Merck’s Novel Indoline Cholesterol Ester Transfer Protein Inhibitors (CETP)

ote


Indoline 7  as in ACS MEDCHEM LETTERS, DOI: 10.1021/acsmedchemlett.5b00404
and
eg 10 as in WO2015054088
(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.
1H-​Indole-​1-​ethanol, 2,​3-​dihydro-​3-​[3-​(trifluoromethoxy)​phenyl]​-​3-​[[3-​ (trifluoromethoxy)​phenyl]​methyl]​-​α-​(trifluoromethyl)​-​, (αR)​-
cas 1699732-96-1 R ISOMER

MF C₂₆ H₂₀ F₉ N O_3, MW 565.43

Merck Sharp & Dohme Corp. INNOVATOR

Using the collective body of known (CETP) inhibitors as inspiration for design, a structurally novel series of tetrahydroquinoxaline CETP inhibitors were discovered. An exemplar from this series, compound 5, displayed potent in vitro CETP inhibition and was efficacious in a transgenic cynomologus-CETP mouse HDL PD (pharmacodynamic) assay. However, an undesirable metabolic profile and chemical instability hampered further development of the series. A three-dimensional structure of tetrahydroquinoxaline inhibitor 6 was proposed from ¹H NMR structural studies, and this model was then used in silico for the design of a new class of compounds based upon an indoline scaffold. This work resulted in the discovery of compound 7, which displayed potent in vitro CETP inhibition, a favorable PK–PD profile relative to tetrahydroquinoxaline 5, and dose-dependent efficacy in the transgenic cynomologus-CETP mouse HDL PD assay.

chemical compounds that inhibit cholesterol ester transfer protein (CETP) and are expected to have utility in raising HDL-C, lowering LDL-C, and in the treatment and prevention of atherosclerosis.
see………….http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.5b00404
http://pubs.acs.org/doi/suppl/10.1021/acsmedchemlett.5b00404/suppl_file/ml5b00404_si_001.pdf

Discovery of Novel Indoline Cholesterol Ester Transfer Protein Inhibitors (CETP) through a Structure-Guided Approach

Jonathan E. Wilson^*†, Ravi Kurukulasuriya†, Mikhail Reibarkh‡, Maud Reiter, Aaron Zwicker, Kake Zhao†, Fengqi Zhang†, Rajan Anand†, Vincent J. Colandrea, Anne-Marie Cumiskey§, Alejandro Crespo‡, Ruth A. Duffy§, Beth Ann Murphy§, Kaushik Mitra∥, Douglas G. Johns⊥, Joseph L. Duffy†, and Petr Vachal†

^†Department of Medicinal Chemistry and ^‡Department of Structural Chemistry, Merck Research Laboratories, Merck & Co, Inc., P.O. Box 2000, Rahway, New Jersey 07065, United States

^§Department of Pharmacology, ^∥Department of Drug Metabolism and Pharmacokinetics, and ^⊥Department of Biology, Merck Research Laboratories, Merck & Co, Inc., P.O. Box 2000, Kenilworth, New Jersey 07033, United States

ACS Med. Chem. Lett., Article ASAP

DOI: 10.1021/acsmedchemlett.5b00404

Publication Date (Web): January 4, 2016

Copyright © 2016 American Chemical Society

*E-mail: jonathan_wilson@merck.com.

 PATENT

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=5D4A5D79894298F5BC0A664CF54B61E9.wapp2nB?docId=WO2013028382&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Atherosclerosis and its clinical consequences, including coronary heart disease
(CHD), stroke and peripheral vascular disease, represent a truly enormous burden to the health care systems of the industrialized world. In the United States alone, approximately 13 million patients have been diagnosed with CHD, and greater than one half million deaths are attributed to CHD each year. Further, this toll is expected to grow over the next quarter century as an epidemic in obesity and diabetes continues to grow.
It has long been recognized that in mammals, variations in circulating lipoprotein profiles correlate with the risk of atherosclerosis and CHD. The clinical success of HMG-CoA reductase inhibitors, especially the statins, in reducing coronary events is based on the reduction of circulating low density lipoprotein cholesterol (LDL-C), levels of which correlate directly with an increased risk for atherosclerosis. More recently, epidemiologic studies have
demonstrated an inverse relationship between high density lipoprotein cholesterol (HDL-C) levels and atherosclerosis, leading to the conclusion that low serum HDL-C levels are associated with an increased risk for CHD.
Metabolic control of lipoprotein levels is a complex and dynamic process involving many factors. One important metabolic control in man is the cholesteryl ester transfer protein (CETP), a plasma glycoprotein that catalyzes the movement of cholesteryl esters from HDL to the apoB containing lipoproteins, especially VLDL (see Hesler, C.B., et. al. (1987) Purification and characterization of human plasma cholesteryl ester transfer protein. J. Biol. Chem. 262(5), 2275-2282)). Under physiological conditions, the net reaction is a heteroexchange in which CETP carries triglyceride to HDL from the apoB lipoprotein and transports cholesterol ester from HDL to the apoB lipoprotein.
In humans, CETP plays a role in reverse cholesterol transport, the process whereby cholesterol is returned to the liver from peripheral tissues. Intriguingly, many animals do not possess CETP, including animals that have high HDL levels and are known to be resistant to coronary heart disease, such as rodents (see Guyard-Dangremont, V., et. al, (1998)
Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 120(3), 517-525). Numerous epidemiologic studies correlating the effects of natural variation in CETP activity with respect to coronary heart disease risk have been performed, including studies on a small number of known human null mutations (see Hirano, K.-L, Yamashita, S. and Matsuzawa, Y. (2000) Pros and cons of inhibiting cholesteryl ester transfer protein, Curr. Opin. Lipidol. 11(6), 589-596). These studies have clearly demonstrated an inverse correlation between plasma HDL-C concentration and CETP activity (see Inazu, A., et. al. (2000) Cholesteryl ester transfer protein and atherosclerosis, Curr. Opin. Lipidol. 11(4), 389-396), leading to the hypothesis that pharmacologic inhibition of CETP lipid transfer activity may be beneficial to humans by increasing levels of HDL-C while lowering LDL-C.
Despite the significant therapeutic advance that statins such as simvastatin and atorvastatin represent, statins only achieve a risk reduction of approximately one-third in the treatment and prevention of atherosclerosis and ensuing atherosclerotic disease events.
Currently, few pharmacologic therapies are available that favorably raise circulating levels of HDL-C. Certain statins and some fibrates offer modest HDL-C gains. Niacin provides an effective therapy for raising HDL-C but suffers from patient compliance issues, due in part to side effects such as flushing. Drugs that inhibit CETP (CETP inhibitors) have been under development with the expectation that they will effectively raise HDL cholesterol levels and also reduce the incidence of atherosclerosis in patients. Torcetrapib was the first drug that was tested in a long-term outcomes clinical trial. The clinical trial of torcetrapib was terminated early due to a higher incidence of mortality in patients to whom torcetrapib and atorvastatin were administered concomitantly compared with patients who were treated with atorvastatin alone. The cause of the increased mortality is not completely understood, but it is not believed to be associated with the CETP inhibiting effects of the drug.
Two other drug candidates, dalcetrapib and anacetrapib, are currently being tested in Phase III clinical trials, including large scale outcomes trials. Data from the recently completed DEFINE Phase III trial of anacetrapib are promising. Patients who were being treated with anacetrapib along with baseline statin therapy showed an increase of HDL-C of 138% and a decrease of LDL-C of 40%> compared with patients who were treated with just a statin. See: N. Engl. J. Med. 2010: 363: 2406-15. The data in the DEFINE trial were sufficient to indicate that an increase in mortality for patients treated with anacetrapib is unlikely. Additional drug candidates are still being sought that may have properties that are advantageous compared with the CETP inhibitors that have so far been studied or are currently being studied. Such properties may include, for example, higher potency, reduced off-target activity, better pharmacodynamics, higher bioavailability, or a reduced food effect compared with many of the highly lipophilic compounds that have so far been studied. “Food effect” refers to the variability in exposure to the active drug that occurs depending on when the patient had last eaten, whether or not the drug is administered with food, and the fat content of the food.

Example 18 as in patent

(R)- 1,1, 1 -trifluoro-3-((R)-4-(3-trifluoromethoxy)benzyl)-2-(3-(l, 1 ,2,2,-tetrafluoroethoxy)phenyl)-3,4- dihydroquinoxalin- 1 (2H)-yl)propan-2-ol
SPA: 15 nM
Example 18 was prepared from 2-bromo-l-(3-(l , 1 ,2,2,-tetrafluoroethoxy)phenyl)ethanone in three steps, using the reactions detailed in Schemes A6, A2 and Al . Spectral data are as follows: 1H NMR (400 MHz, CDC1₃) £2.70 (bd, J=4.1 Hz, IH), 3.24 (dd, J=l 1.3, 3.4 Hz, IH), 3.34 (dd, J=15.5, 9.7 Hz, IH), 3.58 (dd, J=l 1.3, 3.3 Hz, IH), 3.86 (d, J=15.4 Hz, IH), 4.20 (d, J=15.7 Hz, IH), 4.40 (d, J=15.8 Hz, IH), 4.46 (m, IH), 4.927 (t, J=3.3 Hz, IH), 5.90 (tt, J=53.1 , 2.7 Hz, IH), 6.59 (d, J= 7.9 Hz, IH), 6.72 (m, 2H), 6.84 (m, 2H), 6.92 (d, J=7.6 Hz, IH), 7.20 (m, 2H), 7.35 (t, J=7.9 Hz, IH), MS m/z = 613.03.
Scheme A12

Methyl 3 – { 1 – [(R)-3 ,3 ,3 -trifluoro-2-hy droxypropyl] -4- [3 -(trifluoromethoxy) benzyl]-l,2,3,4-tetrahydroquinoxalin-2-yl}benzoate (700 mg, 1.262 mmol) is made as described in Example 16 but with one stereochemical center unresolved. The compound was dissolved in MeOH (12.6mL), lithium hydroxide monohydrate (530 mg, 12.62 mmol) was added, and the reaction mixture was heated to 60°C for 4 hours. The crude mixture was dissolved in saturated ammonium chloride solution and extracted into EtOAc, the organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and purified on a silica gel column with a 0-100% Hex/EtOAc gradient. The major peak was concentrated to afford 3-{l-[(R)-3,3,3-trifluoro-2-hydroxypropyl]-4-[3-(trifluoromethoxy)benzyl]-l,2,3,4-tetra-hydroquinoxalin-2-yl} benzoic acid. MS m/z = 541.09.

1H and 13C NMR spectra for compound 7

(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.

Patent
WO2015054088
http://google.com/patents/WO2015054088A1?cl=en
Scheme Al

Scheme A2

Scheme A3

R = Ar, NR_2l C0₂R, CN, S0₂Me
es
es

SEE EXAMPLE ………SIMILAR BUT NOT SAME

Example 1. (2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethyl)-phenyl)indolin-l-yl)propan-2-ol. This material was prepared according to Scheme Al, as described below.

3-(3-(trifluoromethyl)phenyl)indolin-2-one. Oxindole (1.598 g, 12 mmol), 3-bromo-a,a,a-trifluoromethyltoluene (2.009 ml, 14.40 mmol), potassium carbonate (3.32 g, 24.00 mmol), Pd₂dba₃ (0.220 g, 0.240 mmol), and 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropylbiphenyl (0.458 g, 0.960 mmol) were combined in THF (12 ml) and the mixture was degassed with nitrogen. The solution was then heated to 80 °C for 18h. The mixture was cooled to room temperature, filtered through silica eluting with ethyl acetate, and concentrated. The material was then purified by silica gel chromatography (Biotage lOOg SNAP cartridge, 0-50% ethyl acetate in hexanes) to provide 3-(3-(trifluoromethyl)phenyl)indolin-2-one as a white solid.
1H NMR (500 MHz) δ 8.58 (s, 1H), 7.61 (d, J=7 Hz, 1H), 7.53-7.45 (m, 3H), 7.33-7.29 (m, 1H), 7.16 (d, J=7 Hz, 1H), 7.10 (m, 1H), 7.01-6.90 (m, 1H), 4.73 (s, 1H).

3 -(3 -(trifluoromethoxy)benzyl)-3 -(3 -(trifluoromethyl)phenyl)indolin-2-one . 3 -Trifluoromethoxy-benzylbromide (0.204 ml, 1.255 mmol) was added to a mixture of 3-(3-(trifluoromethyl)-phenyl)indolin-2-one (290 mg, 1.046 mmol) and potassium carbonate (289 mg, 2.092 mmol) (sodium carbonate may be used in place of potassium carbonate) in DMA (2.5 ml). The mixture was stirred at r.t. for 16h. The reaction was diluted with ethyl acetate and washed with water (3×5 mL). The organic layer was dried with Na₂S0₄, filtered, and concentrated. The products were then purified by silica gel chromatography (Biotage 50g SNAP cartridge; 0-40%> ethyl acetate in hexanes) to provide 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)-phenyl)indolin-2-one .
1H NMR (500 MHz) δ 7.79 (s, 1H), 7.73 (d, J=7 Hz, 1H), 7.62-7.60 (m, 2H), 7.51 (t, J=7 Hz, 1H), 7.26- 7.22 (m, 2H), 7.14 (t, J=7.0 Hz, 1H), 7.11 (m, 1H), 6.97 (m, 1H), 6.92 (m, 1H), 6.78 (m, 1H), 6.73 (s, 1H), 3.77 (d, J=13 Hz, 1H), 3.49 (d, J=13 Hz, 1H).
LCMS m/z = 451.8 (M+H)

3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indoline. Borane tetrahydrofuran complex (1.673 ml, 1.673 mmol) was added to a solution of 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indolin-2-one (302 mg, 0.669 mmol) in THF (1.5 ml). The mixture was heated to 70 °C for 20h. The reaction was cooled to room temperature and quenched with saturated NH₄C1 solution, and this mixture was stirred vigorously for 20 minutes. The product was extracted with ethyl acetate. The extracts were dried over Na₂S0₄, filtered, and concentrated. The product was purified by silica gel chromatography (Biotage 25g SNAP cartridge, 0-50% ethyl acetate in hexanes) to provide 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indoline. This material may also be used without purification in the final step of the sequence, epoxide opening.
1H NMR (500 MHz) δ 7.66 (s, IH), 7.59 (d, J=7 Hz, IH), 7.53 (d, J=7 Hz, IH), 7.45 (t, J=8 Hz, IH), 7.18-7.13 (m, 2H), 7.04 (d, J=8 Hz, IH), 6.98 (d, J=7 Hz, IH), 6.81 (t, J=7.5 Hz, IH), 6.71 (m, 2H), 6.60 (s, IH), 3.83 (m, IH), 3.75-3.73 (m, 2H), 3.46 (d, J=13 Hz, IH), 3.41 (d, J=13 Hz, IH).
= 437.9 (M+H)

(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)-phenyl)indolin-l-yl)propan-2-ol. (S)-2-(trifluoromethyl)oxirane (81 μΐ, 0.933 mmol) was added to a solution of 3-(3-(trifluoromethoxy)benzyl)-3-(3-(trifluoromethyl)phenyl)indoline (136 mg, 0.311 mmol) in l,l,l,3,3,3-hexafluoro-2-propanol (412 μΐ, 3.91 mmol). The reaction was stirred at room temperature overnight. The solvent was removed and the product was purified by silica gel chromatography (Biotage 25 g SNAP cartridge; 0-25% ethyl acetate in hexanes) to provide (2R)- 1 ,1,1 -trifluoro-3 -(3 -(3 -(trifluoromethoxy)benzyl)-3 -(3 -(trifluoromethyl)phenyl)indolin- 1 -yl)propan-2-ol.
1H NMR (500 MHz) (mixture of diastereomers) δ 7.72 (s, 0.5 H), 7.69 (s, 0.5 H), 7.65 (d, J=6.5 Hz, 0.5 H), 7.61 (d, J=7.5 Hz, 0.5 H), 7.56 (s, 1H), 7.50 (m, 1H), 7.25-7.17 (m, 2H), 7.07 (broad s, 2H), 6.91-6.89 (m, 1H), 6.79-6.75 (m, 1H), 6.53 (m, 2H), 4.00 (broad s, 1H), 3.83 (d, J= 9 Hz, 0.5H), 3.77 (d, J=9 Hz, 0.5H), 3.59-3.55 (m, 1H), 3.45-3.43 (m, 1H), 3.39-3.29 (m, 2H), 3.21-3.15 (m, 1H), 2.32 (m, 0.5H), 2.15 (m, 0.5H).
LCMS m/z = 549.8 (M+H)
Examples 1-25, in the table below, were prepared according to Scheme Al in a

SEE EG 10…….(2R)- 1,1,1 -trifluoro-3-(3-(3-(trifluoromethoxy)benzyl)-3-(3- (trifluoromethoxy)-phenyl)indolin-l-yl)propan-2-ol.

ABOUT AUTHOR

Jonathan Wilson

Associate Principal Scientist at Merck

https://www.linkedin.com/in/jonathan-wilson-23206523

Experience

Associate Principal Scientist

Merck

October 2013 – Present (2 years 4 months)

Senior scientist

Merck

May 2009 – October 2013 (4 years 6 months)

Postdoctoral researcher

Princeton University

October 2007 – May 2009 (1 year 8 months)

Associate Medicinal Chemist

Merck

2000 – 2002 (2 years)

Education

Massachusetts Institute of Technology

Ph.D., Organic Chemistry

2002 – 2007

Oberlin College

B. A., Chemistry

1996 – 2000

///////CETP inhibition, cholesterol ester transfer protein, HDL,  indoline,  tetrahydroquinoxaline, merck, discovery
c21ccccc1N(C[C@@]2(c3cccc(c3)OC(F)(F)F)Cc4cc(ccc4)OC(F)(F)F)C(C(F)(F)F)O
FC(F)(F)Oc1cccc(c1)C3(CN(C[C@@H](O)C(F)(F)F)c2ccccc23)Cc4cccc(OC(F)(F)F)c4



///////////see..........http://newdrugapprovals.org/2016/01/06/mercks-novel-indoline-cholesterol-ester-transfer-protein-inhibitors-cetp/

1 Vote

• 

MW 422.79,  MF C₁₈ H₁₄ Cl F₃ N₆ O
cas 1627748-32-6
1,​2,​4-​Triazolo[4,​3-​a]​pyrazin-​8(5H)​-​one, 7-​[[2-​chloro-​3-​(trifluoromethyl)​phenyl]​methyl]​-​6,​7-​dihydro-​6-​methyl-​3-​(2-​pyrazinyl)​-​, (6S)​-
(6S)-7-[[2-chloro-3-(trifluoromethyl)phenyl]methyl]-6-methyl-3-pyrazin-2-yl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8-one
(6S)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one

Janssen Pharmaceutica Nv INNOVATOR
Michael K. Ameriks, Jason C. Rech, Brad M. Savall

(6S)-7-[[2-chloro-3-(trifluoromethyl)phenyl]methyl]-6-methyl-3-pyrazin-2-yl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8-one

PAPER

The synthesis, SAR, and preclinical characterization of a series of substituted 6,7-dihydro[1,2,4]triazolo[4,3]pyrazin-8(5H)-one P2X7 receptor antagonists are described. Optimized leads from this series comprise some of the most potent human P2X7R antagonists reported to date (IC₅₀s < 1 nM). They also exhibit sufficient potency and oral bioavailability in rat to enable extensive in vivo profiling. Although many of the disclosed compounds are peripherally restricted, compound 11d is brain penetrant and upon oral administration demonstrated dose-dependent target engagement in rat hippocampus as determined by ex vivo receptor occupancy with radiotracer 5 (ED₅₀ = 0.8 mg/kg).

Volume 26, Issue 2, 15 January 2016, Pages 257–261

Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists

• Michael K. Ameriks, et al

• Janssen Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States

doi:10.1016/j.bmcl.2015.12.052

http://www.sciencedirect.com/science/article/pii/S0960894X15303656

Scheme 3.

Synthesis of compounds 11d and 11l–t. Reagents and conditions: (a) Boc₂O, NaOH, H₂O/MeOH, 0 °C→rt (42%); (b) 2-chloro-3-trifluoromethylbenzaldehyde, Na(OAc)₃BH, DCE, rt (85%); (c) methyl chlorooxoacetate, Et₃N, CH₂Cl₂, 0 °C→rt (97%); (d) 4 N HCl/dioxane, rt, then Et₃N, CH₂Cl₂, rt (100%); (e) Et₃O⁺BF₄⁻, DCM, rt, or Lawesson’s reagent, THF, 55 °C (67–99%); (f) RCONHNH₂, 1-butanol, 130 °C (27–90%).

PATENT
US 20140275096
http://www.google.com/patents/US20140275096

Intermediate 1. 3-(pyrazin-2-yl)-6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one
• 
Step A. tert-butyl 3-ethoxy-5,6-dihydropyrazine-1(2H)-carboxylate

• To a solution of tert-butyl 3-oxopiperazine-1-carboxylate (1 g, 5 mmol) in DCM (15 mL) was added triethyloxonium tetrafluoroborate (2.9 g, 15 mmol). Stirred for 2 h and neutralized with sat. aq NaHCO₃. Layers separated and aqueous layer extracted with DCM. Combined organic layers dried over Na₂SO₄, filtered, and concentrated to give the title compound, which was used directly without further purification.
Step B. tert-butyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate

• To a solution of tert-butyl 3-ethoxy-5,6-dihydropyrazine-1(2H)-carboxylate (1.14 g, 5 mmol) in 1-butanol (30 mL) was added pyrazine-2-carbohydrazide (685 mg, 5 mmol). The reaction mixture was heated at reflux for 16 h. After cooling to rt, the reaction mixture was concentrated and purified by chromatography (SiO2; 2.5% MeOH in DCM) to afford the desired product as a white solid (700 mg, 50% over 2 steps). MS (ESI): mass calcd. for C₁₄H₁₈N₆O₂, 302.2; m/z found, 303.2 [M+H]⁺.
• 1H NMR (500 MHz, CDCl3) d 9.57 (d, J=1.4 Hz, 1H), 8.62 (d, J=2.5 Hz, 1H), 8.59-8.54 (m, 1H), 4.94 (s, 2H), 4.63-4.50 (m, 2H), 3.89 (t, J=5.4 Hz, 2H), 1.51 (s, 9H).
Step C. 3-(pyrazin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine

• To a solution of tert-butyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate (9.3 g, 30 mmol) in DCM (100 mL) was added 1.25M HCl in EtOH (30 mL, 37.5 mmol). After 3 h, the reaction mixture was concentrated, and the resulting solid was purified by chromatography (SiO₂; 10% MeOH in DCM) to provide the desired product as a white solid (3.7 g, 61%). MS (ESI): mass calcd. for C₉H₁₀N₆, 202.1; m/z found, 203.1 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD) δ 9.35 (d, J=1.4 Hz, 1H), 8.72 (dd, J=2.5, 1.6 Hz, 1H), 8.66 (d, J=2.6 Hz, 1H), 4.50 (t, J=5.6 Hz, 2H), 4.22 (s, 2H), 3.24 (t, J=5.6 Hz, 2H).
Step D. 2-(trimethylsilyl)ethyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate

• To a solution of 3-(pyrazin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine (1.0 g, 5.0 mmol) and N,N-diisopropylethylamine (1.7 mL, 9.9 mmol) in DMF (15 mL) was added 1-[2-(trimethylsilyl)ethoxycarbonyloxy]pyrrolidin-2,5-dione (1.5 g, 5.9 mmol). Stirred for 18 h and poured into ice cold brine (150 mL). Precipitate filtered and washed successively with water and ether to afford the desired product as a white solid (1.5 g, 89%). MS (ESI): mass calcd. for C₁₅H₂₂N₆O₂Si, 346.2; m/z found, 347.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 9.50 (d, J=1.4 Hz, 1H), 8.56 (d, J=2.5 Hz, 1H), 8.52-8.48 (m, 1H), 4.91 (s, 2H), 4.60-4.45 (m, 2H), 4.25-4.14 (m, 2H), 3.87 (t, J=5.3 Hz, 2H), 1.07-0.92 (m, 2H), 0.01-0.04 (m, 9H).
Step E. 2-(trimethylsilyl)ethyl 8-oxo-3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate

• To a vigorously stirred solution of 2-(trimethylsilyl)ethyl 3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate (172 mg, 0.5 mmol) in 1:1 CHCl₃:MeCN (3.8 mL) was added a solution of ruthenium (IV) oxide hydrate (9.8 mg, 0.07 mmol) and sodium metaperiodate (504 mg, 2.3 mmol) in water (4.7 mL). After 4 h, the reaction mixture was diluted with water and extracted with CHCl₃ (×3). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford a green oil. Purification by chromatography (SiO₂; EtOAc—10% IPA/EtOAc) provided the desired product as a white solid (663 mg, 63%).
• [0140]
  MS (ESI): mass calcd. for C₁₅H₂₀H₆O₃Si, 360.1; m/z found, 361.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 9.59 (d, J=1.5 Hz, 1H), 8.63 (d, J=2.5 Hz, 1H), 8.55 (dd, J=2.5, 1.6 Hz, 1H), 4.88-4.75 (m, 2H), 4.47-4.33 (m, 2H), 4.33-4.24 (m, 2H), 1.18-1.04 (m, 2H), 0.04-(−0.02) (m, 9H).
Step F. 3-(pyrazin-2-yl)-6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one

• To a solution of 2-(trimethylsilyl)ethyl 8-oxo-3-(pyrazin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate (1.0 g, 2.9 mmol) in DCM (29 mL) was added TFA (5.7 mL, 75 mmol). After 1 h, the reaction mixture was concentrated. The crude residue was diluted with EtOAc, sonicated, and filtered to provide the desired product as a white solid (1.2 g, 95%). MS (ESI): mass calcd. for C₉H₈N₆O, 216.1; m/z found, 217.1 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ 9.39 (d, J=1.1 Hz, 1H), 8.77 (q, J=2.6 Hz, 2H), 8.56 (s, 1H), 4.73-4.60 (m, 2H), 3.67-3.55 (m, 2H).

Intermediate 3. (±)-6-methyl-3-(pyrazin-2-yl)-6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one
• 
• Intermediate 3 was made in a manner analogous to Intermediate 1 substituting (±)-tert-butyl 2-methyl-5-oxopiperazine-1-carboxylate for tert-butyl 3-oxopiperazine-1-carboxylate in Step A. MS (ESI): mass calcd. for C₁₀H₁₀N₆O, 230.1; m/z found, 231.1 [M+H]⁺.
Intermediate 4. (6S)-1-(2-chloro-3-(trifluoromethyl)benzyl)-6-methylpiperazine-2,3-dione

• [0146]
  
Step A. (S)-tert-butyl(2-aminopropyl)carbamate

• To a solution of (S)-1,2-diaminopropane dihydrochloride (16 g, 109 mmol) in MeOH (64 mL) and water (16 mL) was added di-tert-butyl dicarbonate (28.5 g, 131 mmol) in MeOH (16 mL). The resulting solution was cooled in an ice bath, and 4N NaOH (35 mL, 140 mL) was added dropwise over 2 h. The mixture was allowed to warm to rt and stirred for a total of 20 h. The reaction was filtered, and the filtrate concentrated to remove MeOH. 200 mL EtOAc, 200 mL water, and 16 mL 1M HCl were added sequentially. The layers were separated and the aqueous layer washed with EtOAc (200 mL). The combined organic extracts were washed with 0.04M HCl (208 mL). The organic phase was separated and discarded. The aqueous phases were combined, adjusted to pH=14 with 10N NaOH (20 mL), and extracted with DCM (400 mL×2). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford the desired product as a clear oil (8.0 g, 42%). MS (ESI): mass calcd. for C₈H₁₈N₂O₂, 174.1; m/z found, 175.2 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 5.01 (br s, 1H), 3.24-3.09 (m, 1H), 3.09-2.95 (m, 1H), 2.92-2.84 (m, 1H), 1.45 (s, 9H), 1.35-1.19 (m, 2H), 1.07 (d, J=6.4 Hz, 3H).
Step B. (6S)-tert-butyl(2-((2-chloro-3-(trifluoromethyl)benzyl)amino)propyl) carbamate

• A solution of (S)-tert-butyl(2-aminopropyl)carbamate (4.0 g, 23 mmol) and 2-chloro-3-trifluoromethylbenzaldehyde (4.8 g, 23 mmol) in DCE (100 mL) was stirred at rt for 2 h. Sodium triacetoxyborohydride (7.3 g, 34 mmol) was added at once and stirring continued overnight. Saturated aqueous NaHCO₃ was added, and the resulting mixture was extracted with DCM (×2). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford a clear oil. Purification by chromatography (SiO₂; hex—60% EtOAc/hex) provided the desired product as a clear oil (7.2 g, 85%). MS (ESI): mass calcd. for C₁₆H₂₂ClF₃N₂O₂, 366.1; m/z found, 367.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.72-7.56 (m, 2H), 7.35 (t, J=7.7 Hz, 1H), 4.94 (s, 1H), 3.99 (d, J=14.1 Hz, 1H), 3.90 (d, J=14.1 Hz, 1H), 3.29-3.14 (m, 1H), 3.11-2.99 (m, 1H), 2.84 (dd, J=11.1, 6.2 Hz, 1H), 1.44 (s, 9H), 1.11 (d, J=6.4 Hz, 3H).
Step C. (6S)-methyl 2-((1-((tert-butoxycarbonyl)amino)propan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate

• To an ice cold solution of (6S)-tert-butyl(2-((2-chloro-3-(trifluoromethyl)benzyl)amino)propyl) carbamate (7.2 g, 20 mmol) and triethylamine (2.8 mL, 21 mmol) in DCM (121 mL) was added methyl chlorooxoacetate (1.9 mL, 21 mmol) dropwise. The resulting mixture was warmed to rt and stirred overnight. After diluting with brine, the layers were separated, and the aqueous layer washed with DCM. The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford the desired product as a white solid (8.5 g, 97%). ¹H NMR (400 MHz, CDCl₃) δ 7.72-7.56 (m, 1H), 7.49-7.32 (m, 2H), 4.83 (d, J=17.1 Hz, 1H), 4.79-4.62 (m, 1H), 4.51 (d, J=17.1 Hz, 1H), 4.11-3.97 (m, 1H), 3.93 (s, 3H), 3.24-3.13 (m, 2H), 1.44 (s, 9H), 1.16-1.12 (m, 3H).
Step D. (6S)-methyl 2-((1-aminopropan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate hydrochloride

• To a solution of 4M HCl in dioxane (75 mL) was added (6S)-methyl 2-((1-((tert-butoxycarbonyl)amino)propan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate (7.5 g, 16.7 mmol). After 30 minutes, the reaction mixture was concentrated and the product was used in the next step without further purification (6.5 g, 100%). MS (ESI): mass calcd. for C₁₄H₁₆ClF₃N₂O₃, 352.1; m/z found, 353.1 [M+H]⁺.
Step E. (6S)-1-(2-chloro-3-(trifluoromethyl)benzyl)-6-methylpiperazine-2,3-dione

• To a solution of (6S)-methyl 2-((1-aminopropan-2-yl)(2-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate hydrochloride (7.3 g, 18.9 mmol) in DCM (90 mL) was added triethylamine (7.9 mL, 57 mmol) at once. After 2 h, 1N HCl was added and the layers were separated. The aqueous layer was extracted with DCM (×2). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated to afford the desired product as a white solid (5.9 g, 98%). MS (ESI): mass calcd. for C₁₃H₁₁ClF₃N₂O₂, 320.1; m/z found, 321.1 [M+H]⁺. ¹H NMR (600 MHz, CDCl₃) δ 8.24 (d, J=3.6 Hz, 1H), 7.68 (dd, J=7.8, 1.1 Hz, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 5.22 (d, J=15.7 Hz, 1H), 4.52 (d, J=15.7 Hz, 1H), 3.82-3.73 (m, 1H), 3.69-3.61 (m, 1H), 3.31 (ddd, J=13.2, 5.2, 2.3 Hz, 1H), 1.46-1.38 (m, 3H).
• 
  
• Example 14

(±)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one………..
……………………(±) FORM

• 
• Example 14 was made in a manner analogous to Example 2 substituting Intermediate 3 for Intermediate 1 and 1-(bromomethyl)-2-chloro-3-(trifluoromethyl)benzene for 1-(bromomethyl)-2,3-dichlorobenzene to provide the desired compound as a white solid (102 mg, 63%). MS (ESI): mass calcd. for C₁₈H₁₄ClF₃N₆O, 422.1; m/z found, 423.1 [M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) 89.48 (d, J=1.2 Hz, 1H), 8.84-8.82 (m, 2H), 7.85-7.82 (m, 2H), 7.56 (t, J=7.8 Hz, 1H), 5.20 (d, J=16.5 Hz, 1H), 4.98 (dd, J=13.8, 2.2 Hz, 1H), 4.80 (dd, J=13.8, 4.6 Hz, 1H), 4.56 (d, J=16.6 Hz, 1H), 4.23-4.10 (m, 1H), 1.23 (d, J=6.7 Hz, 3H).

Example 15

(6R)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one

……………………UNDESIRED R CONFIGURATION
• 
• Chiral SFC separation of (±)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one on a CHIRALCEL OD-H column (5 μM, 250×20 mm) using 70% CO₂/30% MeOH provided 39 mg of the title compound as the first eluting enantiomer. [α]=+40° (c 2.2, CHCl₃).
• MS (ESI): mass calcd. for C₁₈H₁₄ClF₃N₆O, 422.1; m/z found, 423.1 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃) δ 9.66 (d, J=1.5 Hz, 1H), 8.68 (d, J=2.5 Hz, 1H), 8.59 (dd, J=2.5, 1.5 Hz, 1H), 7.76-7.72 (m, 1H), 7.69 (dd, J=7.9, 1.6 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 5.44 (d, J=15.5 Hz, 1H), 5.17 (dd, J=13.9, 2.1 Hz, 1H), 4.62-4.54 (m, 2H), 4.08-4.02 (m, 1H), 1.36 (d, J=6.8 Hz, 3H).

Example 16

(6S)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one………………  DESIRED
• 
• Chiral SFC separation of (±)-7-[2-Chloro-3-(trifluoromethyl)benzyl]-6-methyl-3-pyrazin-2-yl-6,7-dihydro[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one on a CHIRALCEL OD-H column (5 μM, 250×20 mm) using 70% CO₂/30% MeOH provided 40 mg of the title compound as the second eluting enantiomer.
• [α]=−44° (c 2.2, CHCl₃).
• MS (ESI): mass calcd. for C₁₈H₁₄ClF₃N₆O, 422.1; m/z found, 423.1 [M+H]⁺.
• ¹H NMR (500 MHz, CDCl₃) δ 9.66 (d, J=1.5 Hz, 1H), 8.68 (d, J=2.5 Hz, 1H), 8.59 (dd, J=2.5, 1.5 Hz, 1H), 7.76-7.72 (m, 1H), 7.69 (dd, J=7.9, 1.6 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 5.44 (d, J=15.5 Hz, 1H), 5.17 (dd, J=13.9, 2.1 Hz, 1H), 4.62-4.54 (m, 2H), 4.08-4.02 (m, 1H), 1.36 (d, J=6.8 Hz, 3H).

Patent	Submitted	Granted
P2X7 MODULATORS [US2014275096]	2014-03-14	2014-09-18

//////////////P2X7, 6,7-Dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one, Autoradiography, Depression, CNS, Preclinical characterization, substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one,  P2X7 receptor antagonists, Janssen Pharmaceutical Research & Development L.L.C, 1627748-32-6
FC(F)(F)c4cccc(CN1C(=O)c2nnc(n2C[C@@H]1C)c3cnccn3)c4Cl
CC1CN2C(=NN=C2C(=O)N1CC3=C(C(=CC=C3)C(F)(F)F)Cl)C4=NC=CN=C4

////////see............http://newdrugapprovals.org/2016/01/07/preclinical-characterization-of-substituted-67-dihydro-124triazolo43-apyrazin-85h-one-p2x7-receptor-antagonists/

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THANKS AND REGARD'S DR ANTHONY MELVIN CRASTO Ph.D amcrasto@gmail.com MOBILE-+91 9323115463 GLENMARK SCIENTIST ,  INDIA web link ABOUTME BRAND ANTHONYCRASTO COLLECTION OF SITE LINKS   Dr. Anthony Melvin Crasto Principal Scientist, Glenmark Pharma +919323115463 | amcrasto@gmail.com | http://newdrugapprovals.wordpress.com | India

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FDA approves first drug to treat a rare enzyme disorder in pediatric and adult patients

 

 

 

 

Sebelipase alfa

 

CAS No. 1276027-63-4

 

Synageva... innovator

ALEXION

EMA AUG 28 2015

FDA approves first drug to treat a rare enzyme disorder in pediatric and adult patients

12/08/2015

Today, the U.S. Food and Drug Administration approved Kanuma (sebelipase alfa) as the first treatment for patients with a rare disease known as lysosomal acid lipase (LAL) deficiency.

December 8, 2015

Release

Today, the U.S. Food and Drug Administration approved Kanuma (sebelipase alfa) as the first treatment for patients with a rare disease known as lysosomal acid lipase (LAL) deficiency.
Patients with LAL deficiency (also known as Wolman disease and cholesteryl ester storage disease [CESD]) have no or little LAL enzyme activity. This results in a build-up of fats within the cells of various tissues that can lead to liver and cardiovascular disease and other complications. Wolman disease often presents during infancy (around 2 to 4 months of age) and is a rapidly progressive disease. Patients with Wolman disease rarely survive beyond the first year of life. CESD is a milder, later-onset form of LAL deficiency and presents in early childhood or later. Life expectancy of patients with CESD depends on the severity of the disease and associated complications. Wolman disease affects one to two infants per million births, and CESD affects 25 individuals per million births.
Today’s action involved approvals from two FDA centers. The Center for Veterinary Medicine (CVM) approved an application for a recombinant DNA (rDNA) construct in chickens that are genetically engineered (GE) to produce a recombinant form of human lysosomal acid lipase (rhLAL) protein in their egg whites. The FDA regulates GE animals under the new animal drug provisions of the Federal Food, Drug, and Cosmetic Act, because an rDNA construct introduced into an animal to change its structure or function meets the definition of a drug. The Center for Drug Evaluation and Research (CDER) approved the human therapeutic biologic (Kanuma), which is purified from those egg whites, based on its safety and efficacy in humans with LAL deficiency.
“LAL deficiency is a rare inherited genetic disorder that can lead to serious and life-threatening organ damage, especially when onset begins in infancy,” said CDER Director Janet Woodcock, M.D. “Using this technology, these patients for the first time ever have access to a treatment that may improve their lives and chances of survival.”
The new therapy, Kanuma, provides an rhLAL protein that functions in place of the missing, partially active or inactive LAL protein in the patient. Kanuma is produced by GE chickens containing an rDNA construct responsible for producing rhLAL protein in their egg whites. These egg whites are refined to extract the rhLAL protein that is eventually used to produce Kanuma and treat patients with LAL deficiency. The GE chickens are used only for producing the drug substance, and neither the chicken nor the eggs are allowed in the food supply.
Kanuma is approved for use in patients with LAL deficiency. Treatment is provided via intravenous infusion once weekly in patients with rapidly progressive LAL deficiency presenting in the first six months of life, and once every other week in all other patients.
CDER evaluated the safety and efficacy of Kanuma in an open-label, historically controlled trial in nine infants with rapidly progressive Wolman disease and in a double-blind, placebo-controlled trial in 66 pediatric and adult patients with CESD. In the trial in infants with Wolman disease, six of nine infants (67 percent) treated with Kanuma were alive at 12 months of age, whereas none of the 21 infants in the historical control group survived. In the trial in CESD patients, there was a statistically significant improvement in LDL-cholesterol levels and other disease-related parameters in those treated with Kanuma versus placebo after 20 weeks of treatment.
The most common side effects observed in patients treated with Kanuma are diarrhea, vomiting, fever, rhinitis, anemia, cough, headache, constipation, and nausea.
In its review of the GE chicken application, CVM assessed the safety of the rDNA construct, including the safety of the rDNA construct to the animals, as well as a full review of the construct and its stability in the genome of the chicken over several generations. No adverse outcomes were noted in the chickens. As required by the National Environmental Policy Act and its implementing regulations, CVM evaluated the potential environmental impacts of approval of the sponsor’s GE chickens and determined that the approval does not cause any significant impact on the environment, because the chickens are raised in highly secure indoor facilities.
“We reviewed all of the data to ensure that the hens do produce rhLAL in their egg whites, without suffering any adverse health effects from the introduced rDNA construct. The company has taken rigorous steps to ensure that neither the chickens nor the eggs will enter the food supply, and we have confirmed their containment systems by inspecting the manufacturing facilities,” said CVM Director Bernadette Dunham, D.V.M., Ph.D.
The FDA granted Kanuma orphan drug designation because it treats a rare disease affecting fewer than 200,000 patients in the United States. Orphan drug designation provides financial incentives for rare disease drug development such as clinical trial tax credits, user fee waivers, and eligibility for market exclusivity to promote rare disease drug development. Kanuma was also granted breakthrough therapy designation as it is the first and only treatment available for Wolman disease, the very severe infant form of the disease. The breakthrough therapy designation program encourages the FDA to work collaboratively with sponsors, by providing timely advice and interactive communications, to help expedite the development and review of important new drugs for serious or life-threatening conditions. The Kanuma application was also granted a priority review, which is granted to drug applications that show a significant improvement in safety or effectiveness in the treatment of a serious condition. The manufacturer of Kanuma was granted a rare pediatric disease priority review voucher –– a provision intended to encourage development of new drugs and biologics for the prevention and treatment of rare pediatric diseases.
Kanuma is produced by Alexion Pharmaceuticals Inc., based in Cheshire, Connecticut.

 

 

///////// Kanuma, sebelipase alfa, rare disease, lysosomal acid lipase (LAL) deficiency,