COMPD HAS  cas no 1616882-93-9

MF……….C₁₈ H₁₁ F₃ N₂ O₂

[1]​Benzopyrano[4,​3-​c]​pyrazol-​4(1H)​-​one, 3-​methyl-​1-​[4-​(trifluoromethyl)​phenyl]​-

 3-Methyl-1-(4-(trifluoromethyl)phenylchromeno[4,3-c]pyrazol-4(1H)-one

Synthesis, biological evaluation and docking analysis of 3-methyl-1-phenylchromeno[4,3-c]pyrazol-4(1H)-ones as potential cyclooxygenase-2 (COX-2) inhibitors

DOI: 10.1016/j.bmcl.2014.08.050

Jagdeep Grover, Vivek Kumar, M. Elizabeth Sobhia, Sanjay M. Jachak

http://www.sciencedirect.com/science/article/pii/S0960894X14008944

 Abstract

As a part of our continued efforts to discover new COX inhibitors, a series of 3-methyl-1-phenylchromeno[4,3-c]pyrazol-4(1H)-ones were synthesized and evaluated for in vitro COX inhibitory potential. Within this series, seven compounds (3a–d, 3h, 3k and 3q) were identified as potential and selective COX-2 inhibitors (COX-2 IC₅₀’s in 1.79–4.35 μM range; COX-2 selectivity index (SI) = 6.8–16.7 range). Compound 3b emerged as most potent (COX-2 IC₅₀ = 1.79 μM; COX-1 IC₅₀ >30 μM) and selective COX-2 inhibitor (SI >16.7). Further, compound 3b displayed superior anti-inflammatory activity (59.86% inhibition of edema at 5 h) in comparison to celecoxib (51.44% inhibition of edema at 5 h) in carrageenan-induced rat paw edema assay. Structure–activity relationship studies suggested that N-phenyl ring substituted with p-CF₃ substituent (3b, 3k and 3q) leads to more selective inhibition of COX-2. To corroborate obtained experimental biological data, molecular docking study was carried out which revealed that compound 3b showed stronger binding interaction with COX-2 as compared to COX-1.

Authors

• Jagdeep Grover^a,
• Vivek Kumar^b,
• M. Elizabeth Sobhia^b,
• Sanjay M. Jachak^{a, , ,}

• ^a Department of Natural Products, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar (Mohali) 160062, Punjab, India
• ^b Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar 160062, Punjab, India

Sanjay Corresponding author. Tel.: +91 172 2214683; fax: +91 172 2214692.

 CLICK……….

• Supplementary data

Cyclooxygenase (COX) or prostaglandin endoperoxide synthase (PGHS), catalyzes the conversion of arachidonic acid to inflammatory mediators such as prostaglandins (PGs), prostacyclins and thromboxanes. COX exists in mainly two isoforms: COX-1 and COX-2. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used for relief of fever, pain and inflammation, act by inhibiting COX catalyzed biosynthesis of inflammatory mediators.

However, the therapeutic use of classical NSAIDs is associated with well-known side effects at the gastrointestinal level (mucosal damage, bleeding) and, less frequently, at the renal level.

Two decades after the discovery of COX isoforms, it was recognized that selective inhibition of COX-2 might be endowed with improved anti-inflammatory properties and reduced gastrointestinal toxicity profiles than classical NSAIDs.

Overall, these selective COX-2 inhibitors (coxibs) have fulfilled the hope of possessing reduced risk in gastrointestinal events, but unfortunately cardiovascular concerns regarding the use of these agents have emerged that led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra) from the market in 2004 and 2005, respectively.

Ongoing safety concerns pertaining to the use of non-selective NSAIDs have spurred development of coxibs with improved safety profile.

……………………………………………………………………………………………..

cas no 1616882-93-9

mf……….C₁₈ H₁₁ F₃ N₂ O₂

[1]​Benzopyrano[4,​3-​c]​pyrazol-​4(1H)​-​one, 3-​methyl-​1-​[4-​(trifluoromethyl)​phenyl]​-

 3-Methyl-1-(4-(trifluoromethyl)phenylchromeno[4,3-c]pyrazol-4(1H)-one

Scheme 1.

Reagent and conditions: (a) Piperidine, rt, 20 min; (b) ArNHNH₂, EtOH, reflux, 5 h; (c) K₂CO₃, acetone, reflux, 24 h.

COMPD IS

3b

R1=H

R2= H

4-CF3-C6H4

90

3-Methyl-1-(4-(trifluoromethyl)phenylchromeno[4,3-c]pyrazol-4(1H)-one (3b):

White solid; yield 90%; mp: 224–225 °C;

1H NMR (CDCl3, 400 MHz): δ ppm 7.89 (d, 2H, J = 8.32 Hz, Ar-H), 7.73 (d, 2H, J = 8.24 Hz, Ar-H), 7.45–7.52 (m, 2H, H-6, H-7), 7.16 (dd, 1H, J = 1.4, 8.2 Hz, H-9), 7.10 (td, 1H, J = 1.56, 7.38 Hz, H-8), 2.69 (s, 3H, CH3);

13C NMR (CDCl3, 100 MHz): δ ppm 157.7, 153.3, 151.5, 142.3, 141.8, 131.9, 127.2, 127.1, 127.0, 124.0, 122.2, 118.3, 111.5, 107.1, 12.8;

HRMS (ESI) m/z: Calcd for C18H11F3N2O2Na [M + Na]+ 367.0670; found 367.0676.

Synthetic Communications (2014), 44(13), 1914-1923

DOI:

10.1080/00397911.2013.879184

Jagdeep Grover^a, Somendu Kumar Roy^a & Sanjay Madhukar Jachak^a*

pages 1914-1923

http://www.tandfonline.com/doi/abs/10.1080/00397911.2013.879184#.VCI5f0DgXXM

http://www.tandfonline.com/doi/suppl/10.1080/00397911.2013.879184/suppl_file/lsyc_a_879184_sm8537.pdf

Abstract

Unprecedented cyclization was observed during N-sulfonylation of 3-[1-(phenylhydrazono)-ethyl]-chromen-2-one in pyridine, affording 3-methyl-1-phenylchromeno[4,3-c]pyrazol-4(1H)-ones. To avoid use of noxious pyridine, reaction was tried in different basic conditions and the best results were obtained with potassium carbonate in acetone. A wide range of substrates bearing either electron-donating or electron-withdrawing substituents on phenylhydrazine ring were compatible with the developed methodology. Rapid access of starting material, 3-acetylcoumarin, excellent yields of products, and use of environmentally benign base and solvent for the cyclization make this strategy an efficient and convenient method for synthesis of 3-methyl-1-phenylchromeno[4,3-c]pyrazol-4(1H)-ones.

Methyl-1-(4-(trifluoromethyl)phenylchromeno[4,3-c]pyrazol-4(1H)-one (4b):

Whitesolid;

yield 90%; mp: 224–225 °C;

1H NMR (CDCl3, 400 MHz):δppm 2.69 (s, 3H, CH3),

7.10(td, 1H,J= 1.56, 7.38 Hz, H-8),

7.16 (dd, 1H,J= 1.4, 8.2 Hz, H-9),

7.45–7.52 (m, 2H, H-6, H-7),

7.73 (d, 2H,J= 8.24 Hz, Ar-H),

7.89 (d, 2H,J= 8.32 Hz, Ar-H);

13C NMR (CDCl3, 100MHz):

δppm 12.8, 107.1, 111.5, 118.3, 122.2, 124.0,

127.0, 127.1, 127.2, 131.9, 141.8, 142.3,

151.5, 153.3, 157.7;

HRMS (ESI)m/z: Calcd for C18H11F3N2O2Na [M + Na]+367.0670; found367.0676.

 3-Methyl-1-(4-(trifluoromethyl)phenylchromeno[4,3-c]pyrazol-4(1H)-one

SEE BELOW  1H NMR, 13CNMR, AND MASS SPEC

References
1. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267, 727.
2. Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. F.; Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112, 535.

Zifaxaban

Zifaxaban

cas 1378266-98-8

rotation (-)

C₂₀ H₁₆ Cl N₃ O₄ S

C20H16ClN3O4 S, M = 429.87

Tianjin Institute of Pharmaceutical Research

Deep vein thrombosis; Lung embolism

Factor Xa antagonist

TY-602; zhifeishaban; zifaxaban

天津药物研究院

Chinese J Struc Chem. 2014, 33 (7), 1091-1095.

(S) -5- chloro -N- ((2- oxo _3_ (4_ (2_ oxo _2H_-1-yl) phenyl) oxazolidin-5 -1,3_ yl) methyl) thiophene-2-carboxamide

5-Chloro-N-(5S)-2-oxo-3-[4-(2-oxopyridin-1(2H)-yl)phenyl]oxazolidin-5-ylimethyllthiophene-2-carboxamide]

The title compound(zifaxaban 2, C20H16ClN3O4 S, Mr = 429.87) was synthesized and its crystal structure was determined by single-crystal X-ray diffraction. Zifaxaban crystallizes in monoclinic, space group P21 with a = 5.7900(12), b = 13.086(3), c = 12.889(3) A, β = 100.86(3)°, V = 959.1(3) A3, Z = 2, Dc = 1.489 g/cm3, F(000) = 444, μ = 0.342 mm-1, the final R = 0.0320 and wR = 0.0640 for 2717 observed reflections(I > 2σ(I)).

The absolute configuration of the stereogenic center in the title compound was confirmed to be S by single-crystal X-ray diffraction. Four existing intermolecular hydrogen bonds help to stabilize the lattice and the molecule in the lattice to adopt an L-shape conformation.

Zifaxaban was slightly more active than rivaroxaban 1 in in vitro assay against human FXa and therefore is promising as a drug candidate.

zifaxaban (first disclosed in CN102464658), useful for treating thromboembolic disorders. Zifaxaban, a factor Xa antagonist, is being developed by Tianjin Institute of Pharmaceutical Research, for treating deep vein thrombosis and pulmonary embolism (preclinical, as of November 2014). In May 2014, an IND was filed in China. In June 2014, the institute was seeking to outlicense this product.

In vivo within the cardiovascular, blood coagulation or blood analysis some have formed out of the process of forming a solid mass with the aggregation, called thrombosis, the formation of a solid mass called a thrombus blocks. Thrombosis is an abnormal flow of blood coagulation status due to platelet activation and coagulation factors are activated in accordance therewith.

The blood coagulation was originally a protective mechanism of the organism, there is a mutual antagonism in blood coagulation system and the anti-clotting system. Under physiological conditions, blood clotting factors continue to be activated to produce thrombin, fibrin formation trace, calm on the vascular endothelium, but these traces of fibrin and constantly being activated fibrinolytic system dissolution, while being activated coagulation factors are constantly mononuclear phagocyte system swallowed. The dynamics of the coagulation system and fibrinolysis system, which ensures the blood coagulation potential can also always ensure that the fluid state of the blood.

 Sometimes, however, in certain factors can promote the coagulation process, breaking the above dynamic balance triggered the coagulation process, the blood can form a thrombosis or embolism, such as leading to myocardial infarction, stroke, deep vein thrombosis, pulmonary embolism and other thromboembolic disease.

Thromboembolic disease is cardiovascular disease against the most serious diseases, is the first killer of human health. In China, with the improvement and increased aging of the population’s living standards, the incidence of such diseases, mortality, morbidity is increasing every year.

The existing anti-thromboembolic diseases into anti-platelet drugs, anticoagulants and fibrinolytic drugs. Among them, the anti-clotting drugs are the main contents of antithrombotic therapy, mainly thrombin inhibitors and vitamin K antagonists. Heparin and low molecular weight heparin, represented by the presence of oral thrombin inhibitor invalid, non-selective inhibition and high risk of bleeding and other shortcomings. Although warfarin is representative of vitamin K antagonists can be administered orally, but there are narrow therapeutic index, high risk of bleeding and other shortcomings.

Studies have shown that the coagulation process is usually divided into intrinsic coagulation pathway and the extrinsic coagulation pathway. Coagulation process involves a lot of coagulation factors, coagulation factor activated are each the next inactive clotting factor precursor is converted into the activated form. Endogenous, exogenous pathway final summary, the blood coagulation factor X is converted to Xa.

Therefore, theoretically, the direct inhibition of ¾ factor activity should produce effective anti-clotting effect, without the side effects of thrombin inhibitors with. As direct inhibition) (a factor activity on normal hemostasis reaction / adjustment process produces minimal impact. For example, platelets remain low catalytic activity of thrombin on the ability to respond to, and thus does not affect the formation of platelet thrombi, so bleeding integrated minimize the risk of the levy.

  research also proved this point. Recently reported a variety of compounds can selectively inhibit efficient Xa, which play a preventive and / or treatment of thromboembolic disease effect (W003000256A1; CN00818966; US2007259913A1; US2007259913A1). Among them, rivaroxaban (Rivaroxaban) was listed in 2008 for hip or knee replacement surgery prophylaxis and treatment of venous thrombosis, with oral, fixed dose and other advantages.

  rivaroxaban drawback is the high price of raw materials, low yield preparation, purification of the product is difficult, high production costs. Patent CN00818966 8 reported rivaroxaban synthetic routes as follows:

4

where the first reaction (Preparation of 4- (4-morpholino-3-yl) nitrobenzene) yield of only 17.6%, and rivaroxaban difficult purification.

………………………………

Patent

http://www.google.com/patents/CN103232446A?cl=en

(S) -5- chloro -N- ((2- oxo-3- (4- (2_ oxo -2H- pyridin-1-yl) phenyl) -1, 3_ oxazolidine -5 – yl) methyl) thiophene-2-carboxamide.

[0011] Meanwhile, patent CN201110337461.4 described formula (I) Preparation of the compound:

[0012]

……………………………………..

Patent

CN102464658

http://www.google.com/patents/CN102464658B?cl=en

Example 1

[0046] (S) -5- chloro -N- ((2- oxo-3- (4_ (2_ Batch oxo _2H_ piperidinyl) phenyl) _1,3_ oxazolidin-5-yl) methyl ) thiophene-2-carboxamide (II)

[0048] A, 1- (4- amino-phenyl) -IH- pyridin _2_ -one (Compound VII) is

[0049] The reaction flask was charged with 104g of pyridine -2 (IH) – one (Compound IX), 200g of iodoaniline (compound VIII), 26gCuI, 151g of potassium carbonate, 18g8- hydroxyquinoline, 500mlDMF, nitrogen, heated to reflux, Insulation reaction was stirred 10h. Filtered hot, the filtrate evaporated under reduced pressure to make the solvent, the residue was added ethyl acetate, IL, 0 ° C incubated with stirring lh, filtered and the solid dried, 2L acetonitrile and purified to give 98g dark red solid. Refined liquor was concentrated to 500ml, the ice bath was stirred lh, filtered to give a dark red solid 19g. Total product were 117g, yield 68.9%.

[0050] 1H-NMR (DMSO-Cl6), δ (ppm):… 5 306 (s, 2H), 6 236 (d, 1H), 6 406 (d, 1H), 6 601 (d,. 2H), 6. 977 (d, 2H), 7. 459 (m, 2H).

[0051] B, (R) -2- (2- hydroxy-3- ((2-oxo–2H- pyridin-1-yl) phenyl) amino) propyl) isoindoline-1,3- -dione (Compound V) is

[0052] The reaction flask was added 40gl_ (4- aminophenyl) -IH- pyridin-2-one (Compound VII), 45g (S) _N_ glycidyl phthalimide (Compound VI), 300ml95% ethanol, heating to reflux, the gradual emergence of solid insulation mixing IOh, cooled to room temperature, filtered, and the filter cake washed with ethanol (150ml X 2), and dried to give an off-white solid 38g.

[0053] The mother liquor was taken, evaporated to dryness under reduced pressure, was added 15g (Q-N_ glycidyl phthalimide (Compound VII), 150ml95% ethanol, heated to reflux, stirred incubated 10h, concentrated under reduced pressure, cooled to room temperature , stirred at room temperature for 2h, washed with ethanol and dried to give an off-white solid 33g.

[0054] A total of an off-white solid 71g, yield of 84.8%, without purification, was used directly in the next step.

[0055] 1H-NMR (DMS0_d6), δ (ppm):… 3 053 (m, 1H), 3 194 (m, 1H), 4 644 (m, 2H), 4 020 (m, 1H). , 5. 168 (d, 1H), 5. 851 (t, 1H), 6. 230 (m, 1H), 6. 404 (d, 1H), 6. 665 (d, 2H), 7. 041 ( d, 2H), 7. 435 (m, 1H), 7. 537 (m, 1H), 7. 855 (m, 4H).

[0056] C, ⑶-2- ((2- oxo-3- (4- (2_ oxo _2H_ pyridyl) phenyl) oxazolidin _5_ -1,3_ yl) methyl ) Preparation of isoindoline-1,3-dione (Compound IV) of the

[0057] The reaction flask was charged 50g Compound V, 27gN, N’- carbonyldiimidazole (⑶I), 4_ catalytic amount of dimethylaminopyridine (DMAP), 150mlN, N- dimethylformamide (DMF), stirred for 90 temperature ° C, the reaction was kept for 8 hours to make the solvent was evaporated under reduced pressure, added to IL of water, stirred and dispersed, filtered, washed with water (150mlX “, washed with ethanol (100ml X 1), dried to give a white solid 48g, yield of 90%.

[0058] 1H-NMR (DMSo-CI6), δ (ppm):…. 3 984 (m, 3H), 4 251 (t, 1H), 4 968 (m, 1H), 6 301 (m, 1H), 6. 459 (d, 1H), 7. 423 (d, 2H), 7. 514 (m, 1H), 7. 615 (m, 3H), 7. 892 (m, 4H).

[0059] D, (S) -5- (aminomethyl) -3- (4- (2_ oxo _2H_-1-yl) phenyl) oxazolidin _2_ -1,3_ one hydrochloride (compound III) Synthesis of

[0060] The reaction flask was charged 50g compound IV, 200ml of ethanol, 60ml aqueous methylamine (40%), heated to reflux, stirred incubated 2h, cooled, evaporated under reduced pressure to make the solvent to give a sticky solid.

[0061] added to 300ml of ethanol, 20ml of hydrochloric acid, heated to reflux, stirred incubated lh, cooled to room temperature, incubated with stirring 2h, filtered, washed with ethanol, and dried to obtain;. 34 5g of white solid, yield 88.7%.

 1H-NMR (DMS0_d6), δ (ppm):…. 3 240 (m, 2H), 3 980 (m, 1H), 4 255 (m, 1H), 5 028 (m, 1H) , 6. 321 (m, 1H), 6. 475 (d, 1H), 7. 504 (m, 3H), 7. 634 (m, 3H), 8. 561 (s, 1H).

 Ε, (S) -5- chloro -N – ((2- oxo-3- (4- (2-oxo–2Η- pyridin-1-yl) phenyl) oxazolidin _1,3_ 5-yl) methyl) thiophene-2-carboxamide Preparation of thiophene (II) of

The reaction flask was charged 15g Compound III, 200ml of tetrahydrofuran, 40ml of water was added with stirring 6. 2g of sodium carbonate was added dropwise 10g5- chloro-thiophene-2-carbonyl chloride (Compound II-1) in tetrahydrofuran IOOml, 30~35 ° C insulation stirred 5h, point board to control the reaction was complete.

 to make the solvent was distilled off under reduced pressure, 50ml of water was added, stirring was filtered, the filter cake washed with water and dried to give 18. 5g of white solid.

 200ml of acetic acid and purified room temperature overnight, filtered, and the filter cake washed with ethanol and dried to give a white solid 16g, 80% yield.

Melting point: 204 8 ~205 8 ° C;

 1H-NMR (DMSo-CI6), δ (ppm):…. 3 623 (t, 2H), 3 893 (m, 1H), 4 230 (t, 1H), 4 871 (m, 1H), 6. 308 (t, 1H), 6. 468 (d, 1H), 7. 193 (d, 1H), 7. 426 (m, 2H), 7. 500 (m, 1H), 7. 637 (m, 4H), 8. 967 (t, 1H);

 MS (ESI): m / z = 430 (M + H);

 HPLC: rt (%) = 14. 38 (99. 62);

 [a] 20d = -37 6 ° (c 0. 3004, DMS0);

WO-2014183667Acetic acid solvate of oxazolidinone derivative, preparation method for the solvate, and application thereof

WO-2014183665Oxazolidinone derivative crystal form I and preparation method and use thereof

WO-2014183666Oxazolidinone derivate crystal form II, preparation method therefor, and application thereo

(2E)-N-[2-[2-[(2S)-1-Methyl-2-piperidinyl]ethyl]phenyl]-3-phenyl-2-propenamide (1)

(S)-2′[2-1-(methyl-2-piperidyl) ethyl] cinnamanilide

(2E)-Λ/-[2-[2-[(2S)-1-Methyl-2-piperidinyl]ethyl]phenyl]-3-phenyl-2- propenamide

CAS  951155-17-2

C₂₃H₂₈ N₂O, 348.48

It was reported that the (S)-enantiomer of 2′-[2-(1-methyl-2-piperidyl)ethyl]cinnamanilide (MSA100, 1) is an active 5-HT (5-hydroxytryptamine or serotonin) receptor antagonist; however, its (R)-isomer is totally or substantially devoid of the same activity.(1)

• 1.

  (a) Amer, M. S., U.S. Patent 5,780, 487, 1998.

  (b) Prashad, M.; Liu, Y.; Hu, B.; Girgis, M. J.; Schaefer, F., WO2007/111705, 2007.

  (c) Prashad, M.; Liu, Y.; Hu, B.; Girgis, M. J.; Schaefer, F., WO2007/111706, 2007.

…………………………………………………………………..

http://www.google.com/patents/WO2007111706A2?cl=en

Example 4:

Synthesis of (2E)-/V-[2-[2-[(2S)~1 -Methyl-2-piperidinyl]ethyI]phenyl]-3-phenyl-2- propenamide

(a) Free Base Generation:

A 250-mL, 4-necked, round-bottomed flask, equipped with a mechanical stirrer, digital thermometer and nitrogen inlet-outlet, heating cooling bath, and addition funnel, is charged with 6.28 g (S)-2-[2-(1-methyl-2-piperidinyl)ethyl]-benzenamine (1R,3S)-(+)-camphoric acid salt (1 :1) and 60 mL of isopropyl acetate. Stir the mixture at 20-25 ⁰C under nitrogen and add a solution of 1.60 g of sodium hydroxide in 20 mL of water over a period of 5 min while maintaining an internal temperature at 20-25 ⁰C. Stir the suspension efficiently until all the solid dissolves (5 min). Separate the organic layer and save. Extract the aqueous layer with 20 mL of isopropyl acetate. Combine the organic layers and wash it with 20 mL of water. Separate the organic layer and concentrate it under vacuum (20-100 mbar) at an internal temperature at 20-40 ⁰C (external temperature 30-60 ⁰C) to obtain ~65 mL of a solution of (S)-2-[2-(1-methyl-2-piperidinyl)ethyl]-benzenamine (containing 3.28 g of free base) in isopropyl acetate. Save this solution for the next step and store it under nitrogen.

(b) Reaction:

A 250-mL, 4-necked, round-bottomed flask, equipped with a mechanical stirrer, digital thermometer, nitrogen inlet-outlet, heating mantle, condenser, and addition funnel is charged with -65 mL of a solution of (S)-2-[2-(1-methyl-2-piperidinyl)ethyl]-benzenamine (containing 3.28 g of free base) in isopropyl acetate and 6.22 g of potassium carbonate. Stir the reaction mixture under nitrogen at an internal temperature at 23 ± 3 ⁰C to afford a suspension. Add 3.75 g of cinnamoyl chloride over a period of 5 min while maintaining an internal temperature at 23 ± 3 ⁰C to obtain a slurry. Heat the reaction mixture to an internal temperature at 85 ± 5 ⁰C (external temperature 90-100 ⁰C) over a period of 30-60 min. Stir the reaction mixture at this temperature for an additional 2 h. Cool the reaction mixture to 23 ± 3 ⁰C over a period of 1 h. Add 50 mL of water. Stir the reaction mixture at 23 ± 3 ⁰C for 30-60 min to obtain a bi-phasic solution. Separate the organic layer. Add 80 mL of 0.5 N HCI solution over a period of 10 min while maintaining an internal temperature at 23 ± 3 ⁰C to afford a bi-phasic solution. Separate the aqueous layer. Add 60 mL of isopropyl acetate. Stir the reaction mixture and add a solution of 2.00 g of sodium hydroxide in 25 mL of water over a period of 10 min while maintaining an internal temperature at 23 ± 3 ⁰C to afford a bi- phasic solution. Separate the organic layer and save. Extract the aqueous layer with 60 mL of isopropyl acetate. Combine the organic layers and wash it with 40 mL of water. Separate the organic layer and concentrate it under vacuum (20-100 mbar) at an internal temperature at 20-40 ⁰C (external temperature 30-60 ⁰C) to obtain 22mL (19.3 g) of a solution of (iii) in isopropyl acetate. Stir and heat the reaction mixture to an internal temperature at 85 ± 5 ⁰C (external temperature 90-100 ⁰C) over a period of 30-60 min. Add 96 mL of hepatane over a period of 10 min while maintaining an internal temperature at 85 ± 5 ⁰C. Stir and Cool the reaction mixture to 23 ± 3 ⁰C over a period of 1 h. Stir the resulting slurry at 23 ± 3 ⁰C for an additional 2 h. Collect the solid by filtration over a polypropylene filter paper in a Buchner funnel with suction. Wash the solid with a total of 28 mL of a mixture of isopropyl acetate and heptane (1/6) in two equal portions of 14 mL each. Dry the solid at 45-50 ⁰C under vacuum (13-40 mbar) with nitrogen bleeding to obtain a constant weight (LOD < 1%, 4 h) of 4.06 g of (2£)-A/-[2-[2-[(2S)-1-methyl-2-piperidinyl]ethyl]phenyl]-3-phenyl-2-propenamide as an off white solid.

Theoretical Yield: 5.23 g

Yield: 77.6%

Purity: 99.8% (HPLC area %).

Enantiomeric purity: (R)-(iii) was not detected by Chiral HPLC. Example 5:

Alternative synthesis of (2£)-/V-[2-[2-[(2S)-1-MethyI-2-piperidinyl]ethyl]phenyI]-3- phenyl-2-propenamide

(a) Free base generation:

In a 500 ml round bottomed flask equipped with a mechanical stirrer the resolved camphoric acid salt (IV) (20 g) in isopropyl acetate (120 g) is added at an internal temperature of 20 to 25 ⁰C (external temperature 20 ⁰C). Then, at an internal temperature of 25 to 30 ⁰C (external temperature 20 ⁰C) a solution of sodium hydroxide (38.24 g) in water (60 g) is added to the reaction mixture over a period of 5 minutes. The reaction mixture (suspension) is then stirred for a further 30 minutes. The resulting orange emulsion is then allowed to separate into a two-phase mixture and the water phase is removed. The organic phase is then subjected to a rotary evaporator and the isopropyl acetate is distilled at an internal temperature of 60 ⁰C and under reduced pressure (250 mbar). Approximately 90 g of isopropyl acetate is distilled. Prior to distilling, the organic phase is a clear, bright orange colour and of a volume of approximately 160 ml ( 13Og),

(b) Reaction:

In a 1.5 I flask equipped with a mechanical stirrer and at an internal temperature of 35 ⁰C (external temperature 38 ⁰C) and under inert conditions (nitrogen) 2-butanone (160 g) and isopropyl acetate (20 g) is added to the reaction mixture of part (a). Then, at an internal temperature of 35 ⁰C (external temperature of 38 ⁰C) a solution of cinnamoyl chloride (8.9 g) in 2-butanone (20 g) is added drop wise. Then, the reaction mixture is treated with more 2- butanone (2 x 5 g). The resulting suspension is then stirred for 20 minutes at an internal temperature of 35⁰C. The pH of the mixture is between 6 and 8.

(c) Resolution:

The suspension of step (b) is then cooled to an internal temperature of 25 ⁰C (external temperature 20 ⁰C) and at the same time a mixture of water (200 g) and isopropyl acetate (60 g) is added. The reaction mixture is then stirred for a further 15 minutes at an internal temperature of 25 ⁰C (external temperature 20 ⁰C). The resulting two-phase reaction mixture is then separated and the water phase removed. The resulting yellow upper layer is then treated with 2.5 mol/l hydrochloric acid (200 g). The resulting two-phase mixture is then separated and the water phase is transferred into a 750 ml flask equipped with a mechanical stirrer. The organic phase is then washed with 2.5 mol/l hydrochloric acid (200 g) and the resulting two-phase mixture is separated and the water phase is added to the first water phase. The combined water phases are then treated with acetic acid (300 g) and sodium hydroxide (150 g) is added. The reaction mixture is then stirred at an internal temperature of 25 to 30 ⁰C (external temperature 20 ⁰C) for 15 minutes. The resulting two-phase reaction mixture is then separated.

(d) Crystallization

The organic phase from the above reaction step (c) is reduced in volume on a rorary evaporator at an external temperature of 60 ⁰C and at 250 mbar. Then, the reduced-volume reaction mixture is treated with isopropanol (60 g) and the resulting reaction mixture is reduced in volume on a rotary evaporator at an external temperature of 60 ⁰C and under a vacuum of 150 mbar. Then, at an internal temperature of 50 to 55 ⁰C (external temperature 60 ⁰C) the reaction mixture is treated with water (20 g) and the resulting suspension is further treated with the product (iv) (10 mg) in isopropanol (0.01 g). The reaction mixture is then stirred for a further 15 minutes at an internal temperature of 50 to 55 ⁰C (external temperature 60 ⁰C). Then, further water is added over a period of 15 to 30 minutes and the reaction mixture is maintained at an internal temperature of 50 to 55 ⁰C (external temperature 60 ⁰C). Then, the resulting suspension is cooled to an internal temperature of 22 to 22 ⁰C (external temperature 20 ⁰C. Then, the suspension is stirred for a further 30 minutes at an internal temperature of 22 to 22 ⁰C (external temperature 20 ⁰C) and the resulting solid is collected by filtration and washed with a mixture of water and isopropyl acetate (2 x 20 g), where the water: isopropyl acetate ratio is of 5:1 g/g. The resulting solid may then be dried under a vacuum at a temperature of 55 ⁰C.

Yield: 14.8 g (89.3% of theory). mp: 127.3 to 130.2 ⁰C

Example 6: Recrystallisation of (2E)-Λ/-[2-[2-[(2S)-1-Methyl-2-piperidinyl]ethyl]phenyl]-3-phenyl-2- propenamide

(a) In a 200 ml round bottomed flask equipped with a magnetic stirrer, containing the product (iv) (15 g) is added isopropanol (25 g) and heptane (heptane fraction from petroleum having a boiling point of 65 to 100 ⁰C) (25 g) is added. Then, the reaction mixture is heated to an internal temperature of 75 ⁰C (external temperature 95 ⁰C) and refluxed for approximately 30 minutes, whilst stirring. Then, the reaction mixture is filtered over a glass fibre filter at an internal temperature of 70 to 75 ⁰C (external temperature 85 ⁰C) in to a 350 ml flask equipped with a magnetic stirrer. Then, a mixture of isopropanol (5 g) and heptane (5 g) is added and the reaction mixture is heated to an internal temperature of 70 ⁰C (external temperature 95 ⁰C). Then, further heptane is added drop wise to the reaction mixture at an internal temperature of 65 to 75 ⁰C (external temperature 75 ⁰C).

(b) Crystallization

The solution from step (a) is then cooled to an internal temperature of 40 ⁰C (external temperature 40 ⁰C) over a period of 15 minutes. The, at an internal temperature of 40 ⁰C, the solution is treated with a suspension of the recrystallized product (v) (11 mg) in heptane is added and the reaction mixture is stirred for 30 minutes at an internal temperature of 40 ⁰C (external temperature 40 to 45 ⁰C). Then, the reaction mixture is treated with some further heptane (15 g) at an internal temperature of 40 ⁰C. The resulting suspension is then cooled to an internal temperature of -10 ⁰C (external temperature -10 to -15 ⁰C) over a period of 30 minutes and then further stirred for a further hour. The reaction mixture is then filtered at an internal temperature of -10 ⁰C (external temperature -10 to -15 ⁰C) and the resulting solid may be washed in a mixture of isopropanol and heptane, where the isopropanohheptane ratio is 1 :1.5. The solid may be washed twice (2 x 11.25 g). The solid may then be dried in a vacuum at a temperature of 60 ⁰C.

Yield: 17.8 g (89% of theory) mp: 127.4 to 132.0 ⁰C.

………………………………………………………………..

An efficient process was developed for the manufacture of MSA100, a serotonin receptor antagonist, via a five-step synthetic route furnishing a high quality of active pharmaceutical ingredient. Highlights of this synthesis include: (1) replacing carcinogenic methyl iodide with methyl p-toluenesulfonate as the methylating reagent; (2) a hydrogenation protocol with optimized temperature, pressure, and mass-transfer conditions that avoided one side product and reduced the other one effectively; (3) chemical resolution employing D-camphoric acid in a mixed-solvent system; (4) amidation under anhydrous conditions for controlling a Michael adduct impurity; and (5) plausible mechanisms for the formation of side products.

(2E)-N-[2-[2-[(2S)-1-Methyl-2-piperidinyl]ethyl]phenyl]-3-phenyl-2-propenamide (1)

To a mixture of 14 (6.28 g, 15 mmol) ……………………………………………………to obtain 1 (4.06 g, 78% yield) as an off-white solid: mp 125–127 °C (lit. ref 1a, mp 128 °C);

Chiral HPLC for (S)-1 (t_R = 19.3 min), >99.9% ee; (R)-1 (t_R = 18.5 min): Chiralcel AD-H, 250 × 4.6 mm, flow rate = 1.0 mL/min, 25 °C, 900:100:1 A:B:C isocratic; A = hexanes; B = ethanol; C = diethylamine; UV λ = 230 nm. HPLC for 1 (t_R = 11.2 min) 99.8% purity; 8 (t_R = 5.4 min); 9 (t_R = 12.3 min): Waters Symmetry-C18 150 × 4.6 mm, flow rate = 1 mL/min, 25 °C, gradient elution from 93:7 A–B to 85:15 A–B over 5 min, to 10:90 A–B over 10 min and held for 2 min, to 93:7 A–B over 1 min; A = 0.1% TFA in water; B = acetonitrile; UV λ = 230 nm.

…………………

http://www.google.com/patents/EP0973741A1

Example I PREPARATION AND CONFIRMATION OF S-MPEC

racemic-APEMEP-HI -5/1-

– 6 –

1. 2 – nitrobenzaldehyde

2. 2 – picoline

3. 2 – (o-nitrostyryl) pyridine (NSP)

4. 2 – ^“(o-nitrostyryl)- 1 -methylpyridinium iodide 5. RS-2- (o-aminophenethyl)-l-methylpiperidine. HI

6. S-[2-(o-aminophenethyl)- 1 -methylpiperidine-dibenzoyl-L-tartrate] (S-APEMP. DBLT OR .L-DBT)

7. S-2′- [2-(l-methyl-2-piperidyl) ethyl] cinnamanilide (S-MPEC) 7a. Cinnamoyl chloride S-MPEC CHEMICAL PROCESS

(A) 2-(O-Nitrostyryl) – 1 -Methylpyridinium Iodide fNSMP-P

To a 50 L round bottomed flask was added 2-nitrobenzaldehyde (3,500 g. 23.2 moles), 2-picoline (3.2L., 32.8 moles) and acetic anhydride. The mixture was stirred efficiently under an inert atmosphere (nitrogen or another inert gas) and heated to reflux for 27 hrs. The mixture was cooled to under 100 C, for safe handling, and quenched in a suitable vessel equipped with external cooling and efficient stirring on 10.5 Kg. of ice. The pH was adjusted to 11 with 45% aqueous sodium hydroxide at a rate to keep the temperature below 50°C. After cooling to 20-30°C, the granular solid was collected by filtration, washed well with water. Yield 6572 g. of crude 2-(o-nitrostyryl) pyridine (NSP).

This solid was transferred to a 50L, round bottomed flask, dissolved in acetone (14L.) and iodomethane (2.94L., 47.7 moles) (quaternizing methylating agent) was added. (Other such (alkylating) agents may be used, generally having the formula CH₃X, X being an anion such as sulfate, methyl sulfate, halide (Cl, Br, I), etc.). The mixture was heated to reflux under an inert atmosphere (nitrogen or another inert gas) for 18 hrs. After cooling to 20°C. the precipitate was collected by filtration and washed with acetone or a 1 :1 mixture of acetone:ethyl acetate (3×3.5L.). Drying to constant weight at 50-60°C. yielded 6,839 g. (80%) of NSMP.I. (B) RS- 2-(o-Aminophenethyl)-l-Methylpiperidine. Hvdroiodide

ΓRS-APEMP.HΠ

In a 5 gallon reactor, a solution of NSNP.I (935 g., 2.5 moles) in – 7 – methanol (14L.) was reduced in a hydrogen atmosphere (Psi. 55) in the presence of Pt/C (5 or 10%, 98g.). After removal of the catalyst and evaporation of the filtrate in the usual manner, the residue was dissolved in hot methanol (2.8L.). Ethyl acetate (2.8L) was added to the hot mixture to induce crystallization, yield 516.3 g. (59%) of RS-APEMP.HI.

(C) S-[2-(o-Aminophenethv0- 1 -Methylpiperidine Dibenzoyl-L-Tartrate] (S- APEMP.DBLT)

A solution of RS-APEMP.HI (516g., 1.5 mole) ethyl acetate (5.5g.) (or other low boiling water immiscible solvent such as benzene, toluene etc.) was extracted with 5% aqueous sodium hydroxide to liberate the free base (organic phase), washing the organic phase with water, drying over a suitable drying agent (such as anhydr. sodium sulfate, magnesium sulfate, potassium carbonate etc.) After separating the solvent from the drying agent the solution was evaporated in vacuo and the residual RS-APEMP free base was dissolved in methanol (l .O.L.) and a solution of dibenzoyl-L-tartaric acid (540 g., 1.5 moles) in methanol (2.3 L.) was added. The mixture was held overnight at room temperature. The crystalline precipitate was collected and recrystallized from methanol (3.4 L.), yield 246g. of S-APEMP.DBLT. (28.6%, wt; 57.2% of the S-APEMP). (O) S-2′-r2-π-Methyl-2-Piperidvnethyll Cinnamanilide f S-MPEC) A solution of S-APEMP.DBLT (287 g, 0.5 mole) in ethyl acetate

(3.2 L.) (or other low boiling water immiscible solvent) was extracted with 7.5% aqueous sodium bicarbonate (3.2 L.) to liberate the S-APEMP. After a water wash and drying over a suitable drying agent the solvent was removed in vacuo. The oily residue, S-APEMP, was dissolved in ethyl acetate (1.0 L.) and anhydrous potassium carbonate (412 g, 3.0 moles) (or other suitable acid acceptor such as triethyl amine, pyridine etc.) was added. Cinnamoyl chloride (143 g., 0.7 mole) in 700 ml. of ethyl acetate was added slowly. After the initial reaction, the mixture was refluxed for 14 hrs. After cooling to room temperature the mixture was extracted with water (1.7 L.) and dried over a suitable drying agent. After removing the drying agent the solvent was removed in vacuo and the residue was dissolved in hot ethyl acetate (280 ml.) and allowed to slowly cool to room temperature; filtration yielded S-MPEC, (136 g., 79% yield). Analysis: Calcd. For C, H, N : C, 79.27; H, 8.10;N, 8.04. Found: C, 79.27; H, 8.06; N, 8.07. HPLC(chiral)purity: 99.5%, [oc ]_D25, -46° (c=0.01,EtOH); Melting point: 128°C.
TABLE 2 CERTIFICATE OF ANALYSIS Compound Name: (-)-2′-[2-(l-Methyl-2 piperidyl)ethyl]cinnamanilide(/-MPEC,S- MPEC

……………………………….

http://www.google.com/patents/US20080300410?cl=en

synthesis of 2′[2-1-(methyl -2-piperidyl) ethyl] cinnamanilide (Y1), which is a compound of formula (Y) where R^a is hydrogen and R¹ is methyl:

Processes for the preparation of compounds of formula (Y) are described in U.S. Pat. No. 3,931,195 which comprises the step of alkylating compounds of formula (i) (below) with an alkyl halide, such as methyl iodide for methylation. The same methylation step is described in EP0973741 for the synthesis of compound (Y1).

Thus, the processes described in the prior art involve the use of a highly toxic reagent (e.g. methyl iodide) and provides a yield of about 50%

US4064254 *	Oct 21, 1976	Dec 20, 1977	Mead Johnson & Company	Substituted piperidines therapeutic process and compositions
US5780487 *	Feb 28, 1997	Jul 14, 1998	Amer Moh Samir	S-2′- 2-(1-methyl-2-piperidyl) ethyl! cinnamanilide

N-[2-(7-Benzyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)ethyl]acetamide

N-{2-[7-(Cyclohexylmethyl)-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl]ethyl}acetamide

Acetamide, N-​[2-​[7-​(cyclohexylmethyl)​-​1,​6-​dihydro-​2H-​indeno[5,​4-​b]​furan-​8-​yl]​ethyl]​-

339.47, C₂₂ H₂₉ N O₂

cas 1287785-08-3

Melatonin MT2 Agonists

Takeda……..innovator

Treatment of Sleep Disorders,

• Melatonin (N-acetyl-5-methoxytryptamine), which is a hormone synthesized and secreted principally in the pineal gland, increases in dark circumstances and decreases in light circumstances. Melatonin exerts suppressively on pigment cells and the female gonads, and acts as a synchronous factor of biological clock while taking part in transmittance of photoperiodic code. Therefore, melatonin is expected to be used for the therapy of diseases related with melatonin activity, such as reproduction and endocrinic disorders, sleep-awake rhythm disorders, jet-lag syndrome and various disorders related to aging, etc.
• Recently, it has been reported that the production of melatonin melatonin could reset the body’s aging clock (see Ann. N. Y. Acad. Sci., Vol. 719, pp. 456-460 (1994)). As previously reported, however, melatonin is easily metabolized by metabolic enzymes in vivo (see Clinical Examinations, Vol. 38, No. 11, pp. 282-284 (1994)). Therefore, it cannot be said that melatonin is suitable as a pharmaceutical substance.
• Various melatonin agonists and antagonists such as those mentioned below are known.

  • (1) EP-A-578620 discloses compounds of:

   

  • • (2) EP-A-420064 discloses a compound of:
    • (3) EP-A-447285 discloses a compound of:
    • (4) EP-A-662471 discloses a compound of:
    • (5) EP-A-591057 discloses a compound of:
    • (6) EP-A-527687 discloses compounds of:

      X=S, 0, Y=CH
      X=0, NH, Y=N

    • (7) EP-A-506539 discloses compounds of:
  • Tricyclic or more poly-cyclic compounds with a cyclic ether moiety, such as those mentioned below, are known.

    • (1) Compounds of:

      are disclosed in Tetrahedron Lett., Vol. 36, p. 7019 (1995).

    • (2) Compounds of:

      are disclosed in J. Med. Chem., Vol. 35, p. 3625 (1992).

    • (3) Compounds of:

      are disclosed in Tetrahedron, Vol. 48, p. 1039 (1992).

    • (4) Compounds of:

      are disclosed in Tetrahedron Lett., Vol. 32, p. 3345 (1991).

    • (5) A compound of:

      is disclosed in Bioorg. Chem., Vol. 18, p. 291 (1990).

    • (6) A compound of:

      is disclosed in J. Electroanal. Chem. Interfacial Electrochem., Vol. 278, p. 249 (1990).

     

    see

http://www.google.co.in/patents/EP0885210B1?cl=en

Highly Potent MT₂-Selective Agonists

N-{2-[7-(Cyclohexylmethyl)-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl]ethyl}acetamide (15)

SDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEV SPELVCSMLHLCSG [SEQ ID NO: 2].

BXQ-350

Cincinnati Children’s Hospital  ……..innovator

Bexion Pharmaceuticals……….under license

In February 2015, the US FDA granted saposin C Orphan designation for the treatment of glioblastoma multiforme

SAPOCIN C

Recombinant human Saposin C (SapC) bound to a liposomal formulation of the dioleoylphosphatidylserine

Bexion’s Saposin C – the active ingredient in the brain tumour therapy BXQ-350 – has been awarded Orphan Drug status by US regulators.

Read more at: http://www.pharmatimes.com/Article/15-02-17/US_Orphan_status_for_Bexion_s_brain_tumour_drug.aspx#ixzz3S3zXdHlO

Bexion Pharmaceuticals, under license from the Cincinnati Children’s Hospital, is investigating a human saposin C (SapC)/liposomal dioleoylphosphatidylserine (DOPS) conjugate, SapC-DOPS (BXQ-350), a nanovesicle-formulated pro-apoptotic sphingomyelinase activating molecular imaging agent and anticancer agent, for the potential diagnosis and treatment of cancer , . In October 2013, Bexion was planning a phase I first-in-human trial for the therapy of glioblastoma multiforme

Bexion Pharmaceuticals LLC announced today that the U.S. Food and Drug Administration (FDA) has granted the company Orphan Drug designation for Saposin C, active ingredient in its proprietary drug BXQ-350 for the potential treatment of glioblastoma multiforme.

The FDA’s Office of Orphan Drug Products Development reviews applications for Orphan Drug status to support development of medicines for underserved patient populations, or rare disorders that affect fewer than 200,000 people in the United States. The successful application submitted by Bexion and the FDA granting of Orphan Drug status entitles the company to a seven-year period of marketing exclusivity in the United States for BXQ-350, if it is approved by the FDA for the treatment of glioblastoma multiforme. Orphan Drug status also enables the company to apply for research grant funding for Phase I and II Clinical Trials, tax credits for certain research expenses, and a waiver from the FDA’s application user fee, as well as additional support from FDA and a potentially faster regulatory process.

Bexion was previously awarded a prestigious Phase II Bridge Award (Small Business Innovation Research Grant; SBIR) from the National Cancer Institute (NCI) to support the manufacture and clinical testing of BXQ-350.

“Orphan Drug status for BXQ-350 is an important milestone in the development of this new treatment modality,” stated Dr. Ray Takigiku, founder and CEO of Bexion. “Few treatment options are available for patients suffering from glioblastoma multiforme and this designation recognizes the unmet need that exists with this disease, as well as the unique attributes of BXQ-350. In addition, orphan designation allows Bexion to benefit from important financial, regulatory and commercial considerations and we have seen recently that products with orphan designation have become sought after assets.”

About Orphan Drug Designation
Orphan Drug designation is a status assigned to a medicine intended for use in rare diseases. In the U.S., the Orphan Drug Designation program confers Orphan Drug status to successful applicants for medicines intended for the safe and effective treatment, diagnosis or prevention of rare diseases or disorders that affect fewer than 200,000 people in the U.S. or that are not expected to recover the costs of developing and marketing a treatment.¹

The approval of an orphan designation request does not alter the standard regulatory requirements and process for obtaining marketing approval for investigational use. Sponsors must establish safety and efficacy of a compound in the treatment of a disease through adequate and well-controlled studies. However, the FDA review process may be speedier for Orphan Drugs than those which do not receive Orphan Drug designation.

About BXQ-350
In pre-clinical studies, Bexion’s first-in-class biologic, BXQ-350 has shown promising results in selectively inducing cell death in the laboratory. BXQ-350 is a proprietary nanovesicle formulation of Saposin C (sphingolipid activator protein C, or SapC) and the phospholipid dioleoylphosphatidylserine (DOPS).

About Bexion Pharmaceuticals
Bexion Pharmaceuticals is a privately held biotech company focused on the development and commercialization of innovative cures for cancer.  Initial products are based on a proprietary platform technology licensed from Cincinnati Children’s Hospital Medical Center.  The technology has demonstrated potential for development as a therapeutic, diagnostic and surgical imaging reagent, and as a carrier for other pharmaceutical agents, such as oligonucleotides.  For more information, visit www.bexionpharma.com or contact Margaret van Gilse atmvangilse@bexionpharma.com.

¹ U.S. Food and Drug Administration web site. “Regulatory Information: Orphan Drug Act.”http://www.fda.gov/regulatoryinformation/legislation/federalfooddrugandcosmeticactfdcact/significantamendmentstothefdcact/orphandrugact/default.htm.

Margaret van Gilse859-757-1652mvangilse@bexionpharma.com

SOURCE Bexion Pharmaceuticals LLC

Glioblastoma is the most common primary CNS malignant neoplasm in adults, and accounts for nearly 75% of the cases. Although there has been steady progress in their treatment due to improvements in neuro-imaging, microsurgery, and radiation, glioblastomas remain incurable. The average life expectancy is less than one year from diagnosis, and the five-year survival rate following aggressive therapy, including gross tumor resection, is less than 10%. Glioblastomas cause death due to rapid, aggressive, and infiltrative growth in the brain. The infiltrative growth pattern is responsible for the un-resectable nature of these tumors. Glioblastomas are also relatively resistant to radiation and chemotherapy, and therefore post-treatment recurrence rates are high. In addition, the immune response to the neoplastic cells is mainly ineffective in completely eradicating residual neoplastic cells following resection and radiation therapy.

One problem in treating glioblastoma is the tumor’s protection behind the blood-brain tumor barrier (BBTB). A significant obstacle in the development of therapeutics for glioblastoma is the inability of systemic therapies to efficiently cross the BBTB. Saposin C (SapC) is a sphingolipid- activating protein that functions to catabolize glycosphingolipids. SapC-DOPS forms stable nanovesicles which can efficiently cross the blood-brain tumor barrier and fuse with GBM cells inducing cell death.

Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus, which was discovered first for its properties as an antifungal agent. Streptomyces hygroscopicus has also been implicated as a cancer agent.

There remains a need in the art for new therapeutics for the treatment of glioblastoma.

…………………………………………………………………..

https://www.google.com/patents/US20040229799?cl=en22

Example 1Purification of Recombinant Saposin C

[0106] Recombinant saposin C was overexpressed in E. coli cells by using the isopropyl-1-thio-β-D-galactopyranoside inducing pET system (Qi et al. (1994) J. Biol. Chem. 269:16746-16753, herein incorporated by reference in its entirety). Expressed polypeptides with a His-tag were eluted from nickel columns. After dialysis, the polypeptides were further purified by HPLC chromatography as follows. A C4 reverse phase column was equilibrated with 0.1% trifluoroacetic acid (TFA) for 10 minutes. The proteins were eluted in a linear (0-100%) gradient of 0.1% TFA in acetonitrile over 60 minutes. The major protein peak was collected and lyophilized. Protein concentration was determined as previously described (Qi et al. (1994) J. Biol. Chem. 269:16746-16753).

Example 2Bath Sonication of Sanosin C and Dioleoylphosphatidylserine

[0107] Dioleoylphosphatidylserine (DOPS) was obtained from Avanti Polar Lipids (Alabaster AL). Twenty to thirty imoles of DOPS in chloroform were dried under N₂ and vacuum to lipid films. Five to ten μmoles saposin C polypeptide was added to the dried films and suspended in 50 μl McIlvanine buffer (pH 4.7). The suspension was then brought to a 1 ml volume with either cell culture medium or phosphate buffered saline (PBS) (Ausubel et al. (2002) Current Protocols in Molecular Biology. John Wiley & Sons, New York, New York, herein incorporated by reference). The mixture was sonicated in a bath sonicator for approximately 20 minutes. Ice was added as needed to prevent overheating the samples.

………………………………………………………………

http://www.google.com/patents/WO2014078522A1?cl=en

The SapC-DOPS composition comprises a phospholipid, an isolated saposin C-related polypeptide, wherein the polypeptide comprises an amino acid sequence at least 75% identical to the entire length of SEQ ID NO: 2, and a pharmaceutically acceptable carrier, wherein the phospholipid forms a nano vesicle incorporating the polypeptide. In certain embodiments, the polypeptide comprises an amino acid sequence at least 85% identical to the entire length of SEQ ID NO: 2. In certain embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to the entire length of SEQ ID NO: 2. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99% identical to the entire length of SEQ ID NO: 2.

The Sequence Listing, filed electronically and identified as SEQ_LIST_OSIF-2013- 102.txt, was created on November 12, 2013, is 5,548 in size, and is hereby incorporated by reference.

[0004] SEQ ID NO: 1

siy

J su c n 61y &n

*8 a 210 2iS

t n«

:?e

<H ¾^■ yts ca« ¾»* **u v ΆΧ» s?s ass ¾«¾

:»o

L st S«x ri» r s

SEQ ID NO: 2

BEXION PHARMA

1. 1. Russell Street, Covington, KY 41011, United States

      $2.9 Million Grant Awarded to Covington-Based Bexion for Next Step in Cancer Fight

      921 Spring Street Covington, Kentucky 41016 United States

      112 East 4th Street, Covington, KY 41011.

      1182 Riverhouse Way Covington KY : 427657

cas 1253286-89-3

Spiro[3H-​indole-​3,​4′-​piperidin]​-​2(1H)​-​one, 5-​[6-​amino-​5-​[(1R)​-​1-​(2,​6-​dichloro-​3-​fluorophenyl)​ethoxy]​-​3-​pyridinyl]​-​1′-​methyl-

SMU-B

or is it

1253286-90-6

Spiro[3H-​indole-​3,​4′-​piperidin]​-​2(1H)​-​one, 6-​[6-​amino-​5-​[(1R)​-​1-​(2,​6-​dichloro-​3-​fluorophenyl)​ethoxy]​-​3-​pyridinyl]​-​1′-​methyl-

SMU-B

A series of novel aminopyridyl/pyrazinyl-substituted spiro[indoline-3,4′-piperidine]-2-ones were designed, synthesized, and tested in various in vitro/in vivo pharmacological and antitumor assays. 6-[6-Amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-3-pyridyl]-1′-methylspiro[indoline-3,4′-piperidine]-2-one (compound 5b or SMU-B) was identified as a potent, highly selective, well-tolerated, and orally efficacious c-Met/ALK dual inhibitor, which showed pharmacodynamics effect by inhibiting c-Met phosphorylation in vivo and significant tumor growth inhibitions (>50%) in GTL-16 human gastric carcinoma xenograft models.

see..http://pubs.acs.org/doi/abs/10.1021/ml400203d

cas 1253286-90-6

6-[6-Amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-3-pyridyl]-1′-methylspiro[indoline-3,4′-piperidine]-2-one (compound 5b or SMU-B)

SEE

CN 101851237

南方医科大学

1_4,3_ [(IR) -I- (2,6_ two gas _3_ gas phenyl) ethoxy] -2-nitro-approved P set

  obtained in Step 1-3 (IS) -I- (2,6- dichloro-3-fluorophenyl) ethanol (2. 09g, IOmmol) was dissolved in dry THF (80 ml). Then, at room temperature under a nitrogen atmosphere, a solution of 3-hydroxy-2-nitro-pyridine (1.54g, llmmol) and triphenylphosphine (3. 409g, 13mmol), and so is completely dissolved, cooled to 0 ° C, was added Diisopropyl azodicarboxylate (DIAD, 2.63g, 13mmol), After the addition, the mixture was stirred at 0 ° C for 16 hours, the solvent was removed by rotary evaporation and the oily residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate : 4/1) to give the desired product as a white solid (3. 046g, yield: 92%) o 1H-NMR (CDClySOOMHz): 8 (ppm) I. 86 (d, J = 6. 4Hz, 3H), 6 . 10 (q, J = 6. 4Hz, 1H), 7. 09 (dd, J = 7. 6,8. 8Hz, 1H), 7. 21 (dd, J = 8. 4, I. 2Hz, 1H ), 7. 31 (dd, J = 4. 8,8. 8Hz, 1H),

7. 37 (dd, J = 4. 8,8. OHz, 1H), 8. 04 (dd, J = L 6,4. 4Hz, 1H). Mass spectrum m / z:. 330 94 [M + H, 35C1,35Cl], 332. 92 [M + H, 35Cl, 37Cl].

  1_5,3_ [(IR) -I- (2,6_ two gas -3- gas phenyl) ethoxy] -2_ atmosphere based grant given P

to take steps 1-4 to get the 3 – [(lR) _l- (2,6_ dichloro-3-fluorophenyl) ethoxy] -2_ nitro than Li Jie (2. 649g, 8mmol) was dissolved in ethanol (15mL) was added iron powder (3. 575g, 64mmol) were mixed under nitrogen with vigorous stirring at 90 ° C oil bath, was added via syringe 0.8mL IM HCl (aq), after 10 minutes, was added 0. 8mL IMHCl (aq). Stirring was continued for 30 minutes, TLC showed the reaction. Cooled to room temperature, filtered through Celite, the filter residue washed with ethanol (3X IOmL). The combined organic phase was removed by rotary evaporation of the solvent gave the desired product as a light brown solid (2. 41g, yield: 100%) o 1H-Nmr (Cdci3JOOmHz): 8 (ppm) I. 81 (d, J = 6. 8Hz, 3H ), 5. 03 (s, br, 2H), 6. 01 (q, J = 6. 8Hz, 1H), 6. 47 (dd, J = 4. 8,7. 6Hz, 1H), 6. 70 (d, J = 8. OHz, 1H), 7. 05 (t, J = 8. 8Hz, 1H), 7. 28 (dd, J = 4. 0,8. 0Hz, 1H), 7. 57 ( d, J = 5.2Hz, lH). Mass spectrum m / z:. 301 00 [M + H, 35Cl, 35Cl], 302. 77 [M + H, 35Cl, 37Cl].

  l-6,5_ desert _3_ [(IR) -I- (2,6_ two gas -3- gas phenyl) ethoxy] -2_ atmosphere base than Li Jie

The steps 1-5 obtained 3_ [(IR) -I- (2,6_ two gas _3_ gas phenyl) ethoxy] -2-yl atmosphere than Li Jie (1.506g, 5mmol) dissolved in acetonitrile (20mL) in. Then, at 0 ° C and the degree of stirring in added portionwise N- bromosuccinimide (0.908g, 5. Lmmol), After the addition, stirring was continued for 30 minutes. The solvent was removed by rotary evaporation, the crude product was obtained as a white solid was the desired product (1.045g, yield: 55%) was purified by column chromatography on silica gel. 1H-NMR (⑶Cl3,500MHz): 8 (ppm) I. 81 (d, J = 6. 8Hz, 3H), 4 85 (s, br, 2H), 6 98 (q, J = 6. 8Hz.. , 1H), 6. 82 (d, J =

2. 0Hz, 1H), 7. 08 (t, J = 8. 4Hz, 1H), 7. 31 (dd, J = 4. 8,8. 8Hz, 1H), 7. 65 (d, J = 2 . OHz, 1H). Mass spectrum m / z:… 378 84 [M + H, 35Cl, 35Cl, 79Br], 380 82 [M + H, 35Cl, 35Cl, 81Br or 35Cl, 37Cl, 79Br], 382 80 [M + H, 35Cl , 37Cl, 81Bror 37Cl, 37Cl, 79Br].

Step 2, I ‘- methyl-5- (4,4,5,5-tetramethyl -I, 3,2- dioxolane boron-2-yl) spiro [indoline Spray – 3,4 ‘- piperidin] -2_ one

  2-1,5- bromo -I ‘- methyl-spiro [indoline-3,4’ – piperidin] _2_ one

[0300] 5-bromo – indol-2-one (I. 272g, 6mmol) was suspended THF (15mL) at, and cooled to -78 ° C, added dropwise with stirring IM NaN (SiMe3) THF solution of 2 (30mL, 30mmol). After the addition was stirred at _78 ° C 30 min, then 2-chloro -N- (2- chloro-ethyl) -N- methyl-ethylamine hydrochloride solid (I. 155g, 6mmol). After the addition stirring was continued for 30 minutes, then warmed to room temperature and stirred for two days. TLC showed the reaction was completed, to the pink suspension was carefully added aqueous 4M hydrochloric acid (IOmL), and then adjusted with concentrated aqueous ammonia to pH ^ 9, and extracted with DCM (3 X 80mL). The organic phases were combined, dried (Na2SO4), and concentrated to give the crude product was purified by silica gel column chromatography (7M NH3 in methanol solution / DCM: 5/95) to give the desired product (I. 38g, yield: 78%) was purified. 1H-NMR (CD3ODjOOMHz):. 8 (ppm) I. 86-1 92 (m, 2H), I 94-1 98 (m, 2H), 2 44 (s, 3H), 2 62-…. 2. 68 (m, 2H), 2. 86-2. 91 (m, 2H), 6. 76 (d, J = 7. 6Hz, 1H), 7. 33 (dd, J = I. 2,7 . 6Hz, 1H), 7. 44 (d, J = I. 6Hz, 1H), 7. 81 (s, br, 1H). Mass spectrum m / z:. 294 99 [M + H, 79Br], 296 82 [M + H, 81Br]..

2-2, V – methyl-5- (4,4,5,5-tetramethyl–1,3,2_ dioxolane Borane _2_ yl) spiro [indoline – 3,4 ‘- piperidin] -2_ one

Under nitrogen, obtained in Step 2-1 to 5-bromo -I ‘- methyl-spiro [indoline-_3,4’ – piperidin] _2_ one (147. 6mg, 0. 5mmol) , the United pinacols drop acid unitary purpose (140mg, 0. 55mmol) and acetic acid Bell (147mg, I. 5mmol) in DMSO (0. 2ml) was added in PdCl2 (dppf) • CH2Cl2 (20. 4mg, 0. 025mmol ), to the resulting solution was bubbled with nitrogen for 2 minutes, and then stirred at 80 ° C of 16 hours. LC-MS showed completion of the reaction, after cooling to room temperature, water (2mL), extracted with DCM (3X5mL). The organic phases were combined, dried (Na2SO4), and concentrated to give the desired product (170mg, yield: 100%) o MS m / z:. 342 07 [M + H], 343. 08 [M + H, 100%], 344. 11 [M + H].

  Step 3,5_ [6_ atmosphere base _5_ [(IR) -I- (2,6_ two gas -3- gas phenyl) ethoxy] -3_ batch P fixed base] -I ‘- A group spiro [indoline-3,4 ‘- piperidin] -2_ one

The steps 1-6 5_ desert obtained _3_ [(IR) -I- (2,6_ two gas _3_ gas phenyl) ethoxy] -2-yl batch atmosphere pyridine (75. 8mg , 0. 2mmol), I’- step 2_2 obtained methyl 5- (4,4,5,5-tetramethyl-l, 3,2-dioxolane Borane 2-yl) spiro [ indoline-3,4′-piperidin] -2-one (82mg, 0. 24mmol) and potassium carbonate (82. 9mg, 0. 6mmol) was dissolved in DME / water mixture solution (4 / 1,2. Oml ). Then, under nitrogen, was added Pd (PPh3) 4 (II. 6mg, 0. Olmmol), to the resulting mixture was bubbled with nitrogen for 2 minutes, and then stirred at 80 ° C of 18 hours. LC-MS showed completion of the reaction, after cooling to room temperature, water (5mL), extracted (3 X IOmL) with DCM. The organic phases were combined, dried (Na2SO4), and concentrated to give the crude product was purified by silica gel column chromatography (7M NH3 in methanol solution / DCM: 5/95) to give the desired product (88. 6mg, yield: 86%) was purified. 1H-Nmr (Cdci3JOOmHz): 8 (ppm) I. 86 (d, J = 6. 4Hz, 3H), I 93-2 02 (m, 4H), 2 44 (s, 3H),…

2. 66-2. 72 (m, 2H), 2. 89-2. 93 (m, 2H), 4. 87 (s, br, 2H), 6. ll (q, J = 6. 4Hz, 1H ), 6. 88 (d, J =

8. OHz, 1H), 6. 94 (d, J = I. 2Hz, 1H), 7. 06 (t, J = 8. 4Hz, 1H), 7. 19 (dd, J = I. 2,8 . OHz, 1H),

7. 31 (m, 1H), 7. 36 (s, 1H), 7. 66 (s, br, 1H), 7. 80 (d, J = 2. OHz, 1H). Mass spectrum m / z:.. 515 05 [M + H, 35Cl, 35Cl], 517 03 [M + H, 35Cl, 37Cl].

  Example 2: 6_ [6_ atmosphere base _5_ [(IR) -I- (2,6_ two gas -3- gas phenyl) ethoxy] -3_ than Li Jie base] -I ‘ – methyl-spiro [indoline-3,4 ‘- piperidin] -2_ one

Step I, I ‘- methyl-6- (4,4,5,5-tetramethyl–I, 3,2- dioxolane boron-2-yl) spiro [indoline Spray – 3,4 ‘- piperidin] -2_ one

  1-1,6- bromo -I ‘- methyl-spiro [indoline-3,4’ – piperidin] -2_ one

  As described in Example I steps 2-1 of the method from the commercially available 6-bromo – indol-2-one was prepared, Yield: 82%. Analysis of the data obtained the desired product are = 1H-Nmr (Cd3OdJOOmHz): 8 (ppm) 1.90-1.98 (m, 4H),

2. 44 (s, 3H), 2. 64-2. 68 (m, 2H), 2. 86-2. 92 (m, 2H), 7. 05 (d, J = 2. 0Hz, 1H), 7. 16-7. 21 (m, 2H), 7. 91 (s, br, 1H). Mass spectrum m / z: 295 00 [M + H, 79Br], 296 78 [M + H, 81Br]… [0312] 1-2, 1 ‘- methyl-6- (4,4,5,5-tetramethyl-_1,3,2_ dioxolane Borane _2_ yl) spiro [indoline – 3,4 ‘- piperidin] -2_ one

In the step 1-1 of the obtained 6-bromo -I ‘- methyl-spiro [indoline-_3,4’ – piperidin] -2_ ketone and commercially available linking pinacol boronic ester material, the method of Example I was prepared in accordance with steps 2-2, Yield: 95%. Analysis of the data obtained of the target product are as follows: Mass spectrum m / z:. 342 06 [M + H], 343 04 [M + H, 100%], 344. 12 [M + H]..

  Step 2,6_ [6_ atmosphere base _5_ [(IR) -I- (2,6_ two gas -3- gas phenyl) ethoxy] -3 ratio Li Jie base] -I ‘- methyl-spiro [indoline-3,4 ‘- piperidin] -2_ one

  Example I steps 1-6 to obtain 5-bromo -3 – [(IR) -I- (2,6- dichloro-3-fluorophenyl) ethoxy] -2-amino- pyridine, I obtained in Example 1-2 of the present embodiment in step ‘- methyl-6- (4,4,5,5-tetramethyl-l, 3,2-dioxolane-2-yl borane) spiro [indoline-_3,4 ‘- piperidin] -2-one, prepared as in Example I Step 3. Yield: 82%. 1H-Nmr (Cdci3JOOmHz): 8 (ppm) I. 86 (d, J = 6. 4Hz, 3H), I. 91-1 95 (m, 2H), I 97-2 03 (m, 2H… ), 2. 45 (s, 3H), 2. 65-2. 72 (m, 2H), 2. 89-2. 95 (m, 2H), 5. 12 (s, hr, 2H),

6. 12 (q, J = 6. 4Hz, 1H), 6. 94-7. 00 (m, 3H), 7. 06 (t, J = 8. 4Hz, 1H), 7. 31 (m, 1H ), 7. 35 (d, J = 7. 2Hz, 1H), 7. 90 (d, J = 2. 0Hz, 1H), 9. 28 (s, br, 1H). Mass spectrum m / z:.. 515 05 [M + H, 35Cl, 35Cl], 517 03 [M + H, 35Cl, 37Cl].

5- [6-amino-5 – [(2,6-dichloro-3-fluorophenyl) methoxy] _3_ pyridinyl] -I’–methyl-spiro [indole: 3 [0317] Example morpholine-3,4 ‘- piperidin] -2-one

H2N N

Step I, 5_ desert _3_ (2,6_ two gas -3- integrity oxy) _2_ atmosphere based grant given P

  1-1,2,6_ two gas acid gas _3_

Cl OF

Sodium hydroxide (13g, 325mmol) in water (IlOmL) was cooled to _5 ° C was added dropwise under vigorous stirring of liquid bromine (12. 5g, 78. 2mmol), added after the addition of pre-cooled to 10 ° C dioxane (75mL). The above mixture under vigorous stirring was added dropwise a pre-cooled to 5 ° C of I- (2,6- dichloro-3-fluorophenyl) ethanone (5g, 21. 2mmol) in dioxane (330mL) and water (90mL) was added. After the addition, at room temperature for 2 hours Lan Xiang, Xiang Lan then 90 C for 30 minutes. TLC was not shown with the S starting material disappeared, and was acidified with concentrated hydrochloric acid to PH~9. The resulting mixture was rotary evaporated to dryness, added water (20mL), and extracted with diethyl ether (2X80mL), the organic phases were combined, dried (Na2SO4), and concentrated to give an oily product solidified after cooling to a transparent, slightly yellow solid (3. 4g, Yield: 67%). 1H-Nmr (Cdci3AOOmHz):. 8 (ppm) 7. 21 (. Dd, J = 8. 0,8 8Hz, 1H), 7 35 (. Dd, J = 4. 4,9 2Hz, 1H), 9 . 79 (s, br, 1H). Mass spectrum m / z (ES “:. 207 11 [M_H, 35Clj35Cl], 209 10 [MH, 35Cl, 37Cl]..

  1-2,2,6–dichloro-3-fluoro-benzyl alcohol

^ Coh

F

[0325] To be filled with 2,6-dichloro-3-fluoro benzoic acid (3g, 14. 35mmol) added dropwise to the flask IM BH3. THF (43mL, 43mmol), added after the mixture was stirred under reflux for 24 hours. TLC showed the reaction was complete, methanol (50mL) to destroy excess borane, and the solvent was distilled off under reduced pressure and the resulting trimethyl borate, the process is repeated twice more to give a viscous product 2. I g, yield: 75% . 1H-Nmr (Cdci3JOOmHz): 8 (ppm) 2. 09 (t, J = 6. 4Hz, 1H), 4. 97 (d, J = 6. 4Hz, 2H), 7 09 (t, J = 8. . 8Hz, 1H), 7. 32 (dd, J = 4. 8,9. 1Hz, 1H). Mass spectrum m / z (ES-):.. 193 08 [M_H, 35Cl, 35Cl], 195 12 [MH, 35Cl, 37Cl].

  1-3,3_ (2,6-gas _3_ integrity oxy) _2_ nitro grant given P

Following the procedure of steps 1-4 of Example I, was prepared from 2,6-dichloro-3-fluoro-benzyl alcohol and 3-hydroxy-2-nitropyridine prepared in yield (in this example embodiment steps 1_2) : 90%. 1H-Nmr (Cdci3AOOmHz): 8 (ppm) 5. 45 (s, 2H), 7 20, 7 37 (dd, J = 4. 8. (Dd, J = 8. 0,9 2Hz, 1H.). , 9. 2Hz, 1H), 7. 59 (dd, J = 4. 4,8. 4Hz, 1H),

7. 74 (dd, J = L 2,8. 4Hz, 1H), 8. 17 (dd, J = L 6,4. 4Hz, 1H). Mass spectrum m / z:. 316 89 [M + H, 35Cl, 35Cl], 318. 89 [M + H, 35Cl, 37Cl].

  1_4,3_ (2,6-gas _3_ integrity oxy) _2_ atmosphere based grant given P

The method according to Example I step 1_5 from 3- (2,6-gas -3- integrity oxy) _2_ nitro Jie ratio 唳 preparation (in this case, steps 1-3), that Yield: 95% o 1H-Nmr (Cdci3JOOmHz):. 8 (ppm) 4 65 (s, br, 2H), 5 31 (s, 2H), 6 66 (dd, J = 5. 2,8.. . 0Hz, 1H), 7. 14 (dd, J = I. 2,8. 0Hz, 1H), 7. 18 (dd, J =

8. 4,9. 2Hz, 1H), 7. 37 (dd, J = 4. 8,8. 8Hz, 1H), 7. 73 (dd, J = I. 6,5. 6Hz, 1H). Mass spectrum m / z:. 286 95 [M + H, 35Cl, 35Cl], 288 85 [M + H, 35Cl, 37Cl]..

  1-5,5_ desert -3- (2,6-gas -3_ integrity oxy) ~ 2 ~ atmosphere based grant given P

Following the procedure of Example I step 1_6 embodiment, starting from 3- (2,6-gas _3_ integrity yloxy) _2_ atmosphere group given the preparation of the batch P (in the example of the present embodiment in step 1-4), Yield: 60% o 1H-Nmr (Cdci3JOOmHz):. 8 (ppm) 4 68 (s, br, 2H), 5 28 (s, 2H), 7 21 (dd, J = 8. 0,8.. . 8Hz, lH), 7. 24 (dd, J = 2. OHz, 1H), 7. 39 (dd, J = 4. 8,

9. 2Hz, 1H), 7. 78 (d, J = 2. OHz, 1H). Mass spectrum m / z:. 364 83 [M + H, 35Cl, 36Cl, 79Br], 366 77 [M + H], 368 69 [M + H]…

  Step 2,5_ [6_ atmosphere base _5_ [(2,6_ two gas -3- gas) methoxy] -3_ than Li Jie base] -I-methyl-spiro [indoline _ 3,4 ‘- piperidin] -2-one

The present embodiment 5_ desert steps 1_5 obtained _3_ (2,6_ two gas _3_ integrity yloxy) pyridine ~ 2 ~ atmosphere, Examples 2-2 obtained in step I I ‘- methyl-5- (4,4,5,5-tetramethyl-borane _1,3,2- dioxolane-2-yl) spiro [indoline-_3,4’ – piperidine ] -2-one, prepared as in Example I Step 3. Yield: 85 V0o 1H-Nmr (Cdci3JOOmHz):.. 8 (ppm) I. 92-2 02 (m, 4H), 2. 43 (s, 3H), 2. 65-2 71 (m, 2H) , 2. 90-2. 91 (m, 2H), 4. 92 (s, br, 2H), 5. 52 (s, 2H), 6. 89 (d, J = 8. 4Hz, 1H), 6 . 90 (d, J = L 2Hz, 1H), 7. 06 (t, J = 8. OHz, 1H), 7. 21 (dd, J = L 2,8. OHz, 1H), 7. 31 ( m, 1H),

7. 37 (s, 1H), 7. 79 (s, br, 1H), 7. 80 (d, J = 2.0Hz, lH). MS m / z:. 501 06 [M + H, 35Cl, 35Cl], 503 04 [M + H, 35Cl, 37Cl]..

6- [6-amino-5 – [(2,6-dichloro-3-fluorophenyl) methoxy] _3_ pyridinyl] -I’- methyl-spiro [indole: 4 [0337] Example morpholine _3,4 ‘- piperidin] -2-one

H2N N

  Following the procedure in Example I step of Example 3, the procedure of Example 3 to give 5-bromo-1-5 _3_ (2,6-dichloro-3-fluoro-benzyloxy) -2-amino-pyridine and Step 2 in Example I to give the embodiment 1-2 ‘- methyl-6- (4,4,5,5-tetramethyl-1,3,2-dioxolane Borane 2-yl) spiro [ indoline-3,4 ‘- piperidine] _2_ ester -one, yield:. 78 V0o 1H-Nmr (Cdci3JOOmHz): 8 (ppm) I. 96-2 00 (m, 2H), 2. 01 -2. 12 (m, 2H), 2. 46 (s, 3H), 2. 66-2. 73 (m, 2H), 2. 90-2. 96 (m, 2H), 5. 30 (s , hr, 2H), 6. 94-7. 01 (m, 3H), 7. 07 (t, J =

8. 4Hz, 1H), 7. 30 (m, 1H), 7. 34 (d, J = 7. 2Hz, 1H), 7. 89 (d, J = 2. OHz, 1H), 8. 56 ( s, br, 1H). MS m / z:. 501 06 [M + H, 35Cl, 35Cl], 503 04 [M + H, 35Cl, 37Cl]..

  Example 5: 5_ [5_ atmosphere base -6- [(IR) -I- (2,6_ two gas _3_ gas phenyl) ethoxy] Batch-2-yl] -I ‘ – methyl-spiro [indoline-3,4 ‘- piperidin] -2-one

J0A = o

. | J: too

[0342] Step 1,5_ desert _2_ atmosphere base _3_ [(IR) -I- (2,6_ two gas _3_ gas phenyl) ethoxy] Jie than exposure

Cl 6, / ISL / Br

xy

H2N N

  In at 0 ° C, NaH (80mg of NaH in mineral oil, 2mmol) force the mouth (1R) -1_ (2,6- dichloro-3-fluorophenyl) ethanol (418mg, 2mmol. See example Example I Step 1_3) in anhydrous THF (6mL) and stirred for half an hour, a solution of 2-amino-3,5-dibromo-pyrazine (506mg, 2mmol) in THF (6mL) was added. The resulting mixture was warmed to room temperature, heated under reflux for 20 hours. TLC showed the reaction was substantially complete. After cooling to room temperature, water was added (IOmL), the mixture was extracted three times with ethyl acetate (3x20mL), the organic phases were combined, dried, concentrated, and the residue to give 594mg product was purified by column chromatography (l-3Me0H inhexanes), yield: 78%. 1H-NMR (O) Cl3, 500MHz):. 8 (ppm) I. 83 (d, J = 7. 2Hz, 3H), 5. 12 (s, br, 2H), 6 73 (q, J = 6 . 8Hz, 1H), 7. 05 (t, J = 8. OHz, 1H), 7. 28 (dd, J = 4. 8,

8. 8Hz, 1H), 7. 58 (s, 1H). Mass spectrum m / z:. 379 83 [M + H, 35Cl, 35Cl, 79Br], 381. 81 [M + H, 35Cl, 35Cl, 81Br], 383 79 [M + H, 35Cl, 37Cl, 81Br]..

Step 2,5_ [5_ atmosphere base _6_ [(IR) -I- (2,6_ two gas _3_ gas phenyl) ethoxy] Batch-2-yl] -I ‘- A group spiro [indoline-3,4 ‘- piperidin] -2-one

  5_ bromide present embodiment obtained in step I _2_ amino _3_ [(IR) -I- (2,6_ dichloro _3_ fluorophenyl) ethoxy] pyrazine, implemented I’- methyl step 2-2 obtained in Example I-5 (4,4,5,5-tetramethyl -I, 3,2- dioxolane boron

2-yl) spiro [indoline-3,4 ‘- piperidin] -2-one, prepared as in Example I Step 3. Yield: 54%. 1H-NMR (CD3ODjOOMHz): 8 (ppm) I. 85 (d, J = 6. 8Hz, 3H), I 85-1 88 (m, 2H), I 97-2 04 (m, 2H…. ), 2. 46 (s, 3H), 2. 76-2. 82 (m, 2H), 2. 97-3. 02 (m, 2H), 6. 74 (q, J = 6. 4Hz, 1H ), 6. 85 (d, J = 8. OHz, 1H), 7. 15 (t, J = 8. 4Hz, 1H), 7. 41 (dd, J = 4. 8,9. 2Hz, lH) , 7. 54 (dd, J = I. 6,

8. OHz, 1H), 7. 69 (d, J = I. 8Hz, 1H), 7. 81 (dt, J = 2. 0,8. 0Hz, 1H), 7. 87 (s, 1H). Mass spectrum m / z:. 515 92 [M + H, 35Cl, 35Cl], 517. 90 [M + H, 35Cl, 37Cl].

Example 6: 6- [5-amino -6 – [(lR) -l_ (2,6- dichloro _3_ fluorophenyl) ethoxy] pyrazin-2-yl] -I ‘ – methyl-spiro [indoline-3,4 ‘- piperidin] -2-one

The embodiment of Example 5, 5_ bromo obtained in step I _2_ amino _3_ [(IR) -I- (2,6_ dichloro _3_ fluorophenyl) ethoxy] pyrazine, Example I’- methyl-2 obtained in steps 1-2 6- (4,4,5,5-tetramethyl–I, 3,2- dioxolane boron-2-yl) spiro [indole morpholine -3,4’_ piperidin] -2-one, prepared as in Example I Step 3. Yield: 67% 0

1H-NMR (CD3ODjOOMHz): 8 (ppm) I. 85 (d, J = 6. 8Hz, 3H), I 88-1 96 (m, 4H), 2 48 (s… , 3H), 2. 76-2. 82 (m, 2H), 2. 98-3. 05 (m, 2H), 6. 75 (q, J = 6. 4Hz, 1H), 7. 16 (t , J = 8. 8Hz, 1H), 7. 31 (d, J = 2. OHz, 1H), 7. 36-7. 43 (m, 3H), 7. 88 (s, 1H).

Mass spectrum m / z:. 515 99 [M + H, 35Clj35Cl], 517 90 [M + H, 35Cl, 37Cl]..

SEE

Bioorganic & Medicinal Chemistry Letters (2014), 24(16), 3673-3682.

School of Pharmaceutical Sciences, Southern Medical University,

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AZD 3264

MW 441.50

CAS 1609281-86-8

Inhibition of IkB-kinase IKK2 has been identified as one of the novel pathways to treat inflammatory conditions such as asthma, chronic pulmonary obstructive disorder (COPD) and rheumatoid arthritis

Astrazeneca Ab,

……………………..

PATENT

WO 2003010158

https://www.google.com/patents/WO2003010158A1?cl=en

The synthesis began with the aromatic nucleophilic substitution reaction of 2-fluorobromobenzene (2) with (S)-N-Boc-3-pyrrolidinol 3 to give the bromo intermediate 4, which was borylated via halogen metal exchange using n-hexLi in THF followed by treatment with triisopropyl borate and acidic work-up to give the boronic acid intermediate 5. Suzuki coupling of the boronic acid 5 with bromothiophene 6(2)afforded the intermediate 7. Intermediate 7 was subjected to regioselective bromination using bromine in acetic acid. This reaction was nonregioselective and yielded 17% of the required isomer 8. The bromo compound 8 was coupled with isoxazole boronate ester 9 by another Suzuki reaction to get the title compound. The overall yield of the synthesis was <6%.

………………………..

PAPER

http://pubs.acs.org/doi/full/10.1021/op500105n

An efficient and scalable synthesis of AZD3264 is described in which the differential reactivities of various halogen atoms have been employed. The process involves five linear chemical steps with three isolated stages starting from commercially available fragments.

Purification

To a stirred suspension of crude AZD3264 (1) (1.75 kg, 3.98 mol) in methanol (23.75 L) and water (2.64 L) was added formic acid (0.24 kg, 5.18 mol), and the mixture was heated to 40 °C for 1.5 h, cooled to 25 °C, and basified with aqueous ammonia (12.29 M in water, 1.62 L, 19.92 mol). The product was isolated by filtration.

 ¹H NMR (DMSO-d₆, 400 MHz): δ 1.92–2.10 (m, 2H), 2.28 (s, 3H), 2.46 (s, 3H), 2.75–2.82 (m, 1H), 3.00–3.12 (m, 3H), 5.11–5.12 (m, 1H), 6.90 (br, 2H), 7.00–7.03 (m, 2H), 7.30 (br, 1H), 7.70–7.72 (m, 2H), 7.83 (s, 1H), 10.93 (s, 1H).

 ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 10.54, 11.42, 32.94, 45.51, 53.00, 79.37, 111.76, 114.17, 115.66, 120.70, 121.20, 122.77, 125.39, 126.92, 128.84, 150.12, 152.54, 154.50, 158.13, 165.14, 167.06.

DEPT NMR (DMSO-d₆, 100.6 MHz): δ 10.54, 11.43, 32.94, 45.51, 53.01, 79.35, 114.17, 120.70, 121.20, 126.92.

HRMS calcd for C₂₁H₂₄N₅O₄S (M + H)⁺: 442.1543, found 442.1554.

[α]²⁵_D −13.80 (c 0.5, DMSO)

 

Journal of Medicinal Chemistry (2013), 56(18), 7232-7242 reports similar analogues

A new “flozin” seems to me appearing on the horizon in form of SBM-TFC-039 an SGLT Inhibitor from Sirona Biochem, picked up a list from WO 2012160218,  from TFChem…….see link , Sirona Biochem Announces SGLT2 Inhibitor and Skin Lightening Patent Granted, 29 Jun 2015, Patent entitled “Family of aryl, heteroaryl, o-aryl and o-heteroaryl carbasugars”

This led me to search, “Family of aryl, heteroaryl, o-aryl and o-heteroaryl carbasugars” 
WO 2012160218 A1, IN 2013-DN10635, CN 103649033Tf化学公司

List above as in http://www.google.com/patents/WO2012160218A1?cl=en

FROM THE ABOVE LIST, SBM-TFC-039 MAY BE PREDICTED/OR AS SHOWN BELOW

COMPD 16 as in/WO2012160218

COMPD 16, PREDICTED/LIKELY SBM-TFC-039 has CAS 1413373-30-4, name D-​myo-​Inositol, 1-​[4-​chloro-​3-​[(4-​ethoxyphenyl)​methyl]​phenyl]​-​1,​2,​3-​trideoxy-​2,​2-​difluoro-​3-​(hydroxymethyl)​-

Just scrolling through the patent gave me more insight

MORE EVIDENCE….http://www.google.com/patents/WO2012160218A1?cl=en, this patent descibes compd 16 as follows

Compound 16 according to the invention has been compared to Dapaglifozin to underline the improvement of the duration of action, i.e. the longer duration of glucosuria, of the compound when the intracyclic oxygen atom of the glucose moiety is replaced by a CF₂ moiety.

This assay has been carried out at a dose of 3 mg/ kg.

The results obtained are presented on Figure 5. It appears thus that 16 (3 mg/kg) triggered glucosuria that lasted beyond 24 hours compared to Dapagliflozin.

• Compound 16 according to the invention has been compared to the compound 9 of WO 2009/1076550 to underline the improvement of the duration of action of the compound when a mimic of glucose bearing a CH-OH moiety instead of the intracyclic oxygen atom is replaced by a mimic of glucose bearing a CF₂ in place of the CH-OH moiet .

SBM-TFC-039

PATENT

WO 2012160218

http://www.google.com/patents/WO2012160218A1?cl=en

Examples within this first subclass include but are not limited to:

Synthesis of compound 8

C35H34O5 M = 534.64 g.mol^“

Mass: (ESI ): 535.00 (M + H); 552.00 (M + H₂0); 785.87; 1086.67 (2M + H₂0)

A.

 

Procedure A:

To a solution of 4 (10.5g, 15.89mmol, leq) in toluene (400mL) were added 18-crown-6 (168mg, 0.64mmol, 0.04eq) and potassium carbonate (6.69g, 48.5mmol, 3.05eq.). The mixture was stirred overnight at room temperature, and then the remising insoluble material was filtered off and washed with toluene. The filtrate and the washings were combined, washed with 2N hydrochloric acid aqueous solution followed by saturated sodium hydrogencarbonate aqueous solution, dried over sodium sulphate, filtered and concentrated under reduced pressure. The residue was purified on silica gel chromatography (cyclohexane/ethyl acetate 98:2 to 80:20) to afford cyclohexenone 8 (4.07g; 48% yield) as yellowish oil.

Procedure B:

A solution of 7 (3.27g, 5.92mmol, leq) in pyridine (14mL) was cooled to 0°C before POCl₃ (2.75mL, 29.6mmol, 5eq) was added dropwise. The mixture was stirred at this temperature for 10 min before the cooling bath was removed. The reaction mixture was stirred overnight at room temperature before being re-cooled to 0°C. POCI3 (2.75mL, 29.6mmol, 5eq) was added once again trying to complete the reaction. The mixture was stirred for an additional 20h at room temperature before being diluted with Et₂0 (20mL) and poured onto crushed ice. 1M HC1 aqueous solution (lOOmL) was added, and the mixture was extracted with Et₂0 (200mL & l OOmL). The combined organic extracts were washed with brine (lOOmL), dried over sodium sulphate, filtered and concentrated before being purified on silica gel chromatography (cyclohexane / ethyl acetate 98:2 to 80:20) to afford compound 8 (1.46g, 46% yield) as an orange oil. Synthesis of compound 9

C₁₅H₁₂BrC10₂ M = 339.61 g.moF¹

Mass: (GC-MS): 338-340

 

The synthesis of this product is described in J. Med. Chem. 2008, 51, 1 145—1149.Synthesis of compound 10

C15H14B1CIO M = 325.63 g.mof¹

 

10 The synthesis of this product is described in J. Med. Chem. 2008, 51, 1145-1 149.

Synthesis of compound 11

C50H49CIO6 M = 781.37 g.moF¹

Mass: ESI⁺): 798.20 (M + H₂0)

 

Under inert atmosphere, Mg powder (265mg, 10.9mmol, 2.4eq) was charged into a three necked flask, followed by addition of a portion of 1/3 of a solution of the 4- bromo-l-chloro-2-(4-ethylbenzyl)benzene (2.95g, 9.1mmol; 2eq) in dry THF (25mL) and 1 ,2-dibromoethane (10 mol % of Mg; 85mg; 0.45mmol). The mixture was heated to reflux. After the reaction was initiated (exothermic and consuming of Mg), the remaining solution of 2-(4-ethylbenzyl)-4-bromo-l-chlorobenzene in dry TFIF was added dropwise. The mixture was then allowed to react for another one hour under gentle reflux until most of the Mg was consumed.

The above Grignard reagent was added dropwise into the solution of cyclohexenone 8 (2.42g, 4.53mmol, leq) in dry THF (25mL) under inert atmosphere at room temperature (about 25°C), then allowed to react for 3h. A saturated aqueous solution of ammonium chloride was added into the mixture to quench the reaction. The mixture was extracted with Et₂0, washed with brine, dried over sodium sulphate, filtered and concentrated. The residue was purified on silica gel chromatography (cyclohexane/ethyl acetate 100:0 to 80:20) to afford the target compound 11 as a yellow oil (3.01g, 86%).

Synthesis of compound 12

C₅oH49C10₅ M = 765.37 g.mol^“1

+): 782.13 (M + H₂0)

 

Triethylsilane (0.210mL, 1.30mmol, 3eq) and boron-trifluoride etherate (48% BF₃, O. l lOmL, 0.866mmol, 2eq) were successively added into a solution of alcohol 1 1 (338mg, 0.433mmol, leq) in dichloromethane (5mL) under inert atmosphere at -20°C. After stirring for 2.5h, a saturated aqueous solution of sodium chloride was added to quench the reaction. The mixture was extracted with CH₂C1₂ (10mLx3) and the organic layer was washed with brine, dried over Na₂S0₄, filtrated and concentrated. The residue was purified on silica gel chromatography (cyclohexane/ethyl acetate 9.8:0.2 to 8:2) to afford the target compound 12 as a white powder (278 mg, 0.363mmol, 84%).

Synthesis of compound 13

C₅oH_5tC10₆ M = 783.39g.moF¹

Mass: (ESI⁺): 800 (M + H₂0); 1581 (2M + H₂0)

Under inert atmosphere, borane-dimethyl sulfide complex (2M in THF, 16.7mL, 33mmol, 10.5eq) was added to a solution of 12 (2.41g; 3.15mmol, leq) in dry THF (lOOmL) cooled to 0°C. The reaction mixture was then refluxed for lh,cooled to 0°C and treated carefully with sodium hydroxide (3M in H₂0, 10.5mL, 31.5mmol, lOeq), followed by hydrogen peroxide (30% in H₂0, 3.2mL, 31.5mmol, l Oeq) at room temperature (above 30°C). The mixture was allowed to react overnight at room temperature (~25°C) before a saturated aqueous solution of ammonium chloride was added to quench the reaction. The mixture was extracted with ethyl acetate and the organic layer was washed with brine, dried over Na₂S0₄, filtered, and concentrated. The residue was purified by silica gel chromatography (cyclohexane/ethyl acetate 97:3 to 73:27) to afford the desired compound 13 (1.05g; 43%) as a yellowish oil.

Synthesis of compound 14

C50H49CIO6 M = 781.37g.mol^“1

Mass: (ESI⁺): 798 (M + H₂0); 1471; 1579 (2M + H₂0)

 

13 14

Dess-Martin periodinane (81mg; 1.91mmol; 1.5eq) was added portion wise to a solution of alcohol 13 (l .Og; 1.28mmol, leq) in anhydrous dichloromethane (20mL) at 0°C. The reaction was then stirred overnight at room temperature before being quenched with IN aqueous solution of sodium hydroxide. The organic layer was separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over sodium sulphate, filtered and concentrated. The residue was purified on silica gel chromatography (cyclohexane / ethyl acetate 98:2 to 82: 18), to afford the target ketone 14 (783mg, 79% yield) as a colorless oil. Synthesis of compound 15

C₅oH49ClF₂0₆ M = 803.37g.moF¹

¹⁹ F NMR (CDCU, 282.5MHz): -100.3 (d, J=254Hz, IF, CFF); -1 13.3 (td, Jl=254Hz, J2=29Hz, IF, CFF).

Mass: (ESI⁺): 820.00 (M+H₂0)

 

14 15

A solution of ketone 14 (421mg, 0.539mmol, leq) in DAST (2mL, 16.3mmol, 30eq.) was stirred under inert atmosphere at 70°C for 12h. The mixture was then cooled to room temperature and dichloromethane was added. The solution was poured on a mixture of water, ice and solid NaHC0₃. Agitation was maintained for 30min while reaching room temperature. The aqueous layer was extracted with dichloromethane and the organic phase was dried over Na₂S0₄, filtered and concentrated. The crude product was purified on silica gel chromatography (cyclohexane/ethyl acetate 98:2 to 80:20) to afford the desired compound 15 as a yellowish oil ( 182mg, 42% yield).

Synthesis of compound 16

C22H25CIF2O5 M = 442.88g.mor¹

¹⁹ F NMR (MeOD, 282.5MHz): -96.7 (d, J=254Hz, IF, CFF); 12.2 (td,

Jl=254Hz, J2=28Hz, IF, CFF).

Mass: (ESI⁺): 465.3 (M+Na)

 

o-Dichlorobenzene (0.320mL, 2.82mol, lOeq) followed by Pd/C 10% (0.342g, 0.32mol, l .leq) were added to a solution of 15 (228mg, 0.28mmol, leq) in a mixture of THF and MeOH (2: 1, v/v, 160mL). The reaction was placed under hydrogen atmosphere and stirred at room temperature for 2h. The reaction mixture was filtered and concentrated before being purified on silica gel chromatography (dichloromethane/methanol 100: 1 to 90: 10) to afford compound 16 (105mg, 83% yield).

Sirona Biochem’s SGLT Inhibitor Performs Better Than Johnson and Johnson’s SGLT Inhibitor, According to Study

……………………………………………………………………………….

Shanghai Fosun Pharmaceutical Group Co. Ltd.

//////

Gamendazole

(E) 3-(1-(2,4-Dichlorobenzyl)-6-(trifluoromethyl)-1H-indazol-3-yl)acrylic Acid

trans-3-(1-Benzyl-6-(trifluoromethyl)-1H-indazol-3-yl)acrylic acid)

(E)-3-[1-[(2,4-Dichlorophenyl)methyl]-6-(trifluoromethyl)indazol-3-yl]prop-2-enoic acid

• C₁₈H₁₁Cl₂F₃N₂O₂
• mw415.193
• RC-MC-110

Heat Shock Protein 90 (HSP90) Inhibitors

University Of Kansas …Innovator

Gamendazole is a novel drug candidate for male contraception. It is an indazole carboxylic acid derived from lonidamine (LND). Gamendazole produced 100% antispermatogenic effects at 25 mg/kg i.p. in rats, whereas 200 mg/kg was fatal for 60% of rats tested. Since gamendazole produced 100% efficacy, it was tested orally. At a dose of 6 mg/kg, 100% of rats were infertile 4 weeks after a single administration. Complete infertility was maintained for 2 weeks, followed by complete recovery in 4 of 7 rats. The other 3 never recovered fertility. Upon dosing 6 mg/kg orally for 7 days, it produced similar infertility results, but only 2 of 7 rats recovered fertility. There were no abnormalities in rates of conception or abnormal conception in rats who recovered fertility.

In August 2004, preclinical data were presented at the 228th ACS meeting in Philadelphia, PA. Gamendazole, an indazole-3-acrylic acid derivative

Pathology reports were conducted on gamendazole treated rats. At 25 mg/kg i.p., 6 mg/kg oral, and in animals that survived 200 mg/kg i.p., there were no remarkable findings, with no evidence of inflammation, necrosis, tumors, or hemorrhage. There was also a lack of observable behavioral effects at 25 mg/kg i.p., 6 mg/kg oral, and in animals that survived 200 mg/kg i.p. Gamendazole treatment had no effect on testosterone levels, and was reported to affect Sertoli cell function, leading to decreased levels of inhibin B. Low levels of inhibin B were correlated to the infertility of the rat

Female oral contraceptive drugs are widely available in the market by several trade names, including Altravera, Brevicon, Levora, and i-pill, whereas potentially safer, more convenient, and more effective oral male contraceptives are not yet commercially available. However, there are some experimental drugs.AF-2785 1, gamendazole 2, lonidamine 3, and adjudin 4 are most promising among the experimental

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1.

Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

Synthesis

…………………….

2-Halo benzoic acid is converted into aroyl chloride and then to aroyl cyanide in an overall yield of 82%. Aroyl cyanides 5 are converted to 2-halophenyl glyoxylate ester 7 via ketoamide 6 in 85% yields as shown in Scheme below. Direct conversion of aroyl cyanide 5 to ester 7 is also reported^[ U.S. Patent 4,596,885, 1986 .^] but with lesser yields.

Preparation of (E) 3-(1-(2,4-Dichlorobenzyl)-6-(trifluoromethyl)-1H-indazol-3-yl)acrylic Acid (R = CF₃) (Gamendazole) (2)

trans 3-[l- (l^-dichlorobenzy^-ό-trifluoromethyl-lH-indazol-S-ylj-acrylic acid (RC-MC-110) is provided.

 

EXAMPLE 2: Synthesis of a-ll-fl^-dichlorobenzyn-ό-trifluoromethyl-lH-indazol-S-yll-acrylic acid (RC-MC-110)

Step 1 : 2-(2-nitro-4-trifluoromethylphenyl)-malonic acid dimethyl ester.

 

Dimethyl malonate (59.7 g, 0.44 mol) was added dropwise to a stirred solution of potassium tert-butoxide (51 g, 0.44 mol) in dry t-butanol (500 mL). To the resultant suspension, a warm solution of 2-chloro-5-trifluoromethylnitrobenzene (50 g, 0.22 mol) in t-butanol (100 mL) was added and the mixture was refluxed for 6 h (reaction monitored by TLC). After completion of the reaction, most of the t-butanol was distilled off under vacuum, and chilled water was then added to the reaction mixture. The pH was adjusted to neutral with dilute hydrochloric acid, which resulted in the precipitation of the product. The mixture was stirred for 30 minutes and the product was filtered off (68 g, 95%). This material was used without further purification in the next step. A small amount was crystallized (EtOAc/hexane, 4:6) for analysis, to yield a yellow crystalline material, mp 65-67 ⁰C. ¹H NMR (CDCl₃) 8.30 (s, 1 H), 7.92 (d, J = 8.4 Hz, 1 H), 7.69 (d, J = 8.4 Hz, 1 H), 5.37 (s, 1 H), 3.80 (s, 6 H). MS (FAB) m/z: 322.1 (M⁺ + 1).

Step 2: (2-nitro-4-trifluoromethylphenyl)-acetic acid methyl ester.

 

2-(2-Nitro-4-trifluoromethylphenyl)-malonic acid dimethyl ester (68 g, 0.21 mol) was dissolved in dimethyl sulfoxide (200 mL). Sodium chloride (34 g, 0.58 mol) and water (60 mL) were added and the mixture was stirred for 16-20 h at 120 ⁰C (reaction monitored by TLC). The reaction mixture was then cooled to room temperature and quenched into water, which caused precipitation of the product. After stirring for 30 minutes, the product (45 g, 80%) was isolated by filtration. The product was used without further purification in the next reaction. A small sample was crystallized (EtOAc/hexane, 2:8) for analysis, to yield yellow crystals, mp 104-105 ⁰C. ¹H

NMR (CDCl₃) 8.3 (s, 1 H), 7.88 (d, J = 8.4 Hz, 1 H), 7.50 (d, J = 8.4 Hz, 1 H), 4.12 (s, 2 H), 3.60 (s, 3 H). MS (FAB) m/z: 275.2 (M⁺ + 1).

Step 3: (2-Acetylamino-4-trifluoromethylphenyl)-acetic acid methyl ester.

 

Hydrogenation and acetylation of (2-nitro-4-trifluoromethylphenyl)-acetic acid methyl ester (25 g, 0.095 mol) in the presence of 5% Pd-C (2.5 g, 50% wet) and acetic anhydride (38 g, 0.37 mol) in toluene (200 mL) was carried out under vigorous stirring at room temperature and atmospheric pressure for about 4-5 h (reaction monitored by TLC). The catalyst was removed by filtration and washed with toluene two times. The combined organics were evaporated in vacuo to yield the product (24.8 g, 95%), which was used without further purification in the next step. A small sample was crystallized from hexane to yield the product as a yellow solid, mp 92-94 ⁰C. H NMR (CDCl₃) 8.86 (s, 1 H), 8.21 (s, 1 H), 7.36 (d, J = 8.1 Hz, 1 H), 7.31 (d, J = 8.1 Hz, 1 H), 3.74 (s, 3 H), 3.68 (s, 2 H), 2.23 (s, 3 H). Step 4: ό-Trifluoromethyl-lH-indazole^-carboxylic acid methyl ester.

To a solution of (2-acetylamino-4-trifluoromethylphenyl)-acetic acid methyl ester (16 g, 0.058 mol) in acetic acid (50 mL) was added dropwise t-butyl nitrite (90%) (7.35 g, 0.063 mol) over a period of 20 min. at 90-95 ⁰C. The mixture was then stirred for 0.5 h at 95 ⁰C, poured into cold water and stirred for 1 h. The precipitates were collected by filtration and washed with water. The crude material was dissolved in ethyl acetate and dried over sodium sulfate. The solvent was removed in vacuo. This material (13.4 g, 95%) was used without further purification in the next step. A small sample was crystallized from ethyl acetate to yield a white solid, mp 240-242 ⁰C. H NMR (DMSO-d-6) 8.25 (d, J = 8.5 Hz, 1 H), 8.04 (s, 1 H), 7.58 (d, J = 8.5 Hz, 1 H), 3.95 (s, 3 H). MS (FAB) m/z: 245.1 (M⁺ + 1).

Step 5: l-(2,4-Dichlorobenzyl)-6-trifluoromethyl-lH-indazole-3-carboxylic acid methyl ester.

 

ό-Trifluoromethyl-lH-indazole-S-carboxylic acid methyl ester (2.75 g, 0.0112 mol) was dissolved in acetonitrile (50 mL), and potassium carbonate (1O g, 0.07 mol), 2,4-dichlorobenzyl chloride (2.42 g, 0.01239 mol) and tetrabutylammonium iodide (catalytic) were added. The reaction mixture was heated to reflux and refluxed for 2 h under good stirring. The progress of the reaction was monitored by TLC. After completion of the reaction, potassium carbonate was filtered while hot and then washed with acetone. The combined solvents were distilled off under reduced pressure to afford the crude mixture of Nl and N2 benzylated products. The isomers were separated by column chromatography (silica gel, eluent started with hexane then changed to 8:2 hexane, ethyl acetate). l-(2,4-Dichlorobenzyl)-6-trifluoromethyl-lH-indazole-3-carboxylic acid methyl ester. Yield: 3.62 g (80%), white crystals mp 118-120 ⁰C. ‘ H NMR (CDCl₃) 8.39 (d, J = 8.4 Hz, 1 H) 7.74 (s, 1 H), 7.57 (d, J = 8.4 Hz, 1 H), 7.45 (d, J = 2.1 Hz, 1 H), 7.12 (dd, J = 8.4 and 2.1 Hz, 1 H), 6.78 (d, J = 8.4 Hz, 1 H), 5.82 (s, 2 H), 4.07 (s, 3 H). MS (FAB) m/z: 403 (M⁺ + 1).Z-^^-DichlorobenzylJ-δ-trifluoromethyl-ZH-indazole-S-carboxylic acid methyl ester. Yield: 680 mg (15%), white crystals mp 132-134 ⁰C. ‘ H NMR (DMSO-d-6) 8.27 (s, 1 H), 8.20 (d, J = 8.7 Hz, 1 H), 7.76 (d, J = 1.8 Hz, 1 H), 7.57 (d, J = 8.7 Hz, 1 H), 7.30 (dd, J = 8.3 and 1.8 Hz, 1 H), 6.78 (d, J = 8.3 Hz, 1 H), 6.17 (s, 2 H), 3.96 (s, 3 H).

Step 6: [l-(2.4-Difluorobenzyl)-6-trifluoromethyl-lH-indazol-3-yl1-methanol.

 

l -(2,4-Dichlorobenzyl)-6-trifluoromethyl-lH-indazole-3-carboxylic acid methyl ester (3.0 g, 0.0075 mol) dissolved in CH₂Cl₂(50 mL) was cooled to -78 ⁰C. DIBAL-H (8.18 mL, 0.00818 mol) was added slowly dropwise via a syringe under an argon blanket over a period of 15 minutes. After the complete addition of DIBAL-H, the reaction mixture was stirred at -78°C for another 2 h (reaction monitored by TLC). The reaction was quenched carefully with methanol at -78 ⁰C. The reaction mixture was then carefully poured into water and the layers were separated. The organic layer was washed with water and dried over sodium sulfate. Removal of the solvent yielded the crude alcohol (2.6 g, 93%), which was used without purification in the next step. The alcohol was a white solid, mp 137-139 ⁰C. ¹H NMR (CDCl₃) 7.97 (d, J = 8.4 Hz, 1 H), 7.66 (s, 1 H), 7.44 (d, J = 2.0 Hz, 1 H), 7.42 (d, J = 8.5 Hz, 1 H), 7.12 (dd, J = 8.3 and 2.0 Hz, 1 H), 6.93 (d, J = 8.3 Hz, 1 H), 5.65 (s, 2 H), 5.09 (s, 2 H). MS (FAB) m/z: 375 (M⁺ + 1).Step 7: l-(2,4-Dichlorobenzyl)-6-trifluoromethyl-lH-indazole-3-carbaldehvde.

 

[l-(2,4-Difluorobenzyl)-6-trifluoromethyl-lH-indazol-3-yl]-methanol (3.75 g, 0.01 mol) was dissolved in CH₂Cl₂ (100 mL) and manganese(IV)oxide (8.7 g, 0.1 mol) was added and stirred for 2-3 h at room temperature (reaction monitored by TLC). The solids were removed by filtration and the removal of the CH₂Cl₂ in vacuo yielded the crude aldehyde. The aldehyde was used without further purification in the next step. The aldehyde (3.54 g, 95%) was a white solid, mp 97-98 ⁰C. ¹H NMR (CDCl₃) 10.25 (s, 1 H), 8.45 (d, J = 8.5 Hz, 1 H), 7.79 (s, 1 H), 7.60 (d, J = 8.5 Hz, 1 H), 7.48 (d, J = 2.0 Hz, 1 H), 7.20 (dd, J = 8.3 Hz and 2.0 Hz, 1 H), 6.93 (d, J = 8.3 Hz, 1 H), 5.79 (s, 2 H). MS (FAB) m/z: 373 (M⁺ + 1).

Step 8: 3-ri-(2,4-Dichlorobenzyl)-6-trifluoromethyl-lH-indazol-3-yll-acrylic acid ethyl ester.

 

l-(2,4-Dichlorobenzyl)-6-trifluoromethyl-lH-indazole-3-carbaldehyde (2.0 g, 0.00536 mol) was dissolved in CH₂Cl₂ (50 niL) and Wittig reagent (carbethoxymethylene) triphenylphosphorane (1.06 g, 0.0536 mol) was added to the solution. The homogeneous reaction mixture was heated to reflux in an oil bath for 12 h. The reaction progress was monitored by TLC. The reaction mixture was cooled to room temperature and worked up by quenching into water and separating the organic layer. Removal of the CH₂Cl₂ yielded the crude product, which was purified by column chromatography to yield the pure product (2.25 g, 95%) as a white solid, mp 186-188 ⁰C. ¹H NMR (CDCl₃) 8.08 (d, J = 8.5 Hz, 1 H), 7.99 (d, J = 16.2 Hz, 1 H), 7.74 (s, 1 H), 7.52 (d, J = 8.5 Hz, 1 H), 7.47 (d, J = 2.0 Hz, 1 H), 7.16 (dd, J = 8.3 and 2.0 Hz, 1 H), 6.84 (d, J = 8.3 Hz, 1 H), 6.82 (d, J = 16.2 Hz, 1 H), 5.72 (s, 2 H), 4.32 (q, J = 7.1 Hz, 2 H), 1.38 (t, J = 7.1 Hz, 3 H). MS (FAB) m/z: 443 (M⁺ + 1).It will be appreciated that the acrylic acid ethyl ester can be hydrogenated using 5% Pd-C in the presence of methanol, DCM at RT and 1 atm-pressure to give the propionic acid ester derivative. For example, treatment under such conditions yields 3-[l-(2,4-dichlorobenzyl)-6- trifluoromethyl-lH-indazol-3-yl]-propionic acid ethyl ester (JWS-2-70).

Step 9: l-(2,4-Dichlorobenzyl^‘)-3-r6-trifluoromethyl-lΗ-indazol-3-yll-acrvlic acid.

 

l-(2,4-Dichlorobenzyl)-3-[6-trifluoromethyl-lH-indazol-3-yl]-acrylic acid ethyl ester (2.0 g, 0.0045 mol) was dissolved in a mixture of tetrahydrofuran (50 mL) and methanol (25 mL). A lithium hydroxide solution (0.33 g, 0.013 mol lithium hydroxide in 7.5 mL water) was added slowly at room temperature under good stirring. The reaction mixture was then warmed to 40 ⁰C and held at that temperature for 2 h. The reaction mixture was diluted with water and extracted with ethyl acetate in order to remove neutral impurities. The layers were separated and the aqueous layer was cooled to 0 ⁰C and then acidified with 20% sulfuric acid to pH 2. White solids precipitated and were filtered and dried to constant weight. The crude product was recrystallized from ethyl acetate and hexane (1 :1) to afford the pure product (1.68 g, 90%) as a white solid,

mp 186-188 ⁰C.

¹H NMR (DMSO-d-6) 8.39 (s, 1 H), 8.36 (d, J = 8.5 Hz, 1 H), 7.79 (d, J = 16.2 Hz, 1 H), 7.66 (d, J = 1.6 Hz, 1 H), 7.55 (d, J = 8.5 Hz, 1 H), 7.35 (dd, J = 8.3 and 1.6 Hz, 1 H), 6.93 (d, J = 8.3 Hz, 1 H), 6.76 (d, J = 16.2 Hz, 1 H), 5.89 (s, 2 H).

Anal, calcd. for C₁₈H_nCl₂F₃N₂O₂: C, 52.02; H, 2.65; N, 6.74. Found: C, 50.63; H, 2.63; N, 6.63.

HRMS (FAB +) m/z calcd. for C₁₈HnCl₂F₃N₂O₂ 415.01, found 415.0233.

MS (FAB) m/z: 415 (M⁺ + 1).

1H NMR

13C NMR

• 1. Corsi , G. ; Palazzo , G. ; Germani , C. ; Barcellona , P. S. ; Silvestrini , B. 1-Halobenzyl-1H-indazole-3-carboxylic acids: A new class of antispermatogenic agents . J. Med. Chem. 1976 , 19 , 778 
  
• 2. Palazzo , G. ; Corsi , G. ; Baiocchi , L. ; Silvestrini , B. Synthesis and pharmalogical properties of 1-substituted-3-dimethylaminoalkoxy-1H-indazoles . J. Med. Chem. 1966 , 9 , 38 – 41 . 
  
• 3. Silvestrini , B. Basic and applied research in the study of indazole carboxylic acids . Chemotherapy 1981 , 27 ( Suppl.2 ), 9 – 20 . 
  
• 4. Silvestrini , B. ; Palazzo , G. ; De Gregorio , M. D. 3-Lonidamine and related compounds . Progr. Med. Chem. 1985 , 21 , 111 – 135 .
• 5. Cheng , C. Y. ; Silvestrini , B. ; Grima , J. ; Mo , M. Y. ; Zhu , L. J. ; Johnsson , E. ; Saso , L. ; Leone , M. G. ; Palmery , M. ; Mruk , D. Two new male contraceptives exert their effects by depleting germ cells prematurely from the testes . Biol. Reprod. 2001 , 65 , 449 – 461 . 
  
• 6. Xia , W. ; Mruk , D. D. ; Lee , W. M. ; Ceng , C. Y. Unraveling the molecular targets pertinent to junction restructuring events during spermatogenesis using the Adjudin-induced germ cell depletion model . J. Endocrinol. 2007 , 192 , 563 – 583 .
  
• 7. Cheng , C. Y. ; Mruk , D. D. ; Silvestrini , B. ; Bonanomi , M. ; Wong , C. H. ; Siu , M. K. Y. ; Lee , N. P. Y. ; Mo , M. Y. AF-2364 [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] is a potential male contraceptive: A review of recent data . Contraception2005 , 72 , 251 – 261 . 
  
• 8. Tash , J. S. ; Attardi , B. ; Hild , S. A. ; Chakrasali , R. ; Jakkarg , S. R. ; Georg , G. I. A novel potent indazole carboxylic acid derivative blocks spermatogenesis and is contraceptive in rats after a single oral dose . Biol. Reprod. 2008 , 78 , 1127 – 1138 .
  
• 9. Sarkar , O. ; Mathur , P. P. Adjudin-mediated germ cell depletion alters the anti-oxidant status of adult rat testes . Mol. Reprod. Dev. 2009 , 76 , 31 – 37 . 
  
• 10. Mok , K.-W. ; Mruk , D. D. ; Lie , P. P. Y. ; Lui , W.-Y. ; Cheng , C. Y. Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes. Reproduction. 2011, 141, 571–580. 
  
• 11. Wang , H. ; Chen , X. X. ; Wang , L.-R. ; Mao , Y.-D. ; Zhou , Z. M. ; Sha , J.-H. AF-2364 is a prospective spermicide candidate .Asian J. Androl. 2010 , 12 , 322 – 335 . 
  
• 1.  “Gamendazole”. NextBio. http://www.nextbio.com. Retrieved 31 July 2011.
  2.  Tash, Joseph (July 2008). “A Novel Potent Indazole Carboxylic Acid Derivative Blocks Spermatogenesis and Is Contraceptive in Rats after a Single Oral Dose”. Biology of Reproduction 78 (6): 1127–1138. doi:10.1095/biolreprod.106.057810. PMID 18218612.

Chakrasali, R.; Jakkaraj, S.R.; Tash, J.S.; Hild, S.A.; Attardi, B.; Georg, G.I.
Design, synthesis and in vivo evaluation of Gamendazole(R), a novel orally active male contraceptive agent
228th Am Chem Soc (ACS) Natl Meet (August 22-26, Philadelphia) 2004, Abst MEDI 305

CHENG C.Y. ET AL: “Two New Male Contraceptives Exert Their Effects by Depleting Germ Cells Prematurely from the Testis” BIOLOGY OF REPRODUCTION, SOCIETY FOR THE STUDY OF REPRODUCTION, CHAMPAIGN, IL, US, vol. 65, no. 2, 1 August 2001 (2001-08-01), pages 449-461, XP002547492 ISSN: 0006-3363
2	*	GATTA F. ET AL: “Pyrazolo[3,4-d]pyrimidines. Related to Lonidamine” JOURNAL OF HETEROCYCLIC CHEMISTRY, HETEROCORPORATION. PROVO, US, vol. 26, no. 3, 1 March 1989 (1989-03-01), pages 613-618, XP002547493 ISSN: 0022-152X

US3895026 *	Feb 9, 1973	Jul 15, 1975	Acraf	Substituted 1-benzyl-1h-indazole-3-carboxylic acids and derivatives thereof
WO2003097063A1 *	May 5, 2003	Nov 27, 2003	Bayer Ag	Derivatives of 2-(1-benzyl-1h-pyrazolo (3, 4-b)pyridine-3yl) -5-(4-pyridinyl)-4-pyrimidine amine and the use thereof as guanylate cyclase stimulators
WO2006015263A2 *	Jul 29, 2005	Feb 9, 2006	Duan Jian-Xin	Lonidamine analogs

Gamendazole

Names
IUPAC name (E)-3-[1-[(2,4-Dichlorophenyl)methyl]-6-(trifluoromethyl)indazol-3-yl]prop-2-enoic acid[1]
Other names trans-3-(1-Benzyl-6-(trifluoromethyl)-1H-indazol-3-yl)acrylic acid)
Identifiers
CAS Registry Number	877773-32-5
ChemSpider	9387234
InChI[show]
Jmol-3D images	Image
PubChem	11212172
SMILES[show]
Properties
Chemical formula	C18H11Cl2F3N2O2
Molar mass	415.19 g·mol−1

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Etelcalcetide, AMG 416

AMG-416; Etelcalcetide hydrochloride; KAI-4169; KAI-4169-HCl; ONO-5163; Telcalcetide; Velcalcetide; Velcalcetide hydrochloride

D-Argininamide, N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-, disulfide with L-cysteine,

N-Acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-argininamide disulfide with L-cysteine 

Secondary hyperparathyroidism

• Originator KAI Pharmaceuticals…Kai Pharmaceuticals, Inc.
• Developer Amgen; KAI Pharmaceuticals; Ono Pharmaceutical
• ClassDisulfides; Peptides
• Mechanism of ActionCalcium-sensing receptor agonists

New Parathyroid Disease Drug Seeks FDA Approval

Amgen is seeking FDA approval for etelcalcetide (AMG 461), the first calcimimetic agent administered intravenously after dialysis to treat secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease (CKD).

SHPT is a common and serious condition that is often progressive among CKD patients. It usually manifests as high amounts of parathyroid hormone (PTH) associated with abnormal calcium and phosphorus levels in the body.
– See more at: http://www.pharmacytimes.com/product-news/new-parathyroid-disease-drug-seeks-fda-approval

Etelcalcetide is a D-amino peptide calcimimetic undergoing clinical evaluation for the treatment of secondary hyperparathyroidismfor patients with chronic kidney disease (CKD) on hemodialysis. Etelcalcetide is administered intravenously at the end of each dialysis session.^[1][2] It exerts a pharmacological effect by binding to and activating the calcium-sensing receptor (CaSR) in theparathyroid gland, resulting in parathyroid hormone (PTH) reduction and suppression.^[1] Elevated PTH is often observe in patients with CKD.^[3]

On August 25, 2015 Amgen Inc. announced its submission of a New Drug Application to the Food and Drug Administration for etelcalcetide.^[1]

Etelcalcetide hydrochloride
RN: 1334237-71-6
UNII: 72PT5993DU

The term “AMG 416” refers to the compound having the chemical name: JV-acetyl-D- cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-arginamide disulfide with L- cysteine, which may be represented as:

H-L-Cys-OH

S— S

Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂

The terms “AMG 416 hydrochloride” or “AMG 416 HQ” are interchangeable and refer to the compound having the chemical name: N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl- D-arginyl-D-alanyl-D-arginamide disulfide with L-cysteine hydrochloride, which may be represented as:

H-L-Cys-OH

S— S

Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂ · xHCl

D-Argininamide, N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-, disulfide with L-cysteine, hydrochloride (1:?)

N-Acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-argininamide disulfide with L-cysteine hydrochloride

Amgen  today announced the submission of a New Drug Application (NDA) with the United States Food and Drug Administration (FDA) for etelcalcetide (formerly AMG 416) for the treatment of secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease (CKD) on hemodialysis. If approved, etelcalcetide will be the first calcimimetic agent that can be administered intravenously at the end of the dialysis session.

“Secondary hyperparathyroidism is a serious, progressive disease that can lead to significant clinical consequences and is also associated with a high pill burden for patients,” said Sean E. Harper, M.D., executive vice president of Research and Development at Amgen. “We look forward to working with regulatory authorities during the review process to bring this important treatment to market, helping to fill an unmet need for the many patients impacted by this disease.”

Etelcalcetide is a novel calcimimetic agent that suppresses the secretion of parathyroid hormone and is in clinical development for the treatment of SHPT in patients with CKD on hemodialysis. Etelcalcetide is administered intravenously three times per week at the end of each dialysis session. It acts by binding to and activating the calcium-sensing receptor on the parathyroid gland, thereby causing decreases in parathyroid hormone (PTH). Sustained elevations in PTH are known to be associated with significant clinical consequences for patients with CKD.

The submission includes data from three Phase 3 studies, all of which met the primary endpoints, including two pooled placebo-controlled trials in more than 1,000 patients and a head-to-head study evaluating etelcalcetide compared with cinacalcet.

About Secondary Hyperparathyroidism
SHPT is a common and serious condition that is often progressive among patients with CKD, and it affects many of the approximately two million people throughout the world who are receiving dialysis, including 450,000 people in the U.S. The disorder develops early in the course of CKD and usually manifests as increased levels of PTH as a result of increased production from the parathyroid glands (four small glands in the neck). Patients with end stage renal disease who require maintenance dialysis often have substantial elevations of PTH that are commonly associated with abnormal calcium and phosphorus levels and an increased risk of significant clinical consequences.

About Etelcalcetide (AMG 416)
Etelcalcetide is a novel calcimimetic agent in clinical development for the treatment of SHPT in CKD patients on hemodialysis that is administered intravenously at the end of the dialysis session. Etelcalcetide binds to and activates the calcium-sensing receptor on the parathyroid gland, thereby decreasing PTH levels.

About Sensipar^® (cinacalcet)
Sensipar^® (cinacalcet) is the first oral calcimimetic agent approved by the FDA for the treatment of SHPT in adult patients with CKD on dialysis. Sensipar is not indicated for use in adult patients with CKD who are not on dialysis because of an increased risk of hypocalcemia. The therapy is also approved in the U.S. for treatment of hypercalcemia in adult patients with parathyroid carcinoma and hypercalcemia in adult patients with primary HPT for whom parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy. Sensipar binds to the calcium-sensing receptor, resulting in a drop in PTH levels by inhibiting PTH synthesis and secretion. In addition, the reductions in PTH lower serum calcium and phosphorus levels.

…………………

WO 2011014707

http://www.google.com/patents/WO2011014707A2?cl=en

……………………..

WO 2014210489

http://www.google.com/patents/WO2014210489A1?cl=en

A variety of compounds having activity for lowering parathyroid hormone levels have been described. See International Publication No. WO 2011/014707. In one embodiment, the compound may be represented as follows:

H-L-Cys-OH

S— S

Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂

The main chain has 7 amino acids, all in the D-configuration and the side-chain cysteine residue is in the L-configuration. The amino terminal is acetylated and the carboxyl-terminal is amidated. This compound (“AMG-416”) has utility for the treatment of secondary hyperparathyroidism (SHPT) in hemodialysis patients. A liquid formulation comprising AMG-416 may be administered to a subject intravenously. The hydrochloride salt of AMG-416 may be represented as follows:

H-L-Cys-OH

S— S

Ac-D-Cys-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂ · x(HCl)

Therapeutic peptides pose a number of challenges with respect to their formulation. Peptides in general, and particularly those that contain a disulfide bond, typically have only moderate or poor stability in aqueous solution. Peptides are prone to amide bond hydrolysis at both high and low pH. Disulfide bonds can be unstable even under quite mild conditions (close to neutral pH). In addition, disulfide containing peptides that are not cyclic are particularly prone to dimer formation. Accordingly, therapeutic peptides are often provided in lyophilized form, as a dry powder or cake, for later reconstitution. A lyophilized formulation of a therapeutic peptide has the advantage of providing stability for long periods of time, but is less convenient to use as it requires the addition of one or more diluents and there is the potential risk for errors due to the use of an improper type or amount of diluent, as well as risk of contamination. In addition, the lyophilization process is time consuming and costly.

Accordingly, there is a need for an aqueous liquid formulation comprising a peptide agonist of the calcium sensing receptor, such as AMG 416. It would be desirable for the liquid formulation to remain stable over a relevant period of time under suitable storage conditions and to be suitable for administration by intravenous or other parenteral routes.

…………………………………

Milestones

References

KAI-4169, a novel peptide agonist of the calcium sensing receptor, attenuates PTH and soft tissue calcification and restores parathyroid gland VDR levels in uremic rats
49th Congr Eur Renal Assoc – Eur Dialysis Transpl Assoc (May 24-27, Paris) 2012, Abst SAO014

Long term safety and efficacy of velcalcetide (AMG 416), a calcium-sensing receptor (CaSR) agonist, for the treatment of secondary hyperparathyroidism (SHPT) in hemodialysis (HD) patients
Kidney Week (November 5-10, Atlanta, GA) 2013, Abst SA-PO575

Preclinical PK and PD relationship for KAI-4169, a novel calcimimetic
93rd Annu Meet Endo Soc (June 4-7, Boston) 2011, Abst P1-198

KAI-4169, a novel calcimimetic for the treatment of secondary hyperparathyroidism
93rd Annu Meet Endo Soc (June 4-7, Boston) 2011, Abst P2-98

Characterization of KAI-4169, a novel peptide for the treatment of chronic kidney disease – Mineral and bone disorder, in a phase I study in healthy males
44th Annu Meet Am Soc Nephrol (ASN) (November 8-13, Philadelphia) 2011, Abst FR-PO1238

////Etelcalcetide, Parathyroid Disease,  Amgen Inc, AMG 416, KAI-4169; KAI-4169-HCl,  ONO-5163, Telcalcetide,  Velcalcetide,  Velcalcetide hydrochloride

GSK1904529A, GSK 4529

GSK1904529A is a selective inhibitor of IGF1R with IC50 of 27 nM.

N-(2,6-difluorophenyl)-5-[3-[2-[5-ethyl-2-methoxy-4-[4-(4-methylsulfonylpiperazin-1-yl)piperidin-1-yl]anilino]pyrimidin-4-yl]imidazo[1,2-a]pyridin-2-yl]-2-methoxybenzamide,

N-(2,6-Difluorophenyl)-5-[3-[2-[[5-ethyl-2-(methyloxy)-4-[4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl]phenyl]amino]-4-pyrimidinyl]imidazo[1,2-a]pyridin-2-yl]-2-(methyloxy)benzamide

NMR……http://www.abmole.com/download/gsk1904529a-hnmr.pdf

GSK1904529A, selectively inhibits IGF-IR and IR with IC50s of 27 and 25 nmol/L, respectively. It is a promising candidate for therapeutic use in solid and hematologic cancers. IC50s for GSK1904529A in tumor cell lines ranged from 35 nmol/L to >30 umol/L. The tumor histologic types showing the greatest sensitivity to this compound were Ewing’s sarcoma and multiple myeloma, where IC50s in three of five Ewing’s sarcoma cell lines were <100 nmol/L and IC50s in five of eight multiple myeloma cell lines were <200 nmol/L.

GSK1904529A is a small-molecule inhibitor of the insulin-like growth factor-I receptor (IGF-IR) with IC50 value of 27 nM ¹.

GSK1904529A is a reversible and ATP-competitive inhibitor with Ki value of 1.6 nM. In NIH-3T3/LISN cells, GSK1904529A potently inhibited phosphorylation of IGF-IR with IC50 value of 22 nM. It also demonstrated to be a selective inhibitor since it showed poor inhibitory activity against 45 other serine/threonine and tyrosine kinases. When treated with whole-cell extracts, GSK1904529A significantly inhibited the ligand-induced phosphorylation of IGF-IR and decreased phosphorylation of downstream signaling including AKT, IRS-1 and ERK at concentrations > 0.01μM. GSK1904529A suppressed cell proliferation in a variety of tumor cells. The IC50 values for NCI-H929, TC-71, SK-N-MC, COLO 205, MCF7 and PREC are 81, 35, 43, 124, 137 and 68 nM, respectively. In COLO 205, MCF-7, and NCI-H929 cells, GSK1904529A treatment resulted in cell accumulation in G1 and decrease in S and G2-M phases. Moreover, in NIH-3T3/LISN xenograft model, once daily administration of GSK1904529A at 30 mg/kg inhibited 56% of tumor growt

…………..

Intermediates

, , , 

,

, , 

, 

u can construct your synthesis

http://www.google.com/patents/US20080300242

Intermediate Example 2 5-[3-(2-chloro-4-pyrimidinyl)imidazo[1,2-a]pyridin-2-yl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide

Step A: Methyl 3-formyl-4-hydroxybenzoate

Methyl 4-hydroxybenzoate (3.00 g, 19.7 mmol) and magnesium chloride (2.81 g, 29.5 mmol) were stirred in 100 mL of acetonitrile. TEA (10.3 mL, 73.9 mmol) was added via syringe. Paraformaldehyde (12.0 g, 133 mmol) was added in a single portion and the reaction was heated to reflux. The reaction was stirred at reflux for 24 hours and cooled to rt. The reaction was quenched by the addition of approximately 100 mL of 1N HCl and poured into EtOAc. The layers were separated, and the organic layer was washed with brine. The combined aqueous layers were extracted with EtOAc. The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography. The clean fractions (by TLC) were concentrated in vacuo to afford 2.06 g (58%) of the desired product. ¹H NMR (400 MHz, DMSO-d₆): δ 11.54 (s, 1H), 10.27 (s, 1H), 8.21 (d, J=2.4 Hz, 1H), 8.03 (dd, J=8.8, 2.4 Hz, 1H), 7.07 (d, J=8.8 Hz, 1H), 3.79 (s, 3H).

Step B: methyl 3-formyl-4-(methyloxy)benzoate

Methyl 3-formyl-4-hydroxybenzoate (2.06 g, 11.4 mmol) and K₂CO₃ (2.36 g, 17.1 mmol) were stirred in 50 mL of DMF. Methyl iodide (1.42 mL, 22.8 mmol) was added via syringe, and the reaction was stirred for 6 hours at rt. The reaction was poured into H₂O and diethyl ether, and the layers were separated. The organic layer was washed with brine, and the combined aqueous layers were extracted with diethyl ether. The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo to afford 2.24 g of crude desired product. ¹H NMR (400 MHz, DMSO-d₆): δ 10.33 (s, 1H), 8.23 (d, J=2.2 Hz, 1H), 8.20 (dd, J=8.8, 2.2 Hz, 1H), 7.36 (d, J=8.8 Hz, 1H), 3.99 (s, 3H), 3.83 (s, 3H).

Step C: 2-(methyloxy)-5-[(methyloxy)carbonyl]benzoic acid

Crude methyl 3-formyl-4-(methyloxy)benzoate from the previous step was dissolved in 40 mL of dioxane with stirring. Sulfamic acid (5.87 g, 60.5 mmol) in 20 mL of H₂O was added to the stirring solution. Sodium chlorite (1.68 g, 80% by weight, 18.6 mmol) in 20 mL of H₂O was added dropwise via addition funnel. The reaction was stirred for 40 min and poured into EtOAc and H₂O. The layers were separated, and the organic layer was washed with brine. The combined aqueous layers were extracted with EtOAc, and the combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo. The solid was transferred to an Erlenmeyer flask with the aid of 30-40 mL of DCM. Approximately 50 mL of hexanes was added. Air was blown over the solution to allow most of the DCM to evaporate. Diethyl ether was added (20-30 mL), and the suspension was filtered. The solid was washed with hexanes, collected, and dried to afford 1.96 g (82% over 2 steps) of the desired compound. ¹H NMR (400 MHz, DMSO-d₆): δ 12.92 (brs, 1H), 8.22 (d, J=2.2 Hz, 1H), 8.07 (dd, J=8.8, 2.2 Hz, 1H), 7.24 (d, J=8.8 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H).

Step D: methyl 3-{[(2,6-difluorophenyl)amino]carbonyl}-4-(methyloxy)benzoate

2-(Methyloxy)-5-[(methyloxy)carbonyl]benzoic acid (1.96 g, 9.33 mmol) was suspended in 60 mL of DCM with stirring. DMF (0.036 mL, 0.46 mmol) was added via syringe. Oxalyl chloride (7.0 mL, 2.0M in dichloromethane, 14 mmol) was added dropwise via addition funnel. The addition funnel was rinsed with 10 mL of DCM. The reaction was stirred for 2 hours and concentrated in vacuo. The resultant solid was further dried under high vacuum pressure. The solid was dissolved in 60 mL of DCM with stirring. Pyridine (3.8 mL, 47 mmol), (4-dimethylamino)pyridine (0.0570 g, 0.467 mmol), and 2,6-difluoroaniline (3.0 mL, 28 mmol) were added to the solution. The reaction was stirred for 18 hours and poured into 1N HCl. The layers were separated, and the aqueous layer was washed once with DCM and once with diethyl ether. The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography. The clean fractions (by TLC) were concentrated in vacuo to afford 1.56 g (52%) of the desired product. ¹H NMR (400 MHz, DMSO-d₆): δ 9.81 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.10 (dd, J=8.8, 2.0 Hz, 1H), 7.38 (m, 1H), 7.31 (d, J=88 Hz, 1H), 7.22-7.13 (m, 2H), 3.97 (s, 3H), 3.82 (s, 3H).

Step E: 5-[(2-Chloro-4-pyrimidinyl)acetyl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide and 5-[(E)-2-(2-chloro-4-pyrimidinyl)-1-hydroxyethenyl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide

Methyl 3-{[(2,6-difluorophenyl)amino]carbonyl}-4-(methyloxy)benzoate (1.56 g, 4.86 mmol) was dissolved in 50 mL of THF with stirring and cooled to 0° C. Lithium bis(trimethylsilyl)amide (14.6 mL, 1.0M in THF, 14.6 mmol) was added slowly via syringe. 2-Chloro-4-methylpyrimidine (0.750 g, 5.83 mmol) was dissolved in 10 mL of THF and added dropwise via addition funnel. The addition funnel was rinsed with 10 mL of THF. The reaction was stirred at 0° C. for 1 hour and quenched with saturated ammonium chloride solution. The mixture was poured into H₂O and EtOAc, and the layers were separated. The organic layer was washed with brine, and the combined aqueous layers were extracted with EtOAc. The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography. The clean fractions (by TLC) were concentrated in vacuo to afford 1.26 g (62%) of the desired product. The proton NMR is a mixture of the keto and enol tautomers (˜2:1). ¹H NMR (400 MHz, DMSO-d₆): δ 13.58 (s, 1H, enol), 9.83 (s, 1H, keto), 9.82 (s, 1H, enol), 8.72 (m, 1H, keto), 8.54 (m, 1H, enol), 8.34 (s, 1H, keto), 8.22 (m, 1H, both), 8.06 (m, 1H, enol), 7.56 (m, 1H, keto), 7.42-7.31 (m, 2H, both+1H, enol), 7.22-7.14 (m, 2H, both), 6.55 (s, 1H, enol), 4.66 (s, 2H, keto), 4.00 (s, 3H, keto), 3.97 (s, 3H, enol).

Step F: 5-[3-(2-chloro-4-pyrimidinyl)imidazo[1,2-a]pyridin-2-yl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide

A tautomeric mixture of 5-[(2-Chloro-4-pyrimidinyl)acetyl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide and 5-[(E)-2-(2-chloro-4-pyrimidinyl)-1-hydroxyethenyl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide (1.26 g, 3.02 mmol) was dissolved in 60 mL of DCM with stirring. NBS (0.538 g, 3.02 mmol) was added in a single portion. The reaction was stirred for 20 minutes and concentrated in vacuo. The residue was dissolved in 60 mL of dioxane with stirring, and 2-aminopyridine (0.853 g, 9.06 mmol) was added in a single portion. The reaction was heated at 60° C. with an oil bath for 24 hours and cooled to rt. The reaction was stirred at rt for an additional 40 hours. The reaction was poured into half-saturated NaHCO₃ solution and EtOAc, and the layers were separated. The organic layer was washed with brine, and the combined aqueous layers were extracted twice with EtOAc. The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography. Impure fractions were concentrated and further purified by flash chromatography. The combined clean fractions (by TLC) from both runs were combined and concentrated in vacuo to afford 1.07 g (72%) of the desired product. ¹H NMR (400 MHz, DMSO-d₆): δ 9.80 (s, 1H), 9.40 (d, J=7.0 Hz, 1H), 8.57 (d, J=5.1 Hz, 1H), 8.10 (d, J=1.5 Hz, 1H), 7.84-7.77 (m, 2H), 7.57 (m, 1H), 7.39 (m, 1H), 7.33-7.26 (m, 2H), 7.24-7.14 (m, 3H), 3.99 (s, 3H).

Step A: 1,1-dimethylethyl 4-(methylsulfonyl)-1-piperazinecarboxylate

To 1,1-dimethylethyl 1-piperazinecarboxylate (568 g, 3.05 mol) in DCM (4 L) was added TEA (617 g, 6.10 mol). After stirring for 10 min at 0° C., methanesulfonyl chloride (384 g, 3.35 mol) was added via addition funnel. The mixture was stirred at rt overnight. The mixture was poured into H₂O (1 L) and extracted with DCM (1 L). The organic layer was separated, washed with H₂O (1 L), dried (Na₂SO₄), and rotovapped down to provide the title compound of step A (720 g, 2.72 mol, 90%) which was used without further purification. ¹H NMR (400 MHz, CDCl₃) δ 1.44 (s, 9H), 2.76 (s, 3H), 3.11-3.17 (m, 4H), 3.50-3.53 (m, 4H).

Step B: 1-(methylsulfonyl)piperazine hydrochloride

To 1,1-dimethylethyl 4-(methylsulfonyl)-1-piperazinecarboxylate (360 g, 1.36 mol) in MeOH (1 L) was added HCl (6 M in MeOH, 2 L) dropwise. The mixture was stirred at rt for 1 h. About 1 L of MeOH was rotovapped off. The resultant precipitate was filtered, washed with MeOH, and dried on high vacuum to provide the title compound of Step B (A combination of 2 batches, 570 g) which was used without further purification. ¹H NMR (400 MHz, D₂O) δ 2.95 (s, 3H), 3.27-3.29 (m, 4H), 3.42-3.46 (m, 4H).

Step C: 1-(methylsulfonyl)-4-(4-piperidinyl)piperazine dihydrochloride

To 1-(methylsulfonyl)piperazine hydrochloride (150 g, 632 mmol) in DCE (3.5 L) was added TEA (192 g, 1.90 mol). The mixture was stirred at rt for 1 h and then acetic acid (94.8 g, 1.58 mol) and 1,1-dimethylethyl 4-oxo-1-piperidinecarboxylate (251 g, 1.26 mol) was added. After stirring another h, the reaction was cooled with an ice water bath and NaBH(OAc)₃ (294 g, 1.39 mol) was added in four portions. The mixture was stirred overnight at rt. The reaction mixture was neutralized with saturated Na₂CO₃ to pH 8-9. The organic phase was washed with brine and H₂O, dried (Na₂SO₄), and rotovapped down to provide the crude Boc-protected amine (A combination of 3 batches, 720 g). This amount was split into 2 batches and used without further purification. To 1,1-dimethylethyl 4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinecarboxylate (360 g, 1.04 mol) in MeOH (1 L) was added HCl (6 M in MeOH, 2 L). The mixture was stirred at rt for 30 min. About 1 L of MeOH was rotovapped off. The resultant precipitate was filtered, washed with MeOH, and dried on high vacuum to provide the title compound of Step C (A combination of 2 batches, 600 g, 1.87 mol, 89% over 2 steps). ¹H NMR (400 MHz, D₂O) δ 1.87-1.91 (m, 2H), 2.33-2.36 (m, 2H), 2.97 (s, 3H), 2.99-3.05 (m, 2H), 3.45-3.59 (m, 11H).

Step A: 1-{1-[2-ethyl-5-(methyloxy)-4-nitrophenyl]-4-piperidinyl}-4-(methylsulfonyl)piperazine

A mixture of 1-ethyl-2-fluoro-4-(methyloxy)-5-nitrobenzene (Example 187, step C) (0.93 g, 4.67 mmol), 1-(methylsulfonyl)-4-(4-piperidinyl)piperazine (Example 204, step C) (1.16 g, 4.67 mmol) and K₂CO₃ (0.774 g, 5.60 mmol) in DMSO (20 mL) was heated at 90° C. for 48 h. The reaction had not progressed sufficiently so the reaction was then heated at 120° C. for an additional 4 h. The reaction was cooled to rt, poured into H₂O and extracted with DCM. Some saturated brine solution was added and the resultant was exhaustively extracted with DCM. The combined organics were washed with H₂O then dried over MgSO₄. The resultant solution was concentrated onto silica and purified by flash chromatography to afford 1-{1-[2-ethyl-5-(methyloxy)-4-nitrophenyl]-4-piperidinyl}-4-(methylsulfonyl)piperazine (1.12 g, 56%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.73-7.80 (m, 1H), 6.75 (s, 1H), 3.91 (s, 3H), 3.23-3.30 (m, 1H), 3.05-3.19 (m, 3H), 2.87 (s, 2H), 2.70-2.84 (m, 2H), 2.53-2.67 (m, 5H), 1.77-1.94 (m, 2H), 1.48-1.67 (m, 2H), 1.19 (t, J=7.42 Hz, 3H).

Step B: 5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}aniline

A mixture of 1-{1-[2-ethyl-5-(methyloxy)-4-nitrophenyl]-4-piperidinyl}-4-(methylsulfonyl)piperazine (1.12 g, 2.63 mmol) and sulfided platinum on carbon (0.410 g, 0.105 mmol) in EtOAc (40 mL) was sealed in a round bottom flask with a rubber septum. The reaction mixture was purged with N₂ gas and then a balloon of H₂ gas was connected and the vessel was flushed with the H₂ gas. The reaction was stirred at rt for 2 d. TLC analysis showed the complete consumption of the starting nitro compound so the reaction mixture was filtered through celite to remove the catalyst. The filtrate was concentrated onto silica gel and purified by flash chromatography to afford 5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}aniline (0.479 g, 46%).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 6.60 (s, 1H), 6.46 (s, 1H), 4.35 (br. s., 2H), 3.71 (s, 3H), 3.03-3.16 (m, 4H), 2.81-2.93 (m, 5H), 2.56-2.68 (m, 6H), 2.29-2.42 (m, 1H), 1.72-1.89 (m, 2H), 1.44-1.62 (m, 2H), 1.09 (t, J=7.51 Hz, 3H).

Example 237 N-(2,6-difluorophenyl)-5-(3-{2-[(5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}phenyl)amino]-4-pyrimidinyl}imidazo[1,2-a]pyridin-2-yl)-2-(methyloxy)benzamide

A mixture of 5-[3-(2-chloro-4-pyrimidinyl)imidazo[1,2-a]pyridin-2-yl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide (Intermediate Example 2) (0.60 g, 1.22 mmol), 5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}aniline (Example 206, Step B) (0.48 g, 1.22 mmol) and HCl (4N,1,4-Dioxane, 0.61 mL, 2.44 mmol) in trifluoroethanol (15 mL) was heated at 170° C. for 40 min in the microwave. The reaction mixture was concentrated onto silica gel and purified by flash column chromatography. Recrystallization from DCM and EtOH afforded the title compound N-(2,6-difluorophenyl)-5-(3-{2-[(5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}phenyl)amino]-4-pyrimidinyl}imidazo[1,2-a]pyridin-2-yl)-2-(methyloxy)benzamide (0.61 g, 56%).

¹H NMR (400 MHz, DMSO-d₆)

δ ppm 9.80 (s, 1H), 9.36 (br. s., 1H), 8.50 (s, 1H), 8.26 (d, J=5.22 Hz, 1H), 8.12 (d, J=2.11 Hz, 1H), 7.80 (dd, J=8.80, 2.02 Hz, 1H), 7.71 (d, J=9.07 Hz, 1H), 7.53 (s, 1H), 7.36-7.50 (m, 2H), 7.30 (d, J=8.80 Hz, 1H), 7.14-7.25 (m, 2H), 6.91-7.00 (m, 1H), 6.83 (s, 1H), 6.58 (d, J=5.22 Hz, 1H), 4.00 (s, 3H), 3.80 (s, 3H), 3.08-3.15 (m, 4H), 3.00-3.07 (m, 2H), 2.88 (s, 3H), 2.67-2.76 (m, 2H), 2.61-2.66 (m, 4H), 2.56 (q, J=7.51 Hz, 2H), 2.38-2.46 (m, 1H), 1.80-1.91 (m, 2H), 1.50-1.68 (m, 2H), 1.11 (t, J=7.51 Hz, 3H).

MS (M+H, ES+) 852.

Separately, the Title Compound was Prepared in the Following Manner:

A mixture of 5-[3-(2-chloro-4-pyrimidinyl)imidazo[1,2-a]pyridin-2-yl]-N-(2,6-difluorophenyl)-2-(methyloxy)benzamide (Intermediate Example 2) (23.0 g, 46.8 mmol), 5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}aniline (Example 206, Step B) (18.6 g, 46.8 mmol) and HCl (4N,1,4-Dioxane, 23.4 mL, 93.6 mmol) in trifluoroethanol (200 mL) was heated in a sealed vessel at 85° C. for 48 h. After cooling to rt, the reaction mixture was treated with an excess of 7N NH₃ in MeOH and then subjected to filtration. The filtrate was concentrated onto silica gel and purified by flash chromatography. The chromatographed product was dissolved in DCM and treated with an excess of diethyl ether. The resultant bright yellow precipitate was collected by filtration and then recrystallized from DCM and EtOH to afford the title compound N-(2,6-difluorophenyl)-5-(3-{2-[(5-ethyl-2-(methyloxy)-4-{4-[4-(methylsulfonyl)-1-piperazinyl]-1-piperidinyl}phenyl)amino]-4-pyrimidinyl}imidazo[1,2-a]pyridin-2-yl)-2-(methyloxy)benzamide (28.2 g, 67%).

……………..

Discovery and optimization of imidazo[1,2-a]pyridine inhibitors of insulin-like growth factor-1 receptor (IGF-1R)
Bioorg Med Chem Lett 2009, 19(3): 1004……http://www.sciencedirect.com/science/article/pii/S0960894X08014376

References

Antitumor activity of GSK1904529A, a small-molecule inhibitor of the insulin-like growth factor-I receptor tyrosine kinase.
Sabbatini et al. Clin Cancer Res. 2009 May 1;15(9):3058-67. PMID: 19383820.

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GSK2334470

GSK2334470; 1227911-45-6; GSK-2334470; GSK 2334470;

(3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide

(3S.6/?V1-r6-(3-Amino-1 H-indazol-6-ylV2-(methylaminoV4-pyrimidinyll-Λ/-cvclohexyl-6- methyl-3-piperidinecarboxamide

Glaxosmithkline Llc

Phosphoinositide Dependent Kinase (PDK) 1 Inhibitors

[α]²⁰_D = – 32.6 ^o (c 1.17, MeOH)

[α] D = -27.6 (Concentration = 1.16, Solvent = Methanol)

SOL………DMSO to 100 mM

ethanol to 100 mM

nmr……http://www.chemietek.com/Files/Line2/Chemietek,%20GSK2334470%20(1),%20NMR-DMSO.pdf

http://file.selleckchem.com/downloads/nmr/S708702-GSK2334470-HNMR-Selleck.pdf

GSK2334470 is a potent and selective PDK1 (3-Phosphoinositide dependent protein kinase-1) inhibitor. GSK2334470 blocks the phosphorylation of known PDK1 substrates, but surprisingly find that the potency and kinetics of inhibition vary for different PDK1 targets. GSK2334470 subsequent activation of PDK1 substrates S6K1, SGK and RSK in HEK293, U87 and mouse embryonic fibroblast cell lines.

GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway.

GSK2334470 is a highly specific and potent inhibitor of PDK1 (3-Phosphoinositide dependent protein kinase-1) with IC50 of 10 nM. It does not suppress activity on other 96 kinases, including Aurora, ROCK, p38 MAPK and PI3K. GSK2334470 has been used in cells to ablate T-loop phosphorylation and activate SGK, S6K1 and RSK as well as suppress the activation of Akt.

PATENT

WO  2010059658

http://www.google.com/patents/WO2010059658A1?cl=en

Example 78

(3S.6/?V1-r6-(3-Amino-1 H-indazol-6-ylV2-(methylaminoV4-pyrimidinyll-Λ/-cvclohexyl-6- methyl-3-piperidinecarboxamide

To (3S,6R)-1-[6-(4-cyano-3-fluorophenyl)-2-(methylamino)-4-pyrimidinyl]-Λ/-cyclohexyl-6- methyl-3-piperidinecarboxamide (260 mg, 0.58 mmol) in EtOH (10 ml.) as a suspension at room temperature in a microwave vial was added hydrazine monohydrate (807 uL, 16.7 mmol, 30 equiv) in one portion. The mixture was capped and heated at 100 ⁰C for 48 hours. A duplicate run was performed. The crude reactions from both runs were combined, and concentrated in vacuo. The residue was taken up in 10 ml. of water. The resulting suspension was sonicated briefly, and filtered. The solids collected were dried under vacuum at room temperature over P₂O₅ for 18 hours, and then at 65 ⁰C under vacuum for another 18 hours to afford the title compound (410 mg) as a cream-colored solid. LC-MS (ES) m/z = 463 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): δ 1.16 – 1.32 (m, 3H),1.29 (d, J = 6.8 Hz, 3H), 1.34 – 1.45 (m, 2H), 1.65 – 1.68 (m, 1 H), 1.76 – 1.81 (m, 5H), 1.85 – 1.92 (m, 2H), 1.97 – 2.05 (m, 1 H), 2.35 – 2.42 (m, 1 H), 2.97 (s, 3H), 3.1 1 – 3.15 (m, 1 H),3.64 – 3.70 (m, 1 H), 4.45 – 4.65 (bs, 1 H), 4.72 – 4.92 (bs, 1 H), 6.45 (s, 1 H), 7.52 (dd, J =8.5, 1.14 Hz, 1 H), 7.75 (d, J = 8.3 Hz, 1 H), 7.85 (s, 1 H).

ntermediate 112

Cis- methyl-6-methyl-3-piperidinecarboxylate

A solution of cis-3-methyl 1-(phenylmethyl)-6-methyl-1 ,3-piperidinedicarboxylate (69 g, 237 mol) in EtOH (50 mL) and EtOAc (300 mL) was added to a slurry of 10% Pd/C (3.7 g) in EtOAc (30 mL) and EtOH (10 mL) EtOH under nitrogen in a Parr Shaker bottle. The mixture was hydrogenated under 65 psi at room temperature for 4 hours. The mixture was filtered through celite, and washed with EtOAc. The filtrate was concentrated in vacuo to give 37 g of the title compound as a liquid. LC-MS (ES) m/z = 158 [M+H]⁺.

Intermediate 113

Methyl (3S,6f?)-6-methyl-3-piperidinecarboxylate L-(+)-tartaric acid salt

L-(+)-Tartaric acid salt A suspension of L-(+)-tartaric acid (39 g, 260 mmol, 1.05 equiv) in IPA (200 ml.) and water (13 mL) water was heated in a water bath at 60⁰C until all dissolved. To this hot stirred solution was added neat racemic methyl (3S,6R)-6-methyl-3-piperidinecarboxylate (39 g, 248 mmol), followed by addition of 25 mL of IPA rinse. The resulting mixture was heated to 60 ⁰C, resulting in a clear solution, and then cooled to room temperature, while the hot water bath was removed. This hot solution was seeded with a sample of methyl (3S,6R)-6-methyl-3-piperidinecarboxylate L-(+)-tartaric acid salt that had a chiral purity of 98% ee, and aged at ambient temperature (with the water bath removed) for 20 minutes. The mixture turned into an oily texture with seeds still present. To the mixture was added 5 mL of water, and heated in the warm water bath at 43 ⁰C. The mixture became clear with the seeds still present. The heating was stopped, and the mixture was stirred in the warm water bath. After 20 minutes, the mixture gradually turned into a paste. After another 10 min, the water bath was removed, and the mixture was stirred at ambient temperature for another 1 hour. The resulting paste was filtered. The cake was washed with 50 mL of IPA, giving 62 g of wet solids. This cake was taken up in 150 mL of IPA and 8 mL of water, and stirred as a slurry while being heated in a water bath to 60 ⁰C (internal temp 55 ⁰C) for 5 minutes. The heating was turned off while the mixture was still stirred in the warm water bath. After 30 min, the mixture was filtered. The cake was washed with 100 mL of IPA. Drying under house vacuum at room temperature for 48 hours gave 46.7 g of solids. An analytical sample was derivatised to the corresponding N-Cbz derivative (as in the preparation of intermediate 1 11 ), which was determined by chiral HPLC (methods used to analyze the resolution of intermediate 11 1 above) to have 85% ee. This material was taken up in IPA (420 mL) and water (38 mL) as a suspension. The mixture was heated in a water bath to 65 ⁰C, at which time the mixture became a clear solution. The heating bath was removed. The mixture was seeded and aged at ambient temp for 20 hours. The solids formed were filtered, and washed with 100 mL of IPA. The solids collected were dried under house vacuum at room temperature for 24 h, and then under vacuum at room temperature for another 24 hours to give 28.5 g of the title compound. An analytical sample was converted to the N-Cbz derivative. The ee was determined to be 97.7%. LC-MS (ES) m/z = 158 [M+H]⁺.

Intermediate 114 4,6-Dichloro-Λ/-methyl-2-pyrimidinamine

Methylamine (2M solution, 113 ml_, 217 mmol, 2.05 equiv) was charged to a 1 L 3-neck flask fitted with a magnetic stirrer and a thermometer. The mixture was chilled in an ice bath. To this stirred solution was added via addition funnel a solution of 4,6-dichloro-2-(methylsulfonyl)pyrimidine (25 g, 1 10 mmol) in EtOAc (250 ml.) portionwise over a 25 minutes period. The temp was between 5-10 ⁰C. After completion of addition, the ice bath was removed, and the mixture was stirred for 1 hour at ambient temperature. LCMS showed conversion complete. The suspension was filtered, and washed with EtOAc. The filtrate was concentrated in vacuo. The residue was partitioned between water (100 ml.) and EtOAc (450 ml_). The organic was washed with brine, dried over MgSO₄, filtered and concentrated in vacuo to give white solids, which were triturated in 150 ml. of CH₂CI₂. These solids were collected by filtration and washing with cold CH₂CI₂ (50 ml_). Drying under house vacuum at room temperature for 20 hours, and then high vacuum at room temperature for 3 hours gave 9.31 g of the title compound as a solid. LC-MS (ES) m/z = 179 [M+H]⁺.

Intermediate 121 (3S,6/?)-1-r6-Chloro-2-(methylamino)-4-pyrimidinyll-Λ/-cvclohexyl-6-methyl-3-piperidinecarboxamide

To a suspension of (3S,6/?)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-6-methyl-3-piperidinecarboxylic acid (3.05 g, 10.71 mmol) in CH₂CI₂ (50 ml.) at room temperature was added Hunig’s base (2.70 ml_, 15.43 mmol, 1.3 equiv) and cyclohexylamine (1.60 ml_, 14.2 mmol, 1.2 equiv), and the resulting mixture was chilled in an ice bath. To this stirred solution was added HATU (4.96 g, 13.1 mmol, 1.1 equiv) in one portion, and the resulting suspension was stirred in the ice bath for 30 minutes. LCMS showed conversion complete. The mixture was diluted with CH₂CI₂ (50 ml.) and filtered through celite. The filtrate was washed water (2 X 25 ml.) and then brine. The organic was dried over Na₂SO₄, filtered, and concentrated in vacuo. Silica gel column chromatography using gradient elution of 1 % EtOAc in CHCI₃ to 50% EtOAc in CHCI₃ afforded the title compound (4.26 g) as a foam. LC-MS (ES) m/z = 366 [M+H]⁺.

PAPER

Journal of Medicinal Chemistry (2011), 54(6), 1871-1895.

http://pubs.acs.org/doi/full/10.1021/jm101527u

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

REFERENCES

Najafov, et al., Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem.J. (2011), 433 (2) 357.

For a PDK1 inhibitor, the substrate matters.
Knight ZA. Biochem J. 2011 Jan 15;433(2):e1-2. PMID: 21175429.

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1.
Najafov A, et al. Biochem J. 2011 Jan 15;433(2):357-69. PMID: 21087210.

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2-[4-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-[(3,3-dimethyl-2-oxopyrrolidin-1-yl)methyl]phenyl]-N-(3,4-dimethyl-1,2-oxazol-5-yl)benzenesulfonamide

4‘-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2‘-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1‘-biphenyl]-2-sulfonamide,

4′- . (2-Butyl-4-oxo- 1 ,3-diazaspiro [4.41 non-l-en-3-yl)methyll -N-C3.4- dimethyl-5-isoxazolyl)-2^,-[(3.3-dimethyl-2-oxo-l- pyrrolidinvDmethyll [1.1 ‘-biphenyl] -2-sulfonamide

4‘-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N–(3,4-dimethyl-5-isoxazolyl)-2‘-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1‘-biphenyl]-2-sulfonamide

BMS-248360

PRECLINICAL …..treating hypertension

Bristol Myers Squibb Co, INNOVATOR

Hypertension remains one of the largest unmet medical needs in the 21st century, especially when one considers that hypertension is the portent of future debilitating cardiovascular disease. While many drugs are available for treating the disease, approximately one-third of the hypertensive population is still not adequately treated. Of the more recent avenues explored for treating hypertension, disruption of the effects of either angiotensin II (AII) or endothelin-1 (ET-1) has shown promise. These endogenous vasoactive peptides are among the most potent vasoconstrictors and cell proliferative factors identified to date. AII is the effector molecule of the renin−angiotensin system (RAS), and a large number of AII receptor (AT₁) antagonists, including irbesartan , have been developed for treating hypertension

SYNTHESIS

picked from…….http://www.drugfuture.com/synth/syndata.aspx?ID=324487

EP 1094816; JP 2002519380; US 2002143024; WO 0001389

The intermediate biphenyl aldehyde (XI) is prepared by two related methods. 4-Bromo-3-methylbenzonitrile (I) is oxidized to aldehyde (II) via radical bromination with N-bromosuccinimide/benzoyl peroxide, followed by treatment with trimethylamine N-oxide. Suzuki coupling of aryl bromide (II) with the pinacol boronate (III) affords biphenyl (IV). After protection of the aldehyde moiety of (IV) as the corresponding ethylene ketal (V), its cyano group is reduced to aldehyde (VI) employing DIBAL in THF. Subsequent reduction of (VI) with NaBH4 leads to alcohol (VII), which is further converted into the benzyl bromide (VIII) by means of CBr4/PPh3. Bromide (VIII) is condensed with the spiro imidazolone (IX) in the presence of NaH, to produce (X). Then acidic hydrolysis of the ethylene ketal and SEM groups of (X) gives rise to the intermediate aldehyde (XI)

NEXT

Alternatively, reduction of 4-bromo-3-formylbenzonitrile ethylene ketal (XII) by means of DIBAL leads to aldehyde (XIII), which is further reduced to alcohol (XIV) with NaBH4. After bromination of (XIV) with CBr4/PPh3, the resultant benzyl bromide (XV) is condensed with the spiro imidazolone (IX), yielding (XVI). Then, acidic ketal hydrolysis in (XVI) furnishes aldehyde (XVII). Suzuki coupling between aryl bromide (XVII) and boronic acid (XVIII) gives biphenyl (XIX). The SEM group of (XIX) is then removed under acidic conditions to provide (XI)

Reductive amination of the biphenyl aldehyde (XI) with 4-amino-2,2-dimethylbutanoic acid (XX) in the presence of NaBH(OAc)3 produces aminoacid (XXI). This is finally cyclized to the corresponding lactam by treatment with DIC

Coupling of 2-bromobenzenesulfonyl chloride (I) with 5-amino-3,4-dimethylisoxazole (II) affords sulfonamide (III), which is further protected as the N-methoxyethoxymethyl derivative (IV) employing MEM-chloride in DMF. Lithiation of bromosulfonamide (IV), followed by treatment with trimethyl borate and acidic work up leads to the boronic acid intermediate (V). This is then subjected to Suzuki coupling with 4-bromo-3-methylbenzaldehyde (VI) to yield the biphenyl adduct (VII). After reduction of aldehyde (VII) to the benzylic alcohol (VIII) with NaBH4, reaction with methanesulfonyl chloride and diisopropylethylamine gives rise to the mesylate (IX) (1-3).

Mesylate (IX) is condensed with ethyl 2-propyl-4-ethylimidazole-5-carboxylate (X) yielding (XI). Simultaneous ester group hydrolysis and MEM group deprotection under acidic conditions gives rise to the imidazolecarboxylic acid (XII). This is finally coupled with methylamine via activation with CDI to produce the desired N-methyl carboxamide (1-3).

Reductive amination of the biphenyl aldehyde (XI) with 4-amino-2,2-dimethylbutanoic acid (XX) in the presence of NaBH(OAc)3 produces aminoacid (XXI). This is finally cyclized to the corresponding lactam by treatment with DIC

PAPER

 BMS 248360

The ET_A receptor antagonist (2) (N-(3,4-dimethyl-5-isoxazolyl)-4‘-(2-oxazolyl)-[1,1‘-biphenyl]-2-sulfonamide, BMS-193884) shares the same biphenyl core as a large number of AT₁ receptor antagonists, including irbesartan (3). Thus, it was hypothesized that merging the structural elements of 2 with those of the biphenyl AT₁ antagonists (e.g., irbesartan) would yield a compound with dual activity for both receptors. This strategy led to the design, synthesis, and discovery of (15) (4‘-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2‘-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1‘-biphenyl]-2-sulfonamide, BMS-248360) as a potent and orally active dual antagonist of both AT₁ and ET_Areceptors. Compound 15 represents a new approach to treating hypertension.

Scheme 2 ^a  

^a (a) DIBAL, toluene; (b) NaBH₄, MeOH; (c) (Ph)₃P, CBr₄, THF (51% from 9); (d) compound 7, NaH, DMF; (e) 1 N HCl; (f) compound 4, (Ph₃P)₄Pd, aqueous Na₂CO₃, EtOH/toluene; (g) 6 N aqueous HCl/EtOH (60% from 10); (h) 13, sodium triacetoxy borohydride, AcOH, (i) diisopropylcarbodiimide, CH₂Cl₂ (31% from 12).

15 as a white solid (40 mg, 31%): 

mp 104−110 °C;

¹H NMR (CDCl₃) δ 0.90 (t, J = 7.0 Hz, 3H), 1.08 (s, 3H), 1.14 (s, 3H), 1.36 (m, 2H), 1.61 (m, 2H), 1.75−2.06 (m, 13H), 2.17 (s, 3H), 2.39 (m, 2H), 4.18 (m, 2H), 4.71 (m, 2H), 7.02−7.93 (m, 7H);

¹³CNMR (CDCl₃ ) δ 7.82, 11.91, 14.79, 23.36, 25.50, 25.61, 27.11, 28.81, 29.88, 35.33, 38.42, 41.48, 44.59, 46.24, 46.47, 109.29, 125.15, 125.76, 129.68, 130.58, 131.76, 133.20, 134.07, 137.15, 138.27, 139.11, 139.57, 155.81, 162.68, 162.91, 181.25, 187.83.

Anal. (C₃₆H₄₅N₅O₅S) C, H, N, S.

……………………………

PATENT

US 2002143024

http://www.google.com/patents/US20020143024

Zhang, H.-Y. et al., Tetrahedron, 1994, 50, 11339-11362.

N-(3,4-Dimethyl-5-iso-xazolyl)-2′-formyl-4′-(hydroxy-methyl)-N-[[2-(tri-methylsilyl)ethoxy]- methyl][1,1′- biphenyl]-2- sulfonamide

Example 3 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-[1,1′-biphenyl]-2-sulfonamide

[0414]

Example 3 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-[1,1′-biphenyl]-2-sulfonamide

A. 4′-Cyano-2′-(1,3-dioxolan-2-yl)-N-(3,4-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

A mixture of 2B (1.28 g, 2.73 mmol), ethylene glycol (1.69 g, 27.3 mmol) and p-toluenesulfonic acid (38 mg) in toluene (30 mL) was heated at 130° C. for 5 h, while a Dean-Stark water separator was used. After cooling, the mixture was diluted with EtOAc. The organic liquid was separated and washed with H₂O and brine, dried and concentrated. The residue was chromatographed on silica gel using 5:4 hexane/EtOAc to afford 3A (1.1 g, 79%) as a colorless gum: R_f=0.57, silica gel, 1:2 hexane/EtOAc.

B. 2′-(1,3-Dioxolan-2-yl)-4′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

 To 3A (1.1 g, 2.14 mmol) in THF (21 mL) at 0° C. was added DIBAL-H (1M in CH₂Cl₂, 4.28 mL 4.28 mmol) dropwise. The reaction was stirred at RT overnight. MeOH (20 mL) was added and the reaction was stirred for 5 min. The mixture was poured into cold 0.1 N HCl solution (150 mL), shaken for 5 min, and then extracted with 3:1 EtOAc/hexane. The combined organic extracts were washed with H₂O and brine, dried and concentrated. The residue was chromatographed on silica gel using 3:4 hexane/EtOAc to afford 3B (710 mg, 64%) as a colorless gum: R_f=0.45, silica gel, 2:3 hexane/EtOAc.

 C. 2′-(1,3-Dioxolan-2-yl)-4′-hydroxymethyl-N-(3,4-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl) [1,1′-biphenyl]-2-sulfonamide

 3B (710 mg, 1.4 mmol) was subjected to sodium borohydride reduction according to General Method 11 to afford 3C, which was used for the next reaction step without further purification.

 D. 4′-Bromomethyl-2′-(1,3-dioxolan-2-yl)-N-(3,4′-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl) [1,1′-biphenyl]-2-sulfonamide

3C was treated with carbon tetrabromide and triphenylphosphine according to General Method 2. The crude residue was chromatographed on silica gel using 3:2 hexane/EtOAc to afford 3D (750 mg, 94%) as a colorless gum: R_f=0.74, silica gel, 1:2 hexane/EtOAc.

 E. 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-(1,3-dioxolan-2-yl)-N-(3,4-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

 3D (750 mg, 1.3 mmol) was treated with 2-n-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloride (387 mg, 1.68 mmol) according to General Method 4. The crude residue was chromatographed on silica gel using 100:1.7 CH₂Cl₂/MeOH to afford 3E as a gum (830 mg, 93%): R_f=0.40, silica gel, 100:5 CH₂Cl₂/MeOH.

F. 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-[1,1′-biphenyl]-2-sulfonamide

3E (830 mg, 1.20 mmol) was subjected to deprotection according to General Method 7. The crude residue was chromatographed on silica gel using 100:1.5 and then 100:4 CH₂Cl₂ /MeOH to afford the title compound as a gum (480 mg, 72%): R_f=0.16, silica gel, 100:5 CH₂Cl₂/MeOH.

Example 4 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2′-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl][1,1′-biphenyl]-2-sulfonamide

 To 3F (110 mg, 0.20 mmol) in CH₂Cl₂ (4 mL) was added 4-amino-2,2-dimethylbutanoic acid hydrochloride (98 mg, 0.59 mmol) [Scheinmann, et al., J. Chem. Research (S), 414-415 (1993)] and 3 Å molecular sieves, followed by glacial acetic acid (35 mg, 0.59 mmol) and then sodium acetate (48 mg, 0.59 mmol). The mixture was stirred for 8 minutes, and NaB(AcO)₃H (124 mg, 0.59 mmol) was then added. The reaction mixture was stirred at RT for 2 h, diluted with EtOAc and filtered through celite. The filtrate was washed with H₂O and brine, dried and concentrated. This material was dissolved in CH₂Cl₂ (6 mL) and 1,3-diisopropylcarbodiimide (32 mg, 0.25 mmol) was added. The reaction mixture was stirred at RT for 2 h and diluted with CH₂Cl₂, washed with H₂O and brine, dried and concentrated. The residue was purified by preparative HPLC to provide the title compound as a white solid (40 mg, 31%, for two steps): mp 104-110° C. Analysis calculated for C₃₆H₄₅N₅O₅S.0.8 H₂O: Calc’d: C, 64.13; H, 6.97; N, 10.39; S, 4,75. Found: C, 64.18; H, 6.60; N, 10.23; S, 4.50.

Example 5 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-[1,1′-biphenyl]-2-sulfonamide (Alternative Preparation for 3F)

 A. 2-[(2′-Bromo-5′-formyl)phenyl)]-1,3-dioxolane

DIBAL-H (1.0 M solution in toluene, 445 mL, 445 mmol, 1.1 eq) was added over 30 minutes to a solution of 2-[(2′-bromo-5′-cyano)phenyl)]-1,3-dioxolane (103 g, 404 mmol, 1.0 eq) [Zhang, H.-Y. et al., Tetrahedron, 50, 11339-11362 (1994)] in toluene (2.0 L) at −78° C. The solution was allowed to warm to 0° C. After 1 hour, a solution of Rochelle’s salt (125 g) in water (200 mL) was added, and the mixture was allowed to warm to room temperature and was stirred vigorously for 16 h. The organic layer was concentrated and the residue partitioned between ethyl acetate (1 L) and 1 N hydrochloric acid (800 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (800 mL), dried over sodium sulfate, and then concentrated to give 70.5 g of crude 5A as a yellow solid, which was used without further purification.

 B. 2-[(2′-Bromo-5′-hydroxymethyl)phenyl)]-1,3-dioxolane

Sodium borohydride (3.66 g, 96.7 mmol, 0.5 eq) was added to a solution of crude 5A (49.7 g, approximately 193 mmol, 1.0 eq) in absolute ethanol (1300 mL) at 0° C. After 2 hours, a solution of 10% aqueous sodium dihydrogen phosphate (50 mL) was added and the mixture was stirred and allowed to warm to room temperature. The mixture was concentrated, then partitioned between ethyl acetate (800 mL) and saturated aqueous sodium bicarbonate (500 mL). The organic layer was dried over sodium sulfate and concentrated to give 49.0 g of crude 5B as a yellow oil, which was used without further purification.

 C. 2-[(2′-Bromo-5′-bromomethyl)phenyl)]-1,3-dioxolane

Triphenylphosphine (52.7 g, 199 mmol, 1.05 eq) was added in portions over 15 minutes to a solution of crude 5B (49.0 g, approximately 189 mmol, 1.0 eq) and carbon tetrabromide (69.0 g, 208 mmol, 1.1 eq) in THF at 0° C. After 2 hours, saturated aqueous sodium bicarbonate solution (20 mL) was added, and the mixture was allowed to warm to room temperature and was then concentrated. Ether (500 mL) was added, and the resulting mixture was filtered. The filtrate was dried over magnesium sulfate and concentrated. The residue was chromatographed on silica gel (8:1 hexanes/ethyl acetate as eluant) to give 5C as a white solid (31.1 g, 51% yield from 2-[(2′-bromo-5′-cyano)phenyl)]-1,3-dioxolane).

 D. 2-(1,3-Dioxolan-2-yl)-4-[(2-n-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]bromobenzene

[0436] Sodium hydride (60% dispersion in mineral oil, 9.65 g, 241 mmol, 2.5 eq) was added in portions over 15 minutes to a mixture of 2-n-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloride (18.7 g, 96.5 mmol, 1.0 eq) in DMF (400 mL) at 0° C. The mixture was stirred and allowed to warm to room temperature over 15 minutes. To this mixture was added via canula a solution of 5C (31.1 g, 96.5 mmol, 1.0 eq) in DMF (100 mL). After 14 hours, the mixture was concentrated in vacuo and partitioned between ethyl acetate (500 mL) and 10% aqueous sodium dihydrogen phosphate (300 mL). The organic layer was dried over sodium sulfate and concentrated to give crude 5D as an orange oil (42.7 g), which was used without further purification.

E. 4-[(2-n-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-formyl-bromobenzene

 A solution of crude 5D (6.0 g, approximately 13.6 mmol, 1.0 eq) in THF (180 mL) and 1N hydrochloric acid (30 mL) was heated at 65° C. for 1.5 hours. The mixture was cooled and then treated with saturated aqueous sodium carbonate solution (75 mL) and ethyl acetate (200 mL). The organic layer was removed and dried over sodium sulfate, concentrated, and then further dried azeotropically with toluene to give 5E as a crude yellow oil (8.2 g) which contained a small amount of toluene. This material was used without further purification.

F. 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl)[1,1′-biphenyl]-2-sulfonamide

Palladium catalyzed Suzuki coupling of 5E and [2-[[(3,4-dimethyl-5-isoxazolyl)[(2-methoxyethoxy)methyl]amino]sulfonyl]phenyl]boronic acid was performed according to General Method 1 to yield 5F in 60% yield.

 G. 4′-[(2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2′-formyl-N-(3,4-dimethyl-5-isoxazolyl)-[1,1′-biphenyl]-2-sulfonamide

 Deprotection of 5F according to General Method 7 provided the title compound (5G=3F) in 73% yield: R_f=0.2 (silica gel using CH₂Cl₂/MeOH [100:5]).

EP 1237888; WO 0144239

Example 3 4′-r(2-Butyl-4-oxo-1.3-diazaspiror4.41non-l-en-3-yl)methvn-2′-formyl-N-

(3, 4-dimethyl-5-isoxazolyl)-[ 1,1 ‘-biphenyl] -2-sulfonamide

A. 4′-Cvano-2^>-(1.3-dioxolan-2-yl)-N-(3.4-dimethyl-5-isoxazolyl)-N-(2- methoxyethoxymethyl) [1.1 ‘-biphenyl] -2-sulfonamide

A mixture of 2B (1.28 g, 2.73 mmol), ethylene glycol (1.69 g, 27.3 mmol) and p-toluenesulfonic acid (38 mg) in toluene (30 mL) was heated at 130°C for 5 h, while a Dean-Stark water separator was used. After cooling, the mixture was diluted with EtOAc. The organic liquid was separated and washed with H₂O and brine, dried and concentrated. The residue was chromatographed on silica gel using 5:4 hexane/EtOAc to afford 3A (1.1 g, 79%) as a colorless gum: R^0.57, silica gel, 1:2 hexane EtOAc.

B. 2^,-(1.3-Dioxolan-2-yl)-4′-formyl-N-(3.4-dimethyl-5-isoxazolyl)-N-(2- methoxyethoxymethyl) [1 , l’-biphenyl] -2-sulfonamide To 3A (1.1 g, 2.14 mmol) in THF (21 mL) at 0°C was added DIBAL- H (IM in CH₂C1₂, 4.28 mL 4.28 mmol) dropwise. The reaction was stirred at RT overnight. MeOH (20 mL) was added and the reaction was stirred for 5 min. The mixture was poured into cold 0.1 N HCI solution (150 mL), shaken for 5 min, and then extracted with 3:1 EtOAc/hexane. The combined organic extracts were washed with H₂O and brine, dried and concentrated. The residue was chromatographed on silica gel using 3:4 hexane/EtOAc to afford 3B (710 mg, 64%) as a colorless gum: R^O.45, silica gel, 2:3 hexane/EtOAc. C. 2′-(1.3-Dioxolan-2-yl)-4′-hvdroxymethyl-N-(3.4-dimethyl-5- isoxazolyl)-N-(2-methoxyethoxymethyl) [1.1 ‘-biphenyl] -2- sulfonamide

3B (710 mg, 1.4 mmol) was subjected to sodium borohydride reduction according to General Method 11 to afford 3C, which was used for the next reaction step without further purification.

D. 4^l-Bromomethyl-2^,-(1.3-dioxolan-2-yl)-N-(3.4-dimethyl-5-isoxazolyl)- N-(2-methoxyethoxymethyl) [1 , l’-biphenyl] -2-sulfonamide 3C was treated with carbon tetrabromide and triphenylphosphine according to General Method 2. The crude residue was chromatographed on silica gel using 3:2 hexane/EtOAc to afford 3D (750 mg, 94%) as a colorless gum: R^0.74, silica gel, 1:2 hexane/EtOAc.

E. 4′-[(2-Butyl-4-oxo-1.3-diazaspiro[4.41non-l-en-3-yl)methvn- 2^,-(1.3- dioxolan-2-yl)-N-(3.4-dimethyl-5-isoxazolyl)-N-(2- methoxyethoxymethyl) [ 1. l’-biphenyll -2-sulfonamide 3D (750 mg, 1.3 mmol) was treated with 2-re-butyl-l,3- diazaspiro[4.4]non-l-en-4-one hydrochloride (387 mg, 1.68 mmol) according to General Method 4. The crude residue was chromatographed on silica gel using 100:1.7 CH₂CL/MeOH to afford 3E as a gum (830 mg, 93%): R^O.40, silica gel, 100:5 CH₂Cl₂/MeOH.

F. 4′-r(2-Butyl-4-oxo-1.3-diazaspiro[4.41non-l-en-3-yl)methyl1-2^,– formyl-N-(3.4-dimethyl-5-isoxazolyl)-[l.l’-biphenyl1-2-sulfonamide

3E (830 mg, 1.20 mmol) was subjected to deprotection according to General Method 7. The crude residue was chromatographed on silica gel using 100:1.5 and then 100:4 CH₂C1₂ /MeOH to afford the title compound as a gum (480 mg, 72%): R^O.16, silica gel, 100:5 CH.Cl MeOH.

Example 4

4′- . (2-Butyl-4-oxo- 1 ,3-diazaspiro [4.41 non-l-en-3-yl)methyll -N-C3.4- dimethyl-5-isoxazolyl)-2^,-[(3.3-dimethyl-2-oxo-l- pyrrolidinvDmethyll [1.1 ‘-biphenyl] -2-sulfonamide

To 3F (110 mg, 0.20 mmol) in CH₂C1₂ (4 mL) was added 4-amino- 2,2-dimethylbutanoic acid hydrochloride (98 mg, 0.59 mmol) [Scheinmann, et al., J. Chem. Research (S), 414-415 (1993)] and 3A molecular sieves, followed by glacial acetic acid (35 mg, 0.59 mmol) and then sodium acetate (48 mg, 0.59 mmol). The mixture was stirred for 8 minutes, and NaB(AcO)₃H (124 mg, 0.59 mmol) was then added. The reaction mixture was stirred at RT for 2 h, diluted with EtOAc and filtered through celite. The filtrate was washed with H₂O and brine, dried and concentrated. This material was dissolved in CH₂C1₂ (6 mL) and 1,3-diisopropylcarbodiimide (32 mg, 0.25 mmol) was added. The reaction mixture was stirred at RT for 2 h and diluted with CH₂C1₂, washed with H₂O and brine, dried and concentrated. The residue was purified by preparative HPLC to provide the title compound as a white solid (40 mg, 31%, for two steps): mp 104- 110°C. Analysis calculated for C₃₆H₄₅N₅O₅S • 0.8 H₂O: Calc’d: C, 64.13; H, 6.97; N, 10.39; S, 4,75. Found: C, 64.18; H, 6.60; N, 10.23; S, 4.50.

Example 5

4′-[(2-Butyl-4-oxo-1.3-diazaspiro[4.41non-l-en-3-yl)methyl1-2^,-formyl-N-

(3,4-dimethyl-5-isoxazolyl)-[l,l’-biphenyl]-2-sulfonamide (Alternative

Preparation for 3F)

A. 2-[(2′-Bromo-5′-formyl)phenyl)1-1.3-dioxolane

DIBAL-H (1.0 M solution in toluene, 445 mL, 445 mmol, 1.1 eq) was added over 30 minutes to a solution of 2-[(2′-bromo-5′-cyano)phenyl)]-l,3- dioxolane (103 g, 404 mmol, 1.0 eq) [Zhang, H.-Y. et al., Tetrahedron, 50, 11339-11362 (1994)] in toluene (2.0 L) at -78 °C. The solution was allowed to warm to 0 °C. After 1 hour, a solution of Rochelle’s salt (125 g) in water (200 mL) was added, and the mixture was allowed to warm to room temperature and was stirred vigorously for 16 h. The organic layer was concentrated and the residue partitioned between ethyl acetate (1 L) and 1 N hydrochloric acid (800 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (800 mL), dried over sodium sulfate, and then concentrated to give 70.5 g of crude 5A as a yellow solid, which was used without further purification.

B. 2-[(2′-Bromo-5′-hvdroxymethyl)phenyl)l-1.3-dioxolane

Sodium borohydride (3.66 g, 96.7 mmol, 0.5 eq) was added to a solution of crude 5A (49.7 g, approximately 193 mmol, 1.0 eq) in absolute ethanol (1300 mL) at 0 °C. After 2 hours, a solution of 10% aqueous sodium dihydrogen phosphate (50 mL) was added and the mixture was stirred and allowed to warm to room temperature. The mixture was concentrated, then partitioned between ethyl acetate (800 mL) and saturated aqueous sodium bicarbonate (500 mL). The organic layer was dried over sodium sulfate and concentrated to give 49.0 g of crude 5B as a yellow oil, which was used without further purification. C. 2-[(2′-Bromo-5′-bromomethyl)phenyl)]-l,3-dioxolane Triphenylphosphine (52.7 g, 199 mmol, 1.05 eq) was added in portions over 15 minutes to a solution of crude 5B (49.0 g, approximately 189 mmol, 1.0 eq) and carbon tetrabromide (69.0 g, 208 mmol, 1.1 eq) in THF at 0 °C. After 2 hours, saturated aqueous sodium bicarbonate solution (20 mL) was added, and the mixture was allowed to warm to room temperature and was then concentrated. Ether (500 mL) was added, and the resulting mixture was filtered. The filtrate was dried over magnesium sulfate and concentrated. The residue was chromatographed on silica gel (8:1 hexanes/ethyl acetate as eluant) to give 5C as a white solid (31.1 g, 51% yield from 2-[(2′-bromo-5′-cyano)phenyl)]-l,3-dioxolane).

D. 2-( 1 ,3-Dioxolan-2-yl)-4- [ (2-re-butyl-4-oxo- 1 ,3-diazaspiro [4.4] non- 1- en-3-yl)methyl] bromobenzene Sodium hydride (60% dispersion in mineral oil, 9.65 g, 241 mmol,

2.5 eq) was added in portions over 15 minutes to a mixture of 2-rc-butyl- l,3-diazaspiro[4.4]non-l-en-4-one hydrochloride (18.7 g, 96.5 mmol, 1.0 eq) in DMF (400 mL) at 0°C. The mixture was stirred and allowed to warm to room temperature over 15 minutes. To this mixture was added via canula a solution of 5C (31.1 g, 96.5 mmol, 1.0 eq) in DMF (100 mL). After 14 hours, the mixture was concentrated in vacuo and partitioned between ethyl acetate (500 mL) and 10% aqueous sodium dihydrogen phosphate (300 mL). The organic layer was dried over sodium sulfate and concentrated to give crude 5D as an orange oil (42.7 g), which was used without further purification.

E. 4-[(2-n-Butyl-4-oxo-1.3-diazaspiro[4.41non-l-en-3-yl)methyl1-2- formyl-bromobenzene

A solution of crude 5D (6.0 g, approximately 13.6 mmol, 1.0 eq) in THF (180 mL) and IN hydrochloric acid (30 mL) was heated at 65°C for 1.5 hours. The mixture was cooled and then treated with saturated aqueous sodium carbonate solution (75 mL) and ethyl acetate (200 mL). The organic layer was removed and dried over sodium sulfate, concentrated, and then further dried azeotropically with toluene to give 5E as a crude yellow oil (8.2 g) which contained a small amount of toluene. This material was used without further purification.

F. 4′-.(2-Butyl-4-oxo-1.3-diazaspiro■4.41non-l-en-3-yl)methyl1-2^,– formyl-N-(3,4-dimethyl-5-isoxazolyl)-N-(2-methoxyethoxymethyl) f 1.1 ‘-biphenyl] -2-sulfonamide Palladium catalyzed Suzuki coupling of 5E and [2-[[(3,4-dimethyl-5- isoxazolyl) [(2-methoxyethoxy)methyl] amino] sulfonyl] phenyl]boronic acid was performed according to General Method 1 to yield 5F in 60% yield.

G. 4’-[ 2-Butyl-4-oxo-1.3-diazaspiro[4■41non-l-en-3-yl)methvn-2^,– formyl-N-(3 ,4-dimethyl-5-isoxazolyl)- fi .1 ‘-biphenyl] -2-sulfonamide

Deprotection of 5F according to General Method 7 provided the title compound (5G = 3F) in 73% yield: R^0.2 (silica gel using CH₂ClJ eOH [100:5]).

///////////BMS-248360, Preclinical, SARTAN, BMS, HYPERTENTION

CCCCC1=NC2(CCCC2)C(=O)N1CC3=CC(=C(C=C3)C4=CC=CC=C4S(=O)(=O)NC5=C(C(=NO5)C)C)CN6CCC(C6=O)(C)C

CAS 1452582-16-9, 428.47, C₂₃ H₂₆ F₂ N₄ O₂

1H-​Indazole-​3-​carboxamide, 5-​(2,​3-​difluorophenyl)​-​N-​[[1-​(2-​methoxyethyl)​-​4-​piperidinyl]​methyl]​-

Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.P.A.

1 H-indazole-3-carboxamide compounds acting as glycogen synthase kinase 3 beta (GSK-33) inhibitors and to their use in the treatment of GSK-33-related disorders such as (i) insulin-resistance disorders; (ii) neurodegenerative diseases; (iii) mood disorders; (iv) schizophrenic disorders; (v) cancerous disorders; (vi) inflammation, (vii) substance abuse disorders; (viii) epilepsies; and (ix) neuropathic pain.

Protein kinases constitute a large family of structurally related enzymes, which transfer phosphate groups from high-energy donor molecules (such as adenosine triphosphate, ATP) to specific substrates, usually proteins. After phosphorylation, the substrate undergoes to a functional change, by which kinases can modulate various biological functions.

In general, protein kinases can be divided in several groups, according to the substrate that is phosphorylated. For example, serine/threonine kinase phosphorylates the hydroxyl group on the side chain of serine or threonine aminoacid.

Glycogen synthase kinases 3 (GSK-3) are constitutively active multifunctional enzymes, quite recently discovered, belonging to the serine/threonine kinases group.

Human GSK-3 are encoded by two different and independent genes, which leads to GSK-3a and GSK-33 proteins, with molecular weights of about 51 and 47 kDa, respectively. The two isoforms share nearly identical sequences in their kinase domains, while outside of the kinase domain, their sequences differ substantially (Benedetti et al., Neuroscience Letters, 2004, 368, 123-126). GSK-3a is a multifunctional protein serine kinase and GSK-33 is a serine-threonine kinase.

It has been found that GSK-33 is widely expressed in all tissues, with widespread expression in the adult brain, suggesting a fundamental role in neuronal signaling pathways (Grimes and Jope, Progress in Neurobiology, 2001, 65, 391-426). Interest in glycogen synthase kinases 3 arises from its role in various physiological pathways, such as, for example, metabolism, cell cycle, gene expression, embryonic development oncogenesis and neuroprotection (Geetha et al., British Journal Pharmacology, 2009, 156, 885-898).

GSK-33 was originally identified for its role in the regulation of glycogen synthase for the conversion of glucose to glycogen (Embi et al., Eur J Biochem, 1980, 107, 519-527). GSK-33 showed a high degree of specificity for glycogen synthase.

Type 2 diabetes was the first disease condition implicated with GSK- 3β, due to its negative regulation of several aspects of insulin signaling pathway. In this pathway 3-phosphoinositide-dependent protein kinase 1 (PDK-1 ) activates PKB, which in turn inactivates GSK-33. This inactivation of GSK-33 leads to the dephosphorylation and activation of glycogen synthase, which helps glycogen synthesis (Cohen et al., FEBS Lett, 1997, 410, 3-10). Moreover, selective inhibitors of GSK-33 are expected to enhances insulin signaling in prediabetic insulin- resistant rat skeletal muscle, thus making GSK-33 an attractive target for the treatment of skeletal muscle insulin resistance in the pre-diabetic state (Dokken et al., Am J. Physiol. Endocrinol. Metab., 2005, 288, E1 188-E1 194).

GSK-33 was also found to be a potential drug target in others pathological conditions due to insulin-resistance disorders, such as syndrome X, obesity and polycystic ovary syndrome (Ring DB et al., Diabetes, 2003, 52: 588-595).

It has been found that GSK-33 is involved in the abnormal phosphorylation of pathological tau in Alzheimer’s disease (Hanger et al., Neurosci. Lett, 1992, 147, 58-62; Mazanetz and Fischer, Nat Rev Drug Discov., 2007, 6, 464-479; Hong and Lee, J. Biol. Chem., 1997, 272, 19547- 19553). Moreover, it was proved that early activation of GSK-33, induced by apolipoprotein ApoE4 and β-amyloid, could lead to apoptosis and tau hyperphosphorylation (Cedazo-Minguez et al., Journal of Neurochemistry, 2003, 87, 1 152- 1 164). Among other aspect of Alzheimer’s disease, it was also reported the relevance of activation of GSK-33 at molecular level (Hernandez and Avila, FEBS Letters, 2008, 582, 3848-3854).

Moreover, it was demonstrated that GSK-33 is involved in the genesis and maintenance of neurodegenerative changes associated with Parkinson’s disease (Duka T. et al., The FASEB Journal, 2009; 23, 2820- 2830).

Accordingly to these experimental observations, inhibitors of GSK-33 may find applications in the treatment of the neuropathological consequences and the cognitive and attention deficits associated with tauopathies; Alzheimer’s disease; Parkinson’s disease; Huntington’s disease (the involvement of GSK-33 in such deficits and diseases is disclosed in Meijer L. et al., TRENDS Pharm Sci, 2004; 25, 471 -480); dementia, such as, but not limited to, vascular dementia, post-traumatic dementia, dementia caused by meningitis and the like; acute stroke; traumatic injuries; cerebrovascular accidents; brain and spinal cord trauma; peripheral neuropathies; retinopathies and glaucoma (the involvement of GSK-33 in such conditions is disclosed in WO 2010/109005).

The treatment of spinal neurodegenerative disorders, like amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy and neurodegeneration due to spinal cord injury has been also suggested in several studies related to GSK-33 inhibition, such as, for example in Caldero J. et al., “Lithium prevents excitotoxic cell death of motoneurons in organotypic slice cultures of spinal cord”, Neuroscience. 2010 Feb 17;165(4):1353-69, Leger B. et al., “Atrogin-1 , MuRF1 , and FoXO, as well as phosphorylated GSK-3beta and 4E-BP1 are reduced in skeletal muscle of chronic spinal cord-injured patients”, Muscle Nerve, 2009 Jul; 40(1 ):69-78, and Galimberti D. et al., “GSK33 genetic variability in patients with Multiple Sclerosis”, Neurosci Lett. 201 1 Jun 1 5;497(1 ):46- 8. Furthermore, GSK-33 has been linked to the mood disorders, such as bipolar disorders, depression, and schizophrenia.

Inhibition of GSK-33 may be an important therapeutic target of mood stabilizers, and regulation of GSK-33 may be involved in the therapeutic effects of other drugs used in psychiatry. Dysregulated GSK-33 in mood disorder, bipolar disorder, depression and schizophrenia could have multiple effects that could impair neural plasticity, such as modulation of neuronal architecture, neurogenesis, gene expression and the ability of neurons to respond to stressful, potentially lethal conditions (Jope and Ron, Curr. Drug Targets, 2006, 7, 1421- 1434).

The role of GSK-33 in mood disorder was highlighted by the study of lithium and valproate (Chen et al., J. Neurochem., 1999, 72, 1327- 1330; Klein and Melton, Proc. Natl. Acad. Sci. USA, 1996, 93, 8455-8459), both of which are GSK-33 inhibitors and are used to treat mood disorders. There are also existing reports from the genetic perspective supporting the role of GSK-33 in the disease physiology of bipolar disorder (Gould, Expert. Opin. Ther. Targets, 2006, 10, 377-392).

It was reported a decrease in AKT1 protein levels and its phosphorylation of GSK-33 at Serine-9 in the peripheral lymphocytes and brains of individuals with schizophrenia. Accordingly, this finding supports the proposal that alterations in AKT1 -GSK-33 signaling contribute to schizophrenia pathogenesis (Emamian et al., Nat Genet, 2004, 36, 131- 137).

Additionally, the role of GSK-33 in cancer is a well-accepted phenomenon.

The potential of small molecules that inhibit GSK-33 has been evidenced for some specific cancer treatments (Jia Luo, Cancer Letters, 2009, 273, 194-200). GSK-33 expression and activation are associated with prostate cancer progression (Rinnab et al., Neoplasia, 2008, 10, 624-633) and the inhibition of GSK3b was also proposed as specific target for pancreatic cancer (Garcea et al., Current Cancer Drug Targets, 2007, 7, 209-215) and ovarian cancer (Qi Cao et al., Cell Research, 2006, 16 671 -677). Acute inhibition of GSK-33 in colon-rectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth (Ghosh et al., Clin Cancer Res 2005, 1 1 , 4580-4588).

The identification of a functional role for GSK-33 in MLL-associated leukaemia suggests that GSK-33 inhibition may be a promising therapy that is selective for transformed cells that are dependent on HOX overexpression (Birch et al., Cancer Cell, 2010, 1 7, 529-531 ).

GSK-33 is involved in numerous inflammatory signalling pathways, for example, among others GSK-33 inhibition has been shown to induce secretion of the anti-inflammatory cytokine IL-1 0. According to this finding, GSK-33 inhibitors could be useful to regulate suppression of inflammation (G. Klamer et al., Current Medicinal Chemistry, 2010, 17(26), 2873-2281, Wang et al., Cytokine, 2010, 53, 130-140).

GSK-33 inhibition has been also shown to attenuate cocaine-induced behaviors in mice. The administration of cocaine in mice pretreated with a GSK-33 inhibitor demonstrated that pharmacological inhibition of GSK3 reduced both the acute behavioral responses to cocaine and the long- term neuroadaptations produced by repeated cocaine (Cocaine-induced hyperactivity and sensitization are dependent on GSK3, Miller JS et al. Neuropharmacology. 2009 Jun; 56(8):1 1 16-23, Epub 2009 Mar 27).

The role of GSK-33 in the development of several forms of epilepsies has been demonstrated in several studies, which suggest that inhibition of GSK-33 could be a pathway for the treatment of epilepsy (Novel glycogen synthase kinase 3 and ubiquitination pathways in progressive myoclonus epilepsy, Lohi H et al., Hum Mol Genet. 2005 Sep 15;14(18):2727-36 and Hyperphosphorylation and aggregation of Tau in laforin-deficient mice, an animal model for Lafora disease, Purl R et al., J Biol Chem. 2009 Aug 21 ;284(34) 22657-63). The relationship between GSK-33 inhibition and treatment of neuropathic pain has been demonstrated in Mazzardo-Martins L. et al., “Glycogen synthase kinase 3-specific inhibitor AR-A014418 decreases neuropathic pain in mice: evidence for the mechanisms of action”, Neuroscience. 2012 Dec 13;226, and Xiaoping Gu et al., “The Role of Akt/GSK33 Signaling Pathway in Neuropathic Pain in Mice”, Poster A525, Anesthesiology 2012 October 13-17, 2012 Washington.

A review on GSK-33, its function, its therapeutic potential and its possible inhibitors is given in “GSK-33: role in therapeutic landscape and development of modulators” (S. Phukan et al., British Journal of Pharmacology (2010), 160, 1- 19).

WO 2004/014864 discloses 1 H-indazole-3-carboxamide compounds as selective cyclin-dependant kinases (CDK) inhibitors. Such compounds are assumed to be useful in the treatment of cancer, through a mechanism mediated by CDK₂, and neurodegenerative diseases, in particular Alzheimer’s disease, through a mechanism mediated by CDK₅, and as anti-viral and anti-fungine, through a mechanism mediated by CDK₇, CDK₈ and CDK₉.

Cyclin-dependant kinases (CDKs) are serine/threonine kinases, first discovered for their role in regulating the cell cycle. CDKs are also involved in regulating transcription, mRNA processing, and the differentiation of nerve cells. Such kinases activate only after their interaction and binding with regulatory subunits, namely cyclins.

Moreover, 1 H-indazole-3-carboxamide compounds were also described as analgesics in the treatment of chronic and neuropathic pain (see, for example, WO 2004/074275 and WO 2004/101 548) and as 5-HT₄ receptor antagonists, useful in the treatment of gastrointestinal disorders, central nervous system disorders and cardiovascular disorders (see, for example, WO 1994/101 74).

Patent

WO 2013124158

Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.P.A.

SEE ENTRY 8

DMSO-de; δ 13.09 (s, 1 H), 8.23-8.42 (m, 2H), 7.72 (dd, J=0.82, 8.69 Hz, 1 H), 7.55 (td, J=1.76, 8.74 Hz, 1 H), 7.24-7.49 (m, 3H), 3.40 (t, J=6.04 Hz, 2H), 3.22 (s, 3H), 3.18 (d, J=6.40 Hz, 2H), 2.84 (d, J=11.53 Hz, 2H), 2.42 (t, J=5.95 Hz, 2H), 1.82- 2.02 (m, 2H), 1.41 -1.71 (m, 3H), 1.06-1.31 (m, 2H)

PAPER

Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b01208

Guido Furlotti^*†, Maria Alessandra Alisi^†, Nicola Cazzolla^†, Patrizia Dragone^†, Lucia Durando^†, Gabriele Magarò^†, Francesca Mancini^†, Giorgina Mangano^†, Rosella Ombrato^†, Marco Vitiello^†, Andrea Armirotti^‡, Valeria Capurro^‡, Massimiliano Lanfranco^‡, Giuliana Ottonello^‡, Maria Summa^‡, and Angelo Reggiani^*‡

^† Angelini S.p.A., Angelini Research Center, P.le della Stazione s.n.c., Santa Palomba-Pomezia, 00071 Rome, Italy

^‡ Drug Discovery and Development Department, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.5b01208

Publication Date (Web): October 20, 2015

*(G.F.) Phone: +390691045265. E-mail: g.furlotti@angelini.it..,

Guido Furlotti

• Angelini Acraf S. p. TO.

  Synthetic Chemistry

  Rome, Italy

*(A.G.) Phone: +3901071781571. E-mail: Angelo.Reggiani@iit.it.

Angelo Reggiani

• Italian Institute of Technology

  Department of Drug Discovery and Development

  Genoa, Genoa, Italy

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b01208

Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.P.A.

Angelini S.p.A., Angelini Research Center,

/////

COCCN1CCC(CNC(=O)c2n[nH]c3ccc(cc23)c4cccc(F)c4F)CC1

Cas 1820758-44-8

C₂₄ H₁₈ F N₃ O₄ S

4′-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl-thio)-methyl)-4-fluorobiphenyl-2-carboxamide

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitous serine/threonine kinase that takes part in a number of physiological processes ranging from glycogen metabolism to apoptosis. GSK-3 is a key mediator of various signaling pathways, such as the Wnt and the insulin/AKT signaling pathways.

Therefore, dysregulation of GSK-3 has been linked to various human diseases, such as cancer, diabetes, and neurodegenerative diseases.Two related isoforms of GSK-3 exist in mammals, GSK-3α and -β, which share a sequence identity within their catalytic domains of 98%.

Beyond the catalytic domains they show significant differences. Although these isoforms are structurally related, they are not functionally equivalent, and one cannot compensate for loss of the other.

The debate on the respective contributions of the isoforms GSK-3α and GSK-3β on the pathogenesis of different diseases is ongoing.

Various studies indicate that the therapies of certain diseases benefit from specific targeting of GSK-3α and GSK-3β. GSK-3α was recently identified as a differentiation target in acute myeloid leukemia (AML). AML is a hematopoietic malignancy defined by uncontrolled proliferation and disrupted myeloid differentiation. AML is the second most common form of leukemia in adults.

The current treatment of AML with conventional chemotherapy is very aggressive yet ineffective for the majority of patients with the disease.Thus, alternative targeted treatment approaches for AML are highly desirable. GSK-3α recently emerged as a potential target in this disease.

PAPER

The challenge for glycogen synthase kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML), may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy

Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b01200

http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.5b01200/suppl_file/jm5b01200_si_001.pdf

compound 27 as a colorless solid. HPLC: 96%, t_R = 6.93 min.

¹H NMR (DMSO-d₆, 500 MHz, 300 K): δ (ppm) = 4.32 (td, J = 5.2 Hz, J = 3.7 Hz, 4H), 4.60 (s, 2H), 7.05 (d, J = 8.4 Hz, 1H), 7.25 (dd, J = 9.1 Hz, J = 2.7 Hz, 1H), 7.31 (td, J = 8.6 Hz, J = 2.8 Hz, 1H), 7.38 (m, 3H), 7.41 (d, J = 2.0 Hz, 1H), 7.45 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.73 (s, 1H).

¹³C NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.6, 64.1, 64.4, 114.3 (d, J_C–F = 21 Hz), 115.0, 115.9 (d, J_C–F = 21 Hz), 115.9, 118.1, 120.0, 128.6 (2C), 128.8 (2C), 132.0 (d, J_C–F = 8 Hz), 134.8, 135.5, 138.9, 139.0 (d, J_C–F = 7 Hz), 143.8, 146.7, 160.9 (d, J_C–F = 247 Hz), 162.7, 164.9, 169.5.

EI-MS: m/z = 463 (100, [M⁺]), 464 (26, [M⁺ + H]), 465 (7, [M⁺ + 2H].

ABOUT  Boris Schmidt

………………………………………….

ABOUT Theresa Neumann

////////FC(C=C1C(N)=O)=CC=C1C(C=C2)=CC=C2CSC3=NN=C(O3)C4=CC5=C(OCCO5)C=C4

WCK ?

(5S)-N-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide

(5S)-N- {3-[3,5-difluoro-4-(4-hydroxy-(4-methoxymethyl)-piperidin- lyl)phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide

MF C₁₉ H₂₅ F₂ N₃ O_{5, MW 413.42}

Acetamide, N-​[[(5S)​-​3-​[3,​5-​difluoro-​4-​[4-​hydroxy-​4-​(methoxymethyl)​-​1-​piperidinyl]​phenyl]​-​2-​oxo-​5-​oxazolidinyl]​methyl]​-

CAS 957796-51-9

Antibacterial oxazolidinones

Wockhardt Ltd,  Innovator

Wockhardt Research Center,

THIS MAY BE WCK 4086?????….WATCHOUT THIS POST FOR UPDATION

PATENTS

WO 2015173664, US8217058, WO 2012059823, IN 2011MU03726 

Oxazolidinone represent a novel chemical class of synthetic antimicrobial agents. Linezolid represents the first member of this class to be used clinically. Oxazolidinones display activity against important Gram-positive human and veterinary pathogens including Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE) and β-lactam Resistant Streptococcus pneumoniae (PRSP). The oxazolidinones also show activity against Gram-negative aerobic bacteria, Gram-positive and Gram-negative anaerobes. (Diekema D J et al., Lancet 2001 ; 358: 1975-82).

Various oxazolidinones and their methods of preparation are disclosed in the literature. International Publication No. WO 1995/25106 discloses substituted piperidino phenyloxazolidinones and International Publication No. WO 1996/13502 discloses phenyloxazolidinones having a multisubstituted azetidinyl or pyrrolidinyl moiety. US Patent Publication No. 2004/0063954, International Publication Nos. WO 2004/007489 and WO 2004/007488 disclose piperidinyl phenyl oxazolidinones for antimicrobial use.

Pyrrolidinyl/piperidinyl phenyl oxazohdinone antibacterial agents are also described in Kim H Y et al., Bioorg. & Med. Chem. Lett., (2003), 13:2227-2230. International Publication No. WO 1996/35691 discloses spirocyclic and bicyclic diazinyl and carbazinyl oxazolidinone derivatives. Diazepeno phenyloxazolidinone derivatives are disclosed in the International Publication No. WO 1999/24428. International Publication No. WO 2002/06278 discloses substituted aminopiperidino phenyloxazolidinone derivatives.

Various other methods of preparation of oxazolidinones are reported in US Patent No. 7087784, US Patent No. 6740754, US Patent No. 4948801 , US Patent No. 3654298, US Patent No. 5837870, Canadian Patent No. 681830, J. Med. Chem., 32, 1673 (1989), Tetrahedron, 45, 1323 (1989), J. Med. Chem., 33, 2569 (1990), Tetrahedron Letters, 37, 7937-40 (1996) and Organic Process Research and Development, 11 , 739-741(2007).

Indian Patent Application No. 2534/MUM/2007 discloses a process for the preparation of substituted piperidino phenyloxazolidinones. International Publication No. WO2012/059823 further discloses the process for the preparation of phosphoric acid mono-(L-{4-[(5)-5-(acetylaminomethyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}4-methoxymethyl piperidine-4-yl)ester.

US Patent No. 8217058 discloses (5S)-N-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide as an antibacterial agent and its process for preparation.

PATENT

WO2015173664

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015173664&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

In some embodiments, there is provided a process for preparation of a compound of Formula (I) as shown in Scheme 1

(I I) (I N)

Scheme 1

Example 1

Preparation of (55)-iV-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)- phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide (I)

To a stirred solution of lithium teri-butoxide (59.1 g, 0.74 mol) in tetrahydrofuran (500 ml) was added a solution of [3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)-phenyl]-carbamic acid benzyl ester (II) (100 g, 0.25 mol) in 500 ml of tetrahydrofuran slowly at room temperature. The resulting mixture was stirred for 3 hours at room temperature (formation of lumps observed). The reaction mixture was cooled to temperature of 10°C to 15°C and acetic acid l-(acetylamino-methyl)-2-chloro-ethyl ester (III) (95.2 g, 0.49 mol) was added in one lot, after 5 minutes methanol (2.36 g, 0.075 mol) was added in one portion. The resulting mixture was stirred further at temperature of 10°C to 15°C. After 5 hours the reaction mixture was allowed to warm to room temperature and stirring continued further for 16 hours. An aqueous solution of saturated ammonium chloride (100 ml) was added to the reaction mixture, the resulting mixture was stirred well and the solvent evaporated under reduced pressure (35°C, 150 mm Hg). The residual mixture was diluted with water (1 L stirred well and filtered under suction, the residual solid was washed with additional fresh water (100 ml). The residual mass was suspended in acetone (500 ml), stirred well and the mixture diluted with hexane (1 L), slowly. The mixture was stirred further for 1 hour and filtered under suction. The residual solid was washed with a 2:1 mixture of acetone and water (100 ml). The residual solid was dried at 45°C, for 3.5 hour at 4 mm Hg, to obtain the 78 g of (55)-N-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l -yl)-phenyl]-2-oxo-oxazolidin-5-ylmethylj -acetamide (I) as white solid, in 77% yield.

Analysis:

Mass: 414 (M+l ); for Molecular Weight: 413 and Molecular Formula:

Melting Point: 178-179°C;

1H NMR (400 MHz, DMSO): δ 8.18-8.21 (m, 1H), 7.19-7.25 (d, 2H), 4.07-4.71 (m, 1H), 4.32 (s, 1H), 4.02-4.07 (t, 1H), 3.64-3.68 (t, 1H), 3.14 (s, 2H), 2.81-2.83 (d, 2H), 1.81 (s, 3H), 1.63-1.69 (t, 2H), 1.42-1.45 (d, 2H);

Purity as determined by HPLC: 97.65%.

Example 2

Preparation of acetic acid l-(acetylamino-methyl)-2-chloro-ethyl ester (III)

Step-I: Preparation of l-amino-3-chloro-propan-2-ol hydrochloride (VI)

Benzaldehyde (118.67 g, 1.03 mol) was dissolved in ethanol (297 ml) under stirring and the solution was cooled to 18-19°C. To this solution aqueous ammonia solution (25%) (101.58 ml) was added slowly, followed by slow addition of S-epichlorohydrin (100 g, 1 mol). The resulting mixture was warmed to 40°C and stirred for 7 hours. The mixture was allowed to cool to room temperature and stirred further. After 16 hours, the reaction mixture was concentrated to 50% volume under reduced pressure. Toluene (228 ml) was added to the reaction mixture followed by addition of aqueous hydrochloric acid (162 ml of concentrated hydrochloric acid diluted with 152 ml of water). The mixture thus obtained for 3 hours at 45°C, the resulting mixture was allowed to cool to room temperature and the toluene layer separated. The toluene layer was further extracted with water (56 ml). The combined aqueous layer was diluted with ethanol (56 ml) and the mixture evaporated under reduced pressure. This process was repeated again. To the final concentrate was added ethanol (180 ml), stirred for 10 minutes and the mixture cooled to -28°C to -30°C and maintained at this temperature for 2 hours. The separated solid was filtered under suction and the residue washed with cold (-30°C) ethanol (50 ml). The residue was dried at 45°C, under reduced pressure (4 mm Hg) for 3 hours, to obtain 96 g of l-amino-3-chloro-propan-2-ol hydrochloride (VI) as white solid in 61% yield.

Analysis:

Mass: 110 (M+l) as free base; for Molecular Weight: 145.5 and Molecular Formula:

1H NMR (400 MHz, D₂0): δ 4.02-4.08 (m, 1H), 3.51-3.61 (m, 2H), 3.12-3.16 (dd, 1H), 2.93 -2.99 (dd, 1H).

Step-II: Preparation of acetic acid l-(acetylamino-methyl)-2-chloro-ethyl ester (III).

A stirred solution of dichloromethane (220.8 ml) containing the step-I salt (96 g, 0.66 mol) was cooled to 18-20°C. Acetic anhydride (154.78 g, 1.5175 mol) was added slowly (slight exothermic). Pyridine (67.76 g, 0.8577 mol) was added slowly (exothermic) while maintaining the temperature at 18-20°C. The resulting mixture was heated to 40°C for 5 hours. The reaction mixture was allowed to cool to room temperature and stirring continued for further 16 hours. The reaction mass was cooled to 3-6°C and diluted with 170 ml of fresh water. To this was added an aqueous solution of potassium carbonate (191.2 g of K₂CO₃ in 382 ml water). The reaction mixture was further diluted with additional dichloromethane (170 ml) and water (425 ml). The reaction mass was stirred well and the dichloromethane layer separated. The aqueous layer was further extracted with 2×170 ml dichloromethane. The combined dichloromethane layer was washed with aqueous sodium chloride solution (13.6 g of sodium chloride in 493 ml water). The solvent was evaporated till a volume of 170 ml and the residual layer was diluted with toluene (340 ml), stirred well and the solvent was evaporated completely at 40°C under reduced pressure (4 mm Hg). To the residue ethyl acetate (170 ml) and hexane (187 ml) were added and the mixture stirred for 30 minute. The separated solid was filtered under suction and the residue washed with 50 ml of a 1 :1 mixture of ethyl acetate and hexane. The solid obtained was dried under reduced pressure (4 mm Hg) at 45°C for 3.5 hours, to obtain 96 g of acetic acid l-(acetylamino-methyl)-2-chloro-ethyl ester (III) as a white solid, in 75% yield.

Analysis:

Mass: 194 (M+l); for Molecular Weight: 193 and Molecular Formula: C₇Hi₂ClN0₃; 1H NMR (400 MHz, CDC1₃): 5 5.69 (s, 1H), 5.0-5.1 (m, 1H), 3.4-3.7 (m, 4H), 2.1 (s, 3H), 1.9 (s, 3H).

PATENT

http://www.google.st/patents/WO2007132314A2?cl=en

Wockhardt Ltd,

(3) (4)

Scheme -1

⁽⁶⁾ Formula π Scheme-2

Formula II Formula in

Formula I(a) Scheme-4

Example -11 : (5S)-N- {3-[3,5-difluoro-4-(4-hydroxy-(4-methoxymethyl)-piperidin- lyl)phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide

The example- 10 (54.86 g, 0.144 mol) was suspended in methanol (1100 ml) under stirring at RT. Sodium metal (4 g, 0.174 mol) was added in small lots in 2 min to the above suspension under stirring. The reaction mixture was warmed to 40-42⁰C and was stirred at this temperature for about 40 hrs. After completion of the reaction (TLC), the solvent was evaporated under reduced pressure to obtain a thick slurry. The thick slurry thus obtained was gradually added to water (1100 ml) under stirring. After the complete addition, the pH of the aqueous suspension was adjusted to 7 by adding sufficient quantity of glacial acetic acid. The separated solid was filtered and the residue was washed with water. The obtained solid was further purified by column chromatography over silica gel to obtain the product as a white solid, 32.7 g, 55 % yield.

M.P.: 173-174⁰C;

MS : M+l= 414(MH⁺, 100%); for M.F.: Ci₉H₂₅F₂N₃O₅

¹H-NMR (400 MHz, CDCl₃): δ 7.0-7.1 (m, 2H₅Ar-H), 6.0 (t, IH, NH), 4.70-4.80 (m, IH), 4.00 (t,lH), 3.70-3.75 (m, 2H), 3.5-3.7 (m, IH), 3.43 (s, 3H, OCH₃), 3.37-3.42 (m, 2H), 3.30 (s, 2H, -OCH₂), 3.0-3.05 (m, 2H), 2.22(bs,lH ,-OH),2.04 (s, 3H, COCH₃), 1.70-1.75 (m, 4H).

Patent

INDIAN 3049/MUM/2010

Phosphoric acid mono-(1-{4-[(S)-5-(acetylamino-methyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}-4-methoxy methyl-piperidin-4-yl) ester

Specific intermediate compounds of the invention include:
6-(2,6-difluoro-4-nitrophenyl)-1-oxa-6-azaspiro[2.5]octane;
1-(2,6-Difluoro-4-nitro-phenyl)-4-methoxymethyl-piperidin-4-ol;
[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-carbamic acid benzyl ester;
(5R)-3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-hydroxymethyl-oxazolidin-2-one;
(5R)-Methanesulfonic acid 3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl ester;
(5R)-3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-azidomethyl-oxazolidin-2-one; and
(5S)- N-{3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide.

Examples

Preparation of Intermediate-1: 1-(2,6-Difluoro-4-nitrophenyl)-piperidin-4-one
Chloroform (9.3 L) was charged in a 20 L reaction assembly and 4-piperidone hydrochloride (1.17 Kg, 7.62 mol) was added under stirring followed by triethylamine (2.14 Kg, 2.95 L, 21.1 mol). After 30 minutes of stirring, 3,4,5-trifluoronitrobenzene (1.5 Kg, 8.47 mol) was added to the mixture in one lot and the contents were heated to 65-70ºC for 8 h. After completion of the reaction, chloroform was removed under vacuum to obtain a syrupy mass. At this stage, water (10 L) was added to the mass and the chloroform recovery was continued under vacuum below 65oC till the chloroform was removed completely. The slurry was cooled to RT and filtered. The solid product was washed with water (3 L) followed by hexanes (2 L). The product was dried in a vacuum oven below 70oC to obtain the product as a yellow solid, 1.88 Kg ; Yield 97%.
M.P.: 130-132oC; MS: 257(M+1); M.F.: C11H10F2N2O3.

Preparation of Intermediate 3: 1-(2,6-Difluoro-4-nitro-phenyl)-4-methoxymethyl-piperidin-4-ol

Method A:
Preparation of Intermediate–2: (Stage-I): 6-(2,6-difluoro-4-nitrophenyl)-1-oxa-6-azaspiro[2.5]octane
A solution of trimethylsulfoxonium iodide (1.504kg, 6.836mol) in acetonitrile (7L) was cooled to 0 to 5oC. , under argon atmosphere. Potassium tert-butoxide (0.736kg, 6.552 mol) was added in small lots over 0.5h. The resulting solution was stirred for 2h at the same temperature. To this solution was added 1-(2,6-Difluoro-4-nitrophenyl)-piperidin-4-one ( 1.4kg, 5.46mol) in small lots over a period of 1h, while maintaining the temp. between 5-10oC. The resulting mixture was stirred for 1h. The solvent was evaporated to a minimum amount possible, under reduced pressure while maintaining the temperature below 10oC. The residue was poured in water( 18L) and the pH adjusted to neutral with dilute acetic acid. The resulting slurry was stirred well and the separated solid filtered under suction. The solid was washed with fresh water till the filtrate was free of acetic acid. The solid was dried at 80oC, for 6h, under reduced pressure to obtain the product as pale yellow solid, 1.264kgs, yield 85%.
M.P.: 96-97oC; MS: M+1: 271; M.F.: C12H12F2N2O3,.
Preparation of Intermediate-3: (Stage-II): 1-(2,6-Difluoro-4-nitro-phenyl)-4-methoxymethyl-piperidin-4-ol
To a solution of sodium methoxide (236g, 4.35mol) in methanol (3L), at RT, was added 6-(2,6-difluoro-4-nitrophenyl)-1-oxa-6-azaspiro [2.5]octane (964g, 3.57mol) in small portions and the reaction mixture was stirred for 26h at RT. Acetic acid (265g, 4.44mol) was added slowly to neutralize the pH of the solution. The resulting mixture was poured into chilled water(18L) and stirred for 1h. The separated solid was filtered under suction. The solid was washed with additional water till the filtrate was free of acetic acid. The solid was dried for 10hat RT under reduced pressure, to obtain the product as a pale yellow solid, 973g, yield, 90%
M.P.: 84-86oC; MS: 303 (M+1); M.F.: C13H16F2N2O4

Method B:
Dimethylsulfoxide (DMSO, 100 ml) and methanol (500 ml) were charged in a 1 L glass reaction assembly. Potassium hydroxide (59.2g, 0.898 mol) was charged in the assembly followed by trimethylsulfoxonium iodide (94.5 g, 0.43 mol) and the contents were stirred for 30 minutes and then cooled to 10oC-15oC. To the cooled contents was added 1-(2,6-difluoro-4-nitrophenyl)-piperidin-4-one (100 g, 0.39 mol) in small lots. After the addition, the temperature was allowed to raise to RT and the contents were further stirred for 24 h (ring opening of the epoxide intermediate viz. 6-(2,6-difluoro-4-nitrophenyl)-1-oxa-6-azaspiro[2.5]octane takes place).
[Physical data of the intermediate: M.P.: 96-970C, MS: 271(M+1); M.F.: C12H12F2N2O3, .
After completion of the reaction the contents were poured slowly in ice-water (600g crushed ice in 600 ml water). The precipitated solid product was filtered and was washed with water:methanol, 2:1 (100 ml X 2). The wet product was used in the next step.
M.P.: 84-86oC; MS: 303 (M+1);.M.F.: C13H16F2N2O4,:

Preparation of Intermediate -5: [3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-carbamic acid benzyl ester

Method A: Preparation of Intermediate 4: ( Stage-I)
Water (1.19 L) and methanol (595 ml) were charged in a 3 L glass reaction assembly, followed by 1-(2,6-difluoro-4-nitro-phenyl)-4-methoxymethyl-piperidin-4-ol (85 g, 0.281 mol) and the contents were stirred. Sodium dithionite (288 g, 1.407 mol) was added in one lot and the reaction mixture was heated to 80oC for 8 h. After completion of the reaction (TLC), methanol was recovered under vacuum below 65oC. After the recovery, the aqueous residue was extracted with chloroform (400 ml X 3). The combined chloroform extract (containing the intermediate 1-(4-amino-2,6-difluoro-phenyl)-4-methoxymethyl-piperidin-4-ol) was dried over anhydrous Sodium sulfate and used in the next step (carbamate formation).

Preparation of Intermediate -5: (Stage-II):
The above chloroform extract was charged in a 3 L glass reaction assembly. Sodium bicarbonate (70 g, 0.843 mol) was added to the extract and the contents were cooled to 15oC-20oC. Benzylchloroformate solution (50% in toluene, 48 g, 96 ml, 0.281 mol) was added slowly to the above mixture under stirring. After completion of the addition, the reaction mixture was stirred at RT for 2 h. After completion of the reaction (TLC), the contents were filtered on a Buchner assembly and the solid cake was washed with chloroform (85 ml X 2). The combined filtrate was evaporated under vacuum below 50oC to obtain yellowish oily mass, which was poured slowly in hexanes (850 ml) under stirring to obtain a precipitate. The precipitated product was filtered and washed with hexanes (100 ml X 2). The product was dried in a vacuum oven below 65oC to obtain 60.2 g brownish product (Yield = 38% on the basis of step-I input).
M.P.: 138-140oC; MS: 407(M+1); M.F.: C21H24F2N2O4.:.

Method B: : Preparation of Intermediate 4: ( Stage-I): To a solution of 1-(2,6-difluoro-4-nitro-phenyl)-4-methoxymethyl-piperidin-4-ol (973g, 3.22 mol) in ethyl acetae (10L) was added 10% Pd-C, (250g, 50% wet) and the resulting miture was hydrogenated in a pressure at 30 PSI, 45-55oC, for 3h. The catakyst was filtered and the residue was washed with additional ethyl acetate( 200ml). The combined filtrates were used as such for the next reaction (carbamate formation)

Preparation of Intermediate -5: (Stage-II):
To the above filtrate was added sodium bicarbonate(406g, 4.83 mol) and the mixture warmed to 40-45oC. To this mixture was added a 50% solution of Benzyl chloroformate in toluene(1.373L, 4.025 mol), drop-wise, over a period of 1h. Stir the resulting mixture for 1h and filter the insoluble material. The residue was washed with 300ml of ethyl acetate. The filtrates were combined and the solvent evaporated under reduced pressure, below 55oC.. Cool the residue and dilute it with hexane(10L). The resulting slurry was stirred well and the separated solid was filtered under suction. The residue was washed with additional hexane ( 2L). The solid was dried for 10h at RT, to obtain the product as dark brown solid, 1200g, yield, 96%.
M.P.: 138-140oC; MS: 407( M+1); M.F.: C21H24F2N2O.

Preparation of Intermediate -6:

(5R)-3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-hydroxymethyl-oxazolidin-2-one

To a mixture of [3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-carbamic acid benzyl ester (100g, 0.237 mol) in dry tetrahydrofuran (THF) (2 L) at 40ºC was added drop-wise n-BuLi in hexane (1.6M, 45.5 g, 455 ml, 0.711 mol) under nitrogen atmosphere. The contents were stirred for 1 h at 40ºC and R-(-)-glycidyl butyrate (68.25 g, 0.474 mol) was added gradually. After the addition of R-(-)-glycidyl butyrate, the reaction mixture was stirred for 5-6 h at 40oC till completion of the reaction (TLC). After completion of the reaction, a solution of sodium methoxide (2 g) in methanol (66 ml) was added to the contents followed by water (8 ml) and the contents were stirred for an additional 0.5 h. Water (1 L) was added to the solution and the contents were extracted with ethyl acetate (1 L). The aqueous layer was further extracted with ethyl acetate (3 X 500 ml). The combined organic layer was evaporated under vacuum to obtain a thick residue. tert-Butyl methyl ether (1 L) was added to the residue and the contents were stirred for about 1 h to obtain a solid product, which was filtered and washed with tert-butyl methyl ether (2 X 100 ml). The product was dried under vacuum below 60ºC to obtain the product as a 46.5 g dark brown compound, 46.5g ,yield 51%.
M.P.: 117-119oC; MS: 373(M+1); M.F.: C17H22F2N2O5..

Preparation of Intermediate -7: (5R)-Methanesulfonic acid 3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl ester

To a mixture of (5R)-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-hydroxymethyl-oxazolidin-2-one (45 g, 0.121 mol) in dichloromethane (0.3 L), was added triethylamine (24.5 g, 34 ml, 0.242 mol) while stirring. Methanesulfonyl chloride (18 g, 12.2 ml, 0.157 mol) was added to the above solution over a period of 1 h at 10oC -20oC and the reaction mixture was stirred for additional 2 h at RT. After completion of the reaction (TLC), the contents were evaporated under vacuum at 40oC to obtain an oily residue. Water (450 ml) was added to the residue and the traces of dichloromethane were removed under vacuum. The solid product thus obtained was filtered, washed with water (2 X 50 ml) and dried under vacuum at 70oC to obtain 50.6 g brownish compound. Yield = 93%; M.P.:106-108oC; MS: 451(M+1); M.F.: C18H24F2N2O7S.

Preparation of Intermediate 8a: (5R)-3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-azidomethyl-oxazolidin-2-one

Method A:
To a solution of (R)-3-(3,5-difluoro-4-(4-hydroxy-4-(methoxymethyl)piperidin-1-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (2g, 5.3 mmol),in tetrahydrofuran (20 mL), under argon , was added diphenylphosphoryl azide (1.63mL, 5.9 mmol). The solution was cooled to 0oC in an ice-bath. 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) (0.76mL, 4.9mmol) was added drop-wise over 15min..The reaction was stirred at same temperature for 1 hr, and then warmed to room temperature and stirred under for 16 hr. The reaction mixture was diluted with ethyl acetate (20 mL), and water (20mL). After separation of water layer, the organic layer was washed with water and 0.5M citric acid monohydrate (10 mL). The organic layer was dried over sodium sulfate and the solvent evaporated under reduced pressure.The residue was triturated with ether to obtain the product as a buff colored solid, 1.32g (62%).
M.P.: 106-108oC; M.S.- 398(M+1); M.F.- C17H21F2N5O4,

Method B:
To a solution of (5R)-methanesulfonic acid 3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl ester (20 g, 0.044 mol, wet) in N,N-dimethylformamide (30 ml), was added sodium azide (8.6 g, 0.133 mol) in a single lot. The reaction mixture was gradually heated and the temperature was maintained at 70ºC for 8 h. After completion of the reaction (TLC), the contents were cooled to 20-25ºC and poured slowly into chilled water (300 ml). The solid product thus obtained was filtered and washed with water (2 x 50 ml). The wet product was air dried to obtain 16.5g dark brown compound (being an azide, it was NOT exposed to heat during drying) Yield ~ 93%.
M.P.: 106-108oC; MS : 398(M+1); M.F.: C17H21F2N5O4;:

Preparation of Intermediate 8b: (5S)-N-2-{3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-phthalimide

Method A:
A mixture of (5R)-{3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)phenyl]-2-oxo-oxazolidin-5-yl methyl}-methanesulfonate(10g, 0.022 mol), Potassium phthalimide (12.2g, 0.066 mol) and DMF (50ml) was heated, with stirring, at 90oC for 4h. The resulting mixture was cooled to RT and poured over ice-water mixture. The separated solid was filtered, washed with water and dried under suction to obtain the product as a white solid, 9.46g, in 85% yield.
M.P.: 154-156 oC; MS: 502 (M+1); M.F. C25H25F2N3O6.

Method B:
To tetrahydrofuran (30 ml) were added triphenylphosphine (2.11g, 8 mmol)) and diethyldiazocarboxylate (1.62g, 8 mmol)), and the solution stirred at room temperature. After 10 minute phthalimide (1.18g, 8 mmol)) was added and after a further stirring for 10 minute, (R)-3-(3,5-difluoro-4-(4-hydroxy-4-(methoxymethyl)piperidin-1-yl)phenyl)-5-(hydroxymethyl) oxazolidin-2-one (2g, 5.3 mmol) was added and stirring continued further at room temperature. After 8 hrs ice-cold water (4 ml) was added to the reaction mixture and the resulting mixture was extracted by ethyl acetate (2 x 20ml). The ethyl acetate extract was dried (over sodium sulfate) and concentrated under reduced pressure. The residue was chromatographed on a column of silica gel to obtain the product as an off-white solid, 1.56g, yield 58%.
M.P.: 154-156 oC; MS : 502 (M+1); M.F. C25H25F2N3O6.

Preparation of Intermediate 10: (5S)- N-{3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide
via
Intermediate 9: 5-aminomethyl-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-oxazolidin-2-one

Method A:
To a solution of (5R)-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-azidomethyl-oxazolidin-2-one (10 g, 0.025 mol) in methanol (100 ml), were charged cobalt chloride (0.6 g, 0.0025 mol) followed by sodium borohydride (0.95 g, 0.025 mol) in small lots over a period of 30 minutes. The reaction mixture was stirred at RT for additional 2 h. After completion of the reaction , the contents were evaporated under vacuum below 40oC to obtain a sticky mass. The contents were suspended in a mixture of water (100 ml) and ethyl acetate (50 ml) and stirred for 15 minutes. The contents were filtered through a filter-aid bed and the bed was washed with ethyl acetate (2 X 25 ml). The layers were separated and the aqueous layer was further extracted with ethyl acetate (4 X 50 ml). The combined organic layer was washed with 1% HCl solution (100 ml). The aqueous layer was separated and washed with dichloromethane (4 X 50 ml). The pH of the aqueous layer was adjusted to 8 by adding saturated sodium bicarbonate solution. The contents were extracted with ethyl acetate (6 X 50 ml) till no amine spot was seen in the final organic extract. The combined organic layer (containing the intermediate 5-aminomethyl-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-oxazolidin-2-one) was dried over anhydrous sodium sulfate.

Triethylamine (3.3 g, 4.5 ml, 0.0327 mol) was added to the above organic layer and acetyl chloride (2.17 g, 2 ml, 0.0277 mol) was added gradually over a period of 1 h at RT. The reaction mixture was stirred for 2 h and after completion of the reaction (TLC), the contents were washed with water (50 ml) and the layers separated. Activated carbon (1 g) was added to the organic layer and the contents were stirred for 15 minutes. The contents were filtered on a celite bed and the carbon-celite bed was washed with ethyl acetate (2 X 10 ml). The combined filtrate was evaporated under vacuum to obtain a slurry, which was filtered on a Buchner assembly and the product was washed with ethyl acetate (2 X 10 ml). The product was dried under vacuum at 70oC to obtain 5 g off-white solid. Yield = 48% (on the basis of azide). HPLC Purity ~ 98%.
M.P.: 178-179oC; MS : 414 (M+1); M.F.: C19H25F2N3O5.

Method B:
A solution of (5R)-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-5-azidomethyl-oxazolidin-2-one (50 g, 0.125 mol) in ethyl acetatel (1L ml), were charged with 5g of 10% of Pd-C catalyst(50% wet) and the resulting mixture was hydrogenated at 30psi for 3h at 50oC.. The resulting mixture was cooled and filtered under suction over celite bed. The residue was washed with additional ethyl acetate (200ml). The combined filtrates were concentrated to 500ml volume.

To the above ethyl acetate solution was added Triethyl amine (19.1g, 0.189 mol), and acetic anhydride (16.1g, 1.58mol) in a single lot in few minutes). The reaction mixture was stirred for 16h at R.T. .The resulting mixture was cooled to 0-5oC, stirred for 0.5h and filtered under suction. The residue was washed with cold ethyl acetate(100ml) and dried at 70oC under reduced pressure to obtain the product as a a off-white solid, 43.5g, in 84% yield over two steps.
HPLC Purity ~ 98%
M.P.: 178-179oC; MS : 414 (M+1); M.F.: C19H25F2N3O5.

Method C:
To a solution of (S)-N-2-{3-[3,5-Difluoro-4-(4-methoxymethyl-4-hydroxypiperidine-1yl)phenyl]-2-oxo-oxazolidin-5-yl methyl}-phthalimide (2.77g, 0.0055mol) in ethanol (20ml) was added hydrazine hydrate ( 0.554g, 0.011mol) and the resulting solution stirred at RT for 6h. The solvent was evaporated under reduced pressure, the residue suspended in 3% sodium carbonate solution and extracted in dichloromethane (40ml). The dichloromethane layer was dried and to this solution was added triethylamine(1.11g, 0.011mol) and acetic anhydride (0.67g, 0.007mol) and the solution stirred for 6h at RT. The solvent was evaporated under reduced pressure and the residue purified by flash chromatography to obtain the product as white solid, 1.94g, in 85% yield.
M.P.: 178-179oC; MS: 414 (M+1); M.F.: C19H25F2N3O5.

Method D:
A mixture of (5R)-{3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)phenyl]-2-oxo-oxazolidin-5-yl methyl}-methanesulfonate (1gm, 4.4mmol) and sodium diformylamide (2gms, 22mmol) in DMF (5ml) was stirred at 95 ºC. for 15hrs. Then a mixture of conc. HCl (0.6ml) and water (0.6ml) and ethanol (8ml) were added. The solution was stirred at 75ºC for 5hrs. The mixture was concentrated under reduced pressure at 60-75 ºC. Water (1ml), ammonia solution (0.5ml) and acetic anhydride (1ml) was added to the residue and the mixture stirred at 70-75 ºC for 4-5 hrs. The solution was cooled to room temperature, diluted with water (5ml) and the separated solid filtered. The residue was washed with water (4ml.) and dried in a vacuum oven at 50ºC to obtain the product as an off-white solid, 0.37g, in 41% yield.
M.P.: 178-179oC; MS : 414 (M+1); M.F.: C19H25F2N3O5.

Method E:

To tetrahydrofuran (30 ml) were added triphenylphosphine (2.11g, 8 mmol)) and diethyldiazocarboxylate (1.62g, 8 mmol)), and the solution stirred at room temperature. After 10 min acetamide (0.475g, 8 mmol)) was added and after a further stirring for 10 min, (R)-3-(3,5-difluoro-4-(4-hydroxy-4-(methoxymethyl)piperidin-1-yl)phenyl)-5-(hydroxymethyl) oxazolidin-2-one (2g, 5.3 mmol) was added and stirring continued further at room temperature. After 16 hrs ice-cold water (4ml) was added to the reaction mixture and the resulting mixture was extracted by ethyl acetate (2 x 20ml). The ethyl acetate extract was dried (over sodium sulfate) and concentrated under reduced pressure. The residue was chromatographed on a column of silica gel to obtain the product as an off-white solid, 0.50g, yield 22%.
M.P.: 178-179oC; MS: 414 (M+1); M.F.: C19H25F2N3O5.
Preparation of Intermediate -11: (S)-N-{3-[3,5-Difluoro-4-(4-methoxymethyl-4-di-O-benzylphosphoryloxy-piperi din-1yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide

To a solution of (S)-N-{3-[3,5-difluoro-4-(4-methoxymethyl-4-hydroxypiperidine-1yl)-phenyl]-2-oxo-oxazolidin-5-yl methyl}-acetamide (0.2 mmol) and tetrazole (0.6 mmol) in dichloromethane (5 ml) was added dibenzyl N,N,diisopropylphosphoramidite (0.4 mmol) and the resulting mixture was stirred for 4h. The resulting solution was cooled to 0 oC and 0.6 ml of 0.5M m-chloroperbenzoic acid solution in dichloromethane was added. After 4h, the solvent was evaporated under residue pressure and the residue chromatographed on a column of silica gel to obtain the product as a off-white solid in 75% yield,
MS: 674 (M+1); M.F. C33H38F2N3O8P;

Example A: Phosphoric acid mono-(1-{4-[(S)-5-(acetylamino-methyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}-4-methoxymethyl-piperidin-4-yl) ester

To a suspension of (S)-N-{3-[3,5-difluoro-4-(4-methoxymethyl-4-di-O-benzylphosphoryl- oxypiperidine-1yl)phenyl]-2-oxo-oxazolidin-5-yl methyl}-acetamide (0.15 mmol) and 20 % palladium hydroxide (20 mg) in 20 ml of a mixture of dichloromethane /aqueous methanol was stirred at room temperature for 6h. The catalyst was filtered and the residue evaporated under reduced pressure. The residue obtained was triturated with acetone to obtain a white solid as product in 70% yield.
MP. >140 °C; MS : 494(M+1) M.F.: C19H26F2N3O8P.

PATENT

WO 2012059823

http://www.google.co.in/patents/WO2012059823A1?cl=en

the process comprising the steps of:

a) Converting intermediate of Formula (1) into intermediate of Formula (3)

b) Converting intermediate of Formula (3) into intermediate of Formula (5)

c) Converting intermediate of Formula (5) into intermediate of structure (6)

(5) <⁶> d) Converting intermediate of Formula (6) into intermediate of Formula (10)

e) Converting intermediate of Formula (10) into intermediate of Formula (11),

f) Converting intermediate of Formula (11) into compound of Formula (A) or Pharmaceutically acceptable salts thereof

ormu a-

Scheme-1

Preparation of Intermediate 10: (5S)- N-{ 3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl- piperidin- 1 -yl)-phenyl] -2-oxo-oxazolidin-5-ylmethyl } -acetamide

via

Intermediate 9: 5-aminomethyl-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l- yl)-phenyl] -oxazolidin-2-one

Method A:

To a solution of (5R)-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)- phenyl]-5-azidomethyl-oxazolidin-2-one (10 g, 0.025 mol) in methanol (100 ml), were charged cobalt chloride (0.6 g, 0.0025 mol) followed by sodium borohydride (0.95 g, 0.025 mol) in small lots over a period of 30 minutes. The reaction mixture was stirred at RT for additional 2 h. After completion of the reaction , the contents were evaporated under vacuum below 40°C to obtain a sticky mass. The contents were suspended in a mixture of water (100 ml) and ethyl acetate (50 ml) and stirred for 15 minutes. The contents were filtered through a filter-aid bed and the bed was washed with ethyl acetate (2 X 25 ml). The layers were separated and the aqueous layer was further extracted with ethyl acetate (4 X 50 ml). The combined organic layer was washed with 1% HC1 solution (100 ml). The aqueous layer was separated and washed with dichloromethane (4 X 50 ml). The pH of the aqueous layer was adjusted to 8 by adding saturated sodium bicarbonate solution. The contents were extracted with ethyl acetate (6 X 50 ml) till no amine spot was seen in the final organic extract. The combined organic layer (containing the intermediate 5-aminomethyl-3-[3,5-difluoro-4-(4- hydroxy-4-methoxymethyl-piperidin-l-yl)-phenyl]-oxazolidin-2-one) was dried over anhydrous sodium sulfate.

Triethylamine (3.3 g, 4.5 ml, 0.0327 mol) was added to the above organic layer and acetyl chloride (2.17 g, 2 ml, 0.0277 mol) was added gradually over a period of 1 h at RT. The reaction mixture was stirred for 2 h and after completion of the reaction (TLC), the contents were washed with water (50 ml) and the layers separated. Activated carbon (1 g) was added to the organic layer and the contents were stirred for 15 minutes. The contents were filtered on a celite bed and the carbon-celite bed was washed with ethyl acetate (2 X 10 ml). The combined filtrate was evaporated under vacuum to obtain a slurry, which was filtered on a Buchner assembly and the product was washed with ethyl acetate (2 X 10 ml). The product was dried under vacuum at 70°C to obtain 5 g off-white solid. Yield = 48% (on the basis of azide). HPLC Purity ~ 98%.

M.P.: 178-179°C; MS : 414 (M+l); M.F.: C1₉H₂5F₂N3O5. Method B:

A solution of (5R)-3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)-phenyl]-5- azidomethyl-oxazolidin-2-one (50 g, 0.125 mol) in ethyl acetatel (1L ml), were charged with 5g of 10% of Pd-C catalyst(50% wet) and the resulting mixture was hydrogenated at 30psi for 3h at 50°C. The resulting mixture was cooled and filtered under suction over celite bed. The residue was washed with additional ethyl acetate (200ml). The combined filtrates were concentrated to 500ml volume. To the above ethyl acetate solution was added Triethyl amine (19. lg, 0.189 mol), and acetic anhydride (16. lg, 1.58mol) in a single lot in few minutes). The reaction mixture was stirred for 16h at R.T. .The resulting mixture was cooled to 0-5°C, stirred for 0.5h and filtered under suction. The residue was washed with cold ethyl acetate( 100ml) and dried at 70°C under reduced pressure to obtain the product as a a off-white solid, 43.5g, in 84% yield over two steps.

HPLC Purity ~ 98%

M.P.: 178-179°C; MS : 414 (M+l); M.F.: C1₉H₂5F₂N3O5. Method C:

To a solution of (S)-N-2-{3-[3,5-Difluoro-4-(4-methoxymethyl-4-hydroxypiperidine- lyl)phenyl]-2-oxo-oxazolidin-5-yl methyl }-phthalimide (2.77g, 0.0055mol) in ethanol (20ml) was added hydrazine hydrate ( 0.554g, 0.01 lmol) and the resulting solution stirred at RT for 6h. The solvent was evaporated under reduced pressure, the residue suspended in 3% sodium carbonate solution and extracted in dichloromethane (40ml). The dichloromethane layer was dried and to this solution was added triethylamine(l.l lg, 0.01 lmol) and acetic anhydride (0.67g, 0.007mol) and the solution stirred for 6h at RT. The solvent was evaporated under reduced pressure and the residue purified by flash chromatography to obtain the product as white solid, 1.94g, in 85% yield.

M.P.: 178-179°C; MS: 414 (M+l); M.F.: C1₉H₂5F₂N₃O5. Method D:

A mixture of (5R)-{3-[3,5-Difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)phenyl]- 2-oxo-oxazolidin-5-yl methyl }-methanesulfonate (lgm, 4.4mmol) and sodium diformylamide (2gms, 22mmol) in DMF (5ml) was stirred at 95 °C. for 15hrs. Then a mixture of cone. HC1 (0.6ml) and water (0.6ml) and ethanol (8ml) were added. The solution was stirred at 75°C for 5hrs. The mixture was concentrated under reduced pressure at 60-75 °C. Water (1ml), ammonia solution (0.5ml) and acetic anhydride (1ml) was added to the residue and the mixture stirred at 70-75 °C for 4-5 hrs. The solution was cooled to room temperature, diluted with water (5ml) and the separated solid filtered. The residue was washed with water (4ml.) and dried in a vacuum oven at 50°C to obtain the product as an off-white solid, 0.37g, in 41% yield.

M.P.: 178-179°C; MS : 414 (M+l); M.F.: C1₉H₂5F₂N3O5. Method E:

To tetrahydrofuran (30 ml) were added triphenylphosphine (2.1 lg, 8 mmol)) and diethyldiazocarboxylate (1.62g, 8 mmol)), and the solution stirred at room temperature. After 10 min acetamide (0.475g, 8 mmol)) was added and after a further stirring for 10 min, (R)-3- (3,5-difluoro-4-(4-hydroxy-4-(methoxymethyl)piperidin-l-yl)phenyl)-5-(hydroxymethyl) oxazolidin-2-one (2g, 5.3 mmol) was added and stirring continued further at room temperature. After 16 hrs ice-cold water (4ml) was added to the reaction mixture and the resulting mixture was extracted by ethyl acetate (2 x 20ml). The ethyl acetate extract was dried (over sodium sulfate) and concentrated under reduced pressure. The residue was chromatographed on a column of silica gel to obtain the product as an off-white solid, 0.50g, yield 22%.

M.P.: 178-179°C; MS: 414 (M+l); M.F.: C1₉H₂5F₂N₃O5.

PATENT

http://www.google.co.in/patents/WO2008038092A2?cl=en

Wockhardt Research Center,

IV

V

‘ Scheme-1 ‘

/////////

SEE FULL ZOLID SERIES…………http://drugsynthesisint.blogspot.in/p/zolid.html

PF 04937319

N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)benzofuran-4-yloxy)pyrimidine-2-carboxamide

MW 432.43

CLINICAL TRIALS

SYNTHESIS

Glucokinase is a key regulator of glucose homeostasis and small molecule activators of this enzyme represent a promising opportunity for the treatment of Type 2 diabetes. Several glucokinase activators have advanced to clinical studies and demonstrated promising efficacy; however, many of these early candidates also revealed hypoglycemia as a key risk. In an effort to mitigate this hypoglycemia risk while maintaining the promising efficacy of this mechanism, we have investigated a series of substituted 2-methylbenzofurans as “partial activators” of the glucokinase enzyme leading to the identification of N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)benzofuran-4-yloxy)pyrimidine-2-carboxamide as an early development candidate.

Diabetes is a major public health concern because of its increasing prevalence and associated health risks. The disease is characterized by metabolic defects in the production and utilization of carbohydrates which result in the failure to maintain appropriate blood glucose levels. Two major forms of diabetes are recognized. Type I diabetes, or insulin-dependent diabetes mellitus (IDDM), is the result of an absolute deficiency of insulin. Type Il diabetes, or non-insulin dependent diabetes mellitus (NIDDM), often occurs with normal, or even elevated levels of insulin and appears to be the result of the inability of tissues and cells to respond appropriately to insulin. Aggressive control of NIDDM with medication is essential; otherwise it can progress into IDDM. As blood glucose increases, it is transported into pancreatic beta cells via a glucose transporter. Intracellular mammalian glucokinase (GK) senses the rise in glucose and activates cellular glycolysis, i.e. the conversion of glucose to glucose-6-phosphate, and subsequent insulin release. Glucokinase is found principally in pancreatic β-cells and liver parenchymal cells. Because transfer of glucose from the blood into muscle and fatty tissue is insulin dependent, diabetics lack the ability to utilize glucose adequately which leads to undesired accumulation of blood glucose (hyperglycemia). Chronic hyperglycemia leads to decreases in insulin secretion and contributes to increased insulin resistance. Glucokinase also acts as a sensor in hepatic parenchymal cells which induces glycogen synthesis, thus preventing the release of glucose into the blood. The GK processes are thus critical for the maintenance of whole body glucose homeostasis.

It is expected that an agent that activates cellular GK will facilitate glucose-dependent secretion from pancreatic beta cells, correct postprandial hyperglycemia, increase hepatic glucose utilization and potentially inhibit hepatic glucose release. Consequently, a GK activator may provide therapeutic treatment for NIDDM and associated complications, inter alia, hyperglycemia, dyslipidemia, insulin resistance syndrome, hyperinsulinemia, hypertension, and obesity. Several drugs in five major categories, each acting by different mechanisms, are available for treating hyperglycemia and subsequently, NIDDM (Moller, D. E., “New drug targets for Type 2 diabetes and the metabolic syndrome” Nature 414; 821 -827, (2001 )): (A) Insulin secretogogues, including sulphonyl-ureas (e.g., glipizide, glimepiride, glyburide) and meglitinides (e.g., nateglidine and repaglinide) enhance secretion of insulin by acting on the pancreatic beta-cells. While this therapy can decrease blood glucose level, it has limited efficacy and tolerability, causes weight gain and often induces hypoglycemia. (B) Biguanides (e.g., metformin) are thought to act primarily by decreasing hepatic glucose production. Biguanides often cause gastrointestinal disturbances and lactic acidosis, further limiting their use. (C) Inhibitors of alpha-glucosidase (e.g., acarbose) decrease intestinal glucose absorption. These agents often cause gastrointestinal disturbances. (D) Thiazolidinediones (e.g., pioglitazone, rosiglitazone) act on a specific receptor (peroxisome proliferator-activated receptor-gamma) in the liver, muscle and fat tissues. They regulate lipid metabolism subsequently enhancing the response of these tissues to the actions of insulin. Frequent use of these drugs may lead to weight gain and may induce edema and anemia. (E) Insulin is used in more severe cases, either alone or in combination with the above agents. Ideally, an effective new treatment for NIDDM would meet the following criteria: (a) it would not have significant side effects including induction of hypoglycemia; (b) it would not cause weight gain; (c) it would at least partially replace insulin by acting via mechanism(s) that are independent from the actions of insulin; (d) it would desirably be metabolically stable to allow less frequent usage; and (e) it would be usable in combination with tolerable amounts of any of the categories of drugs listed herein.

Substituted heteroaryls, particularly pyridones, have been implicated in mediating GK and may play a significant role in the treatment of NIDDM. For example, U.S. Patent publication No. 2006/0058353 and PCT publication No’s. WO2007/043638, WO2007/043638, and WO2007/117995 recite certain heterocyclic derivatives with utility for the treatment of diabetes. Although investigations are on-going, there still exists a need for a more effective and safe therapeutic treatment for diabetes, particularly NIDDM.

http://pubs.rsc.org/en/content/articlelanding/2011/md/c1md00116g/unauth#!divAbstract

http://www.rsc.org/suppdata/md/c1/c1md00116g/c1md00116g.pdf

Glucokinase is a key regulator of glucose homeostasis and small molecule activators of this enzyme represent a promising opportunity for the treatment of Type 2 diabetes. Several glucokinase activators have advanced to clinical studies and demonstrated promising efficacy; however, many of these early candidates also revealed hypoglycemia as a key risk. In an effort to mitigate this hypoglycemia risk while maintaining the promising efficacy of this mechanism, we have investigated a series of substituted 2-methylbenzofurans as “partial activators” of the glucokinase enzyme leading to the identification of N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)benzofuran-4-yloxy)pyrimidine-2-carboxamide as an early development candidate.

N,N-Dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)-benzofuran-4- yloxy)pyrimidine-2-carboxamide (28). To a solution of the 5-methyl-2-aminopyrazine (38.9 g, 356 mmol) in dimethoxyethane (315 mL) in a 3-neck flask equipped with overhead stirring and a condenser at 0 o C was added Me2AlCl (1 M solution in hexanes) (715 mL). The mixture was warmed to room temperature and stirred for 1.5 h. In a separate flask, 26 (52.6 g, 142.5 mmol) was dissolved in dimethoxyethane (210 mL). This mixture was then added to the amine mixture. A gum precipitated and upon scratching the flask it dissipated into a solid. The reaction was refluxed for 3.5 h. Aq. Rochelle’s salt (5 L) and 2-MeTHF (2 L) was added to the mixture and this was allowed to stir with overhead stirring for 14 h, after which time, a yellow solid precipitated. The solid was collected by filtration, washing with 2-MeTHF. The resulting solid was dried in a vacuum oven overnight to afford the desired material (50.0g) in 81% yield.

1 H NMR (400MHz, CDCl3) δ 9.54 (d, J = 1.56 Hz, 1H), 8.50 (s, 2H), 8.37 (s, 1H), 8.14 (d, J = 0.78 Hz, 1H), 7.88 – 7.92 (m, 1H), 7.52 (d, J = 1.37 Hz, 1H), 6.28 (t, J = 0.98 Hz, 1H), 3.14 (s, 3H), 2.98 (s, 3H), 2.55 (s, 3H), 2.49 (d, J = 1.17 Hz, 3H);

MS(ES+ ): m/z 433.4 (M+1), MS(ES- ): m/z 431.3 (M-1).

PAPER

http://pubs.rsc.org/en/content/articlelanding/2013/md/c2md20317k#!divAbstract

PAPER

Bioorganic & Medicinal Chemistry Letters (2013), 23(16), 4571-4578

http://www.sciencedirect.com/science/article/pii/S0960894X13007452

PATENT

Pfizer Inc.

WO 2010103437

https://www.google.co.in/patents/WO2010103437A1?cl=en

Scheme I outlines the general procedures one could use to provide compounds of the present invention having Formula (I).

Preparations of Starting Materials and Key Intermediates

Preparation of Intermediate (E)-3-(ethoxycarbonyl)-4-(5-methylfuran-2-yl)but- 3-enoic acid (I- 1a):

(Ma) To a vigorously stirred solution of 5-methyl-2-furaldehyde (264 ml_, 2650 mmol) and diethyl succinate (840 ml_, 5050 mmol) in ethanol (1.820 L) at room temperature was added sodium ethoxide (0.93 L of a 21 weight % solution in ethanol) in one portion. The reaction mixture was then heated at reflux for 13 hours. After cooling to room temperature, the mixture was concentrated in vacuo (all batches were combined at this point). The resulting residue was partitioned between ethyl acetate (1 L) and hydrochloric acid (1 L of a 2M aqueous solution). After separation, the aqueous layer was extracted with ethyl acetate (2 x 1 L). The combined organic extracts were then extracted with sodium hydrogen carbonate (2 x 1 L of a saturated aqueous solution). These aqueous extracts were combined and adjusted to pH 2 with hydrochloric acid (2M aqueous solution) then extracted with ethyl acetate (2 x 1 L). These organic extracts were combined and concentrated in vacuo to give desired (E)-3-(ethoxycarbonyl)-4-(5-methylfuran-2-yl)but-3-enoic acid (J₁ Ia: 34.34 g, 5%). The original organic extract was extracted with sodium hydroxide (2 L of a 2M aqueous solution). This aqueous extract was adjusted to pH 2 with hydrochloric acid (2M aqueous solution) then extracted with ethyl acetate (2 x 1 L). These organic extracts were combined and concentrated in vacuo to give additional desired materials (395.2 gram, 63%) as red liquid. ¹H NMR (CDCI₃, 300 MHz) δ ppm 7.48 (s, 1 H), 6.57 (d, 1 H), 6.09 (d, 1 H), 4.24 (q, 2H), 3.87 (s, 2H), 2.32 (s, 3H), 1.31 (t, 3H).

Preparation of Intermediate ethyl 4-acetoxy-2-methylbenzofuran-6- carboxylate (1-1 b):

(M b) To a vigorously stirred solution of (E)-3-(ethoxycarbonyl)-4-(5- methylfuran-2-yl)but-3-enoic acid (1-1 a: 326.6 g, 1 .371 mol) in acetic anhydride (1 .77 L, 18.72 mol) at room temperature was added sodium acetate (193 g, 2350 mmol) in one portion. The reaction mixture was then heated at reflux for 2.5 hours. After cooling to room temperature, the mixture was concentrated in vacuo (all batches were combined at this point). The resulting residue was suspended in dichloromethane (1 .5 L) and filtered, washing the solids with dichloromethane (3 x 500 ml_). The combined filtrate and washings were then washed with sodium hydrogencarbonate (2 x 1 L of a saturated aqueous solution) and brine (2 L), then concentrated in vacuo to give desired ethyl 4-acetoxy-2-methylbenzofuran-6-carboxylate (H b: 549.03 g, quantitative). ¹H NMR (CDCI₃, 300 MHz) δ ppm 8.00-7.99 (m, 1 H), 7.64 (d, 1 H), 6.32-6.32 (m, 1 H), 4.38 (q, 2H), 2.47 (d, 3H), 2.37 (s, 3H), 1 .39 (t, 3H).

Preparation of Intermediate ethyl 4-hydroxy-2-methylbenzofuran-6- carboxylate (1- 1 c):

(He) To a stirred solution of ethyl 4-acetoxy-2-methylbenzofuran-6- carboxylate (Hb: 549.03 g, 1 .37 mol) in ethanol (4.00 L) at room temperature was added potassium carbonate (266 g, 1 .92 mol) in one portion. The reaction mixture was then heated at 60⁰C for 3 hours. Potassium carbonate (100 g, 0.720 mol) was then added in one portion and the reaction mixture was heated at 60⁰C for a further 3 hours. After cooling to room temperature the mixture was diluted with dichloromethane (2 L) and the suspension filtered, washing the solids with dichloromethane (2 x 1 L) (all batches were combined at this point). The combined filtrate and washings were then washed with citric acid (2.5 L of a 1 M aqueous solution), then concentrated in vacuo and the resulting residue purified by dry flash chromatography (hexane then 2:1 hexane:ethyl acetate). All fractions containing the desired product were combined and concentrated in vacuo. The resulting residue, which solidified on standing, was slurried with cold toluene and filtered. The solids were then stirred with hot toluene and decolourising charcoal for 1 hour, followed by filtration of the hot mixture through a pad of celite. The filtrate was allowed to cool and the resulting precipitate isolated by filtration to give desired ethyl 4-hydroxy-2- methylbenzofuran-6-carboxylate (1-1 c: 360 g, 90%) as orange powder.

¹H NMR (CDCI₃, 300 MHz) δ ppm 7.73-7.73 (m, 1 H), 7.45 (d, 1 H), 6.51 -6.50 (m, 1 H), 5.85 (s, 1 H), 4.39 (q, 2H), 2.48 (d, 3H), 1.40 (t, 3H). LCMS (liquid chromatography mass spectrometry): m/z 221.06 (96.39 % purity).

Preparation of SM-25-bromo-N,N-dimethylpyrimidine-2-carboxamide (SM-

£1:

(SM-2) Oxalyl chloride (47.4g, 369mmol) was added to a suspension of 5-

Bromo-pyrimidine-2-carboxylic acid (5Og, 250mmol) in dichloromethane (821 ml) at room temperature followed by 1 -2 drop of dimethylformamide. The reaction mixture was stirred under nitrogen for 2 hours LCMS in methanol indicated the presence of the methyl ester and some acid. Dimethylformamide (0.2ml) was added to the reaction mixture. The acid dissolved after 30 minutess. LCMS showed corresponding methyl ester and no starting material peak was observed. The solvent was removed and dried in vacuo to afford the crude 5-Bromo-pyrimidine-2-carbonyl chloride (55g, 100%). The 5-Bromo-pyrimidine-2-carbonyl chloride (55g, 250mmol) was dissolved in tetrahydrofuran (828ml) and dimethyl-amine (2M solution in tetrahydrofuran) (373ml, 745mmol) was added portionwise at room temperature. The reaction was stirred at room temperature under nitrogen for 16 hours, after which time, LCMS indicated completion. The mixture was diluted with ethyl acetate (500ml) and washed with H₂O (500ml). The water layer was further extracted with CH₂CI₂ (5x500ml), all organics combined, and dried over magnesium sulfate. The filtrate was concentrated in vacuo and then suspended in methyl-/-butylether (650ml). The solution was then heated to reflux. The hot solution was allowed to cool overnight to afford pink crystals. The crystals were filtered and washed with cold methyl-t-butylether (100ml) the solid was dried in a vacuum oven at 55⁰C for 12 hourrs to afford the title compound 5-bromo-N,N-dimethylpyhmidine-2-carboxamide (SM-2: 44g, 77%) as a pink solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.94 (s, 3 H) 3.13 (s, 3 H) 8.85 (s, 2 H) m/z (M+1 ) = 232.

Preparation of Intermediate Ethyl 4-(2-(dimethylcarbamoyl)Dyrimidin-5- yloxy)-2-methylbenzofuran-6-carboxylate (l-2a):

A mixture of Cs₂CO₃ (62.1 g, 191 mmol), 5-bromo-N,N- dimethylpyrimidine-2-carboxamide (SM-2: 24g, 104mmol) and ethyl 4- hydroxy-2-methylbenzofuran-6-carboxylate (1-1 c: 2Og, 91 mmol); 1 ,10- phenanthroline (1.64g, 9.07mmol) and copper iodide (864mg, 4.54mmol) in dimethylformamide (200ml) was purged with N₂ gas and then heated to 90°C using a mechanical stirrer. The heterogeneous reaction mixture was stirred at this temperature for 18 hours. HPLC indicated near completion. The reaction mixture was cooled to 35⁰C and diluted with ethyl acetate (300ml). The mixture was filtered to remove any cesium carbonate. The filtrate was then partitioned between water (500ml) and ethyl acetate (500ml); however, no separation was observed. Concentrated HCL (20ml) was added to the mixture. When the aqueous phase was about pH1 , the phases separated. The organics were separated and the aqueous layer reextracted with ethyl acetate (2x500ml). All organics were combined and back extracted with water (200ml) and brine (500ml). The organics were separated and treated with activated charcoal (10g) and magnesium sulfate. The mixture was allowed to stir for 10 minutes and then filtered through a plug of celite to afford a crude yellow solution. The filter cake was washed with ethyl acetate (100 ml_). The organics were concentrated in vacuo to afford a crude solid this was dried under high vacuum for 4 days. The dry crude solid was triturated using methanol (80 ml_). The solids were dispersed into a fine light orange crystalline powder with a red liquor. The solids were isolated by filtration and rinsed with methanol (20 ml_). The solid was dried in the vacuum oven at 55⁰C for 12 hours to afford ethyl 4-(2- (dimethylcarbamoyl)pyrimidin-5-yloxy)-2-methylbenzofuran-6-carboxylate (J₁ 2a) as a yellow solid (18.2g, 54%)

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.41 (t, J=7.12 Hz, 3 H) 2.50 (d, J=0.98 Hz, 3 H) 3.00 (s, 3 H) 3.17 (s, 3 H) 4.41 (d, J=7.22 Hz, 2 H) 6.29 (s, 1 H) 7.62 (d, J=1.17 Hz, 1 H) 8.06 (s, 1 H) 8.50 (s, 2 H). m/z (M+1 ) = 370.5

Preparation of Starting material 5-bromo-N-ethyl-N-methylpyrimidine-2- carboxamide (SM-3):

(SM-3) Oxalyl chloride (1 .45g, 1 1 .1 mmol) was added to a suspension of 5-

Bromo-pyrimidine-2-carboxylic acid (1 .5g, 7.4mmol) in dichloromethane (50ml) at room temperature followed by 1 -2 drop of dimethylformamide. The reaction mixture was stirred under nitrogen for 2 hours LCMS in methanol indicated the presence of the methyl ester and some acid. Dimethylformamide (0.2ml) was added to the reaction mixture and all of the acid dissolved after 30 minutes. LCMS showed corresponding methyl ester and no starting material peak was observed. The solvent was removed and dried in vacuo to afford the crude 5-Bromo-pyrimidine-2-carbonyl chloride (1 -6g). 5-Bromo-pyrinnidine-2-carbonyl chloride (1600mg, 7.225mnnol) was dissolved in dichloromethane (25ml) and triethylamine (4.03ml, 28.9mmol) was added followed by ethyl-methyl-amine (0.68 mL, 7.92 mmol). The reaction was stirred at room temperature under nitrogen for 16 ours, after which time, LCMS indicated completion. The mixture was diluted with dichloromethane (50ml) and washed with water (50ml) followed by 10% citric acid (50ml) and brine (50ml). The organic layer was separated and dried over MgSO4, the residue was filtered and the solvent was removed in vacuo to afford the title compound 5-bromo-N-ethyl-N-methylpyrimidine-2- carboxamide (SM-3): (1.4g, 79.4%) as a brown oil.

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.08 – 1.31 (m, 3 H) 2.99 (d, J=79.05 Hz, 3 H) 3.19 (q, J=7.22 Hz, 1 H) 3.59 (q, J=7.22 Hz, 1 H) 8.84 (d, J=3.12 Hz, 2 H)

Example 2

Preparation of N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2- yl)carbamoyl)-benzofuran-4-yloxy)Dyrimidine-2-carboxamide (2):

(2)

To a solution of the 5-methyl-2-aminopyrazine (38.9 g, 356 mmol) in dimethylether (315 ml_) in a 3-neck flask equipped with overhead stirring and a condensor at O⁰C was added Me2AICI (1 M solution in hexanes) (715 ml_). The mixture was warmed at room temperature and stirred for 1.5 hours. In a separate flask, ethyl 4-(2-(dimethylcarbamoyl)pyrimidin-5-yloxy)-2- methylbenzofuran-6-carboxylate (l-2a: 52.6g, 142.5mmol) was dissolved in dimethylether (210 ml_). This mixture was then added to the complexed amine. A gum precipitated upon scratching the flask and dissipated into a solid. The resultant reaction was refluxed for 3.5 hours HPLC indicated 93% complete. Five liters of Rochelles salt made up in water and 2 liters of 2- methyltetrahydrofuran was added to the mixture. The reaction mixture was then poured into the biphasic system. The mixture was allowed to stir with overhead stirring for 14 hours, after which time, a yellow solid precipitated. The solid was collected through filteration. The solid retained was washed with 2-methyltetrahydrofuran. The resultant solid was dried in vacuo oven overnight to afford the title compound N,N-dimethyl-5-(2-methyl-6-((5- methylpyrazin-2-yl)carbamoyl)benzofuran-4-yloxy)pyhmidine-2-carboxamide (2): (49.98g, 81 %)

1H NMR (400 MHz, CHLOROFORM-d) d ppm 2.49 (d, J=1 .17 Hz, 3H) 2.55 (s, 3H) 2.98 (s, 3 H) 3.14 (s, 3 H) 6.28 (t, J=0.98 Hz, 1 H) 7.52 (d, J=1 .37 Hz, 1 H) 7.88 – 7.92 (m, 1 H) 8.14 (d, J=0.78 Hz, 1 H) 8.37 (s, 1 H) 8.50 (s, 2 H) 9.54 (d, J=1 .56 Hz, 1 H).

m/z (M+1 ) = 433.4, m/z (M-1 )= 431 .5

REFERENCES

Beebe, D.A.; Ross, T.T.; Rolph, T.P.; Pfefferkorn, J.A.; Esler, W.P.
The glucokinase activator PF-04937319 improves glycemic control in combination with exercise without causing hypoglycemia in diabetic rats
74th Annu Meet Sci Sess Am Diabetes Assoc (ADA) (June 13-17, San Francisco) 2014, Abst 1113-P

Amin, N.B.; Aggarwal, N.; Pall, D.; Paragh, G.; Denney, W.S.; Le, V.; Riggs, M.; Calle, R.A.
Two dose-ranging studies with PF-04937319, a systemic partial activator of glucokinase, as add-on therapy to metformin in adults with type 2 diabetes
Diabetes Obes Metab 2015, 17(8): 751

Study to compare single dose of three modified release formulations of PF-04937319 with immediate release material-sparing-tablet (IR MST) formulation previously studied in adults with type 2 diabetes mellitus (NCT02206607)

OTHERS

///////////Pfizer , PF 04937319,  glucokinase activators,  Type 2 diabetes

Lefucoxib (乐福昔布)

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

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

CAS 849048-84-6

IND FILED

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

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

cyclooxygenase-2 (COX-2) inhibitor

National Center of Biomedical Analysis

冉允章, 梅世昌

梅世昌, 冉允章

CHINA FLAG

PATENT

CN 1468854

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

Example 1

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

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

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

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

References

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

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

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

Talanta (2011), 85(1), 8-27

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

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

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

China PLA General Hospital

.

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

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

PNQ 370

Advinus Therapeutics Ltd

Adenosine A2a receptor antagonist

for treating disease or disorder susceptible to improvement by antagonism of A_2A receptor.

ONE OF THE ABOVE OR SIMILAR

INTRODUCTION

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=9B4D4A1C3A9C0C5ACBBBA119D16D32E2.wapp2nC?docId=WO2012038980&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

centre: Mr Ratan Tata, Chairman, Tata Sons, flanked by Dr Rashmi Barbhaiya (left), Managing Director and CEO, Advinus, and Mr R. Gopalakrishnan, …

ONE EXAMPLE………..

A1

5-amino-8-(furan-2-yl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin-1-yl]ethyl]-1-methyl-[1,2,4]triazolo[5,1-f]purin-2-one

WO2012038980

Example Al :

5-amino-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin- 1 -yl]ethyl]- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -f]purin-2-one

5-Amino-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin-l-

5-amino-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin- 1 -yl]ethyl]- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -f]purin-2-one

Step-1 : 2-[(2,5-Diamino-6-chloro-pyrimidin-4-yI)amino]ethanol

A mixture of 4,6-dichloropyrimidine-2,5-diamine (28g, 156mmol), ethanolamine (18ml, 312mmol) and ethanol (250ml) were heated at 100-1 10 °C for 16 hours. The mixture was cooled and solvent was removed. To the residue methanol (100ml) was added and stirred for 20 minutes. The solid was filtered off to obtain 2-[(2,5-diamino-6-chloro-pyrimidin-4-yl)amino]ethanol (22.0g, 70%).

‘H MR(400MHz, DMSO d6): δ 3.36-3.40 (m, 2H); 3.50-3.54 (m, 2H); 3.88 (bs, 2H); 4.74 (t, J=5.6Hz, 1H); 5.63 (bs, 2H); 6.51 (t, J=5.6Hz, 1H)

Step-2: 2-Amino-6-chloro-9-(2-hydroxyethyl)-7H-purin-8-one

A mixture of 2-[(2,5-diamino-6-chloro-pyrimidin-4-yl)amino]ethanol obtained in step 1 (l O.Og, 49.26mmol) in acetonitrile (400ml) were cooled to 0 °C. To this reaction mixture K₂C0₃ (20.39gm, 147.7mmol) and 4-nitrophenyl chloroformate (19.8g, 98.52mmol)was added and stirred at 25-27 °C for 24 hours. This reaction mixture was filtered and washed with acetonitrile (300ml) and diethyl ether (300ml) respectively. Solid obtained was dried to obtain crude 2-amino-6-chloro-9-(2-hydroxyethyl)-7H-purin-8-one as a yellow solid. Small amount of crude material was purified by column chromatography to obtain pure product. ‘HNMR(400MHz, DMSO d6): δ 3.61-3.66 (m, 2H); 3.72-3.75 (m, 2H); 4.85 (t, J=6Hz, 1H); 6.60 (s, 2H); 1 1.21 (s, 1 H)

Step-3: 2-Amino-6-chloro-9-(2-hydroxyethyl)-7-methyl-purin-8-one

A mixture of 2-amino-6-chloro-9-(2-hydroxyethyl)-7H-purin-8-one obtained in step 2 (13g, 56.7mmol) , K₂C0₃ (1 1.5g, 84mmol), methyl iodide (12g, 85.15mmol) and DMF (130ml) were stirred at 25-30 °C for 16 hours. The reaction mixture was concentrated and purified by column chromatography using 60-120 silica gel and 4% methanol in DCM as an eluent to obtain 2-amino-6-chloro-9-(2-hydroxyethyl)-7-methyl-purin-8-one (8g, 58%) as an off white solid.

‘HNMR(400MHz, DMSO d6): δ 3.42 (s, 3H); 3.65 (t, J=5.6Hz, 2H); 3.78 (t, J=5.6Hz, 2H); 4.85 (t, J=5.6Hz, 1H); 6.69 (bs, 2H).

Step-4: 2-Amino-6-hydrazino-9-(2-hydroxyethyl)-7-methyI-purin-8-one

A mixture of 2-amino-6-chloro-9-(2-hydroxyethyl)-7-methyl-purin-8-one obtained in step 3 (8g, 32.9mmol) , Hydrazine hydrate (16ml ,32.9mmol) and ethanol (300ml) were heated at 100-1 10 °C for 16 hours. The reaction mixture was concentrated and solid obtained was filtered off and dried to obtain 2-amino-6-hydrazino-9-(2-hydroxyethyl)-7-methyl-purin-8-one (7g, 89 %) as an off white solid.

‘HNMR(400MHz, DMSO d6): δ 3.37 (s, 3H); 3.58-3.61 (m, 2H); 3.71 (t, J=6Hz, 2H); 4.29 (bs, 2H); 4.87 (t, J=5.6Hz, 1H), 6.00 (bs, 2H); 7.63 (s, 1H).

Step-5: N’-[2-Amino-9-(2-hydroxyethyl)-7-methyl-8-oxo-purin-6-yl]furan-2-carbohydrazide

2-amino-6-hydrazino-9-(2-hydroxyethyl)-7-methyl-purin-8-one (4.5g, 18.18mmol) obtained in step 4, 2-furoic acid (2.53g, 22.5mmol), HOBT (2.53g, 18.8 mmol) and N-methylmorpholine were taken in dimethylformamide (40ml). l-Ethyl-3(3′-dimethylaminopropryl)carbodiimide hydrochloride (EDCI.HCl) (5.4g, 28.2mmol) was added to the reaction mixture and stirred at 25-27 °C for 14 hours. The reaction mixture was evaporated and residue was purified by column chromatography to obtain N’-[2-amino-9-(2-hydroxyethyl)-7-methyl-8-oxo-purin-6-yl]furan-2-carbohydrazide (5.3g, 84%) as an off white solid.

‘HNMR (400MHZ, DMSO d6): δ 3.43 (s, 3H); 3.59-3.63 (m, 2H); 3.74 (t, J=6Hz, 2H); 4.88 (t, J=5.6Hz, 1H); 5.98 (bs, 2H); 6.67 (bs, 1H); 7.25 (d, J=3.2Hz, 1H); 7.90 (s, 1H); 8.35 (s, 1H); 10.28 (s, lH).

Step-6: 5-Amino-8-(2-furyl)-3-(2-hydroxyethyl)-l-methyl-[l^,4]triazolo[5,l-flpurin-2-one

A mixture of N’-[2-amino-9-(2-hydroxyethyl)-7-methyl-8-oxo-purin-6-yl]furan-2-carbohydrazide obtained in step 5 (5.3g, 15.9mmol), Ν,Ο-bistrimethylsilylacetamide (27ml, 1 1 1.4mmol) and hexamethyldisilazane (83ml, 397mmol) were heated at 1 10-120 °C for 16 hours. The reaction mixture was quenched with methanol (100ml) and water (100ml) and organic volatiles were evaporated. The solid obtained was filtered off and washed with water (30ml) followed by diethyl ether (100ml) to obtain 5-amino-8-(2-furyl)-3-(2-hydroxyethyl)-l-methyl-[l,2,4]triazolo[5,l-f]purin-2-one (3.50g, 71%) as an off white solid.

‘HNMR (400MHZ, DMSO d6): δ 3.56 (s, 3H); 3.67-3.70 (m, 2H); 3.84-3.87 (m, 2H); 4.88 (t, J=5.6Hz, 1H); 6.73 (bs, 1H); 7.20 (bs, 1H); 7.79 (bs, 2H); 7.94 (bs, 1H).

Step-7: 2-[5-Amino-8-(2-furyl)-l-methyl-2-oxo-[l,2,4]triazolo[5,l-fJpurin-3-yl]ethyl 4-methylbenzenesulfonate

A mixture of 5-amino-8-(2-furyl)-3-(2-hydroxyethyl)-l -methyl-[l,2,4]triazolo[5, l-fJpurin-2-one obtained in step 6 (3.5g, l lmmol), p-toluene sulphonylchloride (5.2 g, 27mmol) were taken in pyridine (30ml)and stirred at 25-27 °C for 16 hours. To the reaction mixture hexane (100ml) was added and solid obtained was filtered off and washed with water (100ml) followed by hexane (100ml) to obtain 2-[5-amino-8-(2-furyl)-l-methyl-2-oxo-[l,2,4]triazolo[5, l-f]purin-3-yl]ethyl 4-methylbenzenesulfonate (4.1g, 78%) as a brown solid. ‘HNMR (400MHz, DMSO d6): δ 2.02 (s, 3H); 3.49 (s, 3H); 3.99 (t, J=4.8Hz, 2H); 4.71 (t, J=4.8Hz, 2H); 6.73-6.75 (m, 1H); 7.01 (d, J=8Hz, 2H); 7.23 (d, J=3.2Hz, 1H); 7.41 (d, J=8.4Hz, 2H); 7.78 (bs, 2H); 7.96 (d, J=1.2Hz, 1H).

Step-8: : 5-Amino-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin-l-yl]ethyl]-l-methyl-[l,2,4]triazolo[5,l-f)purin-2-one

A mixture of 2-[5-amino-8-(2-furyl)-l-methyl-2-oxo-[l ,2,4]triazolo[5, l-f]purin-3-yl]ethyl 4-methylbenzenesulfonate obtained in step 7 (0.25g, 0.533mmol), l-[4-(2-Methoxy-ethoxy)-phenyl]-piperazine (0.188g, 0.799mmol) and DIPEA (0.27ml, 1.599mmol) were taken in DMF (5ml) and stirred at 80 °C for 16 hours. To the reaction mixture water (100ml) was added and solid obtained was filtered off. The crude product was purified by column chromatography to obtain 5-amino-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin- 1 -yl]ethyl]- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -f]purin-2-one (0.135g, 47%) as an off white solid

‘HNMR (400MHz, DMSO d6): δ 2.60 (bs, 4H); 2.68 (t, J=6.4Hz, 2H); 2.96 (bs, 4H); 3.29 (s, 3H); 3.56 (s, 3H); 3.59-3.62 (m, 2H); 3.94-4.00 (m, 4H); 6.71 -6.73 (m, 1H); 6.79-6.86 (m, 4H); 7.19 (dd, J=3.2Hz, 1.2Hz, 1H); 7.80 (bs, 2H); 7.94 (bs, 1H).

ANOTHER……..

Example Gl: 5-Amino-l-ethyl-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin-l-yl]ethyl]-[l,2,4]triazolo[5,l-i]purin-2-one

Step-1 : 2-Amino-6-chloro-7-ethyl-9-(2-hydroxyethyl)purin-8-one

(Procedure is same as step-3 in example Al)

‘HNMR (400MHz, DMSO d6): δ 1.21 (t, J=7.2Hz, 3H); 3.64 (s, 2H); 3.78 (t, J=6Hz, 2H);

3.92 (q, J=7.2Hz, 2H); 4.92 (bs, I H); 6.7 (bs, 2H).

Step-2 : 2-Amino-7-ethyl-6-hydrazino-9-(2-hydroxyethyl)purin-8-one

(Procedure is same as step-4 in example Al)

‘ HNMR (400MHz, DMSO d6): δ 1.07 (t, J=6.8Hz, 3H); 3.59 (q, J=6Hz, 2H); 3.72 (t, J=6Hz,

2H); 3.91 (q, J=6.8Hz, 2H); 4.32 (bs, 2H); 4.86 (t, J=5.6Hz, IH); 5.99 (bs, 2H), 7.55 (bs, IH).

Step-3: N’-[2-Amino-7-ethyl-9-(2-hydroxyethyl)-8-oxo-purin-6-yl]furan- 2carbohydrazide (Procedure is same as step-5 in example Al)

Crude product was used in next step

Step-4: 5-Amino-l-ethyI-8-(2-furyl)-3-(2-hydroxyethyl)-[l,2,4]triazolo[5,l-flpurin-2-one

(Procedure is same as step-6 in example Al)

‘H MR (400MHZ, DMSO d6): δ 1.34 (t, J=7.2Hz, 3H); 3.67 (q, J=5.6Hz, 2H); 3.84 (t, J=5.6Hz, 2H); 4.01 (q, J=7.2Hz, 2H); 4.87 (t, J=6Hz, IH); 6.70 (bs, IH); 7.17 (d, J=2.8Hz, I H); 7.18 (bs, 2H); 7.92 (bs, IH).

Step-5: 2-[5-Amino-l-ethyl-8-(2-furyl)-2-oxo-[l,2,4]triazoIo[5,l-f|purin-3-yl]ethyl 4- methylbenzenesulfonate (procedure is same as step-7 in example Al)

^lHNMR (400MHz, DMSO d6): δ 1.35 (t, J=7.2Hz, 3H); 2.00 (s, 3H); 3.95-4.00 (m, 4H); 4.47 (bs, 2H); 6.74 (s, IH); 7.00 (d, J=7.6Hz, 2H); 7.22 (s, IH); 7.42 (d, J=7.6Hz, 2H); 7.78 (bs, 2H); 7.97 (bs, IH).

Step-6: 5-Amino-l-ethyl-8-(2-furyl)-3-[2-[4-[4-(2-methoxyethoxy)phenyi]piperazin-l- yl]ethyl]-[l,2,4]triazolo[5,l-f]purin-2-one (procedure is same as step-8 in example Al)

HNMR(400MHz, DMSO d6): δ 1.35 (t, J=7.2Hz, 3H); 2.60 (bs, 4H); 2.68 (t, J=6.8Hz, 2H); 2.95 (bs, 4H); 3.28(s, 3H);3.61 (t, J=4.4Hz, 2H); 3.94-4.04 (m, 6H); 6.72 (dd, J=2Hz, 3.6Hz, I H); 6.78-6.85 (m, 4H); 7.19 (d, J=3.2Hz, IH); 7.81(bs, 2H); 7.94 (s, IH).

Representative compounds of the present disclosure were tested and had micromolar to nanomolar activity.

A1 ABOVE

A7 ABOVE

A9 ABOVE

A13 ABOVE

A31 ‘HNMR (400MHz, DMSO d6): δ 2.62 (bs,4H); 2.68 (t, J=6.8Hz, 2H); 2.85 (bs, 4H); 3.28 (s, 3H); 3.57 (s, 3H); 3.59-3.62 (m, 2H); o 3.95 (t, J=6.8Hz, 2H); 4.01-4.04 (m, 2H);

5-Amino-3-[2-[4-[2-fluoro-4-(2- 6.66-6.68 (m, 1H); 6.72 (dd, J=2 Hz,3.6Hz, methoxyethoxy)phenyl]piperazin-l-yl]ethyl]-8- 1H); 6.79 (dd, J=2.8Hz, 14Hz, 1H); 6.92 (t, (2-furyl)- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -f|purin-2- J=9.6Hz, 1H); 7.19 (d, J=3.2Hz, 1 H); 7.93 one (bs, 2H); 7.93-7.94 (m, 1H).

A31 ABOVE

A32 HNM (400MHz, DMSO d6): δ 2.59 (bs,

4H); 2.68(t, J=6.4Hz, 2H); 3.27(t, J=4.8Hz, 4H); 3.56 (s, 3H); 3.96 (t, J=6.4Hz, 2H);

0 6.72(dd, J=2Hz, 3.6Hz, 1H); 6.99 (d, J=8.8Hz,

4-[4-[2-[5-Amino-8-(2-furyl)-l-methyl-2-oxo- 2H); 7.19 (d, J=3.6Hz, 1H);7.56 (d, J=8.8Hz, [ 1 ,2,4]triazolo[5, 1 -f]purin-3-yl]ethyl]piperazin- 2H); 7.80 (bs, 2H); 7.93 (bs, lH).

l-yl]benzonitrile

A32 ABOVE

A36 ‘HNMR(400MHz, CDCI3): δ θ.09 (d,

J=4.4Hz, 2H); 0.50 (d, J=6.8Hz, 2H); 0.82- 0.89 (m, 1H); 2.24 (d, J=6.0Hz, 2H): 2.52- 2.72 (m, 8H); 2.80 (t, J=6.4Hz, 2H); 3.76 (s,

5-Amino-3-[2-[4-(cyclopropylmethyl)piperazin- 3H); 4.07 (t, J=6.8Hz, 2H); 5.89 (bs, 2H); l -yl]ethyl]-8-(2-furyl)-l-methyl- 6.61 (bs, 1H); 7.22 (d, J=2.4Hz, 1H); 7.64 (s, [ 1 ,2,4]triazolo[5, 1 -f]purin-2-one 1H).

A36 ABOVE

A38 ‘HNMR(400MHz, CDCI₃): δ 2.62 . (t,

J=4.4Hz, 4H); 2.79 (t, J=6.4Hz, 2H); 2.81 (s, 6H); 3.22 (t, J=4.4Hz, 4H): 3.77 (s, 3H); 4.06 (t, J=6.8Hz, 2H); 5.74 (bs, 2H); 6.60 (dd,

4-[2-[5-Amino-8-(2-fiiryl)- 1 -methyl-2-oxo- J=2.0Hz, 3.2Hz, 1H); 7.24 (d, J=3.6Hz, 1H);

[ 1 ,2,4]triazolo[5, 1 -f]purin-3-yl]ethyl]-N,N- 7.65 (s, 1H).

dimethy l-piperazine- 1 -sulfonamide

A38 ABOVE

A39 ‘HNMR(400MHZ, DMSO d6): δ 1.89-1.94

im, 1H); 2.09-2.18 .(m, 1 H); 2.60 (bs, 4H); 2.67 (t, J=6.4Hz, 2H); 2.96 (bs, 4H); 3.56 (s, 3H); 3.69-3.85 (m, 4H); 3.95 (t, J=6.4Hz,

2H); 4.89 (bs, 1H); 6.72 (dd, J=2.0, 3.2Hz,

5-Amino-8-(2-furyl)-l -methyl-3-[2-[4-(4- 1H); 6.78 (d, J=9.2Hz, 2H); 6.85 (d, J=9.2Hz, tetrahydrofuran-3-yloxyphenyl)piperazin- 1 – 2H): 7.20 (d, J=3.2Hz, 1 H); 7.80 (bs, 2H); yl]ethyl]-[l ,2,4]triazolo[5,l-f]purin-2-one

7.93 (s, 1H).

A39 ABOVE

A42 ‘HNMR(400MHz, CDCI3): δ

2.26 (s,3H); 2.94-2.97 (m, 6H); 3.72 (s, 2H); 3.75 (s, 3H); 4.17 (t, J=6.4Hz, 2H); 5.74 (bs, 2H); 6.59 (dd, J=1.6Hz, 3.6Hz, 1H);7.13 (s, J=3.6Hz, IH); 7.21-7.24 (m, IH); 7.63 (s,

5-Amino-8-(2-furyl)-l-methyl-3-[2-(3-methyl- IH); 8.20 (bs, IH),

7,8-dihydro-5H- 1 ,6-naphthyridin-6-yl)ethyl]- [ 1 ,2,4]triazolo[5, 1 -f]purin-2-one

A42 ABOVE

A57 HNMR(400MHz, DMSO d6): δ 2.95 (t,

J=8Hz, 2H); 3.52 (s, 3H); 3.69 (s, 3H ), 3.97 (t, J=8Hz, 2H); 6.71 (dd, J=2Hz, 3.6Hz, I H );

5-Amino-8-(2-furyl)-3-[2-(4- 6.80 (dd, J=2Hz, 6.8Hz, 2H); 7.10 (d, methoxyphenyl)ethyl]- 1 -methyl- J=8.8Hz, 2H); 7.18 (dd, J=0.8Hz, 3.2Hz, I H );

[ 1 ,2,4]triazolo[5, 1 -f]purin-2-one 7.80 (bs, 2H), 7.94 (dd, J=lHz, 2Hz, I H ).

A57 ABOVE

A58 HNMR(400MHz, DMSO d6): δ 2.61 (bs,

4H); 2.68 (bs, 2H); 3.05(bs, 4H); 3.57 (s, 3H ), 3.96 (bs, 2H); 6.72 (bs, IH); 6.92 (d, J=8Hz, 2H); 7.01 (d, J=10Hz, 2H );7.03(d, J=148Hz, IH); 7.19 (bs , 1 H); 7.80 (bs, 2H); 7.94 (s,

5-amino-3-[2-[4-[4- IH).

(difluoromethoxy)phenyl]piperazin-l-yl]ethyl]- 8-(2-furyl)- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -fjpurin- 2-one

A58 ABOVE

A62 O ‘HNMR (400MHz, DMSO d6): δ 0.66-0.70

(m, 4H); 1.90-1.94 (m, lH); 2.41 (bs, 4H); 2.65 (t, J=6Hz, 2H); 3.38 (bs, 2H); 3.56 (bs, 5H); 3.93 (t, J=6.4 Hz, 2H); 6.71 (bs, 1H );

5-Amino-3-[2-[4- 7.19 (d, J=2.4Hz, 1H); 7.79 (bs, 2H); 7.93 (bs,

(cyclopropanecarbonyl)piperazin- 1 -yl]ethyl]-8- 1H).

(2-furyl)- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -fjpurin-2- one

A62 ABOVE

A63 ‘HNMR (400MHz, DMSO d6): δ 0.07-0.10

(m, 2H); 0.40-0.44 (m, 2H); 0.88-0.94 (m,lH); 2.21 (d, J=6.4Hz, 2H); 2.41-2.45 (m, 4H); 2.64 (t, J=6.4Hz, 2H); 3.38 (bs,4H); 3.56

5-Amino-3-[2-[4-(2- (s, 3H); 3.93 (t, J=6.4Hz, 2H); 6.72 (dd, cyclopropylacetyl)piperazin-l -yl]ethyl]-8-(2- J=2Hz,3.6 Hz, 1H); 7.19-7.20 (m, 1H); 7.80 fury 1)- 1 -methyl-[ 1 ,2,4]triazolo[5, 1 -fJpurin-2- (bs, 2H); 7.93 (d, J=0.8 Hz, 1H).

one

A63 ABOVE

C1 ABOVE

E1 ABOVE

D3 ABOVE

G1 ABOVE

ETC AS IN TABLE……………..

Dr Nimish Vachharajani, Senior VP & Head (Pharmaceuticals & Agrochemical Development),

/////////

n21c(nc4c(c1nc(n2)c3occc3)N(C(N4CCN5CCN(CC5)c6ccc(cc6)OCCOC)=O)C)N

CN1C2=C(N=C(N3C2=NC(=N3)C4=CC=CO4)N)N(C1=O)CCN5CCN(CC5)C6=CC=C(C=C6)OCCOC

PNQ 201

STRUCTURE COMING……

Adenosine A2b receptor antagonist

Advinus Therapeutics Ltd

KEEP WATCHING THIS POST……………

PNQ-201 is a proprietary orally active A2B Adenosine receptor (A2BAdoR) antagonist, currently in pre-clinical development for potential treatment of IBD. Advinus is looking for partnering/co-development opportunities.