mitogen-activated protein kinase (MAPK) signaling cascades are involved in controlling cellular processes including proliferation, differentiation, apoptosis, and stress responses. Each MAPK module consists of 3 cytoplasmic kinases: a mitogen-activated protein kinase (MAPK), a mitogen-activated protein kinase kinase (MAPKK), and a mitogen-activated protein kinase kinase kinase (MAPKKK). MEK occupies a strategic downstream position in this intracellular signaling cascade catalyzing the phosphorylation of its MAP kinase substrates, ERK1 and ERK2. Anderson et al. “Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase.” Nature 1990, v.343, pp. 651-653. In the ERK pathway, MAPKK corresponds with MEK (MAP kinase ERK Kinase) and the MAPK corresponds with ERK (Extracellular Regulated Kinase). No substrates for MEK have been identified other than ERK1 and ERK2. Seger et al. “Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells.” J. Biol. Chem., 1992, v. 267, pp. 14373-14381. This tight selectivity in addition to the unique ability to act as a dual-specificity kinase is consistent with MEK’s central role in integration of signals into the MAPK pathway. The RAF-MEK-ERK pathway mediates proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes that promote tumor growth, progression, and metastasis. By virtue of its central role in mediating the transmission of growth- promoting signals from multiple growth factor receptors, the Ras-MAP kinase cascade provides molecular targets with potentially broad therapeutic applications.One method of synthesizing Compound I is disclosed in the above-referenced WO 02/06213 andU.S. Patent Application Pub. No. 2004/0054172 A1. This method begins with the reaction of 2-fluoro-4- iodo-phenylamine and 2,3,4-trifluoro-benzoic acid in the presence of an organic base, such as lithium diisopropylamide, to form 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid, which is then reacted with (R)-0-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of a peptide coupling agent (e.g., diphenylphosphinic chloride) and a tertiary amine base (e.g., diisopropylethylamine). The resulting product is hydrolyzed under standard acidic hydrolysis conditions (e.g., p-TsOH in MeOH) to provide Compound 1. (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine is prepared by reaction of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol with N-hydroxyphthalimide in the presence of Ph3P and diethyl azodicarboxylate.Another method of synthesizing Compound I, which is disclosed in the above-referenced U.S.Patent Application No. 10/969,681, comprises reaction of 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzoic acid with (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of N1N1– carbonyldiimidazole. The resulting product is hydrolyzed with aqueous acid and crystallized to provide polymorphic form IV of Compound I.Although the described methods are effective synthetic routes for small-scale synthesis of Compound I, there remains a need in the art for new synthetic routes that are safe, efficient and cost effective when carried out on a commercial scale.The present invention provides a new synthetic route including Steps I through Step III to the MEK inhibitor Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I).Step I: Preparation of 0-{r(4RV2.2-dimethyl-1.3-dioxolan-4-ynmethyl}hydroxylanπine (6) The method of the present invention comprises a novel Step I of preparing of 0-{[(4R)-2,2- dimethyl-1 ,3-dioxolan-4-yl]methyl}hydroxylamine (6) from [(4S)-2,2-dimethyl-1 ,3-dioxoIan-4-yl]methanol (1) through the formation of [(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl trifluoromethanesulfonate (3) and its coupling with N-hydroxyphthalimide (4) to afford 2-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methoxy}-1 H- isoindole-1 ,3(2H)-dione (5), which is subsequently de-protected to give 6 as shown in Scheme 1.Scheme 1

Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003

The reaction of compound (1) with trifluoromethanesulfonic anhydride (2) is carried out in the presence of a non-nucleophilic base, such as, for example, a tertiary organic amine, in an aprotic solvent at a temperature of from -5O0C to 50C, preferably, at a temperature less than -150C, to form triflate (3). A preferred tertiary organic amine is triethylamine, and a preferred solvent is toluene. Treatment of triflate (3) with N-hydroxyphthalimide (4) furnishes phthalimide (5), which can be isolated if desired. However, in order to minimize processing time and increase overall yield, 0-{[(4R)- 2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) can be prepared in a one-pot process with no phthalimide (S) isolation. Cleavage of the phthalimide function could be achieved by methods known in the art, for example, by hydrazinolysis. However, the use of less hazardous aqueous or anhydrous ammonia instead of methyl hydrazine (CH3NHNH2) is preferred.Step II: Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) As shown in Scheme 2, Step Il of the method of the present invention provides 3,4-difluoro-2-(2- fluoro-4-iodophenylamino)-benzoic acid (9).Scheme 2

Figure imgf000010_0001

Preparation of compound (9) can be carried out by reacting compound (7), wherein X is halogen, or O-SC^R^ or 0-P(3O)(OR^, wherein R^ is alkyl or aryl, with compound (8) optionally in a solvent, and in the presence of from about 1 mol equivalent to about 10 mol equivalents of at least one base, wherein the base is selected from: a Group I metal cation hydride or a Group 2 metal cation hydride, including lithium hydride, sodium hydride, potassium hydride, and calcium hydride, a Group I metal cation dialkylamide or a Group 2 metal cation dialkylamide, including lithium diisopropylamide, a Group I metal cation amide or a Group 2 metal cation amide, including lithium amide, sodium amide, potassium amide, a Group I metal cation alkoxide or a Group 2 metal cation alkoxide, including sodium ethoxide, potassium terf-butoxide, and magnesium ethoxide, and a Group I metal cation hexamethyldisilazide, including lithium hexamethyldisilazide; for a time, and at a temperature, sufficient to yield compound (9).Preferably, preparation of compound (9) is carried out by reacting compound (7), wherein X is halogen, more preferably, X is fluorine, in an aprotic solvent with compound (8) in the presence of from about 3 mol equivalents to about 5 mol equivalents of a Group I metal cation amide at a temperature of from 2O C to 55°C, more preferably, at a temperature from 45°C to 55°C. A catalytic amount of Group I metal cation dialkylamide can be added if necessary. A preferred Group I metal cation amide is lithium amide, a preferred Group I metal cation dialkylamide is lithium diisopropylamide, and a preferred solvent is tetrahydrofuran. Preferably, the reaction is performed by adding a small amount of compound (7) and compound (8) to lithium amide in tetrahydrofuran followed by slow continuous addition of the remaining portion. This procedure minimizes the risk of reactor over-pressurization due to gas side product (ammonia) generation.Step III: Preparation of N-((RV2.3-dihydroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound I)Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using a carboxylic acid activating reagent such as, for example, COCI2, S(O)C^, S(O)2Cl2, P(O)Cl3, triphenylphosphine/diethylazodicarboxylate, diphenylphosphinic chloride, N, N’-dicyclohexylcarbodiimide, (benzotriazol-1 -yloxy)tripyrolidinophosphonium hexafluorophosphate, (benzotriazol-1 – yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, N-ethyl-N’-(3- dimethylaminopropyl)carbodiimide hydrochloride, or 1,1′-carbonyldiimidazole (CDI).A preferred carboxylic acid activating reagent is 1,1′-carbonyldimidazole (CDI) shown in Scheme 3. Preparation of the desirable polymorphic Form IV of Compound I using CDI is described in the above- referenced U.S. Patent Application No. 10/969,681.Scheme 3

Figure imgf000011_0001

10

Figure imgf000011_0002

10 11 Compound IIn according to the present invention, the method was modified to include the advantageous procedure for product purification and isolation, which procedure is performed in single-phase systems such as, for example, toluene/acetonitrile for the first isolation/crystallization and ethanol/toluene for the second recrystallization. Water addition, implemented in the previous procedure, was omitted to avoid the two-phase crystallization from the immiscible water-toluene system that caused inconsistent product purity. The one-phase procedure of the present invention provides consistent control and removal of un- reacted starting material and side products. Alternatively, Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using thionyl chloride (SOCI2) as shown in Scheme 4.Scheme 4

Figure imgf000012_0002
Figure imgf000012_0001

Compound IExamplesThe reagents and conditions of the reactions described herein are merely illustrative of the wide variety of starting materials, their amounts and conditions which may be suitably employed in the present invention as would be appreciated by those skilled in the art, and are not intended to be limiting in any way.HPLC (Conditions A): 10 μL injection volume onto Agilent Zorbax RX-C18 150 mm x 4.6 mm x 3.5 μm column at 30°C column temperature, 1.0 mL/min flow rate and detection at 246 nm. Mobile phase A (v/v): 25 mM Acetate Buffer, pH 6.0; Mobile phase B (v/v): Acetonitrile, and Linear Gradient Table:

Figure imgf000012_0003

Sample Preparation: Dilute 100 μL reaction mixture to 10 mL with acetonitrile. Mix in a vial 200 μL of this sample solution with 300 μL carbonate buffer pH 10.0 and 300 μL solution of 2-mercaptopyridine in acetonitrile (18 mM), heat the vial for 10 minutes at 500C and dilute to 1:1 ratio in mobile phase A.GC (Conditions B): 1 μL injection onto an RTX-5 column (30 m x 0.25 mm x 0.25 μm) with initial oven temperature of 120°C for 2 min. to final temperature of 250°C in 15°C/minute ramping and a final time of 2.33 min; Flow rate: 1 mL/min.HPLC (Conditions C): 5 μL injection onto Phenomenex Luna C18(2) 150 mm x 4.6 mm x 3μm column ; flow rate : 1.0 mL/min; detection at 225 nm; mobile phase A: 95/5 v/v Water/Acetonitrile with 0.1% Trifluoroacetic acid (TFA), mobile phase B: 5/95 v/v Water/Acetonitriie with 0.1% TFA; Linear Gradient Table:

Figure imgf000013_0001

Sample preparation: Dilute 1 ml_ reaction mixture to 100 mL with acetonitrile and dilute 1 mL of this solution to 10 mL with 50:50 Water/Acetonitrile.HPLC (Conditions D): 5 μL injection onto Waters SymmetryShield RP 18, 150 mm x 4.6 mm x 3.5 μm column; flow rate: 1.0 mL/min; detection at 235 nm; mobile phase A: 25 mM Acetate Buffer adjusted to pH 5.5, mobile phase B: Acetonitrile; Linear Gradient Table:

Figure imgf000013_0002

Sample preparation: Dilute 40 μL of reaction mixture in 20 mL acetonitrile.HPLC (Conditions E): 10 μL sample injection onto YMC ODS-AQ 5 μm, 250 mm x 4.6 mm column; flow rate: 1.0 ml_/min; detection at 280 nm; temperature 30°C; mobile phase : 75/25 v/v Acetonitrile/Water with 0.1% Formic acid.Sample preparation: Quench reaction mixture sample with dipropylamine and stir for about 5 minutes before further dilution with mobile phase.DSC measurement was performed using a Mettler-Toledo DSC 822, temperature range 25° to 150°C with 5°C/min heating rate in a 40 μL aluminum pan. Experimental Conditions for Powder X-Rav Diffraction (XRD):A Rigaku Miniflex+ X-ray diffractometer was used for the acquisition of the powder XRD patterns. The instrument operates using the Cu Ka1 emission with a nickel filter at 1.50451 units. The major instrumental parameters are set or fixed at:X-ray: Cu / 30 kV (fixed) / 15 mA (fixed)Divergence Slit: Variable Scattering Slit: 4.2° (fixed) Receiving Slit: 0.3 mm (fixed) Scan Mode: FT Preset Time: 2.0 s Scan Width: 0.050° Scan Axis: 2Theta/Theta Scan Range: 3.000° to 40.000°Jade Software Version: 5.0.36(SP1) 01/05/01 (Materials Data, Inc.) Rigaku Software: Rigaku Standard Measurement for Windows 3.1 Version 3.6(1994-1995) Example 1. Preparation of 0-ffl4R)-2.2-dimethyl-1.3-dioxolan-4-vπmethyl}hvdroxylamine (6)A solution containing [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) (13.54 ml_, 0.109 mol) (DAISO Co., Ltd., CAS# 22323-82-6) and triethylamine (18.2 ml_, 0.131 mol) in 115 mL toluene was cooled to -15 C, then trifluoromethanesulfonic anhydride (2) (18.34 mL, 30.75 g, 0.109 mol) (Aldrich, Catalog # 17,617-6 ) was added drop wise while maintaining the temperature at less than -15°C. The mixture was then stirred for 2 hours, and transferred to a separate flask containing a mixture (slurry) of N- hydroxyphthalimide (4) (18.99 g, 0.116 mol) (Aldrich, Catalog # H5.370-4) and 18.2 mL (0.13 mol) triethylamine in 95 mL toluene. The resulting mixture was warmed to 20-25°C and stirred for at least 5 hours or until reaction completion (determined by HPLC (Conditions A)). Water (93 mL) was then added to quench the reaction mixture, the phases were separated, and the bottom aqueous layer was discarded. The water quench was repeated two more times resulting in a pale yellow organic layer. The organic layer was heated to 35 C and treated with 36.7 mL ammonium hydroxide solution (contains about 28-29% wt/wt ammonia). The mixture was stirred for at least 12 hours or until the reaction was deemed complete as determined by GC (Conditions B). The water was then removed under reduced pressure by co- distilling it with toluene to about half of the original volume at temperatures around 35-45 C. Toluene (170 mL) was added to the concentrated solution and the distillation was repeated. A sample was drawn for water content determination by Karl Fisher method (using EM Science Aquastar AQV-2000 Titrator with a sample injected to a pot containing methanol and salicylic acid). The distillation was repeated ifl water content was more than 0.1%. The concentrated solution was filtered to remove the white solid side product, and the filtrate was stored as 112mL (98 g) product solution containing 9.7% w/w compound 6 in toluene. This solution was ready for use in the final coupling step (Example 3). Overall chemical yield was 59%. A small sample was evaporated to yield a sample for NMR identification.1H NMR (400 MHz, CDCI3): δ 5.5 (bs, 2H), 4.35 (m, 1H), 4.07 (dd, 1H), 3.77 (m, 2H), 3.69 (dd, 1H), 1.44 (s, 3H), 1.37 (s, 3H).Example 2. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9)A solution of 2-fluoro-4-iodoaniline (8) (16.4 g, 0.069 mol) (Aldrich, Catalog # 30,660-6) and 2,3,4- trifluorobenzoic acid (7) (11.98 g, 0.068 mol) (Aldrich, Cat # 33,382-4) in 38 mL tetrahydrofuran (THF) was prepared and a portion (about 5%) of this solution was added to a stirring slurry of lithium amide (5 g, 0.22 mol) in 40 mL THF at 50-55 C. After about 15-30 min. an exotherm followed by gas release and color change are observed. The remaining portion of the (8) and (7) solution was added slowly over 1-2 hr while maintaining temperatures within 45-55°C. The mixture was stirred until the reaction was deemed complete (by HPLC (Conditions C). The final mixture was then cooled to 20-25°C and transferred to another reactor containing 6 N hydrochloric acid (47 mL) followed by 25 mL acetonitrile, stirred, and the bottom aqueous phase was discarded after treatment with 40 mL 50% sodium hydroxide solution. The organic phase was concentrated under reduced pressure and 57 mL acetone was added. The mixture was heated to 50°C, stirred, and added with 25 mL warm (40-50°C) water and cooled to 25-30°C to allow crystallization to occur (within 1-4 hours). Once the crystallization occurred, the mixture was further cooled to 0 to -5°C and stirred for about 2 hours. The solid product was filtered and the wet cake was dried in vacuum oven at about 55°C. Overall chemical yield was 21.4 g, 80%. 1H NMR (400 MHz, (CD3)2SO): δ 13.74 (bs, 1H), 9.15 (m, 1 H), 7.80 (dd, 1H), 7.62 (d, 1H), 7.41 (d, 1H), 7.10 (q, 1H), 6.81 (m, 1H).Example 2B. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) by the solid addition of lithium amide methodTo a stirring solution of 2,3,4-trifluorobenzoic acid (13) (5.0 g, 28.4 mmol) and 2-fluoro-4- iodoaniline (14) (6.73 g, 28.4 mmol) in MeCN (100 mL), under N2 atmosphere was added lithium amide (2.61 g, 113.6 mmol) in small portions. The reaction mixture was heated to reflux for 45 minutes, cooled to ambient temperature and quenched with 1 N HCI and then water. The yellowish white precipitate was filtered, washed with water. The solid was triturated in CH2CI2 (30 mL) for 1h, filtered and dried in a vacuum oven at 45°C for 14 hours to give 8.Og (72%) of compound (9) as an off-white solid, mp 201.5-203 °C.Example 3. Preparation of N-((R)-2.3-dihvdroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound \)3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (20 g, 0.051 mol) in 100 mL acetonitrile was treated with 1,1′-carbonyldiimidazole (CDI) (8.66 g, 0.053 mol) (Aldrich, Cat # 11,553-3) and stirred for about 2 hours at 20-25°C until the reaction was deemed complete by HPLC (Conditions D). 94 mL (84.9 g) of 9.7% w/w solution of O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) in toluene was then added and stirred for about 4 hours or until the reaction was deemed complete by HPLC (Conditions D). To this mixture was added 66 mL of 5.6 % hydrochloric acid solution, and after stirring, the bottom aqueous phase was discarded. Again 66 mL of 5.6 % hydrochloric acid solution was added to the organic phase and stirred at 20-25°C for 12-18 hours or until the reaction was deemed complete by HPLC (Conditions D). The bottom layer was then discarded and the remaining organic layer was concentrated under reduced pressure to remove about 10-20% solvent, and the volume was adjusted to about 9-11 mL/g with toluene (80 mL). Crude product was then crystallized at 10-15°C. The slurry was allowed to stir for about 2 hours and the crude solid product was filtered, and dried. The dried crude product was recharged to the reactor and dissolved into 150 mL of 5% v/v ethanol/toluene mixture at 55- 67°C. The solution was then clarified at this temperature through filter (line filter) to remove any remaining particulate matter. The solution was then cooled slowly to 5°C to crystallize and stirred for at least 2 h, filtered and dried. The dried solid product was redissolved in EtOH (60 mL) at 35°C, and product was precipitated out by adding water (300 mL) at 35°C followed by cooling to 200C. The slurry was stirred for at least 2 hours to transform the crystals to the desired polymorphic Form IV as determined by DSC and Powder X-ray Diffraction pattern (PXRD). The slurry was filtered and dried under vacuum oven at 70- 90°C to yield the final N-((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound I) product. Overall chemical yield was 13 g, 53%. Melting point (DSC): 112+1° C. Appearance: White to off-white crystals.Shown in Figure 1, PXRD conforms to polymorphic crystal Form IV disclosed in the above mentioned U.S. Patent Application No. 10/969,681 1H NMR (400 MHz, (CD3)2SO): δ 11.89 (bs, 1H), 8.71 (bs, 1H), 7.57 (d, 1H), 7.37 (m, 2H), 7.20 (q, 1H), 6.67 (m, 1H), 4.84 (bs, 1H), 4.60 (m, 1H), 3.87 (m, 1 H), 3.7 (m, 2H), 3.34 (m, 2H).Example 4. Preparation of N-((R)-2.3-dihydroxypropoxyV3.4-difluoro-2-(2-fluoro-4-iodo-phenylanrιinoV benzamide (Compound \)To a stirring solution of 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (120 g, 0.30 mol) in a mixture of 1 mL N,N-dimethylformamide and 1000 mL toluene was added thionyl chloride (55 g, 0.462 mol). The mixture was heated to 50-65 C and stirred for 2 hours or until reaction completion as determined by HPLC (Conditions E). The final reaction mixture was then cooled and concentrated under reduced pressure to a slurry keeping the temperature below 35°C. Toluene (600 mL) was added to dissolve the slurry and vacuum distillation was repeated. Additional toluene (600 mL) was added to the slurry dissolving all solids and the solution was then cooled to 5° -10°C. The solution was then treated with O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) (63 g, 0.43 mol) solution in 207 mL toluene followed by potassium carbonate (65 g) and water (200 mL), stirred for at least 2 hours at 20- 25°C. The stirring was stopped to allow phase separation and the bottom phase was discarded. The remaining organic layer was treated with hydrochloric acid solution (7.4%, 240 mL) until pH was less than 1 and stirred for 2 hours. The final reaction mixture was slightly concentrated under vacuum collecting about 100 mL distillate and the resulting organic solution was cooled to 5°C to crystallize the product and filtered. The filter cake was washed with toluene (1000 mL) followed by water (100 mL) and the wet cake (crude product Compound I) was charged back to the flask. Toluene (100 mL), ethanol (100 mL) and water (100 mL) are then added, stirred at 30-35°C for about 15 min, and the bottom aqueous phase was discarded. Water (200 mL) was then added to the organic solution and the mixture was stirred at about 3O C to allow for crystallization. The stirring was continued for 2 hours after product crystallized, then it was further cooled to about 0°C and stirred for at least 2 hours. The slurry was filtered and wet cake was dried under reduced pressure at 55-85°C to yield the final product N-((R)-2,3-dihydroxypropoxy)-3,4- difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I) product. Overall chemical yield was 86 g, 58%.

PATENT

WO2002/006213 describes crystalline Forms I and II. U.S. Pat. No. 7,060,856 (“the ‘856 patent”)

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2002006213

////////MIRDAMETINIB, Orphan Drug Status, Neurofibromatosis 1, PHASE 2, PD0325901, PD 0325901, PD-325901, 

O=C(NOC[C@H](O)CO)C1=CC=C(F)C(F)=C1NC2=CC=C(I)C=C2F

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Nanatinostat


Nanatinostat Chemical Structure
ChemSpider 2D Image | CHR-3996 | C20H19FN6O2
Hdac inhibitor CHR-3996.png

Nanatinostat

Tractinostat

CHR-3996, CHR 3996, VRx 3996,

C20H19FN6O2, 394.41

CAS 1256448-47-1

2-[(1α,5α,6α)-6-[[(6-Fluoro-2-q

2-[(1R,5S,6R)-6-{[(6-fluoroquinolin-2-yl)methyl]amino}-3-azabicyclo[3.1.0]hexan-3-yl]-N-hydroxypyrimidine-5-carboxamide2-[(1R,5S,6s)-6-{[(6-Fluoro-2-quinolinyl)methyl]amino}-3-azabicyclo[3.1.0]hex-3-yl]-N-hydroxy-5-pyrimidinecarboxamide5-Pyrimidinecarboxamide, 2-[(1R,5S)-6-[[(6-fluoro-2-quinolinyl)methyl]amino]-3-azabicyclo[3.1.0]hex-3-yl]-N-hydroxy-Chroma Therapeutics Ltd. (Originator)

  • OriginatorChroma Therapeutics
  • DeveloperChroma Therapeutics; Viracta Therapeutics
  • ClassAmides; Antineoplastics; Pyrimidines; Quinolines; Small molecules
  • Mechanism of ActionHistone deacetylase inhibitors
  • Orphan Drug StatusYes – Post-transplant lymphoproliferative disorder; Plasmablastic lymphoma; T-cell lymphoma
  • Phase IILymphoma
  • Phase I/IIMultiple myeloma
  • Phase ISolid tumours
  • No development reportedGastric cancer; Nasopharyngeal cancer; Post-transplant lymphoproliferative disorder
  • 01 Jun 2021Phase-II clinical trials in Lymphoma (Combination therapy, Second-line therapy or greater) in North America, Europe, Asia (PO)
  • 18 May 2021Ninatinostat is still in phase I trials for Solid tumour in United Kingdom and Netherlands (Viracta Therapeutics pipeline, May 2021)
  • 18 May 2021Virata Therapeutics has patent protection for dose regimen in NAVAL-1 trial in USA

Nanatinostat is under investigation in clinical trial NCT00697879 (Safety Study of the Histone Deacetylase Inhibitor, CHR-3996, in Patients With Advanced Solid Tumours).

Nanatinostat is an orally bioavailable, second-generation hydroxamic acid-based inhibitor of histone deacetylase (HDAC), with potential antineoplastic activity. Nanatinostat targets and inhibits HDAC, resulting in an accumulation of highly acetylated histones, the induction of chromatin remodeling, and the selective transcription of tumor suppressor genes; these events result in the inhibition of tumor cell division and the induction of tumor cell apoptosis. This agent may upregulate HSP70 and downregulate anti-apoptotic Bcl-2 proteins more substantially than some first-generation HDAC inhibitors. HDACs, upregulated in many tumor cell types, are a family of metalloenzymes responsible for the deacetylation of chromatin histone proteins.

Patent

WO2006123121

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2006123121

Example 44: N-Hvdroxy 2-(6-fr(6-fluoroαuinolin-2-yl)methvnamino)-3-azabicvclorS.I.OIhex-S-vDpyrimidine-δ-carboxamide

LCMS purity >98%, m/z 395 [M+H]+1H NMR (300 MHz, c/6-DMSO) δ: 2.30 (2H, s), 2.75 (1 H, s), 3.60 (2H, dm, J = 11.7 Hz), 3.88 (2H, d, J = 11.7 Hz), 4.69 (2H, br s), 7.66 (1 H, d, J = 8.4 Hz), 7.75 (1 H, td, J = 8.7, 3.0 Hz), 7.88 (1 H, dd, J = 9.3, 2.7 Hz), 8.48 (1 H, d, J = 8.4 Hz), 8.67 (2H, s), 9.01 (1 H, br s), 9.61 (1 H, br s), 11.09 (1 H, br s).

PATENT

WO-2021113694

Crystalline hydrate form A of N-hydroxy 2-{6-[(6-fluoro-quinolin-2-ylmethyl)-amino]-3-aza-bicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxamide ( nanatinostat ) .

Compound 1 is also known as nanatinostat, VRx-3996, or CHR-3996. It has been previously described in patents and patent applications, e.g. US patent 7,932,246 and US patent application 15/959,482, each of which is incorporated by reference in their entirety.

Compound 1

PATENT

WO2021071809 , claiming dosages for HDAC treatment with reduced side effects.

/////////Nanatinostat, CHR-3996, CHR 3996, VRx 3996, CHROMA, ORPHAN DRUG, Tractinostat, PHASE 2

FC1=CC=C2N=C(CN[C@H]3[C@]4([H])CN(C5=NC=CC(C(NO)=O)=N5)C[C@]34[H])C=CC2=C1
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TROPIFEXOR


Tropifexor (USAN).png
Tropifexor.svg
Tropifexor CAS 1383816-29-2

TROPIFEXOR

トロピフェクサー;

 PHASE 2, NASH, PBC, liver fibrosis, bile acid diarrhea, non-alcoholic fatty liver disease

FormulaC29H25F4N3O5S
CAS1383816-29-2
Mol weight603.5845

TROPIFEXORLJN 452;LJN-452;LJN452;CS-2712;CPD1549;Tropifexor;Tropifexor (LJN452);LJN452;LJN452,Tropifexor;2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acidтропифексор [Russian] [INN]
تروبيفيكسور [Arabic] [INN]
曲匹法索 [Chinese] [INN]2-[(3-endo)-3-({5-Cyclopropyl-3-[2-(trifluormethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]oct-8-yl]-4-fluor-1,3-benzothiazol-6-carbonsäure [German] [ACD/IUPAC Name]
2-[(3-endo)-3-({5-Cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]oct-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid [ACD/IUPAC Name]
6-Benzothiazolecarboxylic acid, 2-[(3-endo)-3-[[5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-4-isoxazolyl]methoxy]-8-azabicyclo[3.2.1]oct-8-yl]-4-fluoro- [ACD/Index Name]
Acide 2-[(3-endo)-3-({5-cyclopropyl-3-[2-(trifluorométhoxy)phényl]-1,2-oxazol-4-yl}méthoxy)-8-azabicyclo[3.2.1]oct-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylique [French] [ACD/IUPAC Name]
NMZ08KM76Z

Tropifexor fast facts

CAS Reg. No.1383816-29-2
Molar mass603.58 g/mol
Empirical formulaC29H25F4N3O5S
AppearanceWhite crystals
Melting point221 ºC
Water solubility6 mg/L
EfficacyAnti-inflammatory, Farnesoid X receptor (FXR) agonist
CommentTreatment of non-alcoholic steatohepatitis

Novartis is developing tropifexor, a non-bile acid farnesoid X receptor agonist, and its analog LJP-305, for treating NASH, PBC, liver fibrosis, bile acid diarrhea and non-alcoholic fatty liver disease. In June 2021, this drug was reported to be in phase 2 clinical development.

Nonalcoholic steatohepatitis (NASH) is a liver disease that is becoming more prevalent as worldwide obesity and type 2 diabetes increase. It is characterized by accumulation of fat in the liver, inflammation, hepatocyte ballooning, and fibrosis.

Another liver disease, primary biliary cholangitis (PBC), is a cholestatic condition in which bile flow from the liver to the intestine is reduced or interrupted. It is thought to be autoimmune.

PBC is associated with decreased expression of the farnesoid X receptor (FXR), a ligand-activated nuclear receptor that is highly expressed in the liver and other organs. FXR is a key regulator of bile acid production, conjugation, and transport. FXR activation also suppresses lipogenesis; thus, it has been proposed as a treatment for NASH.

Recently, David C. Tully and colleagues at the Genomics Institute of the Novartis Research Foundation (San Diego) and the Novartis Institutes for Biomedical Research (Emeryville, CA) discovered tropifexor, a highly potent FXR agonist. They began by replacing an indole group in an existing partial FXR agonist with a 2-substituted benzothiazole-6-carboxylic acid, a change that resulted in a dramatic increase in potency. Further changes, including optimization of the benzothiazole substituent, resulted in more potent, orally bioavailable tropifexor.

Tropifexor is an investigational drug which acts as an agonist of the farnesoid X receptor (FXR). It was discovered by researchers from Novartis and Genomics Institute of the Novartis Research Foundation. Its synthesis and pharmacological properties were published in 2017.[1] It was developed for the treatment of cholestatic liver diseases and nonalcoholic steatohepatitis (NASH). In combination with cenicriviroc, a CCR2 and CCR5 receptor inhibitor, it is undergoing a phase II clinical trial for NASH and liver fibrosis.[2]

Rats treated orally with tropifexor (0.03 to 1 mg/kg) showed an upregulation of the FXR target genes, BSEP and SHP, and a down-regulation of CYP8B1. Its EC50 for FXR is between 0.2 and 0.26 nM depending on the biochemical assay.

The patent which covers tropifexor and related compounds was published in 2010.[3]

PATENT

WO-2021104022

Novel, stable crystalline polymorphic form II of tropifexor , useful for treating non-alcoholic steatohepatitis (NASH), fatty liver and primary biliary cholangitis (PBC).Tropifexor was originally developed by Novartis and then licensed to Pfizer for cooperative development. It is a non-steroidal FXR (farnesoid receptor) agonist, currently in clinical phase II of indications for NASH (non-alcoholic steatohepatitis), fatty liver and primary biliary cholangitis. 
The structure of Tropifexor is shown in the following formula (1): 

Drug polymorphism is a common phenomenon in drug development and an important factor affecting drug quality. Different crystal forms of the same drug may have significant differences in physical and chemical properties such as appearance, fluidity, solubility, storage stability, bioavailability, etc., and there may be great differences, which will affect the storage transfer, application, stability, and efficacy of the drug In order to obtain an effective crystal form that is conducive to production or pharmaceutical preparations, it is necessary to conduct a comprehensive investigation of the crystallization behavior of the drug to obtain a crystal form that meets the production requirements. 
At present, there is no literature that discloses the crystal form of Tropifexor, and there is no related literature report. 
The present invention obtains a new crystal form of the compound through a large number of experimental studies on the Tropifexor compound. The new crystal form has the advantages of high solubility, good stability, low moisture absorption, simple preparation process and easy operation, etc., and has excellent properties in industrial production. Superiority.Example 1 Preparation method of Tropifexor crystal form II[0049]After mixing 60.3 mg of Tropifexor and p-aminobenzoic acid (13.7 mg), they were added to ethanol (3.0 ml), stirred at 27° C. to obtain a clear solution, and then allowed to stand at room temperature for about 2 days to precipitate a solid product. It was filtered with suction and placed in a drying box at 50°C and vacuum dried to constant weight to obtain 51.3 mg of solid powder. The obtained crystal was detected by XPRD and confirmed to be Tropifexor crystal form II; its X-ray powder diffraction pattern was basically consistent with Fig. 1, its DSC pattern was basically the same as Fig. 2, and its TGA pattern was basically the same as Fig. 3.[0050]Example 2 Preparation method of Tropifexor crystal form II[0051]After mixing 60.3 mg of Tropifexor and p-hydroxybenzoic acid (13.8 mg), they were added to ethanol (3.0 ml), stirred at 27° C. to obtain a clear solution, and then allowed to stand at room temperature for about 2 days to precipitate a solid product. It was filtered with suction and placed in a drying box at 50°C and vacuum dried to constant weight to obtain 48.5 mg of solid powder. The obtained crystal was detected by XPRD and confirmed to be Tropifexor crystal form II; its X-ray powder diffraction pattern was basically consistent with Fig. 1, its DSC pattern was basically the same as Fig. 2, and its TGA pattern was basically the same as Fig. 3.[0052]Example 3 Preparation method of Tropifexor crystal form II[0053]After mixing 60.3 mg of Tropifexor and salicylic acid (13.8 mg), they were added to ethanol (3.0 ml), stirred at 27°C to obtain a clear solution, and then allowed to stand at room temperature for about 2 days to precipitate a solid product. Filter with suction and place in a drying box at 50°C and vacuum dry to constant weight to obtain 50.0 mg of solid powder. The obtained crystal was detected by XPRD and confirmed to be Tropifexor crystal form II; its X-ray powder diffraction pattern was basically consistent with Fig. 1, its DSC pattern was basically the same as Fig. 2, and its TGA pattern was basically the same as Fig. 3.[0054]Example 4 Preparation method of Tropifexor crystal form II[0055]After mixing 60.3 mg of Tropifexor and 2,4-dihydroxybenzoic acid (15.4 mg), they were added to ethanol (3.0 ml), stirred at 27°C to obtain a clear solution, and then allowed to stand at room temperature for about 2 days to precipitate a solid product. It was filtered with suction and placed in a drying box at 50°C and vacuum dried to constant weight to obtain 49.5 mg of solid powder. The obtained crystal was detected by XPRD and confirmed to be Tropifexor crystal form II; its X-ray powder diffraction pattern was basically consistent with Fig. 1, its DSC pattern was basically the same as Fig. 2, and its TGA pattern was basically the same as Fig. 3.

PATENT

WO2021104021 ,

claiming crystalline polymorphic form I of tropifexor,Example 1 Preparation method of Tropifexor crystal form I 
50.0 mg of Tropifexor was added to ethanol (1.0 ml), heated to 60° C. and stirred to obtain a clear solution, and then water (3 ml) was added dropwise to the Tropifexor solution. Stir and precipitate solid product. It was filtered with suction and placed in a drying box at 50°C and vacuum dried to constant weight to obtain 38.5 mg of solid powder. The obtained crystal was detected by XPRD and confirmed to be Tropifexor crystal form I; its X-ray powder diffraction pattern was basically consistent with Figure 1, its DSC pattern was basically consistent with Figure 2, and its TGA pattern was basically consistent with Figure 3

PATENT

product pat, WO2012087519 , https://patents.google.com/patent/WO2012087519A1/en

has protection in the EU  until November 2031, and expire in  US in February 2032 with US154 extension.

PATENT

WO 2016097933

Example 1

2-r(1 R,3r,5S)-3-(f5-cvclopropyl-3-r2-(trifluoromethoxy)phenyll-1 ,2-oxazol-4-yl)methoxy)-8- azabicvcloi3.2.1 loctan-8-yll-4-fluoro-1 ,3-benzothiazole-6-carboxylic acid (1 -1 B) and

-r(1 R,3r,5S)-3-(f5-cvclopropyl-3-r2-(trifluoromethyl)phenyll-1 ,2-oxazol-4-yl)methoxy)-8-

R1a = OCF3 (1 -1A, 1 -1 B)

a = CF3 (1-2A, 1-2B)

Methyl 2-[(1 R,3r,5S)-3-(i5-cvclopropyl-3-r2-(trifluoromethoxy)phenyll-1 ,2-oxazol-4- yl}methoxy)-8-azabicvcloi3.2.1 loctan-8-yll-4-fluoro-1 ,3-benzothiazole-6-carboxylate (1 -1 A). Into a 25-mL round-bottom flask equipped with a stir bar was added sequentially 4-(((1 R,3r,5S)- 8-azabicyclo[3.2.1 ]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (1 .29 mmol), N,N-dimethylacetamide (3.6 mL), cesium carbonate (3.31 mmol), and methyl 2- bromo-4-fluorobenzo[d]thiazole-6-carboxylate (3.87 mmol). After stirring the resulting slurry at room temperature for 10 minutes, the mixture was then warmed to 60 °C and stirred for 1 h. The reaction slurry was allowed to cool to room temperature, and was diluted with 200 mL of ethyl acetate and washed with water (3 χ 30 mL). The organic extracts were concentrated under vacuum and directly purified using normal phase silica gel chromatography (40 g silica column) with a 15 min gradient of 10 % to 60 % ethyl acetate/hexanes. Desired fractions were concentrated in vacuo, and the resulting residue crystallized upon standing to give methyl 2- [(1 R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1 ,2-oxazol-4-yl}methoxy)-8- azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1 ,3-benzothiazole-6-carboxylate (1-1 A) as a white crystalline solid. MS (m/z) : 618.2 (M+1 ).

2-r(1 R,3r,5S)-3-(i5-cvclopropyl-3-r2-(trifluoromethoxy)phenyll-1 ,2-oxazol-4-yl}methoxy)- 8-azabicvcloi3.2.1 loctan-8-yll-4-fluoro-1 ,3-benzothiazole-6-carboxylic acid (1 -1 B). To a 25-mL round-bottom flask equipped with a stir bar was added the ester (0.89 mmol), THF (4 mL),

MeOH (2 mL), and 3 N aqueous KOH solution (1 mL, 3 mmol). The resulting homogenous solution was stirred for 1 hour at 70 °C, cooled to room temperature, and then quenched with AcOH (roughly 0.2 mL of glacial acetic, 3 mmol) until pH=6 was achieved (Whatman class pH strip paper). At this time the reaction was diluted with ethyl acetate (40 mL) and washed with water (3 5 mL). The ethyl acetate fraction was concentrated under vacuum to give to an oily residue. To the resulting oil was then added MeOH (6 mL). The oil quickly dissolved, then immediately began to crystallize. Upon standing for 2.5 hrs, the mother liquor was withdrawn and crystals washed (3 x 2 mL of ice cold MeOH). The crystals were dried via vacuum (10 mm Hg pressure at 45 °C overnight) and then recrystallized from acetonitrile, filtered, and dried under vacuum to give 2-[(1 R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1 ,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1 ,3-benzothiazole-6-carboxylic acid (1 -1 B). 2-[(1 R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1 ,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1 ,3-benzothiazole-6-carboxylic acid (1 -2B).

Examples 1 -2A and the corresponding acid 1 -2B can be prepared following the same procedures, from the reaction of intermediate 4-((8-azabicyclo[3.2.1 ]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethyl)phenyl)isoxazole.

PAPER

 European journal of medicinal chemistry (2021), 209, 112910

https://www.sciencedirect.com/science/article/abs/pii/S0223523420308825

Image 1

Abstract

Farnesoid X receptor (FXR) agonists are emerging as potential therapeutics for the treatment of various metabolic diseases, as they display multiple effects on bile acid, lipid, and glucose homeostasis. Although the steroidal obeticholic acid, a full FXR agonist, was recently approved, several side effects probably due to insufficient pharmacological selectivity impede its further clinical application. Activating FXR in a partial manner is therefore crucial in the development of novel FXR modulators. Our efforts focusing on isoxazole-type FXR agonists, common nonsteroidal agonists for FXR, led to the discovery a series of novel FXR agonists bearing aryl urea moieties through structural simplification of LJN452 (phase 2). Encouragingly, compound 11k was discovered as a potent FXR agonist which exhibited similar FXR agonism potency but lower maximum efficacy compared to full agonists GW4064 and LJN452 in cell-based FXR transactivation assay. Extensive in vitro evaluation further confirmed partial efficacy of 11k in cellular FXR-dependent gene modulation, and revealed its lipid-reducing activity. More importantly, orally administration of 11k in mice exhibited desirable pharmacokinetic characters resulting in promising in vivo FXR agonistic activity.

References

  1. ^ Tully DC, Rucker PV, Chianelli D, Williams J, Vidal A, Alper PB, et al. (December 2017). “Discovery of Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH)”Journal of Medicinal Chemistry60 (24): 9960–9973. doi:10.1021/acs.jmedchem.7b00907PMID 29148806.
  2. ^ Clinical trial number NCT03517540 for “Safety, Tolerability, and Efficacy of a Combination Treatment of Tropifexor (LJN452) and Cenicriviroc (CVC) in Adult Patients With Nonalcoholic Steatohepatitis (NASH) and Liver Fibrosis. (TANDEM)” at ClinicalTrials.gov
  3. ^ WO Application Filing 2012087519, Alper PB, Chianelli D, Mutnick D, Vincent P, Tully DC, “Compositions and methods for modulating fxr”, published 2012-06-28, assigned to Genomics Institute of the Novartis Research Foundation. Retrieved 17 May 2019.
 
Clinical data
ATC codeNone
Identifiers
showIUPAC name
CAS Number1383816-29-2
PubChem CID121418176
UNIINMZ08KM76Z
KEGGD11548
Chemical and physical data
FormulaC29H25F4N3O5S
Molar mass603.59 g·mol−1
3D model (JSmol)Interactive image
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///////////TROPIFEXOR, トロピフェクサー, NOVARTIS, PHASE 2, тропифексор , تروبيفيكسور , 曲匹法索 , LJN 452, LJN-452, LJN452, CS-2712, CPD1549, Tropifexor, Tropifexor (LJN452), LJN452, LJN452, PHASE 2, NASH, PBC, liver fibrosis, bile acid diarrhea, non-alcoholic fatty liver disease

1ccc(c(c1)c2c(c(on2)C3CC3)CO[C@H]4C[C@H]5CC[C@@H](C4)N5c6nc7c(cc(cc7s6)C(=O)O)F)OC(F)(F)F

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TROFINETIDE


ChemSpider 2D Image | Trofinetide | C13H21N3O6
Trofinetide structure.png

Trofinetide

  • Molecular FormulaC13H21N3O6
  • Average mass315.322 Da

Tofinetide , NNZ-256610076853400-76-7[RN]
glycyl-2-methyl-L-prolyl-L-glutamic acid
H-Gly-PMe-Glu-OHL-Glutamic acid, glycyl-2-methyl-L-prolyl-UNII-Z2ME8F52QLZ2ME8F52QLтрофинетид [Russian] [INN]تروفينيتيد [Arabic] [INN]曲非奈肽 [Chinese] [INN]

IUPAC CondensedH-Gly-aMePro-Glu-OH
SequenceGXE
HELMPEPTIDE1{G.[*C(=O)[C@@]1(CCCN1*)C |$_R2;;;;;;;;_R1;$|].E}$$$$
IUPACglycyl-alpha-methyl-L-prolyl-L-glutamic acid

An (1-3) IGF-1 analog with neuroprotective activity.

OPTICAL ROT; -52.4 °   Conc: 0.19 g/100mL;  water ;  589.3 nm; Temp: 20 °C; Len: 1.0 dm…Tetrahedron 2005, V61(42), P10018-10035 

EU Customs Code CN, 29339980

Harmonized Tariff Code, 293399

  • L-Glutamic acid, glycyl-2-methyl-L-prolyl-
  • glycyl-2-methyl-L-prolyl-L-glutamic acid
  • Glycyl-L-2-methylprolyl-L-glutamic acid
2D chemical structure of 853400-76-7

Trofinetide (NNZ-2566) is a drug developed by Neuren Pharmaceuticals that acts as an analogue of the neuropeptide (1-3) IGF-1, which is a simple tripeptide with sequence GlyProGlu formed by enzymatic cleavage of the growth factor IGF-1 within the brain. Trofinetide has anti-inflammatory properties and was originally developed as a potential treatment for stroke,[1][2] but has subsequently been developed for other applications and is now in Phase II clinical trials against Fragile X syndrome and Rett syndrome.[3][4][5]

Trofinetide (NNZ-2566), a neuroprotective analogue of glypromate, is a novel molecule that has a profile suitable for both intravenous infusion and chronic oral delivery. It is currently in development to treat traumatic brain injury.

In February 2021, Neuren is developing trofinetide (NNZ-2566, phase 2 clinical ), a small-molecule analog of the naturally occurring neuroprotectant and N-terminus IGF-1 tripeptide Glypromate (glycine-proline-glutamate), for intravenous infusion treatment of various neurological conditions, including moderate to severe traumatic brain injury (TBI), stroke, chronic neurodegenerative disorders and peripheral neuropathies. At the same time, Neuren is also investigating an oral formulation of trofinetide (phase 3 clinical) for similar neurological indications, including mild TBI.

Autism Spectrum Disorders and neurodevelopment disorders (NDDs) are becoming increasingly diagnosed. According to the fourth edition of the American Psychiatric Association’s (APA) Diagnostic and Statistical Manual oƒ Mental Disorders (DSM-4), Autism spectrum disorders (ASD) are a collection of linked developmental disorders, characterized by abnormalities in social interaction and communication, restricted interests and repetitive behaviours. Current classification of ASD according to the DSM-4 recognises five distinct forms: classical autism or Autistic Disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD-NOS). A sixth syndrome, pathological demand avoidance (PDA), is a further specific pervasive developmental disorder.

More recently, the fifth edition of the American Psychiatric Association’s (APA) Diagnostic and Statistical Manual oƒ Mental Disorders (DSM-5) recognizes recognises Asperger syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS) as ASDs.

This invention applies to treatment of disorders, regardless of their classification as either DSM-4 or DSM-5.

Neurodevelopment Disorders (NDDs) include Fragile X Syndrome (FXS), Angelman Syndrome, Tuberous Sclerosis Complex, Phelan McDermid Syndrome, Rett Syndrome, CDKL5 mutations (which also are associated with Rett Syndrome and X-Linked Infantile Spasm Disorder) and others. Many but not all NDDs are caused by genetic mutations and, as such, are sometimes referred to as monogenic disorders. Some patients with NDDs exhibit behaviors and symptoms of autism.

As an example of a NDD, Fragile X Syndrome is an X-linked genetic disorder in which affected individuals are intellectually handicapped to varying degrees and display a variety of associated psychiatric symptoms. Clinically, Fragile X Syndrome is characterized by intellectual handicap, hyperactivity and attentional problems, autism spectrum symptoms, emotional lability and epilepsy (Hagerman, 1997a). The epilepsy seen in Fragile X Syndrome is most commonly present in childhood, but then gradually remits towards adulthood. Hyperactivity is present in approximately 80 percent of affected males (Hagerman, 1997b). Physical features such as prominent ears and jaw and hyper-extensibility of joints are frequently present but are not diagnostic. Intellectual handicap is the most common feature defining the phenotype. Generally, males are more severely affected than females. Early impressions that females are unaffected have been replaced by an understanding of the presence of specific learning difficulties and other neuropsychiatric features in females. The learning disability present in males becomes more defined with age, although this longitudinal effect is more likely a reflection of a flattening of developmental trajectories rather than an explicit neurodegenerative process.

The compromise of brain function seen in Fragile X Syndrome is paralleled by changes in brain structure in humans. MRI scanning studies reveal that Fragile X Syndrome is associated with larger brain volumes than would be expected in matched controls and that this change correlates with trinucleotide expansion in the FMRP promoter region (Jakala et al, 1997). At the microscopic level, humans with Fragile X Syndrome show abnormalities of neuronal dendritic structure, in particular, an abnormally high number of immature dendritic spines (Irwin et al, , 2000).

Currently available treatments for NDDs are symptomatic – focusing on the management of symptoms – and supportive, requiring a multidisciplinary approach. Educational and social skills training and therapies are implemented early to address core issues of learning delay and social impairments. Special academic, social, vocational, and support services are often required. Medication, psychotherapy or behavioral therapy may be used for management of co-occurring anxiety, ADHD, depression, maladaptive behaviors (such as aggression) and sleep issues, Antiepileptic drugs may be used to control seizures.

Patent

WO 2014085480,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014085480

str1-1

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro-Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

WO95/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS to increase TH-mediated dopamine production in the treatment of diseases such as Parkinson’s disease.

WO02/16408 discloses certain GPE analogs having amino acid substitutions and certain other modification that are capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including injury or disease in the CNS.

EXAMPLES

The following examples are intended to illustrate embodiments of this invention, and are not intended to limit the scope to these specific examples. Persons of ordinary skill in the art can apply the disclosures and teachings presented herein to develop other embodiments without undue experimentation and with a likelihood of success. All such embodiments are considered part of this invention.

Example 1: Synthesis of N,N-Dimethylglycyl-L-prolyl)-L-glutamic acid

The following non-limiting example illustrates the synthesis of a compound of the invention, N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

All starting materials and other reagents were purchased from Aldrich; BOC=tert-butoxycarbonyl; Bn=benzyl.

BOC-L-proline-(P-benzyl)-L-glutamic acid benzyl ester

To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem. Soc: 79, 6810, 1994] (10 mmol) in dichloromethane (50 mi), cooled to 0°C, was added triethylamine (1 .39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl-L-glutamate (10 mmol) was then added and the mixture stirred at 0° C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol 1-1) then dried (MgSO4) and concentrated at reduced pressure to give BOC-L-proline-L-glutamic acid dibenzyl ester (5.0 g, 95%).

L-proline-L-glutamic acid dibenzyl ester

A solution of BOC-L-glutamyl-L-proline dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 h. at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give L-proline-L-glutamic acid dibenzyl ester.

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of L-proline-L-glutamic acid dibenzyl ester (10 mmol), N,N-dimethylglycine (10 mmol) and triethylamine ( 10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0°C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallised from ethyl acetate to yield the tripeptide derivative.

It can be appreciated that following the method of the Examples, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

Eample 2: Synthesis of Glycyl-L-2-Methyl-L-Prolyl-L-Glutamate

L-2-Methylproline and L-glutamic acid dibenzyl ester p-toluenesulphonate were purchased from Bachem, N-benzyloxycarbonyl-glycine from Acros Organics and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) from Aldrich Chem. Co.

Methyl L-2-methylprolinate hydrochloride 2

Thionyl chloride (5.84 cm3, 80.1 mmol) was cautiously added dropwise to a stirred solution of (L)-2-methylproline 1 (0.43 g, 3.33 mmol) in anhydrous methanol (30 cm3) at -5 °C under an atmosphere of nitrogen. The reaction mixture was heated under reflux for 24 h, and the resultant pale yellow-coloured solution was. concentrated to dryness in vacuo. The residue was dissolved in a 1 : 1 mixture of methanol and toluene (30 cm3) then concentrated to dryness to remove residual thionyl chloride. This procedure was repeated twice more, yielding hydrochloride 2 (0.62 g, 104%) as an hygroscopic, spectroscopically pure, off-white solid: mp 127- 131 °C; [α]D -59.8 (c 0.24 in CH2Cl2); vmax (film)/cm-1 3579, 3398 br, 2885, 2717, 2681 , 2623, 2507, 1743, 1584, 1447, 1432, 1374, 1317, 1294, 1237, 1212, 1172, 1123, 981 , 894, 861 and 764; δH (300 MHz; CDCl3; Me4Si) 1.88 (3H, s, Proα-CH3), 1 .70-2.30 (3H, br m, Proβ-HAΗΒ and Proγ-H2), 2.30-2.60 (1H, br m, Proβ-HAΗΒ), 3.40-3.84 (2H, br m, Proδ-H2), 3.87 (3H, s, CO2CH3), 9.43 (1H, br s, NH) and 10.49 ( 1H, br s, HCl); δC (75 MHz; CDCl3) 21.1 (CH3, Proα-CH3), 22.4 (CH2, Proγ-C), 35.6 (CH2, Proβ-C), 45.2 (CH2, Proδ-C), 53.7 (CH3, CO2CH3), 68.4 (quat., Proα-C) and 170.7 (quat, CO); m/z (FAB+) 323.1745 [M2.H35Cl.H+: (C7H13NO2)2. H35Cl.H requires 323.1738] and 325.1718 [M2.H37Cl.H+: (C7H13NOz)2. H37Cl.H requires 325.1708],

N-Benxyloxycarbonyl-glycyl-L-2-methylproline 5

Anhydrous triethylamine (0.45 cm3, 3.23 mmol) was added dropwise to a mixture of methyl L-2-methylprolinate hydrochloride 2 (0.42 g, 2.34 mmol) and N-benzyloxycarbonyl-glycine (98.5%) 3 (0.52 g, 2.45 mmol) in methylene chloride (16 cm3), at 0 °C, under an atmosphere of nitrogen. The resultant solution was stirred for 20 min and a solution of 1 ,3-dicyclohexylcarbodiimide (0.56 g, 2.71 mmol) in methylene chloride (8 cm3) at 0 °C was added dropwise and the reaction mixture was warmed to room temperature and stirred for a further 20 h. The resultant white mixture was filtered through a Celite™ pad to partially remove 1 ,3-dicyclohexylurea, and the pad was washed with methylene chloride (50 cm3). The filtrate was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and concentrated to dryness in vacuo. Further purification of the residue by flash column chromatography (35 g SiO2; 30-70% ethyl acetate – hexane; gradient elution) afforded tentatively methyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 4 (0.56 g), containing 1 ,3-dicyclohexylurea, as a white semi-solid: Rf 0.65 (EtOAc); m/z (ΕI+) 334.1534 (M+. C17H22N2O5 requires 334.1529) and 224 ( 1 ,3-dicyclohexylurea).

To a solution of impure prolinate 4 (0.56 g, ca. 1.67 mmol) in 1,4-dioxane (33 cm3) was added dropwise 1 M aqueous sodium hydroxide (10 cm3, 10 mmol) and the mixture was stirred for 19 h at room temperature. Methylene chloride ( 100 cm3) was then added and the organic layer extracted with saturated aqueous sodium hydrogen carbonate (2 x 100 cm3). The combined aqueous layers were carefully acidified with hydrochloric acid (32%), extracted with methylene chloride (2 x 100 cm3), and the combined organic layers dried (MgSO4), filtered, and

concentrated to dryness in vacuo. Purification of the ensuing residue (0.47 g) by flash column chromatography ( 17 g SiO2; 50% ethyl acetate – hexane to 30% methanol – dichloromethane; gradient elution) gave N-protected dipeptide 5 (0.45 g, 60%) as a white foam in two steps from hydrochloride 2. Dipeptide 5 was shown to be exclusively the frafw-orientated conformer by NMR analysis: Rf 0.50 (20% MeOH – CH2Cl2); [α]D -62.3 (c 0.20 in CH2Cl2); vmax (film)/cm-1 3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337, 1251 , 1219, 1179, 1053, 1027, 965, 912, 735 and 698; δH (300 MHz; CDCl3; Me4Si) 1.59 (3H, s, Proα-CH3), 1 .89 (1H, 6 lines, J 18.8, 6.2 and 6.2, Proβ-HAHB), 2.01 (2H, dtt, J 18.7, 6.2 and 6.2, Proγ-H2), 2.25-2.40 (1H, m, Proβ-HAΗΒ), 3.54 (2H, t, J 6.6, Proδ-H2), 3.89 (1H, dd, J 17.1 and 3.9, Glyα-HAHB), 4.04 (1H, dd, J 17.2 and 5.3, Glyα-HAΗΒ), 5.11 (2H, s, OCH2Ph), 5.84 (I H, br t, J 4.2, N-H), 7.22-7.43 (5H, m, Ph) and 7.89 (1 H, br s, -COOH); δC (75 MHz; CDCl3) 21.3 (CH3, Proα-CH3), 23.8 (CH2, Proγ-C), 38.2 (CH2, Proβ-C), 43.6 (CH2, Glyα-C), 47.2 (CH2, Proδ-C), 66.7 (quat, Proα-C), 66.8 (CH2, OCH2Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH, Ph), 136.4 (quat., Ph), 156.4 (quat., NCO2), 167.5 (quat., Gly-CON) and 176.7 (quat., CO); m/z (EI+) 320.1368 (M+. C16Η20Ν2Ο5 requires 320.1372).

Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamate 7

Triethylamine (0.50 cm3, 3.59 mmol) was added dropwise to a solution of dipeptide 5 (0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl ester /Moluenesulphonate 6 (0.73 g, 1.46 mmol) in methylene chloride (60 cm3) under nitrogen at room temperature, and the reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazoIidinyl)phosphinic chloride (BoPCl, 97%) (0.37 g, 1.41 mmol) was added and the colourless solution stirred for 17 h. The methylene chloride solution was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and evaporated to dryness in vacuo. Purification of the resultant residue by repeated (2x) flash column chromatography (24 g SiO2; 30-70% ethyl acetate – hexane; gradient elution) yielded ƒully protected tripeptide 7 (0.63 g, 89%) as a colourless oil. Tripeptide 7 was shown to be exclusively the trans-orientated conformer by NMR analysis: Rf 0.55 (EtOAc); [α]D -41.9 (c 0.29 in CH2Cl2); vmax (film)/cm-1 3583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1166, 1051, 911, 737 and 697; δH (400 MHz; CDCl3; Me4Si) 1.64 (3H, s, Proot-CH3), 1.72 (1H, dt, J 12.8, 7.6 and 7.6, Proβ-HAHB), 1.92 (2H, 5 lines, J 6.7, Proγ-H2), 2.04 (1H, 6 lines, J 7.3 Gluβ-HAHB), 2.17-2.27 (1H, m, Gluβ-HAΗΒ), 2.35-2.51 (3H, m, Proβ-HAΗΒ and Gluγ-H2), 3.37-3.57 (2H, m, Proδ-H2), 3.90 (1 H, dd, J 17.0 and 3.6, Glyα-HAHB), 4.00 (1H, dd, J 17.1 and 5.1, Glyα-HAΗΒ), 4.56 (1H, td, J 7.7 and 4.9, Glyα-H), 5.05-5.20 (6H, m, 3 x OCH2Ph), 5.66-5.72 (1H, br m, Gly-NH), 7.26-7.37 (15H, m, 3 x Ph) and 7.44 (1H, d, J 7.2, Glu-NH); δC (100 MHz; CDCl3) 21.9 (CH3, Proα-CH3), 23.4 (CH2, Proγ-C), 26.6 (CH2, Gluβ-C), 30.1 (CH2, Gluγ-C), 38.3 (CH2, Proβ-C),

43.9 (CH2, Glyα-C), 47.6 (CH2, Proδ-C), 52.2 (CH, Glua-C), 66.4 (CH2, OCH2Ph), 66.8 (CH2, OCH2Ph), 67.1 (CH2, OCH2Ph), 68.2 (quat, Proα-C), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1, (CH, Ph), 128.2, (CH, Ph), 128.2, (CH, Ph), 128.3, (CH, Ph), 128.4, (CH, Ph), 128.5, (CH, Ph), 128.5, (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat, Ph), 156.1 (quat, NCO2), 167.3 (quat., Gly-CO), 171.4 (quat., CO), 172.9 (quat., CO) and 173.4 (quat., CO); m/z (FAB+) 630.2809 (MH+. C35H40N3O8 requires 630.2815).

Glycyl-L-2-methylprolyl-L-glutamic acid (G-2-MePE)

A mixture of the protected tripeptide 7 (0.63 g, 1.00 mmol) and 10 wt % palladium on activated carbon (0.32 g, 0.30 mmol) in 91 :9 methanol – water (22 cm3) was stirred under an atmosphere of hydrogen at room temperature, protected from light, for 23 h. The reaction mixture was filtered through a Celite™ pad and the pad washed with 75 :25 methanol – water (200 cm3). The filtrate was concentrated to dryness under reduced pressure and the residue triturated with anhydrous diethyl ether to afford a 38: 1 mixture of G-2-MePE and tentatively methylamine 8 (0.27 g, 86%) as an extremely hygroscopic white solid. Analytical reverse-phase HPLC studies on the mixture [Altech Econosphere C 18 Si column, 150 x 4.6 mm, 5 ☐m; 5 min flush with H2O (0.05% TFA) then steady gradient over 25 min to MeCN as eluent at flow rate of 1 ml/min; detection using diode array] indicated it was a 38: 1 mixture of two eluting peaks with retention times of 13.64 and 14.44 min at 207 and 197 nm, respectively. G-2-MePE was shown to be a 73 :27 trans:cis mixture of conformers by 1H NMR analysis (the ratio was estimated from the relative intensities of the double doublet and triplet at δ 4.18 and 3.71 , assigned to the Gluα-H protons of the major and minor conformers, respectively):

mp 144 °Cɸ;

[ α]D -52.4 (c 0.19 in H2O);

δα (300 MHz; D2O; internal MeOH) 1.52 (3H, s, Proα-CH3), 1.81-2.21 (6H, m, Proβ-H2, Proγ-H, and Gluβ-H2), 2.34 (1.46H, t, J 7.2, Gluy-H2), 2.42* (0.54H, t, 77.3, Gluγ-H2), 3.50-3.66 (2H, m, Pro6-H2), 3.71 * (0.27H, t, J 6.2, Gluoc-H), 3.85 (1H, d, J 16.6, Glyα-HAHB), 3.92 (1H, d, J 16.6, Glyα-HAΗΒ) and 4.18 (0.73H, dd, J 8.4 and 4.7, Glua-H);

δC (75 MHz; D2O; internal MeOH) 21.8 (CH3, Proα-CH3), 25.0 (CH2, Proγ-C), 27.8* (CH2: Gluβ-C), 28.8 (CH2, Gluβ-C), 32.9 (CH2, Gluγ-C), 40.8 (CH2, Proβ-C), 42.7 (CH2, Glyα-C), 49.5 (CH2, Proδ-C), 56.0* (CH, Gluα-C), 56.4 (CH, Gluα-C), 69.8 (quat, Proα-C), 166.5 (quat., Gly-CO), 177.3 (quat., Pro-CON), 179.2 (quat., Gluα-CO), 180.2* (quat., Gluγ-CO) and 180.6 (quat., Gluγ-CO);

m/z (FAB+) 3 16.1508 (MH+. C13H22N3O6 requires 316.1509).

PATENT

WO02094856

Example

The following non-limiting example illustrates the synthesis of a compound of the invention, NN-dimethylglycyl-L-prolyl-L-glutamic acid.

All starting materials and other reagents were purchased from Aldrich;
BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-(γ-benzyl)-L-prolyl-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.

Soc: 79, 6180, 1957] (10 mmol) in dichloromethane (50 ml), cooled to 0 °C, was added triethylamine (1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl L-glutamate (10 mmol) was then added and the mixture stirred at 0 °C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol l“1) then dried (MgS04) and concentrated at reduced pressure to give BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (5.0 g, 95%).

(7-Benzyl)-L-prolyl-L-glutamic acid dibenzyl ester
A solution of BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 hr at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give (γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (I).

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of (7-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (10 mmol), TVN-dimethylglycine (10 mmol) and triethylamine
(10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0 °C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to yield the tri-peptide derivative.

It will be evident that following the method of the Example, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

Testing; Material and Methods
The following experimental protocol followed guidelines approved by the

University of Auckland animal ethics committee.
Preparation of cortical astrocyte cultures for harvest of metabolised cell culture supernatant

One cortical hemisphere from a postnatal day 1 rat was used and collected into

4ml of DMEM. Trituration was done with a 5ml glass pipette and subsequently through an 18 gauge needle. Afterwards, the cell suspension was sieved through a lOOμm cell strainer and washed in 50ml DMEM (centrifugation for 5min at 250g). The sediment was resuspended into 20ml DMEM+10% fetal calf serum. 10 Milliliters of suspension was added into each of two 25cm3 flasks and cultivated at 37°C in the presence of 10% C02, with a medium change twice weekly. After cells reached confluence, they were washed three times with PBS and adjusted to Neurobasal/B27 and incubated for another 3 days. This supernatant was frozen for transient storage until usage at -80°C.

Preparation of striatal and cortical tissue from rat E18/E19 embryos
A dam was sacrificed by C02-treatment in a chamber for up to 4 minutes and was prepared then for cesarean section. After surgery, the embryos were removed from their amniotic sacs, decapitated and the heads put on ice in DMEM/F12 medium for striatum and PBS + 0.65% D(+)-glucose for cortex.

Striatal tissue extraction procedure and preparation of cells
Whole brain was removed from the skull with the ventral side facing upside in DMEM/F12 medium. The striatum was dissected out from both hemispheres under a stereomicroscope and the striatal tissue was placed into the Falcon tube on ice.

The collected striatal tissue was triturated by using a PI 000 pipettor in 1ml of volume. The tissue was triturated by gently pipetting the solution up and down into the pipette tip about 15 times, using shearing force on alternate outflows. The tissue pieces settled to the bottom of the Falcon tube within 30 seconds, subsequently the supernatant was transferred to a new sterile Falcon tube on ice. The supernatant contained a suspension of dissociated single cells. The tissue pieces underwent a second trituration to avoid excessively damaging cells already dissociated by over triturating them. 1 Milliliter of ice-cold DMEM/F12 medium was added to the tissue pieces in the first tube and triturated as before. The tissue pieces were allowed to settle and the supernatant was removed to a new sterile Falcon tube on ice. The cells were centrifuged at 250g for 5 minutes at 4°C. The resuspended cell pellet was ready for cell counting.

Plating and cultivation of striatal cells
Striatal cells were plated into Poly-L-Lysine (O.lmg/ml) coated 96-well plates (the inner 60 wells only) at a density of 200,000 cells /cm2 in Neurobasal/B27 medium (Invitrogen). The cells were cultivated in the presence of 5% C02 at 37°C under 100% humidity. Complete medium was changed on days 1, 3 and 6.

Cortical tissue extraction procedure and preparation of cells
The two cortical hemispheres were carefully removed by a spatula from the whole brain with the ventral side facing upside into a PBS +0.65% D(+)-glucose containing petri dish. Forcips were put into the rostral part (near B. olfactorius) of the cortex for fixing the tissue and two lateral – sagittal oriented cuttings were done to remove the paraform and entorhinal cortices. The next cut involved a frontal oriented cut at the posterior end to remove the hippocampal formation. A final frontal cut was done a few millimeters away from the last cut in order to get hold of area 17/18 of the visual cortex.

The collected cortices on ice in PBS+0.65% D(+)-glucose were centrifuged at 350g for 5min. The supernatant was removed and trypsin/EDTA (0.05%/0.53mM) was added for 8min at 37°C. The reaction was stopped by adding an equal amount of DMEM+10%) fetal calf serum. The supernatant was removed by centrifugation followed by two subsequent washes in Neurobasal/B27 medium.

The cells were triturated once with a glass Pasteur pipette in 1 ml of
Neurobasal/B27 medium and subsequently twice by using a 1ml insulin syringe with a 22 gauge needle. The cell suspension was passed through a lOOμm cell strainer and subsequently rinsed by 1ml of Neurobasal B27 medium. Cells were counted and adjusted to 50,000 cells per 60μl.

Plating and cultivation of cortical cells

96-well plates were coated with 0.2mg/ml Poly-L-Lysine and subsequently coated with 2μg/ml laminin in PBS, after which 60μl of cortical astrocyte-conditioned medium was added to each well. Subsequently, 60μl of cortical cell suspension was added. The cells were cultivated in the presence of 10% C02 at 37°C under 100%) humidity. At day 1, there was a complete medium change (1:1- Neurobasal/B27 and astrocyte-conditioned medium) with addition of lμM cytosine-β-D-arabino-furanoside (mitosis inhibitor). On the second day, 2/3 of medium was changed. On day 5, 2/3 of the medium was changed again.

Cerebellar microexplants from P8 animals: preparation, cultivation and fixation

The laminated cerebellar cortices of the two hemispheres were explanted from a P8 rat, cut into small pieces in PBS + 0.65% D(+)glucose solution and triturated by a 23gauge needle and subsequently pressed through a 125 μm pore size sieve. The microexplants that were obtained were centrifuged (60 g) twice (media exchange) into serum-free BSA-supplemented START V-medium (Biochrom). Finally, the
microexplants were reconstituted in 1500 μl STARTV-medium (Biochrom). For cultivation, 40μl of cell suspension was adhered for 3 hours on a Poly-D-Lysine
(O.lmg/ml) coated cover slip placed in 35mm sized 6-well plates in the presence of 5% C02 under 100% humidity at 34°C. Subsequently, 1ml of STARTV-medium was added together with the toxins and drugs. The cultures were monitored (evaluated) after 2-3 days of cultivation in the presence of 5% C02 under 100% humidity. For cell counting analysis, the cultures were fixed in rising concentrations of paraformaldehyde (0.4%, 1.2%, 3% and 4% for 3min each) followed by a wash in PBS.
Toxin and drug administration for cerebellar, cortical and striatal cells: analysis

All toxin and drug administration experiments were designed that 1/100 parts of okadaic acid (30nM and lOOnM concentration and 0.5mM 3-nitropropionic acid for cerebellar microexplants only), GPE (InM -ImM) and G-2Methyl-PE (InM-lmM) were used respectively at 8DIV for cortical cultures and 9DIV for striatal cultures. The incubation time was 24hrs. The survival rate was determined by a colorimetric end-point MTT-assay at 595nm in a multi-well plate reader. For the cerebellar microexplants four windows (field of 0.65 mm2) with highest cell density were chosen and cells displaying neurite outgrowth were counted.

Results
The GPE analogue G-2Methyl-PE exhibited comparable neuroprotective capabilities within all three tested in vitro systems (Figures 12-15).

The cortical cultures responded to higher concentrations of GPE (Figure 12) /or

G-2Methyl-PE (lOμM, Figure 13) with 64% and 59% neuroprotection, respectively.

Whereas the other 2 types of cultures demonstrated neuroprotection at lower doses of G-2Methyl-PE (Figures 14 and 15). The striatal cells demonstrated
neuroprotection within the range of InM to ImM of G-2Methyl-PE (Figure 15) while the postnatal cerebellar microexplants demonstrated neuroprotection with G-2Methyl-PE in the dose range between InM and lOOnM (Figure 14).

While this invention has been described in terms of certain preferred embodiments, it will be apparent to a person of ordinary skill in the art having regard to that knowledge and this disclosure that equivalents of the compounds of this invention may be prepared and administered for the conditions described in this application, and all such equivalents are intended to be included within the claims of this application.

PATENT

WO-2021026066

Composition and kits comprising trofinetide and other related substances. Also claims a process for preparing trofinetide and the dosage form comprising the same. Disclosed to be useful in treating neurodegenerative conditions, autism spectrum disorders and neurodevelopmental disorders.

Trofinetide is a synthetic compound, having a similar core structure to Glycyl-Prolyl-Glutamic acid (or “GPE”). Trofinetide has been found to be useful in treating neurodegenerative conditions and recently has been found to be effective in treating Autism Spectrum disorders and Neurodevelopmental disorders.

Formula (Ila),

Example 1: Trofinetide Manufacturing Process

In general, trofinetide and related compounds can be manufactured from a precursor peptide or amino acid reacted with a silylating or persilylating agent at one or more steps. In the present invention, one can use silylating agents, such as N-trialkylsilyl amines or N-trialkylsilyl amides, not containing a cyano group.

Examples of such silylating reagents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, 3-(trimethylsilyl)-2-oxazolidone.

Step 1: Preparation of Z-Gly-OSu

Several alternative procedures can be used for this step.

Procedure 1A

One (1) eq of Z-Gly-OH and 1.1 eq of Suc-OH were solubilized in 27 eq of iPrOH and 4 eq of CH2Cl2 at 21 °C. The mixture was cooled and when the temperature reached -4 °C, 1.1 eq of EDC.HCl was added gradually, keeping the temperature below 10 °C. During the reaction a dense solid appeared. After addition of EDC.HCl, the mixture was allowed to warm to 20 °C. The suspension was cooled to 11 °C and filtered. The cake was washed with 4.9 eq of cold iPrOH and 11 eq of IPE before drying at 34 °C (Z-Gly-OSu dried product -Purity: 99.5%; NMR assay: 96%; Yield: 84%).

Procedure 1B

This Procedure is for a variant of Procedure 1A, and differs by replacing iPrOH with ACN. One (1) eq of Z-Gly-OH and 1.1 eq of Suc-OH were solubilized in 22 eq of ACN at 35 °C. The mixture was cooled in an ice bath. When the temperature reached 1 °C, 0.9 eq of DCC in 5.5 eq of ACN was added gradually to keep the temperature below 5 °C. The coupling reaction took about 20 hrs. During the reaction, DCU precipitated and was removed by filtration at the end of the coupling. After filtration, DCU was washed with ACN to recover the product. The mixture of Z-Gly-OSu was then concentrated to reach 60% by weight. iPrOH (17 eq) was added to initiate the crystallization. Quickly after iPrOH addition a dense solid appeared. An additional 17 eq of iPrOH was needed to liquify the suspension. The suspension was cooled in an ice bath and filtered. The solid was washed with 9 eq of iPrOH before drying at 45 °C (Z-Gly-OSu dried product – Purity: 99.2%; HPLC assay: 99.6%; Yield: 71%).

Step 2: Preparation of Z-Gly-MePro-OH

Several alternative procedures can be used for this step.

Procedure 2A

 One (1) eq of MePro.HCl was partially solubilized in 29 eq of CH2Cl2 at 35 °C with 1.04 eq of TEA and 1.6 eq of TMA. The mixture was heated at 35 °C for 2 hrs to perform the silylation. Then 1.02 eq of Z-Gly-OSu was added to the mixture. The mixture was kept at 35 °C for 3 hrs and then 0.075 eq of butylamine was added to quench the reaction. The mixture was allowed to return to room temperature and mixed for at least 15 min. The Z-Gly-MePro-OH was extracted once with 5% w/w NaHCO3 in 186 eq of water, then three times successively with 5% w/w NaHCO3 in 62 eq of water. The aqueous layers were pooled and the pH was brought to 2.2 by addition of 34 eq of HCl as 12N HCl at room temperature. At this pH, Z-Gly-MePro-OH formed a sticky solid that was solubilized at 45 °C with approximately 33 eq of EtOAc and 2.3 eq of iButOH. Z-Gly-MePro-OH was extracted into the organic layer and washed with 62 eq of demineralized water. The organic layer was then dried by azeotropic distillation with 11.5 eq of EtOAc until the peptide began to precipitate. Cyclohexane (12 eq) was added to the mixture to complete the precipitation. The suspension was cooled at 5 °C for 2 hrs and filtered. The solid was washed with 10 eq of cyclohexane before drying at 45 °C (Z-Gly-MePro-OH dried product – Purity: 100%; HPLC assay: 100%; Yield 79%).

Procedure 2B

This Procedure is for a variant of Procedure 2A. One (1) eq of MePro.HCl was partially solubilized in 36.6 eq of CH2Cl2 at 34 °C with 1.01 eq of TEA and 0.1 eq of TMA. Then 1.05 eq of Z-Gly-OSu was added to the mixture, followed by 1.0 eq of TEA. The mixture was maintained at 35 °C for approximately 1 hr, cooled to 25 to 30 °C and 0.075 eq of DMAPA was added to stop the reaction. One hundred (100) eq of water, 8.6 eq of HCl as 12N HCl and 0.3 eq of KHSO4 were added to the mixture (no precipitation was observed, pH=1.7). Z-Gly-MePro-OH was extracted into the organic layer and washed twice with 97 eq of demineralized water with 0.3 eq of KHSO4, then 100 eq of demineralized water, respectively. EtOAc (23 eq) was added to the mixture and CH2Cl2 was removed by distillation until the peptide began to precipitate. Cyclohexane (25 eq) was added to the mixture to complete the precipitation. The suspension was cooled at -2 °C overnight and filtered. The solid was washed with 21 eq of cyclohexane before drying at 39 °C (Z-Gly-MePro-OH dried product – Purity: 98.7%; NMR assay: 98%; Yield 86%).

Procedure 2C


In reactor 1, MePro.HCl (1 eq) was suspended in EtOAc (about 7 eq). DIPEA (1 eq) and TMA (2 eq) were added, and the mixture heated to dissolve solids. After dissolution, the solution was cooled to 0 °C. In reactor 2, Z-Gly-OH (1 eq) was suspended in EtOAc (about 15 eq). DIPEA (1 eq), and pyridine (1 eq) were added. After mixing, a solution was obtained, and cooled to -5 °C. Piv-Cl (1 eq) was added to reactor 2, and the contents of reactor 1 added to reactor 2. Upon completed addition, the contents of reactor 2 were taken to room temperature. The conversion from Z-Gly-OH to Z-Gly-MePro-OH was monitored by HPLC. When the reaction was complete, the reaction mixture was quenched with DMAPA (0.1 eq), and washed with an aqueous solution comprised of KHSO4, (about 2.5 wt%), NaCl (about 4 wt%), and conc. HCl (about 6 wt%) in 100 eq H2O. The aqueous layer was re-extracted with EtOAc, and the combined organic layers washed with an aqueous solution comprised of KHSO4 (about 2.5 wt%) and NaCl (about 2.5 wt%) in 100 eq H2O, and then with water (100 eq). Residual water was removed from the organic solution of Z-Gly-MePro-OH by vacuum distillation with EtOAc. The resulting suspension was diluted with heptane (about 15 eq) and cooled to 0 °C. The product was isolated by filtration, washed with cold heptane (about 7 eq), and dried under vacuum at 45 °C. Z-Gly-MePro-OH (85% yield) was obtained.

Step 3: Preparation of Z-Gly-MePro-Glu-OH

Several alternative procedures can be used in this step.

Procedure 3A

 H-Glu-OH (1.05 eq) was silylated in 2 eq of CH2Cl2 with 3.5 eq of TMA at 65 °C. Silylation was completed after 2 hrs. While the silylation was ongoing, 1.0 eq of Z-Gly-MePro-OH and 1.0 eq of Oxyma Pure were solubilized in 24 eq of CH2Cl2 and 1.0 eq of DMA at room temperature in another reactor. EDC.HCl (1.0 eq.) was added. The activation rate reached 97% after 15 min. The activated Oxyma Pure solution, was then added to silylated H-Glu-OH at 40 °C and cooled at room temperature. Coupling duration was approximately 15 min, with a coupling rate of 97%. Addition of 8.2% w/w NaHCO3 in 156 eq of water to the mixture at room temperature (with the emission of CO2) was performed to reach pH 8. Z-Gly-MePro-Glu-OH was extracted in water. The aqueous layer was washed twice with 29 eq of CH2Cl2. Residual CH2Cl2 was removed by concentration. The pH was brought to 2.5 with 2.5N HCl, followed by 1.4 eq of solid KHSO4 to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 3 x 52 eq of water. The filtered solid was added to 311 eq of demineralized water and heated to 55-60 °C. iPrOH (29 eq) was added gradually until total solubilization of the product. The mixture was slowly cooled to 10 °C under moderate mixing during 40 min to initiate the crystallization. The peptide was filtered and washed with 2 x 52 eq of water before drying at 45 °C (Z-Gly-MePro-Glu-OH dried product – Purity: 99.5%; NMR assay: 96%; Yield 74%).

Procedure 3B

One (1) eq of Z-Gly-MePro-OH and 1.05 eq of Suc-OH were solubilized in 40 eq of ACN and 30 eq of CH2Cl2 at room temperature. The mixture was cooled in an ice bath, and when the temperature was near 0 °C, 1.05 eq of DCC dissolved in 8 eq of ACN was added gradually, keeping the temperature below 5 °C. After addition of DCC, the mixture was progressively heated from 0 °C to 5 °C over 1 hr, then to 20 °C between 1 to 2 hrs and then to 45 °C between 2 to 5 hrs. After 5 hrs, the mixture was cooled to 5 °C and maintained overnight. The activation rate reached 98% after approximately 24 hrs. DCU was removed by filtration and washed with 13.5 eq of ACN. During the activation step, 1.1 eq of H-Glu-OH was silylated in 30 eq of ACN with 2.64 eq of TMA at 65 °C. Silylation was completed after 2 hrs. Z-Gly-MePro-OSu was then added gradually to the silylated H-Glu-OH at room temperature, with 0.4 eq of TMA added to maintain the solubility of the H-Glu-OH. The mixture was heated to 45 °C and 0.7 eq of TMA was added if precipitation occurred. The coupling duration was about 24 hrs to achieve a coupling rate of approximately 91%. The reaction was quenched by addition of 0.15 eq of butylamine and 2.0 eq of TEA. Water (233 eq) was added and the mixture concentrated until gelation occurred. Z-Gly-MePro-Glu-OH was extracted in water by addition of 5% w/w NaHCO3 in 233 eq of water and 132 eq of CH2Cl2. The aqueous layer was washed twice with 44 eq of CH2Cl2. Residual CH2Cl2 was removed by distillation. The pH was brought to 2.0 with 24 eq of HCl as 12N HCl followed by 75 eq of HCl as 4N HCl. At this pH, Z-Gly-MePro-Glu-OH precipitated. The mixture was cooled in an ice bath over 1 hr and filtered. The solid was washed with 186 eq of cold water before drying at 45 °C (Z-Gly-MePro-Glu-OH dried product – HPLC Purity: 98.4%; NMR assay: 100%; Yield 55%).

Procedure 3C

This Procedure is for a variant of Procedure 3A. H-Glu-OH (1.05 eq) was silylated in 3.7 eq of CH2Cl2 with 3.5 eq of TMA at 62 °C. Silylation was completed after approximately 1.5 to 2 hrs, as evidenced by solubilization. During the silylation step, 1.0 eq of Z-Gly-MePro-OH and 1.0 eq of Oxyma Pure were solubilized in 31.5 eq of CH2Cl2 at 22 °C. One (1.06) eq of EDC.HCl was added to complete the activation. The silylated H-Glu-OH was then added to the activated Oxyma Pure solution. The temperature was controlled during the addition to stay below 45 °C. Desilylation was performed by addition of a mixture of 2.5% w/w KHSO4 in 153 eq of water and 9 eq of iPrOH to reach a pH of 1.65. Residual CH2Cl2 was removed by concentration. The mixture was cooled to 12 °C to precipitate the Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 90 eq of water before drying at 36 °C.

Procedure 3D

This Procedure is for a variant of Procedure 3A. H-Glu-OH (1.05 eq.) was silylated in 3.9 eq of CH2Cl2 with 3.5 eq of TMA at 62 °C. Silylation was completed after 2 hrs, as evidenced by Solubilization. During the silylation step, 1 eq of Z-Gly-MePro-OH and 1 eq of Oxyma Pure were solubilized in 25 eq of CH2Cl2 at 23 °C. One (1) eq of EDC.HCl was added. To complete the activation, an additional 0.07 eq of EDC. HCl was added. Silylated H-Glu-OH was then added to the activated Oxyma Pure solution. Temperature was controlled during the addition to stay below 45 °C. Desilylation was performed by addition of a mixture of 2.5% w/w KHSO4 in 160 eq of water and 9.6 eq of iPrOH to reach pH 1.63.

Residual CH2Cl2 was removed by concentration. The mixture was cooled to 20 °C to precipitate the Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 192 eq of water before drying at about 25 °C for 2.5 days. The solid was then solubilized at 64 °C by addition of 55 eq of water and 31 eq of iPrOH. After solubilization, the mixture was diluted with 275 eq of water and cooled to 10 °C for crystallization. The mixture was filtered and the solid was washed with 60 eq of water before drying at 27 °C (Z-Gly-MePro-Glu-OH dried product – Purity: 99.6%; NMR assay: 98%; Yield 74%).

Procedure 3E

 In reactor 1, H-Glu-OH (1.05 eq) was suspended in ACN (about 2.2 eq). TMA (about 3.5 eq) added, and the mixture was heated to dissolve solids. After dissolution, the solution was cooled to room temperature. In reactor 2, Z-Gly-MePro-OH (1 eq) was suspended in ACN (14 eq). Oxyma Pure (1 eq) and EDC.HCl (1 eq) were added. The mixture was stirred at room temperature until the solids dissolved. The contents of reactor 2 were added to reactor 1. The conversion from Z-Gly-MePro-OH to Z-Gly-MePro-Glu-OH was monitored by HPLC. Upon completion the reaction mixture was added to an aqueous solution comprised of KHSO4 (about 2.5 wt%) dissolved in about 100 eq H2O. ACN was removed from the aqueous suspension of Z-Gly-MePro-Glu-OH by vacuum distillation with H2O. After stirring at room temperature, the product in the resulting suspension was isolated by filtration and washed with water. The solid obtained was dissolved in an aqueous solution comprised of NaHCO3 (about 5 wt%) in 110 eq H2O, and recrystallized by addition of an aqueous solution comprised of KHSO4 (about 10 wt%) in 90 eq H2O. The product was isolated by filtration, washed with water, and dried under vacuum at 45 °C. Z-Gly-MePro-Glu-OH (75% yield) was obtained.

Step 4: Deprotection and Isolation of Trofinetide

Several alternative procedures can be used in this step.

Procedure 4A

 Z-Gly-MePro-Glu-OH (1 eq) was suspended in water (about 25 eq) and EtOAc (about 15 eq). Pd/C (0.025 eq by weight and containing 10% Pd by weight) was added, and the reaction mixture hydrogenated by bubbling hydrogen through the reaction mixture at room temperature. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and upon reaction completion the catalyst was removed by filtration, and the layers separated. Residual EtOAc was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trofinetide (90% yield) was obtained. Alternatively, deprotection can be accomplished using MeOH only, or a combination of iPrOH and MeOH, or by use of ethyl acetate in water.

Procedure 4B

This Procedure is for a variant of Procedure 4A, excluding EtOAc. Z-Gly-MePro-Glu-OH (1 eq) was suspended in water (about 50 eq). Pd/C (0.05 eq, 5% Pd by weight) was added, and the reaction mixture hydrogenated at room temperature with a pressure of 5 bar. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Upon

reaction completion the catalyst was removed by filtration, and the aqueous layer washed with EtOAc (about 5 eq). Residual EtOAc was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trofinetide (90% yield) was obtained.

Procedure 4C

This Procedure is for a variant of Procedure 4A, replacing EtOAc with MeOH. Z-Gly-MePro-Glu-OH (1 eq) was suspended in MeOH (100 eq) and water (12 eq). Pd/Si (0.02 eq by weight) was added and the mixture was heated at 23 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and upon reaction completion the catalyst was removed by filtration and the layers were washed with MeOH and iPrOH. The solvents were concentrated under vacuum at 45 °C, and trofinetide precipitated. The precipitate was filtered and dried at 45 °C to provide trofinetide.

Procedure 4D

This Procedure is for a variant of Procedure 4A, replacing Pd/C with Pd/Si. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 105 eq of MeOH and 12 eq of water. Pd/Si (0.02 eq by weight) was added and the mixture was heated at 23 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate approximately 99% after 1 hr), the catalyst was filtered off and washed with 20-30 eq of MeOH. iPrOH (93 eq) was added and MeOH was replaced by iPrOH by concentration at 45 °C under vacuum. The peptide was concentrated until it began to precipitate. The peptide was filtered and dried at 45 °C (H-Gly-MePro-Glu-OH dried product: Purity: 98.1%; NMR assay: 90%; Yield 81%).

Procedure 4E

This Procedure is for a variant of Procedure 4A, removing H2O and replacing Pd/C with Pd/Si. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 44 eq of MeOH. Pd/Si type 340 (0.02 eq by weight) was added and the mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate about 99.9%, after 3-3.5 hrs), the catalyst was filtered off and washed with 8 eq of MeOH. Deprotected peptide was then precipitated in 56 eq of iPrOH. After 30 min at 5 °C, the peptide was filtered and washed with three times with 11 eq of iPrOH before drying at 25 °C (H-Gly-MePro-Glu-OH dried product: Purity: 99.4%; HPLC assay: ~98%; Yield: 81%).

Procedure 4F

This Procedure is for a variant of Procedure 4A. One (1) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 14 eq of EtOAc and 25 eq of water. Pd/C (0.01 eq by weight) was added and the mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate about 100%, after about 3.5 hrs), the catalyst was filtered off and washed with a mixture of 3.5 eq of EtOAc and 6 eq of water. The aqueous layer was then ready for spray-drying (Aqueous H-Gly-MePro-Glu-OH peptide solution: Purity: 98.6%; Yield: ~95%).

Procedure 4G

This Procedure is for a variant of Procedure 4A, replacing Pd/C with Pd/Si, EtOAc with MeOH, and removing H2O. Pd/Si type 340 (0.02 eq by weight) was added to 2.9 vols of MeOH for pre-reduction during 30 min. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 34 eq of MeOH. The reduced palladium was then transferred to the peptide mixture. The mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. Pd/C type 39 (0.007 eq by weight) was added to the mixture to increase reaction kinetics. At the end of the deprotection, the catalyst was filtered off and washed with 13.6 eq of MeOH. The deprotected peptide was then precipitated in 71 eq of iPrOH. After about 40 min, the peptide was filtered and washed with 35 eq of iPrOH. The peptide was dried below 20 °C and was then ready for solubilization in water and spray-drying.

Procedure 4H

This Procedure is for a variant of Procedure 4A. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 24.8 eq of water and 13.6 eq of EtOAc. Pd/C type 39 (0.025 eq by weight) was added to the peptide mixture. The mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (19 hrs), the catalyst was removed by filtration and washed with 5.3 eq of water and 2.9 eq of EtOAc. The biphasic mixture was then decanted to remove the upper organic layer. The aqueous layer was diluted with water to reach an H-Gly-MePro-Glu-OH concentration suitable for spray-drying the solution.

Example 2: Alternative Trofinetide Manufacturing Process

An alternative method for synthesis of Trofinetide is based on U.S. Patent No.

8,546,530 adapted for a tripeptide as follows.

The persilylated compounds used to synthesis Formula (Ia) (trofinetide) are obtained by silylating a corresponding peptide or amino acid by reaction with a silylating agent, optionally in an organic solvent. The persilylated peptide or amino acid can be isolated and purified if desired. One can use the persilylated peptide or amino acid in situ, e.g. by combining a solution containing persilylated peptide or amino acid with a solution containing, optionally activated, peptide or amino acid.

In step 2, the persilylated compound of an amino acid is obtained by silylating a corresponding amino acid (for example, H-MePro-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid can be isolated and purified if desired. One can use the persilylated amino acid in situ, e.g. by combining a solution containing the persilylated amino acid with a solution containing, optionally activated, amino acid (for example, Z-Gly-OH).

In step 3, the persilylated compound of an amino acid is obtained by silylating a corresponding amino acid (for example, H-Glu-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid or peptide can be isolated and purified if desired. It is however useful to use the persilylated amino acid or peptide in situ, e.g. by combining a solution containing the persilylated amino acid with a solution containing, optionally activated (for example, by using EDC.HCl and Oxyma Pure), peptide (for example, Z-Gly-MePro-OH).

In the present invention, it is useful to use silylating agents, such as N-trialkylsilyl amines or N-trialkylsilyl amides, not containing a cyano group. Examples of such silylating reagents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, 3-(trimethylsilyl)-2-oxazolidone.

The reaction of step 2 is generally carried out at a temperature from 0 °C to 100 °C, optionally from 10 °C to 40 °C, and optionally from 15 °C to 30 °C.

The reaction of step 3 is generally carried out at a temperature from 0 °C to 100 °C, optionally from 10 °C to 60 °C, optionally from 15 °C to 50 °C.

In the reaction of step 2, generally 0.5 to 5 equivalents, optionally 1 to 3 equivalents, optionally about 1.5 to 2.5 equivalents of silylating agent are used relative to the molar amount of functional groups to be silylated. Use of 2 to 4 equivalents of silylating agent relative to the molar amount of functional groups to be silylated is also possible. “Functional groups to be silylated” means particular groups having an active hydrogen atom that can react with the silylating agent such as amino, hydroxyl, mercapto or carboxyl groups.

In the reaction of step 3, generally 0.5 to 5 equivalents, optionally 2 to 4.5 equivalents, optionally about 3 to 4 equivalents of silylating agent are used relative to the molar amount of functional groups to be silylated. Use of 2.5 to 4.5 equivalents of silylating agent relative to the molar amount of functional groups to be silylated is also possible.

It is understood that “persilylated” means an amino acid or peptide or amino acid analogue or peptide analogue in which the groups having an active hydrogen atom that can react with the silylating agent are sufficiently silylated to ensure that a homogeneous reaction medium for a coupling step is obtained.

In the process according to the invention, the reaction between the amino acid or peptide and the persilylated amino acid or peptide is often carried out in the presence of a carboxyl group activating agent. In that case the carboxylic activating reagent is suitably selected from carbodiimides, acyl halides, phosphonium salts and uronium or guanidinium salts. More optionally, the carboxylic activating agent is an acyl halide, such as isobutyl chloroformate or pivaloyl chloride or a carbodiimide, such as EDC.HC1 or DCC.

Good results are often obtained when using additional carboxylic activating reagents which reduce side reactions and/or increase reaction efficiency. For example, phosphonium and uronium salts can, in the presence of a tertiary base, for example, N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA), convert protected amino acids into activated species. Other reagents help prevent racemization by providing a protecting reagent. These reagents include carbodiimides (for example, DCC) with an added auxiliary nucleophile (for example, 1-hydroxy-benzo triazole (HOBt), 1-hydroxy-azabenzotriazole (HOAt), or Suc-OH) or derivatives thereof. Another reagent that can be utilized is TBTU. The mixed anhydride method, using isobutyl chloroformate, with or without an added auxiliary nucleophile, is also used, as is the azide method, due to the low racemization associated with it. These types of compounds can also increase the rate of carbodiimide-mediated couplings. Typical additional reagents include also bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM).

When the silylation is carried out in the presence of a solvent, said solvent is optionally a polar organic solvent, more optionally a polar aprotic organic solvent. An amide type solvent such as N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAC)

can be used. In the present invention for step 2, one can use an alkyl acetate solvent, in particular ethyl acetate is more particularly optional.

In the present invention for step 3, one can use a chlorinated hydrocarbon solvent or alkyl cyanide solvent, in particular dichloromethane or acetonitrile are more particularly optional.

In another embodiment, silylation is carried out in a liquid silylation medium consisting essentially of silylating agent and amino acid or peptide.

In the present invention, amino acid or peptide is understood to denote in particular an amino acid or peptide or amino acid analogue or peptide analogue which is bonded at its N-terminus or optionally another position, to a carboxylic group of an amino protected amino acid or peptide.

Example 3: Specifications for Compositions Containing Compounds of Formula (I)

1 ICH guideline Q3C on impurities: guideline for residual solvents

Example 4: Alternative Manufacturing of Trofinetide Example 1, Step 4, Procedure 4B

This Procedure is for a variant of Step 4, Procedure 4B. Z-Gly-MePro-Glu-OH (1 eq) was added in portions to Pd/C (0.027 eq by weight and containing 5% Pd by weight) in about 50 eq of water. The reaction mixture was hydrogenated at 20 °C at a pressure of 5 bar for at least 4 cycles of 4 hrs each. Pd/C (0.0027 eq by weight) was charged between cycles, as needed, to speed up the reaction. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Upon reaction completion the catalyst was removed by filtration, washed with water (12.5 eq) and the aqueous layer washed with EtOAc (about 14 eq). After phase separation, residual EtOAc was removed from the aqueous solution containing

trofinetide by sparging with nitrogen under vacuum at 20 °C for about 3 hrs. The aqueous solution was filtered. The final concentration of trofinetide was about 25 wt% and the solution was then ready for spray-drying to isolate the product.

Example 5: Alternative Composition of Trofinetide

A composition comprising a compound of Formula (I)

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (II):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and/or a compound of Formula (III):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 independently are selected from the group consisting of hydrogen and C1-4 alkyl, provided that least one of R1, R2, R3 and R4 is C1-4 alkyl, and wherein the composition comprises at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, or 97 wt% of the compound of Formula (I) on an anhydrous basis.

Example 6: Alternative Composition of Trofinetide

A composition comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (II):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and/or a compound of Formula (III):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 independently are selected from the group consisting of hydrogen and C1-4 alkyl, provided that least one of R1, R2, R3 and R4 is C1-4 alkyl, and wherein the composition comprises at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, or 97 wt% of the compound of Formula (Ia) on an anhydrous basis.

Example 7: A Product of Trofinetide

A product, including a kit containing a dosage form with instructions for use, comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (IIa)


or a hydrate, or pharmaceutically acceptable salt thereof, wherein the product comprises between 95 wt% and 105 wt%, such as 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101

wt%, 102 wt%, 103 wt%, or 104 wt% of the specified amount of the compound of Formula (Ia) in the product.

Example 8: A Product of Trofinetide

A product, including a kit containing a dosage form with instructions for use, comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (IIa)

 or a hydrate, or pharmaceutically acceptable salt thereof, and additionally comprising one or more compounds selected from the group consisting of Formula (III), Formula (IIIa), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), and Formula (IX), wherein the composition comprises between 95 wt% and 105 wt%, such as 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt% of the specified amount of the compound of Formula (Ia) in the product.

Example 9: Analysis of Products and Compositions

The products and compositions disclosed herein may be analyzed by liquid chromatography, a suitable chromatographic method using UPLC, e.g. using materials and conditions such as Waters Acquity CSH C18, 1.7 µm, 150 x 2.1 mm column, water with 0.1 % TFA (mobile phase A), and water/ACN 70/30 + 0.1 % TFA (mobile phase B), ranging from (4% phase A/6% phase B to 100% phase B and flushed with 4% phase A/6% phase B).

Flow rate: 0.35 ml/min, Column temperature: 40 °C, autosampler temperature: 4 °C, injection volume: 4 ml (e.g. prepared by weighing about 10 mg of powder in a 10 ml volumetric flask and diluted to volume with water). Examples of detectors are UV (ultraviolet, UV 220 nm) and MS (mass spectrometry).

INDUSTRIAL APPLICABILITY

This invention finds use in the pharmaceutical, medical, and other health care fields.

PATENT

WO2014085480 ,

claiming use of trofinetide for treating autism spectrum disorders including autism, Fragile X Syndrome or Rett Syndrome.

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro- Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

W095/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS for increasing TH-mediated dopamine production in the treatment of diseases such as Parkinson’s disease.

WO02/16408 discloses GPE analogs capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including but not limited to, injury or disease in the CNS.

Example

The following non-limiting example illustrates the synthesis of a compound of the invention, NN-dimethylglycyl-L-prolyl-L-glutamic acid.

All starting materials and other reagents were purchased from Aldrich;
BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-(γ-benzyl)-L-prolyl-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.

Soc: 79, 6180, 1957] (10 mmol) in dichloromethane (50 ml), cooled to 0 °C, was added triethylamine (1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl L-glutamate (10 mmol) was then added and the mixture stirred at 0 °C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol l“1) then dried (MgS04) and concentrated at reduced pressure to give BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (5.0 g, 95%).

(7-Benzyl)-L-prolyl-L-glutamic acid dibenzyl ester
A solution of BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 hr at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give (γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (I).

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of (7-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (10 mmol), TVN-dimethylglycine (10 mmol) and triethylamine
(10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0 °C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to yield the tri-peptide derivative.

It will be evident that following the method of the Example, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

PAPER

Tetrahedron (2005), 61(42), 10018-10035.  (CLICK HERE)

The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring system. The pyrrolidine ring was also modified by replacing the γ-CH2 group with sulfur and/or incorporation of two methyl groups at C-5.

Graphical Abstract

PAPER

Bioorganic & Medicinal Chemistry Letters (2005), 15(9), 2279-2283

A series of GPE analogues, including modifications at the Pro and/or Glu residues, was prepared and evaluated for their NMDA binding and neuroprotective effects. Main results suggest that the pyrrolidine ring puckering of the Pro residue plays a key role in the biological responses, while the preference for cis or trans rotamers around the Gly-Pro peptide bond is not important.

Graphical abstract

A series of Pro and/or Glu modified GPE analogues is described. Compounds incorporating PMe and dmP showed higher affinity for glutamate receptors than GPE and neuroprotective effects similar to those of this endogenous tripeptide in culture hippocampal neurons exposed to NMDA.

PATENT

US 20060251649

WO 2006127702

US 20070004641

US 20080145335

WO 2012102832

WO 2014085480

US 20140147491

References

  1. ^ Bickerdike MJ, Thomas GB, Batchelor DC, Sirimanne ES, Leong W, Lin H, et al. (March 2009). “NNZ-2566: a Gly-Pro-Glu analogue with neuroprotective efficacy in a rat model of acute focal stroke”. Journal of the Neurological Sciences278 (1–2): 85–90. doi:10.1016/j.jns.2008.12.003PMID 19157421S2CID 7789415.
  2. ^ Cartagena CM, Phillips KL, Williams GL, Konopko M, Tortella FC, Dave JR, Schmid KE (September 2013). “Mechanism of action for NNZ-2566 anti-inflammatory effects following PBBI involves upregulation of immunomodulator ATF3”Neuromolecular Medicine15 (3): 504–14. doi:10.1007/s12017-013-8236-zPMID 23765588S2CID 12522580.
  3. ^ Deacon RM, Glass L, Snape M, Hurley MJ, Altimiras FJ, Biekofsky RR, Cogram P (March 2015). “NNZ-2566, a novel analog of (1-3) IGF-1, as a potential therapeutic agent for fragile X syndrome”. Neuromolecular Medicine17 (1): 71–82. doi:10.1007/s12017-015-8341-2PMID 25613838S2CID 11964380.
  4. ^ Study Details – Rett Syndrome Study
  5. ^ Neuren’s trofinetide successful in Phase 2 clinical trial in Fragile X
PHASESTATUSPURPOSECONDITIONSCOUNT
3Enrolling by InvitationTreatmentRett’s Syndrome1
3RecruitingTreatmentRett’s Syndrome1
2CompletedSupportive CareInjuries, Brain1
2CompletedTreatmentFragile X Syndrome (FXS)1
2CompletedTreatmentInjuries, Brain1
2CompletedTreatmentRett’s Syndrome2
2TerminatedTreatmentConcussions1
1CompletedTreatmentBrain Injuries,Traumatic2
Legal status
Legal statusUS: Investigational New Drug
Identifiers
IUPAC name[show]
CAS Number853400-76-7 
PubChem CID11318905
ChemSpider9493869
UNIIZ2ME8F52QL
Chemical and physical data
FormulaC13H21N3O6
Molar mass315.322 g·mol−1
3D model (JSmol)Interactive image
SMILES[hide]C[C@]1(CCCN1C(=O)CN)C(=O)N[C@@H](CCC(=O)O)C(=O)O
InChI[hide]InChI=1S/C13H21N3O6/c1-13(5-2-6-16(13)9(17)7-14)12(22)15-8(11(20)21)3-4-10(18)19/h8H,2-7,14H2,1H3,(H,15,22)(H,18,19)(H,20,21)/t8-,13-/m0/s1Key:BUSXWGRAOZQTEY-SDBXPKJASA-N

////////////Tofinetide , NNZ 2566, PHASE 2, PHASE 3. NEUREN, Amino Acids, Peptides, Proteins,

CC1(CCCN1C(=O)CN)C(=O)NC(CCC(=O)O)C(=O)O

Telacebec


ChemSpider 2D Image | Telacebec | C29H28ClF3N4O2
Image result for Telacebec
Image result for Telacebec

Telacebec

  • Molecular FormulaC29H28ClF3N4O2
  • Average mass557.006 Da

Telacebec, IAP6, CAS No. 1334719-95-7телацебек [Russian] [INN]تيلاسيبيك [Arabic] [INN]特雷贝克105731334719-95-7[RN]55G92WGH3X
6-Chloro-2-ethyl-N-(4-{4-[4-(trifluoromethoxy)phenyl]-1-piperidinyl}benzyl)imidazo[1,2-a]pyridine-3-carboxamide
Imidazo[1,2-a]pyridine-3-carboxamide, 6-chloro-2-ethyl-N-[[4-[4-[4-(trifluoromethoxy)phenyl]-1-piperidinyl]phenyl]methyl]-Q203Q-203T56 AN DNJ C2 HG BVM1R D- AT6NTJ DR DOXFFF

Qurient Therapeutics and Russia licensee Infectex are developing telacebec, an oral formulation which targets QcrB subunit of the cytochrome bc1 complex, for treating multi drug resistant or extensively drug resistant Mycobacterium tuberculosis infection. Qurient is also investigating telacebec for treating buruli ulcer (an infection caused by Mycobacterium ulcerans ). In January 2021, a global phase II trial was expected to begin by December 2021 for the treatment of buruli ulcer.

syn

Angewandte Chemie, International Edition, 57(4), 1108-1111; 2018

PATENT

WO-2021018387

Novel crystalline forms of telacebec , processes for their preparation and compositions comprising them are claimed. Also claimed is their use for treating bacterial infection.

Different forms of 6-chloro-2-ethyl-AT-(4-(4-(4- (trifluoromethoxy)phenvDpiperidine-i-vDbenzvDimidazolT.2-alpyridine- 3-carboxamide

The present invention relates to different forms of the compound 6-chloro-2-ethyl-lV-(4-(4-(4-(trifhioromethoxy)phenyl)piperidine-i-yl)benzyl)imidazo[i,2-a]pyridine-3-carboxamide and to methods of making such forms/compounds. The present invention furthermore relates to mono-acid addition salts thereof, to methods of making such mono-acid addition salts and to pharmaceutical compositions comprising any of the aforementioned compounds. Furthermore, the present invention relates to uses of any of these compounds.

Tuberculosis as a disease continues to result in millions of deaths each year. Inadequate use of chemotherapy has led to an increasing number of drug resistant cases. This situation is likely to worsen with the emergence of extremely resistant strains to all currently known drugs. Current chemotherapy consists of compounds that directly target Mycobacterium tuberculosis, either by neutralizing general information pathways and critical processes such as RNA polymerization and protein synthesis inhibition or by interfering with mycobacterial specific cell envelop synthesis. The most widely used dedicated anti-tubercular drugs isoniazid, ethionamide, and pyriazin amide are pro-drugs that first require activation. They are administered to a patient for a course of several months. Patients infected with multi-drug resistant strains of M. tuberculosis may have to undergo combination therapies for extended periods of time.

WO 2011/113606 describes various anti-tubercular compounds and their use in the treatment of bacterial infections, including compound“Q203” which chemically is 6-chloro-2-ethyl-!V-(4-(4-(4-(trifluoromethoxy)phenyl)piperidine-i-yl)benzyl)imidazo[i,2-a]pyridine-3-carboxamide. In a publication by Pethe et al. (Nature Medicine, 19, 1157-1160 (2013), this compound is reported to be active against tuberculosis by interfering with the bacterial energy metabolism, inhibiting cytochrome bci activity which is an essential component of the electron transport chain required for synthesis of ATP.

Whilst the compound shows promise for future therapy of tuberculosis and related infections, there continues to be a need for forms thereof that are particularly suitable for pharmaceutical administration. In particular there is a need to provide forms that are showing an improved solubility in comparison to the free base of this compound. Furthermore, there is a need in the art to provide for forms that show an improved stability.

In a first aspect the present invention relates to a compound 6-chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidine-i-yl)benzyl)imidazo[i,2-a]pyridine-3-carboxamide ditosylate having the structure

PATENT

WO2011113606 .

WO 2017049321

WO 2012143796

PAPER

Scientific reports (2019), 9(1), 8608.

Angewandte Chemie, International Edition (2018), 57(4), 1108-1111.

European journal of medicinal chemistry (2017), 136, 420-427.

European Journal of Medicinal Chemistry (2017), 136, 420-427.

 European journal of medicinal chemistry (2017), 125, 807-815.

Nature communications (2016), 7, 12393.

Nature medicine (2013), 19(9), 1157-60

PAPER

Journal of Medicinal Chemistry (2014), 57(12), 5293-5305.

https://pubs.acs.org/doi/10.1021/jm5003606J. Med. Chem. 2014, 57, 12, 5293–5305

Publication Date:May 28, 2014
https://doi.org/10.1021/jm5003606

Abstract Image

A critical unmet clinical need to combat the global tuberculosis epidemic is the development of potent agents capable of reducing the time of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis therapy. In this paper, we report on the optimization of imidazo[1,2-a]pyridine amide (IPA) lead compound 1, which led to the design and synthesis of Q203 (50). We found that the amide linker with IPA core is very important for activity against Mycobacterium tuberculosis H37Rv. Linearity and lipophilicity of the amine part in the IPA series play a critical role in improving in vitro and in vivo efficacy and pharmacokinetic profile. The optimized IPAs 49 and 50 showed not only excellent oral bioavailability (80.2% and 90.7%, respectively) with high exposure of the area under curve (AUC) but also displayed significant colony-forming unit (CFU) reduction (1.52 and 3.13 log10 reduction at 10 mg/kg dosing level, respectively) in mouse lung.

6-Chloro-2-ethyl-N-(4-{4-[4-(trifluoromethoxy)phenyl]piperidin-1-yl}benzyl)imidazo[1,2-a]pyridine-3-carboxamide (50)

Mp = 164.0 °C; 1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 1.82–1.97 (m, 4H), 2.64–2.70 (m, 1H), 2.80–2.87 (m, 2H), 2.93 (q, J = 7.6 Hz, 2H), 3.80–3.83 (m, 2H), 4.61 (d, J = 5.2 Hz, 2H), 6.00 (br t, J = 5.2 Hz, 1H), 6.96–6.99 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.24–7.30 (m, 5H), 7.52 (dd, J = 9.6, 0.8 Hz, 1H), 9.53 (dd, J = 2.0, 0.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 13.3, 23.6, 33.4, 42.0, 43.3, 50.4, 115.4, 117.0, 121.2, 121.6, 121.9, 126.3, 128.2, 128.3, 128.7, 128.9, 144.5, 144.7, 147.7, 151.4, 151.5, 161.2; 19F NMR (376 MHz, CDCl3) δ 58.31 (s, 3F); LC/MS (ESI) m/z 557 [M + H]+; HRESIMS calcd for C29H29ClF3N4O2 [M + H]+ 557.1926, found 557.1918.

str1
str2
str3

19F NMR (376 MHz, CDCl3) δ 58.31 (s, 3F); 

13C NMR (100 MHz, CDCl3) δ 13.3, 23.6, 33.4, 42.0, 43.3, 50.4, 115.4, 117.0, 121.2, 121.6, 121.9, 126.3, 128.2, 128.3, 128.7, 128.9, 144.5, 144.7, 147.7, 151.4, 151.5, 161.2; 

1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 1.82–1.97 (m, 4H), 2.64–2.70 (m, 1H), 2.80–2.87 (m, 2H), 2.93 (q, J = 7.6 Hz, 2H), 3.80–3.83 (m, 2H), 4.61 (d, J = 5.2 Hz, 2H), 6.00 (br t, J = 5.2 Hz, 1H), 6.96–6.99 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.24–7.30 (m, 5H), 7.52 (dd, J = 9.6, 0.8 Hz, 1H), 9.53 (dd, J = 2.0, 0.8 Hz, 1H);

CLIP

June 3, 2019.  Qurient press release:

SEONGNAM-SI, South Korea–(BUSINESS WIRE)– Qurient Co. Ltd. today announced positive results from the Phase 2a EBA (early bactericidal activity) clinical trial for telacebec (Q203), a first-in-class, orally-available antibiotic for the treatment of tuberculosis (TB). Telacebec is a selective inhibitor with high specificity for the cytochrome bc1 complex of Mycobacterium tuberculosis. This complex is a critical component of the electron transport chain, and inhibition disrupts the bacterium’s ability to generate energy.

The EBA trial assessed the pharmacokinetics, safety, and activity of telacebec in three dose strength (100 mg, 200 mg and 300 mg) in the treatment of adult patients with pulmonary TB. Telacebec met the primary objective of rate of change in the time to positivity (TTP) in sputum over days 0 to 14. Telacebec was safe and well tolerated throughout the different dose strengths. Full results from EBA trial are expected to be presented at future scientific meetings.


Phase 2. EBA began July 2018 in South Africa.  As of March 2019, study is active, not enrolling.


June 2018. Q203 has a non-proprietary name assigned: telacebec. USAN: -cebec Cytochrome bc1 complex inhibitors in Mycobacterium tuberculosis.


Phase 1. Description from clinicaltrials.gov:  Randomized, double-blind, placebo-controlled, dose-escalation study in healthy male and female volunteers. Subjects randomly assigned to 1 of 7 treatment cohorts (Cohorts 1 – 7) of 8 subjects each, receiving either Q203 or placebo (6 active treatment : 2 placebo) in a fasting state. Dose escalation to the next cohort may be considered when at least 6 out of 8 subjects, in a cohort, completes all procedures and none of the subjects has a clinically significant adverse event (AE) that is being followed, or at the discretion of the PI if no drug-related serious adverse events (SAEs) have occurred. A food effect cohort will be enrolled to test administration of Q203 in a fed state, at 100 mg dose level (this dose level may change based on PK analysis results). Subjects who received 100mg dose in a fasting state will return and receive the second dose, with food. Subjects will be followed up for AEs, SAE or pregnancy for 30 days postdrug administration.

Related Links


Qurient Press Release. June 2019.Kalia NP et al. 2017. Exploiting the synthetic lethality between terminal respiratory oxidases to kill M. tuberculosis and clear host infection.. PNAS.114.7426

Related Links


//////////////Telacebec,  IAP6, 1334719-95-7, PHASE 2, QURIENT, TUBERCULOSIS, телацебек , تيلاسيبيك , 特雷贝克 , Q 203

Catequentinib, Anlotinib


Anlotinib.png
ChemSpider 2D Image | ANLOTINIB | C23H22FN3O3

Catequentinib

C23H22FN3O3  407.4 g/mol

1-[[4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxyquinolin-7-yl]oxymethyl]cyclopropan-1-amine

1058156-90-3

CAS No. 1360460-82-7 DI HCL

Molecular Weight480.36
FormulaC23H22FN3O3 • 2HCl

Anlotinib

AL3818

UNII-GKF8S4C432

Chia Tai Tianqing Pharmaceutical Group Co Ltd

Launched (Metastatic non small cell lung cancer – China – May-2018)

Orphan Drug; Priority Review

MOA:VEGFR inhibitor

Indication:advanced gastric adenocarcinoma; Advanced renal cell carcinoma (RCC); Medullary thyroid cancer (MTC); Metastatic colorectal cancer (CRC); Non small cell lung cancer (NSCLC); Soft tissue sarcoma; Ovarian cancerStatus:Phase III (Active)

AL-3818 ; AL-3818, Jiangsu Chia-tai Tianqing Pharmaceutical ; FOCUS-V ; FuKeWei ; VEGFR2/VEGFR3 inhibitor (capsule, cancer), Jiangsu Chia Tai Tianqing Pharmaceutical ; anlotinib ; anlotinib dihydrochloride ; catequentinib ; catequentinib ; catequentinib dihydrochloride

NMR  https://file.selleckchem.com/downloads/nmr/S872601-Anlotinib-AL3818-hnmr-selleck.pdf

Anlotinib (AL3818) is a highly potent and selective VEGFR2 inhibitor with IC50 less than 1 nM. It has broad-spectrum antitumor potential in clinical trials.

Anlotinib dihydrochloride is in phase II/III clinical trials for the treatment of metastatic colorectal cancer and advanced gastric adenocarcinoma. The compound was co-developed by CTTQ Pharmaceutical (正大天晴) and Advenchen Laboratory.

It is also in phase II clinical trials for the treatment of ovarian cancer, endometrial cancer, non small cell lung cancer (NSCLC), medullary thyroid cancer (MTC), soft tissue sarcoma and advanced renal cell carcinoma (RCC).

In 2015, orphan drug designation was received in the U.S. for the treatment of ovarian cancer.

PATENT

WO 2016179123

https://patents.google.com/patent/WO2016179123A1/en

new process to synthesize l-((4-(4-Fluoro-2-methyl- lH- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by condensing intermediate (XI) with (Yl) in a solvent at the presence of KI or Nal, or intermediate (X2) with (Y2) in a solvent to form intermediate (Z) which is deprotected to give the final compound (AL3818) in Scheme I. A stable crystalline form of l-((4-(4-Fluoro-2 -methyl- lH-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)-methyl)cyclopropanamine and its salts as well as crystalline forms of salts have also been prepared.

Figure imgf000004_0001

Wherein, R is selected from H and Ci-Cealkoxy.

Process A

Figure imgf000007_0001

R is selected from H and C1 -C6 alkoxy

The final compound (AL3818) was prepared according to Process Al when R is H by deprotecting intermediate (Z-l) with HCOONH4 (ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25°C-80°C for 0.1-4 hours. (Z-l) was prepared by reacting intermediate (XI) with (Yl-1) at the presence of KI or Nal with K2CO3 in a solvent, such as acetone or DMF, at a temperature of 60°C-160°C for 2-24 hours.

Process Al (R=H)

Figure imgf000008_0001

The final compound (AL3818) was prepared according to Process A2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0°C-30°C for 1-24 hours. (Z-2) was prepared by reacting intermediate (XI) with (Y 1-2) at the presence of KI or Nal with K2C03 in a solvent, such as acetone or DMF, at a temperature of 60°C -160°C for 2-24 hours.

Process A2 (R=4-OMe)

Figure imgf000008_0002

The present invention relates a new process to synthesize l-((4-(4-Fluoro-2 -methyl- 1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by reacting intermediate (X2) with (Y2) in a solvent to form intermediate (Z) which is deprotected to give the final compound (AL3818) according to Process B. Proce B

Figure imgf000009_0001

R is selected from H and C1-C6 alkoxy

The final compound (AL3818) was prepared according to Process Bl when R is H by deprotecting intermediate (Z-1) with HCOONH4 (ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25°C-80°C for 0.1-4 hours. (Z-1) was prepared by reacting intermediate (X2-1) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60°C – 160°C for 1-12 hours.

Process Bl R=H)

Figure imgf000009_0002

The final compound (AL3818) was prepared according to Process B2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0°C-30°C for 1-24 hours. (Z-2) was prepared by reacting intermediate (X2-2) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60°C -160°C for 1-12 hours.

Process B2 (R=4-OMe)

Figure imgf000009_0003

The following examples further illustrate the present invention, but should not be construed as in any way to limit its scope.

Example 1

Representation of Process A, Process Al

Process for preparation of l-((4-(4-Fluoro-2 -methyl- lH-indol-5-yloxy)-6-methoxy- quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

To a stirred mixture of benzyl l-(hydroxymethyl)cyclopropylcarbamate (50 g) and DCM (200 ml) was added DIPEA (39g). The result solution was cooled to 0-5 °C with ice/water and further stirred under this temperature for 15 min. MsCl (30g) was added via an addition funnel dropwise keeping temperature below 5°C for about 1.5 hours. After completion of addition, the reaction mixture was allowed stirring at 0-5°C for 30 min and quenched with saturated NaHC03 (150 ml). The solution was extracted with 150 ml DCM twice. The combined DCM layer was washed with 0.1 N HCl (400 ml) followed by brine. It was dried over Na2S04 and concentrated to obtain an off-white solid 60 gram as (l-(benzyloxycarbonylamino)cyclopropyl)methyl methanesulfonate (Yl-1), MS: (M+l) 300.

To a stirred mixture of (Yl-1) (16 g), XI [(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6- methoxy-7-hydroxyquinoline, 12 g] , K2CO3 (21 g) and KI (21 g) was added DMF (100 ml), the reaction suspension was heated at 80°C for 10 hours and (Yl-l) (10 g) was added to continuously heated 80°C for 10 hours. The reaction then was quenched with water (150 ml) and extracted with 150 ml DCM twice. The combined DCM layer was washed with 2 N NaOH (100 ml) followed by water and brine. It was dried over Na2SC>4 and concentrated, further recrystallized from EtOH to obtain a yellow solid as benzyl l-((4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin- 7-yloxy)methyl)cyclopropylcarbamate (Z-l) 9.5 g. MS: (M+l) 542.

To a stirred mixture of (Z-l) (9.5 g), HCOONH4 (4.7 g) and Pd/C (10%, wet 50%, 4.7g) was added MeOH, the reaction mixture was heated at 45°C for 1.5 hours. It was then cooled and filtered through Celite, further evaporated. 2N HCl (200 ml) was added and extracted with DCM/MeOH (10/1, 100 ml) twice. The aqueous layer was basified with 3N NaOH to adjust pH 11-12 to generate a solid precipitation. The solid was filtered and washed with water to neutral, further suction dry. The solid was dissolved into a mixture of DCM/MeOH (250 ml, 10/1) and further washed with water and brine. It was dried with MgS04 and filtered, further evaporated to give a light yellow solid 5.5 g crude product. Further purification was conducted by dissolving the crude product into DCM/MeOH (40 ml, 10/1) to triturate with petroleum ether (40 ml) for 2 hours slow stirring. The precipitate was filtered and dried in an oven to give the final crystalline product 4.4 g (MP: 203-208 C) and it can be further purified by recrystallizing from EtOH to give purer final product as a same crystalline form. MS: (M+l) 408; ¾ NMR(DMSO-dg) δ 0.60- 0.63(d, 4H), 2.41(s, 1H), 2.42-2.5 l(t, 2H), 3.3 l(s, 2H), 3.96(s, 3H), 4.04(s, 2H), 6.27(s, 1H), 6.31-6.32(m, 1H), 6.97-7.02(t, 1H), 7.20-7.22(d, 1H), 7.36(s, 1H), 7.60(s, 1H), 8.40-8.42(d, 1H), 1 1.41(s, 1H). MP: 208-210°C; DSC Melting Range (Endo): 207-220°C with Peak Temp=216°CPATENTWO 2019154273https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=11C1DF5485B11ADA40E45C9488AB5679.wapp1nB?docId=WO2019154273&tab=FULLTEXT
Tyrosine kinases are a group of enzymes that catalyze the phosphorylation of protein tyrosine residues. They play an important role in intracellular signal transduction. They are involved in the regulation, signal transmission and development of normal cells, and are also related to tumor cells. Proliferation, differentiation, migration and apoptosis are closely related. Many receptor tyrosine kinases are related to the formation of tumors, and can be divided into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and vascular endothelial cell growth factor receptor according to the structure of their extracellular region. Body (VEGFR), Fibroblast Growth Factor Receptor (FGFR), etc.[0003]WO2008112407 discloses the compound 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy in Example 24 )Methyl)cyclopropylamine and its preparation method, its structural formula is shown in formula I:[0004]

[0005]It is a multi-target receptor tyrosine kinase inhibitor that can inhibit the activity of vascular endothelial cell growth factor receptors (VEGFR1, VEGFR2/KDR and VEGFR3), stem cell factor receptors, platelet-derived growth factor receptors and other kinase activities. Inhibit the downstream signal transduction mediated by VEGFR2, thereby inhibiting tumor angiogenesis.[0006]Solid drugs generally have multiple crystal forms, such as polymorphs, solvates (hydrates), salts, and co-crystals. The change in the crystal form of the same drug usually results in different melting points, solubility, stability, biological activity, etc., which are important factors that affect the difficulty of drug preparation, storage stability, preparation difficulty, and bioavailability. . When the compound has multiple crystal forms, due to the specific thermodynamic properties and stability of the specific crystal form of the drug, it is important to understand the crystal form of the compound used in each dosage form during the preparation process to ensure the production process Use the same form of medicine. Therefore, it is necessary to ensure that the compound is a single crystal form or a known mixture of some crystal forms.[0007]WO2016179123 discloses the crystalline form 1 of the free base anhydrate of the compound of formula I and a preparation method thereof. CN201010245688.1 discloses the anhydrate and dihydrate crystals of quinoline derivative dihydrochloride and the preparation method thereof.[0008]The discovery of a variety of new crystal forms of medicinal compounds provides an opportunity to improve the physical properties of the drug, that is, to expand all the properties of the substance, which can better guide the research of the compound and its preparation. Therefore, the quinoline derivative provided in this application The crystals and pharmaceutical compositions containing the crystals have commercial value in the manufacture of medicines and other applications.Example 1 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropylamine (Formula I compound) preparation[0081]

[0082]Put intermediate 1 (its chemical name is (1-((4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxy Quinolin-7-yl)oxy)methyl)cyclopropyl)benzyl carbamate) 100g, 10% palladium on carbon 30g, ammonium formate 50g and methanol 800ml. Incubate the reaction at 45-55°C, TLC tracking showed that the reaction was complete, filtered, the filter cake was washed with a small amount of methanol, the filtrate was concentrated to dryness under reduced pressure, ethyl acetate and 2mol/L hydrochloric acid were added, stirred for 10 minutes, and then stood for 10 minutes. Separate the aqueous phase, adjust the pH to above 12 with 4N sodium hydroxide, and a large amount of solids will precipitate out. After washing with water until neutral, the aqueous phase is filtered to obtain the crude product of the title compound.[0083]Example 2 Preparation of amorphous compound of formula I[0084]According to the preparation method disclosed in Example 24 of WO2008112407, 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yl (Oxy)methyl)cyclopropylamine is composed of (1-(((4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl )Oxy)methyl)cyclopropyl)benzyl carbamate (Intermediate 1) was prepared according to the following methods 2.1 and 2.2.[0085]2.1 Take 100 mg of Intermediate 1 and Pd/C (10%, 40 mg) into ethanol (20 ml), and hydrogenate at 50 psi for 12 hours. The reaction solution was filtered with diatomaceous earth, and evaporated to obtain an amorphous compound of formula I, and its X-ray powder diffraction (XRD) pattern was obtained as shown in FIG. 11.[0086] 
2.2 Take 100 mg of Intermediate 1, acetic acid (1ml) and 33% hydrobromic acid/acetic acid (1ml) and mix. The reaction was stirred for 1 hour at room temperature, diluted with ethyl acetate/water, and then basified with sodium carbonate. The organic layer is dried, concentrated, and purified by silica gel column to obtain the amorphous compound of formula I.PATENTUS 20160326138https://patents.google.com/patent/US20160326138A1/enNew process has been outlined in Scheme I.

Figure US20160326138A1-20161110-C00001
  • The present invention relates a new process to synthesize 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by condensing intermediate (X1) with (Y1) in a solvent at the presence of KI or NaI to form intermediate (Z) which is deprotected to give the final compound (AL3818) according to Process A.
  • [0040]
    The final compound (AL3818) was prepared according to Process A1 when R is H by deprotecting intermediate (Z-1) with HCOONH(ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25° C.-80° C. for 0.1-4 hours. (Z-1) was prepared by reacting intermediate (X1) with (Y1-1) at the presence of KI or NaI with K2COin a solvent, such as acetone or DMF, at a temperature of 60° C.-160° C. for 2-24 hours.
  • [0041]
    The final compound (AL3818) was prepared according to Process A2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0° C.-30° C. for 1-24 hours. (Z-2) was prepared by reacting intermediate (X1) with (Y1-2) at the presence of KI or NaI with K2COin a solvent, such as acetone or DMF, at a temperature of 60° C.-160° C. for 2-24 hours.
  • [0042]
    The present invention relates a new process to synthesize 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by reacting intermediate (X2) with (Y2) in a solvent to form intermediate (Z) which is deprotected to give the final compound (AL3818) according to Process B.
  • [0043]
    The final compound (AL3818) was prepared according to Process B1 when R is H by deprotecting intermediate (Z-1) with HCOONH(ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25° C.-80° C. for 0.1-4 hours. (Z-1) was prepared by reacting intermediate (X2-1) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60° C.-160° C. for 1-12 hours.
  • [0044]
    The final compound (AL3818) was prepared according to Process B2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0° C.-30° C. for 1-24 hours. (Z-2) was prepared by reacting intermediate (X2-2) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60° C.-160° C. for 1-12 hours.
  • [0045]
    The following examples further illustrate the present invention, but should not be construed as in any way to limit its scope.

Example 1Representation of Process A, Process A1Process for preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

  • [0046]
    To a stirred mixture of benzyl 1-(hydroxymethyl)cyclopropylcarbamate (50 g) and DCM (200 ml) was added DIPEA (39 g). The result solution was cooled to 0-5° C. with ice/water and further stirred under this temperature for 15 min. MsCl (30 g) was added via an addition funnel dropwise keeping temperature below 5° C. for about 1.5 hours. After completion of addition, the reaction mixture was allowed stirring at 0-5° C. for 30 min and quenched with saturated NaHCO(150 ml). The solution was extracted with 150 ml DCM twice. The combined DCM layer was washed with 0.1 N HCl (400 ml) followed by brine. It was dried over Na2SOand concentrated to obtain an off-white solid 60 gram as (1-(benzyloxycarbonylamino)cyclopropyl)methyl methanesulfonate (Y1-1), MS: (M+1) 300.
  • [0047]
    To a stirred mixture of (Y1-1) (16 g), X1 [(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-7-hydroxyquinoline, 12 g], K2CO(21 g) and KI (21 g) was added DMF (100 ml), the reaction suspension was heated at 80° C. for 10 hours and (Y1-1) (10 g) was added to continuously heated 80° C. for 10 hours. The reaction then was quenched with water (150 ml) and extracted with 150 ml DCM twice. The combined DCM layer was washed with 2 N NaOH (100 ml) followed by water and brine. It was dried over Na2SOand concentrated, further recrystallized from EtOH to obtain a yellow solid as benzyl 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropylcarbamate (Z-1) 9.5 g. MS: (M+1) 542.
  • [0048]
    To a stirred mixture of (Z-1) (9.5 g), HCOONH(4.7 g) and Pd/C (10%, wet 50%, 4.7 g) was added MeOH, the reaction mixture was heated at 45° C. for 1.5 hours. It was then cooled and filtered through Celite, further evaporated. 2N HCl (200 ml) was added and extracted with DCM/MeOH (10/1, 100 ml) twice. The aqueous layer was basified with 3N NaOH to adjust pH 11-12 to generate a solid precipitation. The solid was filtered and washed with water to neutral, further suction dry. The solid was dissolved into a mixture of DCM/MeOH (250 ml, 10/1) and further washed with water and brine. It was dried with MgSOand filtered, further evaporated to give a light yellow solid 5.5 g crude product. Further purification was conducted by dissolving the crude product into DCM/MeOH (40 ml, 10/1) to triturate with petroleum ether (40 ml) for 2 hours slow stirring. The precipitate was filtered and dried in an oven to give the final crystalline product 4.4 g (MP: 203-208° C.) and it can be further purified by recrystallizing from EtOH to give purer final product as a same crystalline form. MS: (M+1) 408; 1H NMR (DMSO-d6) δ 0.60-0.63 (d, 4H), 2.41 (s, 1H), 2.42-2.51 (t, 2H), 3.31 (s, 2H), 3.96 (s, 3H), 4.04 (s, 2H), 6.27 (s, 1H), 6.31-6.32 (m, 1H), 6.97-7.02 (t, 1H), 7.20-7.22 (d, 1H), 7.36 (s, 1H), 7.60 (s, 1H), 8.40-8.42 (d, 1H), 11.41 (s, 1H). MP: 208-210° C.; DSC Melting Range (Endo): 207-220° C. with Peak Temp=216° C. TGA demonstrating as an unsolvated material with weight loss at about 210° C. (between 205-215° C.). XRPD having pattern comprising characteristic 10 peaks with intensity % greater than 10% expressed in d values and angles as follows:
  • Angle d value 13.344 6.62986 15.858 5.58405 16.799 5.27326 17.640 5.02377 18.770 4.72373 20.650 4.29771 21.633 4.10463 23.087 3.84934 25.128 3.54112 26.607 3.34755
  • [0049]
    It was similar prepared according to the preparation procedures of (Z-1) described in Example 1 by using 4-methoxybenzyl 1-(hydroxymethyl)cyclopropylcarbamate to first generate (1-((4-methoxybenzyloxy)carbonylamino)cyclopropyl)methyl methanesulfonate (Y1-2) then to give 4-methoxybenzyl 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)-methyl)cyclopropylcarbamate (Z-2), MS: (M+1) 572
  • [0050]
    To a stirred mixture of (Z-2) (1.5 g) in DCM (15 ml) at 0° C. was added TFA (1.5 ml) for about 30 min and warmed up to RT. The reaction was stirred at RT for 2 hours and added into water (30 ml). The aqueous layer was extracted with DCM twice (100 ml×2) and basified with 2N NaOH to adjust pH 11-12. The mixture was extracted with DCM (100 ml×3) and further washed with brine (100 ml). It was dried with MgSOand filtered. The solution was evaporated to give 1.05 g crude final product. Further purification was conducted to dissolve the crude product into DCM/MeOH and triturated with petroleum ether and dried in an oven to give the final pure product 0.8 g AL3818 with the same crystalline form.

Example 3Representation of Process A, Process B1Process for preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

  • [0051]
    To a mixture of benzyl 1-((4-chloro-6-methoxyquinolin-7-yloxy)methyl)cyclopropyl-carbamate (X2-1) (5 g), 4-fluoro-2-methyl-1H-indol-5-ol (Y2) (5 g) and DMAP (4 g) was added 1,6-lutidine (15 ml). The reaction was stirred and heated at 135° C. for 5 hours and was cooled followed by adding IPA with slow stirring for 2 hours at RT. The solid was filtered and further washed with IPA, dried to give (Z-1) 5.2 g as a solid. It was then similarly prepared according to deprotection procedures described of (Z-1) in Example 1 to give the final compound AL3818 with the same crystalline form.

Example 4Representation of Process A, Process B2Process for preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

  • [0052]
    (Z-2) was similarly prepared according to the procedures described in Example 3 by using 4-methoxybenzyl 1-((4-chloro-6-methoxyquinolin-7-yloxy)methyl)cyclopropylcarbamate (X2-2) and (Y2). It was then similarly prepared according to deprotection procedures of (Z-2) described in Example 2 to give the final compound AL3818 with the same crystalline form.

Example 5

  • [0053]
    Preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)-methyl)cyclopropanamine bishydrochloride acid salt and its crystalline
  • [0054]
    To a 25 ml flask was added 250 mg free base (AL3818), 4N HCl in dioxane 0.625 mL (2.5 mmol, 4 eq.) in 10 ml EtOH, the reaction was heated at 75° C. for 30 minutes, cooled to RT and stirred for O.N. The solid was filtered and rinsed with acetone twice. It was dried in oven at 50° C. for 4 hours to give 126 mg white solid as the bishydrochloride salt as a crystalline and further recrystallized from EtOH to give a purer product as a same crystalline form. 1H NMR (DMSO-d6) δ 1.09-1.24 (m, 4H), 2.43 (s, 3H), 4.08 (s, 3H), 4.40 (s, 2H), 6.32 (s, 1H), 6.76 (s, 1H), 7.05-7.11 (t, 1H), 7.27-7.30 (d, 1H), 7.65 (s, 1H), 7.82 (s, 1H), 8.64 (s, 2H), 8.70-8.73 (m, 1H), 11.51 (s, 1H). Chloride ion chromatography showed 2 molecular ratio ions (16.1%). DSC Melting Range (Exo): 249-280 with Peak Temp=268° C.
  • [0055]
    To a 10 mL flask, charged 140 mg of 3818-2HCl salt from above Example 4 and 0.7 mL (×5 with salt volume) of 80% MeOH in H2O. The result suspension was heated to 70° C. to form a solution and cooled to RT and further stirred for O.N. The solid was filtered and rinsed with acetone twice. It was dried in oven at 50° C. for 4 hours to obtain off-white solid 110 mg as the crystalline bishydrochloride hydrate salt. 1H NMR (DMSO-d6) δ 1.09 (s, 2H), 1.22 (s, 2H), 2.44 (s, 1H), 2.52 (s, 2H), 4.09 (s, 3H), 4.44 (s, 2H), 6.32 (s, 1H), 6.81-6.82 (d, 1H), 7.08-7.14 (t, 1H), 7.29-7.32 (d, 1H), 7.79 (s, 1H), 7.85 (s, 1H), 8.75-8.78 (d, 1H), 8.85 (s, 2H), 11.66 (s. 1H). Chloride ion chromatography showed 2 molecular ratio ions (17.8%). DSC Melting Range (Exo): 207-260° C. with Peak Temp=226° C. TGA demonstrating 2.68% (˜3%, 1 water) weight loss till 120° C. (between 115-125° C.) and further weight loss at about 170° C. (between 165-175° C.).

PATENT

US8148532B2.

https://patents.google.com/patent/US8148532B2/en

Patent

2. US20080227811A1.

/////////////catequentinib, ANLOTINIB, AL3818, AL 3818, PHASE 2, CHINA 2018

NC1(CC1)COc1cc2nccc(Oc3ccc4[NH]c(C)cc4c3F)c2cc1OC

TILDACERFONT


Tildacerfont.png

img

TILDACERFONT

Synonyms:

Tildacerfont

1014983-00-6

3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(1-ethyl-propyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidine

7-(1-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidine

MW/ MF 420 g/mol/ C20H26ClN5OS
  • Originator Spruce Biosciences
  • Class2 ring heterocyclic compounds; Morpholines; Pyrazoles; Pyrimidines; Small molecules; Thiazoles
  • Mechanism of Action Corticotropin receptor antagonists
  • Orphan Drug Status Yes – Congenital adrenal hyperplasia
  • New Molecular Entity Yes
  • Phase II Congenital adrenal hyperplasia
  • 09 Jul 2020 Spruce Biosciences initiates a phase II trial in Congenital adrenal hyperplasia in USA (PO) (NCT04457336)
  • 24 Sep 2019 Spruce Biosciences completes a phase II trial in Congenital adrenal hyperplasia in USA (NCT03687242)
  • 19 Sep 2019 Updated safety and efficacy data from a phase II trial in Congenital adrenal hyperplasia release by Spruce Biosciences

Deuterated pyrazolo[1,5-a]pyrimidine derivatives, particularly tildacerfont (SPR-001), useful as CRF antagonists for treating congenital adrenal hyperplasia.  Spruce Bioscience is developing tildacerfont under license from Lilly as an oral capsule formulation for the treatment of congenital adrenal hyperplasia; in July 2017, a phase II trial for CAH was initiated.

Corticotropin releasing factor (CRF) is a 41 amino acid peptide that is the primary physiological regulator of proopiomelanocortin (POMC) derived peptide secretion from the anterior pituitary gland. In addition to its endocrine role at the pituitary gland, immunohistochemical localization of CRF has demonstrated that the hormone has a broad extrahypothalamic distribution in the central nervous system and produces a wide spectrum of autonomic, electrophysiological and behavioral effects consistent with a neurotransmitter or neuromodulator role in the brain. There is also evidence that CRF plays a significant role in integrating the response in the immune system to physiological, psychological, and immunological stressors.

PATENT

Product case, WO2008036579 ,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2008036579

Example 16
3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl- pyrazolo [ 1 ,5 -α]pyrimidine

Under a nitrogen atmosphere dissolve 3-(4-bromo-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl-pyrazolo[l,5-α]pyrimidine (116 mg, 0.25 mmol) in THF (1.5 mL) and chill to -78 0C. Add n-butyl lithium (0.1 mL. 2.5 M in hexane, 0.25 mmol) and stir at -78 0C for 30 min. Add N-chlorosuccinimide (33.4 mg, 0.25 mmol) and stir for another 30 min, slowly warming to room temperature. After stirring overnight, quench the reaction by adding a solution of saturated ammonia chloride and extract with ethyl acetate. Wash the organic layer with brine, dry over sodium sulfate, filter, and concentrate to a residue. Purify the crude material by flash chromatography, eluting with hexanes:dichloromethane: ethyl acetate (5:5:2) to provide the title compound (54 mg). MS (APCI) m/z (35Cl) 420.6 (M+l)+1H NMR (400 MHz, CDCl3): 6.44 (s, IH), 3.79 (t, 4H, J=4.8 Hz), 3.63-3.56 (m, IH), 3.47 (t, 4H, J=4.8 Hz), 2.55 (s, 3H), 2.45 (s, 3H), 1.88-1.75 (m, 4H), 0.87 (t, 6H, J=7.5 Hz).
Alternate Preparation from Preparation 6:
Combine 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-α]pyrimidine, (9 g,

26.2 mmol) and 4-chloro-2-morpholino-thiazole (7.5 g, 36.7 mmol) in
dimethylformamide (90 mL) previously degassed with nitrogen. Add cesium carbonate (17.8 g, 55 mmol), copper iodide (250 mg, 1.31 mmol), triphenylphosphine (550 mg, 2.09 mmol) and palladium acetate (117 mg, 0.52 mmol). Heat the mixture to 125 0C for 16 h and then cool to 22 0C. Add water (900 mL) and extract with methyl-?-butyl ether (3 x 200 mL). Combine the organic portions and evaporate the solvent. Purify by silica gel chromatography eluting with hexanes/ethyl acetate (4/1) to afford the title compound (6.4 g, 62%). ES/MS m/z (35Cl) 420 (M+l)+.

Example 16a
3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl- pyrazolo[l,5-α]pyrimidine, hydrochloride
Dissolve 3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl-pyrazolo[l,5-α]pyrimidine (1.40 g, 3.33 mmol) in acetone (10 mL) at 50 0C and cool to room temperature. Add hydrogen chloride (2 M in diethyl ether, 2.0 mL, 4.0 mmol) and stir well in a sonicator. Concentrate the solution a little and add a minimal amount of diethyl ether to crystallize the HCl salt. Cool the mixture in a refrigerator overnight. Add additional hydrogen chloride (2 M in diethyl ether, 2.0 mL, 4.0 mmol) and cool in a refrigerator. Filter the crystalline material and dry to obtain the title compound (1.15 g, 75%). ES/MS m/z (35Cl) 420 (M+l)+1H NMR(CDCO): 9.18 (br, IH), 6.86 (s, IH), 3.72 ( m, 4H), 3.49(m, IH), 3.39 (m, 4H), 2.48 (s, 3H), 2.38(s, 3H), 1.79 (m, 4H), 0.79 (m, 6H).

PATENT

US-20200255436

https://patentscope.wipo.int/search/en/detail.jsf?docId=US301567348&tab=PCTDESCRIPTION&_cid=P22-KE0UZI-30504-1

PATENT

WO2019210266

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019210266

claiming the use of CRF-1 antagonists (eg tildacerfont).

PATENT

WO 2010039678

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2010039678

EXAMPLES

Example 1 : 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolori ,5-alpyrimidine nthroline 

Charge 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (1.03 g, 3.00 mmoles), K3PO4 (1.95 g, 9.00 mmoles), 2,4-dichlorothiazole (0.58 g, 3.75 mmoles), 1,10 phenanthroline (0.05 g, 0.30 mmoles) and anhydrous DMAC (5 mL) to a round bottom flask equipped with a magnetic stir bar, thermal couple and N2 inlet. Degas the yellow heterogeneous reaction mixture with N2 (gas) for 30 min. and then add CuI (0.06 g, 0.30 mmoles) in one portion followed by additional 30 min. degassing with N2 (gas). Stir the reaction mixture at 120 0C for about 6 hr. Cool the reaction mixture to room temperature overnight, add toluene (10 mL) and stir for 1 hr. Purify the mixture through silica gel eluting with toluene (10ml). Extract with 1 M HCl (10 mL), water (10 mL), brine (10 mL) and concentrate under reduced pressure to give a yellow solid. Recrystallize the solid from methanol (5ml) to yield the title compound as a yellow crystalline solid. (0.78 g, 70% yield, >99% pure by LC) MS(ES) = 369 (M+ 1). 1H NMR (CDCl3)= 6.5 (IH, s); 3.6 (IH, m); 2.6 (3H, s); 2.5 (3H, s); 1.9 (4H, m); 0.9 (6H, t).

Example 2: 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolol! ,5-aipyrimidine

Charge 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (0.37 g, 1.00 mmoles), K2CO3 (0.28 g, 2.00 mmoles) and anhydrous morpholine (3 mL) to a round bottom flask equipped with a magnetic stir bar and N2 inlet. Stir the yellow mixture at 100 0C for about 4 hr., during which time the reaction becomes homogeneous. Cool the reaction mixture to room temperature, add H2O (10 mL) and stir the heterogeneous reaction mixture overnight at room temperature. Collected the yellow solid by filtration, wash with H2O and allowed to air dry overnight to give the crude title compound (391mg). Recrystallize from isopropyl alcohol (3 mL) to yield the title compound as a light yellow crystalline solid (380 mg, 90.6% yield, >99% by LC). MS(ES) = 420 (M+l). 1H NMR (CDCl3)= 6.45 (IH, s); 3.81 (m, 4H); 3.62 (IH, m); 3.50 (m, 4H); 2.6 (3H, s); 2.45 (3 H, s); 1.85 (4H, m); 0.9 (6H, t).

Example 3 :

The reactions of Example 1 are run with various other catalysts, ligands, bases and solvents, which are found to have the following effects on yield of 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine. (See Tables 1 – 4).

Table 1 : Evaluation of different li ands

(Reactions are carried out in parallel reactors with 1.2 mmol 2,4-dichlorothiazole, 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.5 mmol CuI, 0.5 mmol ligand and 2.1 mmol Cs2CO3 in 4 mL DMAC. The reactions are degassed under N2 for 30 min. and then heated at between 80 and

1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak. Longer reaction times are shown in parenthesis) Table 2: Evaluation of various solvents


(Reactions are carried out in parallel reactors with 1.2 mmol 2,4-dichlorothiazole 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.25 mmol CuI, 0.25 mmol 1,10-phenanthroline and 2.1 mmol Cs2CO3 in 3 mL specified solvent. The reactions are degassed under N2 for 30 minutes and then heated at 1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak.)

Table 3 : Evaluation of different copper sources

(Reactions are carried out in in parallel reactors with 1 mmol 2,4-dichlorothiazole 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.05 mmol CuX, 0.01 mmol 1,10-phenanthroline and 3 equivalents K3PO4 in 3 mL DMAC. The reactions are degassed under N2 for 30 minutes and then heated at 1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak.)

Table 4: Evaluation of various inorganic bases

(Reactions are carried out in in parallel reactors with 1 mmol 2,4-dichlorothiazole 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.1 mmol CuI, 0.1 mmol 1,10-phenanthroline and 2.1 mmol base and degassed for 30 minutes prior to the addition of 3 mL DMAC. The reactions are degassed under N2 for 10 minutes and then heated at 1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak.)

Example 4. Use of morpholine both as a reactant and base in 2-MeTHF as solvent.

solvent

7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-ajpyrimidine (15.2 g, 41.16 mmoles) is charged into a 250 mL 3-necked round bottomed flask, followed by addition of 2-MeTHF (61 mL, 4.0 volumes), the yellowish brown slurry is stirred at about 20 0C for 5 min. Then morpholine (19 g, 218.18 mmoles) is added over 2-5 minutes. Contents are heated to reflux and maintained at reflux for 12 hr. The slurry is cooled to 25 0C, followed by addition of 2-MeTHF (53 mL, 3.5 volumes) and water ( 38 mL 2.5 volumes). The reaction mixture is warmed to 40 0C, where upon a homogenous solution with two distinct layers formed. The layers are separated, the organic layer is filtered and concentrated to ~3 volumes at atmospheric pressure. Four volumes 2-propanol (61 mL) are added. The solution is concentrated to ~3 volumes followed by addition of 4 volumes 2-propanol (61 mL), re-concentrated to ~3 volumes, followed by addition of another 6 volumes 2-propanol (91 mL), and refluxed for 15 min. The clear solution is gradually cooled to 75 0C, seeded with 0.45 g 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine slurried in 2 mL 2-propanol, rinsed with an additional 2 mL 2-propanol and transferred to a crystallization flask. The slurry is cooled to between 0-5 0C, maintained for 1 hr, filtered and the product rinsed with 2-propanol (30 mL, 2 volumes). The solid is dried at 60 0C in a vacuum oven to afford 16.92 g 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine. Purity of product by HPLC assay is 100.00 %. XRPD and DSC data of product is consistant with reference sample. MS(ES) = 420 (M+ 1).

Example 5. Use of morpholine as both reactant and base in 2-propanol as solvent.

7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-ajpyrimidine (11.64 mmoles) is charged into a 100 mL 3 -necked round bottomed flask followed by addition of 2-propanol ( 16 mL, 3.72 volumes). The yellowish brown slurry is stirred at about 20 0C for 5 min. Then morpholine (3.3 g, 37.84 mmoles) is added over 2-5 minutes. Contents are refluxed for 6 hr. The slurry is cooled to 25 0C. 2-Propanol ( 32 mL, 7.44 volumes) and water ( 8.6 mL, 2.0 volumes) are added and the mixture warmed to 70-75 0C, filtered and concentrated to ~ 9 volumes at atmospheric pressure. The clear solution is gradually cooled to 55 0C, seeded with 0.06 g of crystalline 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine slurried in 0.5 mL 2-propanol, rinsed with additional 0.5 mL 2-propanol and added to crystallization flask. The slurry is cooled to 0-5 0C, maintained for 1 hr., filtered and the product rinsed with 2-propanol ( 9 mL, 2.1 volumes). Suctioned dried under vacuum at 60 0C to afford 4.6 g of dry 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (88.8 % yield, purity by HPLC assay is 99.88 % ). MS(ES) = 420 (M+ 1).

Example 6: 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolori ,5-alpyrimidine

7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (10 g, 29.17 mmoles), 2, 4-dichlorothiazole (5.2 g , 33.76 mmoles), cesium carbonate(19.9g, 61.07 mmoles) and 1,10-phenanthroline (1 g, 5.5 mmoles) are charged into a 250 mL 3-necked round bottomed flask, followed by 2-MeTHF (36 mL, 3.6 volumes). The reaction mixture is degassed with nitrogen and then evacuated. Cuprous chloride (0.57 g, 5.7 mmoles), DMAC (10 mL, 1 volume) and 2-MeTHF (4 mL, 0.4 volumes) are added in succession. The reaction mixture is degassed with nitrogen and then evacuated. The contents are refluxed for 20 hr. The reaction mixture is cooled to -70 0C and 2-MeTHF (100 mL, 10 volumes) is added. The contents are filtered at ~70 0C and the residual cake is washed with 2-MeTHF (80 mL, 8 volumes) at about 65-72°C. The filtrate is transferred into a separatory funnel and extracted with water. The organic layer is separated and washed with dilute HCl. The resulting organic layer is treated with Darco G60, filtered hot (600C). The filtrate is concentrated at atmospheric pressure to -2.8 volumes. 25 mL 2-propanol is added, followed by re-concentration to -2.8 volumes. An additional 25 mL 2-propanol is added, followed again by re-concentration to -2.8 volumes. Finally, 48 mL 2-propanol is added. The contents are cooled to -7 0C, maintained at -7 0C for 1 hr., filtered and rinsed with 20 mL chilled 2-propanol. Product is suction dried and then vacuum dried at 60 0C to afford 9.41 g 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (purity of product by HPLC assay is 95.88 %). MS(ES) = 369 (M+ 1).

Example 7. Synthesis of 7-(l-ethyl-propyl)-3-(2, 4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolori,5-a1pyrimidine using 1,4-Dioxane solvent and CuCl catalyst

Add dioxane (9.06X), Cs2CO3 (2.00X), 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (1.0 equivalent), 2,4-dichlorothiazole (0.54 equivalent) to a reactor under N2. Purge the reactor with N2 three times, degas with N2 for 0.5-1 hr., and then add 1,10-phenanthroline (0.3 eq) and CuCl (0.3eq) under N2 , degassing with N2 for 0.5-1 hr. Heat the reactor to 1000C -1100C under N2 . Stir the mixture for 22-24 hr. at 100 0C -1100C. Cool to 10~20°C and add water (10V) and CH3OH (5V), stir the mixture for 1-1.5 hr. at 10~20°C. Filter the suspension, resuspend the wet cake in water, stirr for 1-1.5 hr. at 10~20°C, and filter the suspension again. Charge the wet cake to n-heptane (16V) and EtOAc (2V) under N2. Heat the reactor to 40 °C~500C under N2.

Active carbon (0. IX) is added at 40 °C~500C. The reactor is heated to 55°C~650C under N2 and stirred at 55 °C~650C for 1-1.5 hr. The suspension is filtered at 40~55°C through diatomite (0.4 X). The cake is washed with n-heptane (2.5V). The filtrate is transferred to another reactor. EtOAc (10V) is added and the the organic layer washed with 2 N HCl (10V) three times, followed by washing two times with water (10X, 10V). The organic layer is concentrated to 3-4V below 500C. The mixture is heated to 80-90 0C. The mixture is stirred at this temperature for 40-60 min. The mixture is cooled to 0~5°C, stirred for 1-1.5 hr. at 0~5°C and filtered. The cake is washed with n-heptane (IV) and vacuum dried at 45-500C for 8-10 hr. The crude product is dissolved in 2-propanol (7.5V) under N2, and re-crystallized with 2-propanol. The cake is dried in a vacuum oven at 45°C~50°C for 10-12 hr. (55-80% yield). 1H NMR56.537 (s, IH) 3.591-3.659 (m, IH, J=6.8Hz), 2.593 (s, 3H), 2.512 (s, 3H), 1.793-1.921(m, 4H), 0.885-0.903 (m, 6H).

REFERENCES

1: Zorrilla EP, Logrip ML, Koob GF. Corticotropin releasing factor: a key role in the neurobiology of addiction. Front Neuroendocrinol. 2014 Apr;35(2):234-44. doi: 10.1016/j.yfrne.2014.01.001. Epub 2014 Jan 20. Review. PubMed PMID: 24456850; PubMed Central PMCID: PMC4213066.

/////////////tildacerfont, SPR 001, Orphan Drug Status, Congenital adrenal hyperplasia, SPRUCE BIOSCIENCES, PHASE 2

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