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MAVORIXAFOR

April 19, 2023 3:14 am / Leave a comment

MAVORIXAFOR

AMD 070

N1-(1H-BENZIMIDAZOL-2-YLMETHYL)-N1-((S)-5,6,7,8-TETRAHYDROQUINOLIN-8-YL)-BUTANE-1,4-DIAMINE

fda approved 4/26/2024, To treat WHIM syndrome (warts, hypogammaglobulinemia, infections and myelokathexis), Xolremdi

Mavorixafor (AMD-070) is a potent, selective and orally available CXCR4 antagonist, with an IC₅₀ value of 13 nM against CXCR4 ¹²⁵I-SDF binding, and also inhibits the replication of T-tropic HIV-1 (NL4.3 strain) in MT-4 cells and PBMCs with an IC₅₀ of 1 and 9 nM, respectively.

Molecular Weight	349.47
Appearance	Solid
Formula	C₂₁H₂₇N₅
CAS No.	558447-26-0
SMILES	NCCCCN(CC1=NC2=C(N1)C=CC=C2)[C@@H]3C4=C(CCC3)C=CC=N4

PHASE 2

Mavorixafor trihydrochloride

Molecular Weight	458.86
Appearance	Solid
Formula	C₂₁H₃₀Cl₃N₅
CAS No.	2309699-17-8
SMILES	[H]Cl.[H]Cl.[H]Cl.NCCCCN(CC1=NC2=CC=CC=C2N1)[C@@H]3C4=NC=CC=C4CCC3

AMD-070 is a small molecule drug candidate that belongs to a new investigational class of anti-HIV drugs known as entry (fusion) inhibitors. Approximately 76% of HIV-patients with measurable viral load are infected with a strain of virus that is resistant to one or more classes of antiretroviral agents, thus reducing treatment options. Unlike many existing HIV drugs that target the virus after it has infected a healthy cell, AMD-070 blocks the virus from entering a healthy cell, thus preventing the replication process. AMD-070 targets the CXCR4 receptor on HIV and prevents the virus from entering and infecting healthy cells. AMD-070 is specific for the CXCR4 receptor and does not interact with any other chemokine receptors in vitro. AMD-070 strongly inhibits viral infection by all CXCR4 using virus (including virus using CXCR4 alone and/or virus using CXCR4 and CCR5) in vitro. AMD-070 is orally bioavailable in animals, it has suitable PK and toxicity profile for oral dosing. AMD-070 shows additive or synergistic effects in vitro in combination with other known anti-HIV agents. AMD-070 is active against CXCR4 using HIV strains that are resistant to existing antiretroviral therapies in vitro, reveals potent anti-HIV activity against CXCR4-using laboratory strains and clinical isolates. MD-070 had been in phase II clinical trials by Genzyme for the treatment of HIV infection. However, this research has been discontinued. AMD-070 has been studied in Phase I/II clinical trials for the treatment of Renal cell carcinoma and Phase I clinical trials for the treatment of malignant melanoma and solid tumours.

PAPER

https://pubs.acs.org/doi/10.1021/acs.oprd.2c00076

Org. Process Res. Dev. 2022, 26, 6, 1831–1836

A novel and practical synthesis of mavorixafor (1) is reported. The novelty of this synthetic route is the use of 8-chloro-5,6,7,8-tetrahydroquinoline (9) and 1,4-diaminobutane as the materials, instead of 8-amino-5,6,7,8-tetrahydroquinoline (4) and N,N-diprotected aminobutyraldehyde (6a or 6b). The preparation of (S)-8-(4-aminobutylamino)-5,6,7,8-tetrahydroquinoline (13) by resolution with N-acetyl-l-leucine was first achieved. Then the one-pot synthesis of 1 from 13 involving protection, condensation, and subsequent hydrolysis was successfully developed. In addition, the final product with a satisfactory purity (>99.5%, detected by both achiral and chiral HPLC) was obtained by a simple operation (salification) without column chromatographic purification.

NEW PAT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2023059903&_gid=202315

Scheme I

Mavorixafor

EXEMPLIFICATION

Example 1: Synthesis of Sulfonate adduct F-2d:

Scheme V:

1) AcOH, NaCI, water 1) Na ₂ S ₂ O ₅ , THF, water

2) n-Heptane, THF 2) THF/n-heptane, acetonitrile

Step 1C Step 1 D

Step 1A: Preparation of Dl

Charge diethyl-4-aminobutyl acetal (E) (1.00 wt, 1.00 eq) to vessel A. Charge acetonitrile (10.0 vol, 7.8 wt) and adjust temperature to 20°C. Heat the mixture to 40°C. Concentrate the reaction mixture to 6.0 vol under reduced pressure at 35 to 45°C.

[0098] Acetonitrile filler (5.0 vol, 3.9wt) at 35 to 45°C. Concentrate the reaction mixture to 6.0 vol under reduced pressure 35 to 45°C. This step is repeated once as described below.

[0099] Acetonitrile filler (5.0 vol, 3.9wt) at 35 to 45°C. Concentrate the reaction mixture to 6.0 vol under reduced pressure at 35 to 45°C. Cool to 20°C.

[00100] Charge di-tert-butyl dicarbonate (1.1 eq, 1.5 wt) to a drum, followed by acetonitrile (0.4 vol, 0.3 wt) and agitate until fully dissolved. Concentrate the reaction mixture to 6.0 vol under reduced pressure at 35 to 45°C.

[00101] Charge this di-tert-butyl dicarbonate solution in acetonitrile to vessel A maintaining 20°C. Charge acetonitrile (1.5 vol, 1.1 wt) to the solution as a line rinse and stir at 20°C for 30 to 60 min..

[00102] Charge 4-dimethylaminopyridine (0.076 wt, 0.10 eq) to the vessel A at 20°C. Heat the solution to 40°C. Concentrate the reaction mixture to 5.0 vol under reduced pressure. Charge acetonitrile (5.0 vol, 3.9 wt) to the solution. Concentrate the reaction mixture to 5.0 vol under reduced pressure.

[00103] Take the resulting solution of Dl into next reaction without isolation.

Step IB: Preparation of Cl

[00104] Charge acetonitrile (2.0 vol, 1.6 wt) at 35 to 45°C to vessel A containing solution of D-1 from Step 1A.

[00105] Charge di-tert-butyl dicarbonate (1.4 eq, 1.9 wt) to a drum, followed by acetonitrile (10.0 vol, 7.8 wt) and agitate until fully dissolved. Charge this di-tert-butyl dicarbonate solution to vessel A, 2 to 6 h while distilling under vacuum at 35 to 45°C maintaining the volume of the reaction at 7.0 vol. Load acetonitrile (3.0 vol, 2.4 wt) over 20 to 40 min. as a line rinse while distilling under vacuum at 35 to 45°C, maintaining the volume of the reaction at 7.0 vol.

[00106] Charge di-tert-butyl dicarbonate, (0.14 eq, 0.19 wt) to a drum, followed by acetonitrile (1.0 vol, 0.74 wt) and agitate until fully dissolved. Charge this di-tert-butyl dicarbonate solution to vessel A over 20 to 40 min.. Charge acetonitrile (0.3 vol, 0.24 wt) over 10 to 20 min as a line rinse while distilling under vacuum at 35 to 45°C, maintaining the volume of the reaction at 7.0 vol.

[00107] Concentrate the reaction mixture to 5.0 vol distilling under vacuum at 35 to 45°C.

[00108] Charge n-heptane, (7.5 vol, 5.1 wt) to the reaction mixture, and concentrate the reaction mixture to 5.0 vol under reduced pressure at 40°C. This step is repeated once as described below.

[00109] Charge n-heptane, (7.5 vol, 5.1 wt) to the reaction mixture, and concentrate the reaction mixture to 5.0 vol under reduced pressure at 40°C.

[00110] Charge decolorizing, activated charcoal (0.2 wt) to the solution and stir for 1 to 2 h at 40°C. Filter the reaction mixture at 40°C. Charge n-heptane, (2.0 vol, 1.4 wt) to the reactor vessel and stir for 5 to 15 min. at 20°C before charging to the filter as a line rinse. Combine the filtrate and wash, and as required adjust to 20°C.

[00111] Take the resulting solution of Cl into next reaction without isolation.

Step 1C: Preparation of Bl

[00112] Charge 15% v/v acetic acid (2.0 vol) caution gas evolution, to vessel A containing solution of Cl from Step IB, maintaining the temperature at 20°C and stir for 10 min. at 20°C. Allow the phases to separate for 15 min. at 20°C. Discharge the aqueous phase to waste, retaining the organic phase in vessel A. This step is repeated once as described below.

[00113] Charge 15% v/v acetic acid (2.0 vol) maintaining 20°C and stir for 10 min. at 20°C. Allow the phases to separate for 15 min. at 20°C. Discharge the aqueous phase to waste, retaining the organic phase in vessel A.

[00114] Adjust the reaction to 30°C. Charge 4% w/w sodium chloride solution (2.1 vol) to the vessel maintaining the temperature at 30°C. Charge glacial acetic acid (4.1 vol, 4.3 wt) to the vessel maintaining 30°C. Stir the reaction mixture for 2 h maintaining the temperature at 30°C.

[00115] Charge purified water, (6.0 vol) at 30°C. Stir the contents for 5 to 10 min. at 30°C, and separate the phases, retaining the upper organic phase in vessel A. Charge the lower aqueous phase to vessel B.

[00116] Charge purified water (4.0 vol) at 30°C and stir for 5 to 10 min. maintaining the temperature at 30°C. Separate the phases at 30°C, retaining the upper organic phase in vessel A. Charge the lower aqueous phase to vessel B.

[00117] Adjust the temperature to 30°C of vessel B containing combined aqueous phases. Charge n-heptane, (2.0 vol, 1.4 wt) to vessel B and stir for 5 to 10 min. maintaining the temperature at 30°C. Separate the phases at 30°C, over 15 min.. Charge the upper organic phase to vessel A and recharge the lower aqueous phase to vessel B. This step is repeated two additional times as described below.

[00118] Charge n-heptane, (2.0 vol, 1.4 wt) to vessel B and stir for 5 to 10 min. maintaining the temperature at 30°C. Separate the phases at 30°C, over 15 min.. Charge the upper organic phase to vessel A and recharge the lower aqueous phase to vessel B.

[00119] Charge n-heptane, (2.0 vol, 1.4 wt) to vessel B and stir for 5 to 10 min. maintaining the temperature at 30°C. Separate the phases at 30°C, over 15 min., discharge the lower aqueous phase to waste and charge the upper organic layer to vessel A.

[00120] Concentrate the combined organic phases in vessel A to 3.0 vol at 10 to 20°C under reduced pressure. Offload the solution to new HDPE drum(s) and line rinse with n-heptane (0.5

vol, 0.4 wt) at 20°C. Homogenize the drum and store as “Bl solution in n-heptane,” and take into next reaction without isolation.

Step ID: Preparation of F-2d

[00121] Calculate a new 1.00 wt based on the above assay.

[00122] Charge “Bl solution in n-heptane” from Step 1C (1.00 wt, 1.00 eq, corrected for w/w assay, ca. 3.0 vol), into an appropriate vessel. THF load (3.0 vol, 2.7 wt). Heat the reaction mixture to 40°C.

[00123] Charge purified water, (0.02 vol, 0.02 wt) followed by sodium metabisulphite, (0.125 eq, 0.08 wt) as a solid via the charge hole at 40°C. Stir the resulting mixture for 30 to 35 min. at 40°C. This step was repeated four additional times to add the reagent in five portions total, as detailed below.

[00124] Charge purified water, (0.02 vol, 0.02 wt) followed by sodium metabisulphite, (0.125 eq, 0.08 wt) as a solid via the charge hole at 40°C. Stir the resulting mixture for 30 to 35 min. at 40°C.

[00125] Charge purified water, (0.02 vol, 0.02 wt) followed by sodium metabisulphite, (0.125 eq, 0.08 wt) as a solid via the charge hole at 40°C. Stir the resulting mixture for 30 to 35 min. at 40°C.

[00126] Charge purified water, (0.02 vol, 0.02 wt) followed by sodium metabisulphite, (0.125 eq, 0.08 wt) as a solid via the charge hole at 40°C. Stir the resulting mixture for 30 to 35 min. at 40°C.

[00127] Charge purified water, (0.02 vol, 0.02 wt) followed by sodium metabisulphite, (0.125 eq, 0.08 wt) as a solid via the charge hole at 40°C. Stir the resulting mixture for 36 hours at 40°C.

[00128] Cool the reaction mixture to 20°C over 3 to 4 h at a target constant rate. Filter the reaction mixture at 20°C on a 1-2 pm cloth.

[00129] Wash the solid with a pre-mixed mixture of THF (0.5 vol, 0.5 wt) and n-heptane (0.5 vol, 0.3 wt) maintaining the temperature at 20°C. This step was repeated an additional three times, as detailed below.

[00130] Wash the solid with n-heptane, (2.0 vol, 1.4 wt) as a line rinse and apply to the filtercake at 20°C.

[00131] Wash the solid with n-heptane, (2.0 vol, 1.4 wt) as a line rinse and apply to the filtercake at 20°C.

[00132] Wash the solid with acetonitrile, (2.0 vol, 1.6 wt) as a line rinse and apply to the filtercake at 20°C.

[00133] Dry the solid at 38°C under a flow of nitrogen for 12 h.

[00134] Determine residual solvent content. Pass criteria acetonitrile <2.0% w/w, n-heptane <2.0% w/w and tetrahydrofuran <2.0% w/w.

[00135] Yield of compound F-2d: 52-69%.

[00136] ‘H NMR (400 MHz, d ₆ -DMSO): 8 5.22 (s, 1H), 3.77 (s, 1H), 3.45 (t, 2H), 1.70 (m, 2H), 1.44 (m, 20H) ). ¹³ C NMR (400 MHz, d ₆ -DMSO): 8 152.6, 83.2, 82.0, 46.5, 29.6, 28.1, 26.0. FTIR (wavenumber, cm’ ¹ ) 3294, 1721, 1738, 1367, 1233, 1180, 1135, 1109, 1045.

Example 2: Synthesis of F-3a:

Scheme VI:

Step 2A: Preparation of Gl

[00137] Charge J, (1.00 wt, 1.00 eq) to vessel A. Charge purified water, (1.0 vol, 1.0 wt) to vessel A and as necessary adjust the temperature to 20°C. Charge concentrated hydrochloric acid, (4.0 eq, 3.0 vol, 3.6 wt) to vessel A maintaining the temperature at 20°C. Line rinse with purified water, (0.5 vol, 0.5 wt) maintaining the contents of vessel A at 15 to 25°C.

[00138] Charge chloroacetic acid, (1.3 wt, 1.5 eq) and purified water, (1.0 vol, 1.0 wt) to vessel B and as necessary, adjust the temperature to 20°C. Stir until fully dissolved, expected 10 to 20 min.

[00139] Charge the chloroacetic acid solution to vessel A maintaining the temperature of vessel A at 20°C. Line rinse vessel A with purified water, (0.5 vol, 0.5 wt) at 15 to 25°C and charge to vessel B at 20°C. Heat the reaction mixture to 80°C. Stir the reaction mixture at 80°C for 20 h.

[00140] Cool the reaction mixture to 10°C over 1.5 h. Load 47% w/w potassium phosphate solution (6.0 vol) over 60 min. targeting a constant rate maintaining 10°C. Adjust the pH of the reaction mixture by charging 47% w/w potassium phosphate solution to pH 7.0 maintaining the reaction temperature at 10°C. Expected charge is 2.0 to 3.5 vol 47% w/w potassium phosphate solution.

[00141] Stir the slurry for >30 min. maintaining 10°C and rechecking the pH, pass criterion pH 7.0. Filter the reaction mixture through 20 pm cloth at 10°C. Wash the filter-cake with purified water, (1.0 vol, 1.0 wt) at 10°C. This step is repeated additional three times as described below.

[00142] Slurry wash the filter-cake in the reactor vessel with purified water, (10.0 vol, 10.0 wt) for 45 min. to 90 min. at 10°C. Filter the mixture through 20 pm cloth at 10°C.

[00143] Slurry wash the filter-cake in the reactor vessel with purified water, (10.0 vol, 10.0 wt) for 45 min. to 90 min. at 10°C. Filter the mixture through 20 pm cloth at 10°C.

[00144] Slurry wash the filter-cake in the reactor vessel with purified water, (10.0 vol, 10.0 wt) for 45 min. to 90 min. at 10°C. Filter the mixture through 20 pm cloth at 10°C.

[00145] Wash the filter-cake with purified water, (1.0 vol, 1.0 wt) at 10°C. The filter-cake was washed with purified water additional five times as described below.

[00146] Wash the filter-cake with purified water, (1.0 vol, 1.0 wt) at 10°C.

[00147] Wash the filter-cake with acetonitrile, (2×1.3 vol, 2×1.0 wt) at 10°C.

[00148] Dry the filter-cake on the filter under vacuum and strong nitrogen flow through the filter cake at 20°C until the water content is <15.0% w/w by Karl-Fisher analysis.

[00149] Dry the filter-cake on the filter under vacuum and strong nitrogen flow through the filter cake at 30°C until the water content is <5.0% w/w by Karl-Fisher analysis.

[00150] Dry the filter-cake on the filter under vacuum and strong nitrogen flow through the filter cake at 50°C until the water content is <1.0% w/w by Karl-Fisher analysis.

[00151] Yield of compound Gl: about 75%.

Step 2B: Preparation of F-3a

Charge di-/c/7-butyl dicarbonate, (1.85 wt, 1.4 eq) to vessel A followed by N,N-dimethylformamide, (2.6 wt, 2.7 vol) and stir at 20°C for 20 min. until dissolution achieved. Add A,A-diisopropylethylamine, (0.08 wt, 0.11 vol, 0.1 eq) to contents of vessel A at 20°C. Heat the contents of vessel A to 40°C.

[00153] Charge Gl, (1.00 wt) to vessel B followed by YW-di methyl form am ide, (5.2 wt, 5.5 vol) and adjust to 14°C.

[00154] Charge the Gl/DMF solution from vessel B to vessel A over 5 h at 40°C, at an approximately constant rate. Line rinse with Y,Y-di methyl form am ide, (0.4 wt, 0.4 vol), maintaining vessel A at 40°C. Stir the resulting reaction mixture at 40°C for 16 h.

[00155] Charge decolorizing charcoal activated, (0.20 wt). Adjust the mixture to 40°C and stir at 40°C for 60 to 90 min..

[00156] Clarify (filter) the reaction mixture into vessel B at 40°C. Charge N,N-dimethylformamide, (0.9 wt, 1.0 vol) via vessel A and filter at 40°C. Charge purified water, (3.5 vol) to the combined filtrates, over 60 min., maintaining the temperature at 40°C. As required, cool the mixture to 35°C over 30 to 60 min..

[00157] Filler F-3a, (0.02 wt) as seed material at 35°C. Stir at 34°C for 1.5 h then check for crystallization. Cool slurry to 30°C over 40 min.

[00158] Filler F-3a, (0.02 wt) as seed material at 30°C. Stir at 30°C for 1.5 h then check for crystallization.

[00159] Cool slurry at 20°C over 3.5 h at a targeted constant rate. Stir at 20°C for 3 hours. Charge purified water, (1.0 vol), maintaining the temperature at 20°C over 60 min..

Stir at 20°C for 3 hours.

[00160] Cool slurry to 2°C over 2.5 h. Stir at 2°C for 2.5 hours. Filter through 20 pm cloth and pull dry until no further filtrate passes. Wash the solid with pre-mixed Y,Y-di methyl form am ide / purified water, (2.0 vol, 1:2 v:v) at 2°C. Wash the solid with purified water, (2 x 3.0 vol) at 2°C. Dry under vacuum at 28°C until KF <0.2% w/w, and Y,Y-di methyl form am ide <0.4% w/w.

[00161] Yield of compound F-3a: 62-70%.

Example 3: Synthesis of Mavorixafor:

Scheme VI:

nce

Step 3A: Preparation of imine Q-1

[00162] To vessel A charge purified water, (8.7 vol, 8.7 wt) followed by potassium phosphate, (5.52 eq, 5.3 wt) portion-wise and cool to 15°C. Charge tetrahydrofuran, (4.3 vol, 3.8 wt) and n-heptane, (2.2 vol, 1.5 wt) to vessel A and cool the biphasic mixture to 0°C. Charge Fl, (1.00 eq, 1.00 wt) to the vessel in 2 portions maintaining 0°C.

[00163] Charge F-2d, (1.10 eq, 1.95 wt) to the vessel in 4 portions maintaining 0°C, ensuring portions are spaced by 10 min.. Stir the resulting biphasic mixture for 1.5 h at 0°C. Allow the layers to separate for 45 min. at 0°C before separating the layers. Retain the upper organic phase within vessel A.

[00164] Take the resulting solution of Ql into next reaction without isolation.

Step 3B: Preparation of amine P-1

[00165] To vessel B, charge tetrahydrofuran, (6.0 vol, 5.3 wt) and adjust to 15°C. Charge zinc chloride, (1.5 eq, 0.92 wt) to vessel B in 4 portions, maintaining 10 to 30°C. Adjust the reaction mixture in vessel B to 15°C. Stir the mixture at 15°C for 1 hour. Charge sodium borohydride,(1.0 eq, 0.17 wt) to vessel B in 2 portions maintaining 15°C. Cool the reaction mixture in vessel B to 15°C. Stir the mixture for 1 hour maintaining 15°C. Cool the reaction mixture in vessel B to -5°C.

[00166] Cool the retained organic solution of Ql in vessel A, from Step 3A, to -5°C.

[00167] Charge the organic solution in vessel A into vessel B over 1 to 2 h maintaining -5°C. Charge tetrahydrofuran, (1.0 vol, 0.9 wt) to vessel A as a line rinse and adjust to -5°C. Transfer the contents of vessel A to vessel B maintaining -5°C.

[00168] Stir the resulting reaction mixture in vessel B for 1.5 h maintaining -5°C.

[00169] Charge purified water, (4.5 vol, 4.5 wt) and glacial acetic acid, (1.0 eq, 0.27 wt, 0.26 vol) to the cleaned vessel A and cool to 0°C. Charge the contents of vessel B to vessel A over 1 to 2 h maintaining 0°C. Charge tetrahydrofuran, (1.0 vol, 0.9 wt) to vessel B as a vessel rinse, cool to 0°C and transfer to vessel A maintaining 0°C.

[00170] Warm the resulting mixture in vessel A to 30°C. Stir the resulting mixture in vessel A at 30°C for 1 h. Allow the layers to settle for 15 min. at 30°C before separating the layers. Retain the upper organic phase.

[00171] Cool the retained organic phase to 15°C. Charge to the vessel 25% w/w ammonia solution (3.0 vol) at 10 to 30°C. Cool the reaction mixture to 20°C. Charge to the vessel 25% w/w ammonium chloride solution (3.0 vol) at 20°C and stir for 1 h. Separate the layers for 15 min. at 20°C, retain the upper organic phase. Wash the retained organic phase with 10% w/w sodium chloride solution (3.0 vol) at 20°C for 10 min.. Allow the layers to settle for 10 min. at 20°C before separating and retaining the upper organic phase within the vessel.

[00172] Charge tert-butyl methyl ether, (0.5 vol, 0.4 wt) to the organic phase. Cool the mixture to 5°C. Adjust the pH of the reaction mixture to pH 5 with hydrochloric acid aqueous solution (expected ca. 9.0 vol) over 1 h at a targeted constant rate at 5°C. Stir the mixture at 5°C for 45 min.. Measure the pH of the aqueous phase to confirm the value is pH 5.

[00173] Charge sodium chloride, (2.1 wt) to the reaction mixture at 5°C and stir the mixture until everything is dissolved. Adjust the temperature of the reaction mixture to 20°C. Separate the layers at 20°C and retain the organic phase within the vessel. Tetrahydrofuran charge, (1.5 vol, 1.3 wt) maintaining 20°C.

[00174] Charge to the vessel 24% w/w sodium chloride solution (7.5 vol) at 20°C and stir for 10 min.. Separate the layers at 20°C and retain the organic phase in the vessel. This step is repeated additional one more time as described below.

[00175] Charge to the vessel 24% w/w sodium chloride solution (7.5 vol) at 20°C and stir for 10 min.. Separate the layers at 20°C and retain the organic phase in the vessel.

[00176] Heat the retained organic phase to 35°C and concentrate the mixture to 6.0 vol under reduced pressure maintaining 35°C.

[00177] Tetrahydrofuran charge, (15.0 vol, 13.2 wt) maintaining 35°C. Concentrate the mixture to 6.0 vol under reduced pressure maintaining 35°C.

[00178] Tetrahydrofuran charge, (15.0 vol, 13.2 wt) maintaining 35°C. Concentrate the mixture to 11.0 vol under reduced pressure maintaining 35°C.

[00179] Cool the mixture to -5°C. Load tert-butyl methyl ether, (10.0 vol, 7.4 wt) over 1 h maintaining -5°C. Stir the mixture at -5°C for 1.5 hours. Filter the solid on 1 to 2 pm filter cloth at -5°C. Wash the solid with pre-mixed tetrahydrofuran, (1.9 vol, 1.7 wt) and tert-butyl methyl ether, (3.1 vol, 1.9 wt) at -5°C as a displacement wash.

[00180] Wash the solid with tert-butyl methyl ether, (5.0 vol, 3.7 wt) at -5°C.

[00181] Dry the solid on the filter under a flow of nitrogen at 23°C.

[00182] Yield of compound P-1: 76-87%.

Step 3C: Preparation of compound 0-1

[00183] Charge purified water, (2.0 vol, 2.0 wt) followed by potassium phosphate, (3.3 eq, 1.54 wt), carefully portion-wise, maintaining <15°C, to vessel A. Charge toluene, (4.5 vol, 3.9 wt) to the vessel maintaining <15°C. As necessary, adjust the temperature to 10°C.

[00184] Charge P-1, (1.00 eq, 1.00 wt) to the vessel in two portions maintaining 10°C. Stir the reaction mixture at 10°C for 15 min..

[00185] Load F-3a, (1.1 eq, 0.64 wt) in 4 equal portions ensuring portions are spaced by 10 min. at 10°C.

[00186] Tetrabutylammonium iodide (TBAI) filler (0.20 eq, 0.16 wt). Heat the reaction mixture to 40°C. Stir the reaction mixture at 40°C for 30 h.

[00187] Charge pre-mixed 2-mercaptoacetic acid, (0.40 eq, 0.08 wt, 0.06 vol), and toluene, (0.5 vol, 0.4 wt) over 20 min. to Vessel A at 40°C. Line rinse with toluene, (0.5 vol, 0.4 wt) at 40°C. Adjust the temperature of the reaction mixture to 50°C. Stir the mixture at 50°C for 2.5 hours.

[00188] Adjust the temperature of Vessel A to 20°C. Charge purified water, (3.0 vol, 3.0 wt) maintaining 20°C. Stir the reaction mixture at 20°C for 15 min. and transfer to a new, clean HDPE container. Line/vessel rinse with toluene, (0.5 vol, 0.4 wt) at 20°C. Clarify (filter) the reaction mixture via a 1 pm filter at 20°C into clean Vessel A. Wash the vessel and the filter with toluene, (0.5 vol, 0.4 wt) at 20°C. Allow the layers to separate for 15 min. at 20°C, retaining the upper organic layer (organic layer 1).

[00189] Wash the aqueous layer with toluene, (2.5 vol, 2.2 wt) at 20°C for 15 min.. Allow the layers to separate for 15 min. at 20°C. Retain the upper organic layer (organic layer 2).

[00190] Combine the organic layer 1 and organic layer 2 and adjust the temperature to 20°C. Wash the combined organic layers with 10% w/w sodium chloride solution (5.0 vol) at 20°C for 15 min.. Allow the layers to settle for 15 min. at 20°C. Retain the upper organic layer.

[00191] Take the resulting solution of Ol into next reaction without isolation.

Step 3D: Preparation of compound Kl

[00192] Charge n-butanol, (2.4 wt, 3.0 vol) to vessel B and adjust to 5°C. Charge concentrated sulfuric acid, (1.1 wt, 5.0 eq, 0.6 vol) slowly to Vessel B maintaining <15°C. Line rinse with toluene, (0.4 wt, 0.5 vol) maintaining <15°C. Adjust the temperature of Vessel B to 25°C.

[00193] Heat the n-butanol/ sulfuric acid solution in Vessel B to 55°C. Charge the organic layer from Vessel A (from Step 3C) to the butanol/ sulfuric acid solution in Vessel B over 60 to 90 min. maintaining 55°C. Charge toluene, (1.3 wt, 1.5 vol) to Vessel A as a line rinse and transfer to Vessel B maintaining 55°C. Stir the contents of Vessel B at 55°C for 1.5 h.

[00194] Stir the mixture in Vessel B for 4.5 h at 55°C. Cool the contents of Vessel B to 20°C over 10 h. Filter the slurry over 1-2 pm filter cloth under nitrogen at 20°C. Wash the filter cake with pre-mixed toluene, (3.5 wt, 4.0 vol) and n-butanol, (1.0 vol, 0.8 wt) at 20°C. Wash the filter cake with toluene, (4.3 wt, 5.0 vol) at 20°C. Dry the solid at 30°C under vacuum.

[00195] Correct the output weight for assay. Expected 50-55% w/w.

[00196] Yield of compound K1: 89-92%.

Step 3E: Preparation of Mavorixafor Drug Substance

[00197] Charge Kl, (1.00 eq, 1.00 wt, corrected for HPLC assay) in vessel A followed by nitrogen-purged purified water, (2.0 wt, 2.0 vol) and if necessary, adjust the temperature to 20°C. Charge nitrogen-purged toluene, (12.0 wt, 14.0 vol) to the solution maintaining 20°C. Charge nitrogen-purged n-butanol, (0.8 wt, 1.0 vol) to the solution maintaining 20°C. Heat the biphasic mixture to 30°C. Charge nitrogen-purged 3.0 M aqueous sodium hydroxide solution (6.2 eq, 5.9 vol) maintaining 30°C. Check the pH (expected 12 to 13). Adjust the pH of the aqueous layer to pH 10.0 with nitrogen-purged 0.3 M sulfuric acid solution (expected up to 2.5 vol) maintaining 30°C. Stir the mixture at 30°C for 45 min..

[00198] Measure the pH to confirm the value is pH 10.0.

[00199] Allow the layers to settle at 30°C for 30 min. and separate the layers retaining the organic phase in the vessel, and discharge the aqueous layer into a separate container (container C).

[00200] Charge pre-mixed toluene, (4.1 wt, 4.7 vol) and n-butanol, (0.24 wt, 0.3 vol) to a separate vessel; heat the contents to 30°C and charge the aqueous layer from container C. As required adjust the temperature to 30°C and stir for 5 to 10 min. at 30°C. Allow the phases to separate for 10 to 15 min. at 30°C. Discharge the aqueous phase to waste and combine the organic phase to the organic phase in vessel A.

[00201] Charge nitrogen-purged purified water, (2.0 wt, 2.0 vol) to the organic layer maintaining the temperature at 30°C and stir for 5 to 10 min. at 30°C. Allow the phases to separate for 10 to 15 min. at 30°C. Discharge the aqueous phase to waste retaining the organic phase in the vessel. Heat the retained organic solution to 40°C. Concentrate the resulting organic phase to 7.0 vol by vacuum distillation at 40°C.

[00202] Charge nitrogen -purged toluene, (13.0 wt, 15.0 vol) to the mixture and concentrate the solution 7.0 vol by vacuum distillation at 40°C. This step is repeated additional one time as described below.

[00203] Charge nitrogen -purged toluene, (13.0 wt, 15.0 vol) to the mixture and concentrate the solution 7.0 vol by vacuum distillation at 40°C.

[00204] Charge nitrogen-purged toluene, (7.0 wt, 8.0 vol) to the mixture at 40°C, heat to 55°C and clarify the hot reaction mixture under nitrogen via a 1 pm filter.

[00205] Charge clarified nitrogen-purged toluene, (1.7 wt, 2.0 vol) to the mixture as a line and vessel rinse at 40°C. Concentrate the solution to 7.0 vol by vacuum distillation at 40°C. At the end of the distillation the product is expected to have precipitated. Heat the mixture to 63°C.

[00206] Adjust the temperature to 60.5°C. This batch will be referred to as the main batch.

[00207] Load seed material, (0.02 wt) to a new clean container. Charge clarified nitrogen-purged toluene, (0.09 wt, 0.10 vol) to this seed material and gently shake.

[00208] Seed the main batch with the slurry maintaining the temperature at 60.5 ± 2°C. Stir the reaction at the 60.5± 2°C for 1 hour.

[00209] Cool to 40°C for 2.5 h. Stir the reaction at 40°C for 1 hour.

[00210] Cool to 30°C over 2 h.. Stir the reaction at 30°C for 1 h.

[00211] Cool to 25°C 50 min. Stir the reaction at 25°C over 2 hours.

[00212] Cool to 2°C over 4 h. Stir the mixture for 12 hours at 2°C.

[00213] Filter the mixture at 2°C over 1 to 2 pm cloth. Wash the filter cake with clarified nitrogen-purged toluene, (2.0 vol, 1.7 wt) at 2°C. Dry the filter cake under vacuum and a flow of nitrogen for 1.5 h.

[00214] Dry the solid at 40°C under vacuum and a flow of nitrogen until drying specification is achieved.

[00215] Yield of the final compound mavorixafor: 72%.

[00216] When toluene is used as the recrystallization solvent, optionally with a dissolution aid such butanol or methanol, for maxorixa for recrystallization, advantages were found compared to using dichloromethane and isopropyl acetate. We have found that these solvents do not react with the API, and accordingly we believe that this change has caused the significant reduction of impurities A (imine), B (N-formyl) and C (acetamide) that we have observed.

[00217] In some embodiments, the mavorixafor composition included 7000, 6000, 5000, 4500, 4450, 4000, 3500, 3000, 2500, 2000, 1750, 1700, 1650, 1600, 1550, 1500, 1450, 1400, 1400, 1400, 1400 gold 50 ppm of toluene or less. In some embodiments, the mavorixafor composition comprises a detectable amount of toluene. In some embodiments, the mavorixafor composition comprises from a detectable amount of toluene to 1350 ppm of toluene.

////////

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Description	Mavorixafor (AMD-070) is a potent, selective and orally available CXCR4 antagonist, with an IC₅₀ value of 13 nM against CXCR4 ¹²⁵I-SDF binding, and also inhibits the replication of T-tropic HIV-1 (NL4.3 strain) in MT-4 cells and PBMCs with an IC₅₀ of 1 and 9 nM, respectively.
IC₅₀ & Target^[1]	¹²⁵I-SDF-CXCR413 nM (IC₅₀)HIV-1 (NL4.3 strain)1 nM (IC₅₀, in MT-4 cells)HIV-1 (NL4.3 strain)9 nM (IC₅₀, in PBMCs)HIV-1 (NL4.3 strain)3 nM (IC90, in MT-4 cells)HIV-1 (NL4.3 strain)26 nM (IC90, in PBMCs)
In Vitro	Mavorixafor (AMD-070) is a potent and orally available CXCR4 antagonist, with an IC₅₀ value of 13 nM against CXCR4 ¹²⁵I-SDF binding, and also inhibits the replication of T-tropic HIV-1 (NL4.3 strain) in MT-4 cells and PBMCs with an IC₅₀ of 1 and 9 nM, respectively. Mavorixafor (AMD-070) shows no effect on other chemokine receptors (CCR1, CCR2b, CCR4, CCR5, CXCR1, and CXCR2)^[1]. Mavorixafor (AMD-070) (6.6 µM) significantly suppresses the anchorage-dependent growth, the migration and matrigel invasion of the B88-SDF-1 cells^[2].MCE has not independently confirmed the accuracy of these methods. They are for reference only.
In Vivo	Mavorixafor (AMD-070) (2 mg/kg, p.o.) significantly reduces the number of metastatic lung nodules in mice, and lowers the expression of human Alu DNA in mice, without body weight loss^[2].MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Clinical Trial	NCT NumberSponsorConditionStart DatePhaseNCT00089466National Institute of Allergy and Infectious Diseases (NIAID)\|AIDS Clinical Trials GroupHIV InfectionsNovember 2004Phase 1\|Phase 2NCT02667886X4 PharmaceuticalsClear Cell Renal Cell CarcinomaJanuary 2016Phase 1\|Phase 2NCT02823405X4 PharmaceuticalsMelanomaSeptember 15, 2016Phase 1NCT00361101Genzyme, a Sanofi Company\|SanofiHIV Infections\|X4 Tropic VirusOctober 2005Phase 1NCT03005327X4 PharmaceuticalsWHIM SyndromeDecember 2016Phase 2NCT04274738X4 PharmaceuticalsWaldenstrom´s MacroglobulinemiaApril 30, 2020Phase 1NCT04154488X4 PharmaceuticalsNeutropeniaOctober 16, 2020Phase 1NCT03995108X4 PharmaceuticalsWHIM SyndromeOctober 17, 2019Phase 3NCT05103917Abbisko Therapeutics Co, LtdTriple Negative Breast CancerJuly 21, 2021Phase 1\|Phase 2NCT00063804National Institute of Allergy and Infectious Diseases (NIAID)\|AIDS Clinical Trials GroupHIV Infections Phase 1NCT02923531X4 PharmaceuticalsClear Cell Renal Cell CarcinomaDecember 7, 2016Phase 1\|Phase 2NCT02680782X4 Pharmaceuticals\|CovanceHealthyJanuary 12, 2016Phase 1

REF

[1]. Skerlj RT, et al. Discovery of novel small molecule orally bioavailable C-X-C chemokine receptor 4 antagonists that are potent inhibitors of T-tropic (X4) HIV-1 replication. J Med Chem. 2010 Apr 22;53(8):3376-88. [Content Brief][2]. Chow LN, et al. Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice. PLoS One. 2016 Mar 21;11(3):e0151765. [Content Brief][3]. Uchida D, et al. Effect of a novel orally bioavailable CXCR4 inhibitor, AMD070, on the metastasis of oral cancer cells. Oncol Rep. 2018 Jul;40(1):303-308. [Content Brief]

/////////////////////////////////////////////////////////////////////////////MAVORIXAFOR, AMD 070, PHASE 2

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LORPUCITINIB

September 21, 2022 3:12 am / Leave a comment

LORPUCITINIB

JNJ 64251330

2230282-02-5

UNII-OE1QTY7C25

Molecular Weight	408.50
Formula	C₂₂H₂₈N₆O₂

1-(TRANS-4-(CYANOMETHYL)CYCLOHEXYL)-1,6-DIHYDRO-N-(2-HYDROXY-2-METHYLPROPYL)IMIDAZO(4,5-D)PYRROLO(2,3-B)PYRIDINE-2-ACETAMIDE

2-[3-[4-(cyanomethyl)cyclohexyl]-3,5,8,10-tetrazatricyclo[7.3.0.0^2,6]dodeca-1,4,6,8,11-pentaen-4-yl]-N-(2-hydroxy-2-methylpropyl)acetamide

is a Gut-Restricted JAK Inhibitor for the research of Inflammatory Bowel Disease.

Lorpucitinib is an orally bioavailable pan-inhibitor of the Janus associated-kinases (JAKs), with potential immunomodulatory and anti-inflammatory activities. Upon oral administration, lorpucitinib works in the gastrointestinal (GI) tract where it targets, binds to and inhibits the activity of the JAKs, thereby disrupting JAK-signal transducer and activator of transcription (STAT) signaling pathways and the phosphorylation of STAT proteins. This may inhibit the release of pro-inflammatory cytokines and chemokines, reducing inflammatory responses and preventing inflammation-induced damage. The Janus kinase family of non-receptor tyrosine kinases, which includes tyrosine-protein kinase JAK1 (Janus kinase 1; JAK1), tyrosine-protein kinase JAK2 (Janus kinase 2; JAK2), tyrosine-protein kinase JAK3 (Janus kinase 3; JAK3) and non-receptor tyrosine-protein kinase TYK2 (tyrosine kinase 2), plays a key role in cytokine signaling and inflammaton.

PATENT

WO2019239387

WO2018112379

WO2018112382

PATENT

WO/2022/189496LORPUCITINIB FOR USE IN THE TREATMENT OF JAK MEDIATED DISORDERS

Example 1

[0117] 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide

Step A: 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide. To ensure dry starting material, ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (Intermediate 3) was heated under vacuum at 50 °C for 18 h prior to the reaction. In a 1 L flask, ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (Intermediate 3, 52.585 g, 104.01 mmol) was suspended in DMA (50 mL). 1-Amino-2-methylpropan-2-ol (50 mL) was added and the reaction was heated to 110 °C for 45 minutes, then to 125 °C for 5 hours. The reaction was cooled to room temperature and diluted with EtOAc (800 mL). The organic layer was extracted three times with a solution of water/ brine wherein the solution was made up of 1 L water plus 50 mL brine. The aqueous layers were back extracted with EtOAc (2 × 600 mL). The combined organic layers were dried over anhydrous MgSO₄,

concentrated to dryness, and then dried for 3 days under vacuum to provide the title compound (65.9 g, 98% yield) as a yellow foam. The product was taken to the next step with no further purification. MS (ESI): mass calcd. for C₂₈H₃₂N₆O₄S, 548.22; m/z found, 549.2 [M+H]⁺.¹H NMR (400 MHz, CDCl₃): δ 8.76 (s, 1H), 8.26 – 8.19 (m, 2H), 7.84 (d, J = 4.1 Hz, 1H), 7.60 – 7.53 (m, 1H), 7.50 – 7.44 (m, 2H), 6.84 (d, J = 4.2 Hz, 1H), 4.76 – 4.61 (m, 1H), 3.97 (s, 2H), 3.45 (s, 1H), 3.27 (d, J = 5.9 Hz, 2H), 2.41 (d, J = 6.5 Hz, 2H), 2.38 – 2.25 (m, 2H), 2.23 – 2.12 (m, 2H), 2.09 -1.94 (m, 4H), 1.48 (qd, J = 13.6, 4.0 Hz, 2H), 1.21 (s, 6H).

[0118] Step B: 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide. 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide (65.90 g, 102.1 mmol) was added to a 1 L flask containing a stir bar. 1,4-dioxane (300 mL) was added, followed by aq KOH (3 M, 150 mL). The reaction was heated at 80 °C for 2 h. The reaction was cooled to room temperature and the solvent volume was reduced to about 200 mL on a rotovap. The residue was treated with a solution of water/brine (100 mL/100mL), then extracted with 10% MeOH in CH₂Cl₂ (2 x 1L). The organic layers were combined, dried over anhydrous MgSO₄, and concentrated to dryness to provide a yellow solid. The solid was suspended in CH₂Cl₂ (200 mL), stirred vigorously for 30 minutes, and then collected by filtration. The solid was rinsed with CH₂Cl₂ (100 mL), dried by pulling air through the filter, and then further dried under vacuum at room temperature for 16 h to provide the title compound (41.59 g, 89% yield) as a white solid. MS (ESI): mass calcd. for C₂₂H₂₈N₆O₂, 408.23; m/z found, 409.2 [M+H]⁺. ¹H NMR (600 MHz, DMSO-d₆): δ 11.85 (s, 1H), 8.50 (s, 1H), 8.21 – 8.10 (m, 1H), 7.49 – 7.43 (m, 1H), 6.74 – 6.65 (m, 1H), 4.53 – 4.42 (m, 2H), 4.07 (s, 2H), 3.08 (d, J = 6.0 Hz, 2H), 2.58 (d, J = 6.1 Hz, 2H), 2.41 – 2.28 (m, 2H), 2.09 – 1.92 (m, 5H), 1.42 – 1.31 (m, 2H), 1.09 (s, 6H). The synthesis and active compound characterization of each of the aspects of this invention are provided herein in the form of examples. Due to the crystal structure of some of the aspects of this invention, polymorph screening may be pursued to further characterize specific forms of any such compound. This is illustrated in a non-limiting manner for compound of Formula I by the example under the heading polymorph screening.

[0119] The following compounds were prepared in reference to the foregoing synthesis:

Intermediate 1

[0120] 2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile

[0121] Step A: tert-butyl N-[(1r,4r)-4-(Hydroxymethyl)cyclohexyl]carbamate. To a 20-L 4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed (1r,4r)-4-[[(tert-butoxy)carbonyl]amino]cyclohexane-1-carboxylic acid (1066 g, 4.38 mol, 1.00 equiv) and THF (10 L). This was followed by the dropwise addition of BH₃-Me₂S (10 M, 660 mL) at -10 °C over 1 h. The resulting solution was stirred for 3 h at 15 °C. This reaction was performed three times in parallel and the reaction mixtures were combined. The reaction was then quenched by the addition of methanol (2 L). The resulting mixture was concentrated under vacuum. This resulted in of tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (3000 g, 99.6%) as a white solid. MS (ESI): mass calcd. for C₁₂H₂₃NO₃, 229.32; m/z found, 215.2 [M-tBu+MeCN+H]⁺; ¹H NMR: (300 MHz, CDCl₃): δ 4.40 (s, 1H), 3.45 (d, J = 6.3 Hz, 2H), 3.38 (s, 1H), 2.05-2.02 (m, 2H), 1.84-1.81 (m, 2H), 1.44 (s, 11H), 1.17-1.01 (m, 4H).

[0122] Step B: tert-butyl N-[(1r,4r)-4-[(Methanesulfonyloxy)methyl]cyclohexyl]carbamate. To a 20 L 4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (1000 g, 4.36 mol, 1.00 equiv.), dichloromethane (10 L), pyridine (1380 g, 17.5 mol, 4.00 equiv.). This was followed by the dropwise addition of MsCl (1000 g, 8.73 mol, 2.00 equiv.) at -15 °C. The resulting solution was stirred overnight at 25 °C. This reaction was performed in parallel for 3 times and the reaction mixtures were combined. The reaction was then quenched by the addition of 2 L of water. The

water phase was extracted with ethyl acetate (1 x 9 L). The organic layer was separated and washed with 1 M HCl (3 x 10 L), NaHCO₃ (saturated aq.) (2 x 10 L), water (1 x 10 L) and brine (1 x 10 L). The mixture was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. This resulted in of tert-butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (3300 g, 82%) as a white solid. LC-MS: MS (ESI): mass calcd. for C₁₃H₂₅NO₅S, 307.15; m/z found 292.1, [M-tBu+MeCN+H]⁺; ¹H NMR: (300 MHz, CDCl₃): δ 4.03 (d, J = 6.6 Hz, 2H), 3.38 (s, 1H), 3.00 (s, 3H), 2.07-2.05 (m, 2H), 1.87-1.84 (m, 2H), 1.72-1.69 (m, 1H), 1.44 (s, 9H), 1.19-1.04 (m, 4H).

[0123] Step C: tert-butyl N-[(1r,4r)-4-(Cyanomethyl)cyclohexyl]carbamate. To a 10 L 4-necked round-bottom flask, was placed tert-butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (1100 g, 3.58 mol, 1.00 equiv.), DMSO (5500 mL) and NaCN (406 g, 8.29 mol, 2.30 equiv.). The resulting mixture was stirred for 5 h at 90 °C. This reaction was performed in parallel 3 times and the reaction mixtures were combined. The reaction was then quenched by the addition of 15 L of water/ice. The solids were collected by filtration. The solids were washed with water (3 x 10 L). This resulted in tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (2480 g, 97%) as a white solid. MS (ESI): mass calcd. for C₁₃H₂₂N₂O₂, 238.17; m/z found 224 [M-tBu+MeCN+H]⁺; ¹H NMR: (300 MHz, CDCl₃): δ 4.39 (s, 1H), 3.38 (s, 1H), 2.26 (d, J = 6.9 Hz, 2H), 2.08-2.04 (m, 2H), 1.92-1.88 (m, 2H), 1.67-1.61 (m, 1H), 1.44 (s, 9H), 1.26-1.06 (m, 4H).

[0124] Step D: 2-[(1r,4r)-4-Aminocyclohexyl]acetonitrile hydrochloride. To a 10-L round-bottom flask was placed tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (620 g, 2.60 mol, 1.00 equiv.), and 1,4-dioxane (2 L). This was followed by the addition of a solution of HCl in 1,4-dioxane (5 L, 4 M) dropwise with stirring at 10 °C. The resulting solution was stirred overnight at 25 °C. This reaction was performed for 4 times and the reaction mixtures were combined. The solids were collected by filtration. The solids were washed with 1,4-dioxane (3 x 3 L), ethyl acetate (3 x 3 L) and hexane (3 x 3 L). This resulted in 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride (1753 g, 96%) as a white solid. MS (ESI): mass calcd. for C₈H₁₄N₂, 138.12; m/z found 139.25, [M+H]⁺; ¹H NMR: (300 MHz, DMSO-d₆): δ 8.14 (s, 3H), 2.96-2.84 (m, 1H), 2.46 (d, J = 6.3 Hz, 2H), 1.98 (d, J = 11.1 Hz, 2H), 1.79 (d, J = 12.0 Hz, 2H), 1.64-1.49 (m, 1H), 1.42-1.29 (m, 2H), 1.18-1.04 (m, 2H).

[0125] Step E: 2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile. To a 1000 mL round bottom flask containing 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride (29.10 g, 166.6 mmol) was added DMA (400 mL). The resulting suspension was treated with 4-chloro-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (51.53 g, 152.6 mmol), followed by DIPEA (63.0 mL, 366 mmol). The reaction mixture was placed under N₂ and heated at 80 °C for 4 h. The crude reaction mixture was cooled to room temperature and slowly poured into a vigorously stirred 2 L flask containing 1.6 L water. The resulting suspension was stirred for 15 minutes at room temperature, then filtered and dried for 16 h in a vacuum oven with heating at 70 °C to provide the title compound (63.37 g, 95%) as a yellow solid. MS (ESI): mass calcd. for C21H21N5O4S, 439.1; m/z found, 440.1 [M+H]⁺. ¹H NMR (500 MHz, CDCl₃): δ 9.10 (s, 1H), 8.99 (d, J = 7.8 Hz, 1H), 8.23 – 8.15 (m, 2H), 7.66 – 7.59 (m, 2H), 7.56 – 7.49 (m, 2H), 6.67 (d, J = 4.2 Hz, 1H), 3.95 – 3.79 (m, 1H), 2.38 (d, J = 6.2 Hz, 2H), 2.32 -2.21 (m, 2H), 2.08 – 1.98 (m, 2H), 1.88 – 1.76 (m, 1H), 1.60 – 1.32 (m, 4H).

Intermediate 2

[0126] 2-((1r,4r)-4-((5-Amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile

[0127] 2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile (Intermediate 1, 58.60 g, 133.3 mmol) was dissolved in THF/MeOH (1:1, 4800 mL). The mixture was passed through a continuous-flow hydrogenation reactor (10% Pd/C), such as a Thales Nano H-Cube®, at 10 mL/min with 100 % hydrogen (atmospheric pressure, 80 °C), then the solution was concentrated to provide the product as a purple solid. The solid was triturated with EtOAc (400 mL) and then triturated again with MeOH (200 mL) then filtered and dried under vacuum to provide the title compound (50.2 g, 91.9% yield).

MS (ESI): mass calcd. for C₂₁H₂₃N₅O₂S, 409.2; m/z found, 410.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.10 – 8.03 (m, 2H), 7.76 (s, 1H), 7.51 – 7.43 (m, 1H), 7.43 – 7.34 (m, 3H), 6.44 (d, J = 4.2 Hz, 1H), 4.61 (d, J = 8.5 Hz, 1H), 3.65 – 3.51 (m, 1H), 2.74 (s, 2H), 2.26 (d, J = 6.4 Hz, 2H), 2.19 – 2.05 (m, 2H), 1.97 – 1.86 (m, 2H), 1.76 – 1.59 (m, 1H), 1.33 – 1.12 (m, 4H).

Intermediate 3

[0128] Ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate

[0129] To a 1L round bottom flask containing a stir bar and 2-((1r,4r)-4-((5-amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile (Intermediate 2, 58.31 g, 142.4 mmol) was added ethyl 3-ethoxy-3-iminopropanoate (60.51 g, 309.3 mmol), followed by EtOH (600 mL, dried over 3Å molecular sieves for 48 h). A reflux condenser was attached to the reaction flask, the reaction was purged with N₂, and was heated at 90 °C for 9 h. The reaction mixture was cooled to room temperature and left to stand for 30 h where the product crystallized out as brown needles. The solids were broken up with a spatula and the reaction mixture was transferred to a 2 L flask. Water (1.4 L) was added slowly via separatory funnel with vigorous stirring. After addition of the water was complete, the suspension was stirred for 30 minutes. The brown needles were isolated by filtration and then dried by pulling air through the filter for 1 h. The product was transferred to a 500 mL flask and treated with EtOAc (200 mL). A small quantity of seed crystals were added, which induced the formation of a white solid precipitate. The suspension was stirred for 30 minutes at room temperature, filtered, rinsed with EtOAc (25 mL), and dried under vacuum to provide the product as a white solid (48.65 g, 68% yield). MS (ESI): mass calcd. for C₂₆H₂₇N₅O₄S, 505.2; m/z found, 506.2 [M+H]⁺. ¹H NMR (400

MHz, CDCl₃) δ 8.85 (s, 1H), 8.28 – 8.19 (m, 2H), 7.84 (d, J = 4.0 Hz, 1H), 7.61 – 7.53 (m, 1H), 7.52 – 7.43 (m, 2H), 6.84 (d, J = 4.1 Hz, 1H), 4.32 (s, 1H), 4.20 (q, J = 7.1 Hz, 2H), 4.09 (s, 2H), 2.44 (d, J = 6.2 Hz, 2H), 2.40 – 2.27 (m, 2H), 2.16 (d, J = 13.3 Hz, 2H), 2.12 – 1.96 (m, 3H), 1.54 – 1.38 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H).

Polymorph screening example

[0130] Some embodiments of compound of Formula I as free bases present multiple crystalline configurations that have a complex solid-state behavior, some of which in turn can present distinguishing features among themselves due to different amounts of incorporated solvent. Some embodiments of compound of Formula I are in the form of pseudopolymorphs, which are embodiments of the same compound that present crystal lattice compositional differences due to different amounts of solvent in the crystal lattice itself. In addition, channel solvation can also be present in some crystalline embodiments of compound of Formula I, in which solvent is incorporated within channels or voids that are present in the crystal lattice. For example, the various crystalline configurations given in Table 2 were found for compound of Formula I. Because of these features, non-stoichiometric solvates were often observed, as illustrated in Table 2. Furthermore, the presence of such channels or voids in the crystal structure of some embodiments according to this invention enables the presence of water and/or solvent molecules that are held within the crystal structure with varying degrees of bonding strength. Consequently, changes in the specific ambient conditions can readily lead to some loss or gain of water molecules and/or solvent molecules in some embodiments according to this invention. It is understood that “solvation” (third column in Table 2) for each of the embodiments listed in Table 2 is the formula solvation, and that the actual determination of the same as a stoichiometry number (fourth column in Table 2) can slightly vary from the formula solvation depending on the actual ambient conditions when it is experimentally determined. For example, if about half of the water molecules in an embodiment may be present as hydrogen-bonded to the active compound in the crystal lattice, while about the other half of water molecules may be in channels or voids in the crystal lattice, then changes in ambient conditions may alter the amount of such loosely contained water molecules in voids or channels, and hence lead to a slight difference between the formula solvation that is assigned according to, for example, single crystal diffraction, and the

stoichiometry that is determined by, for example, thermogravimetric analysis coupled with mass spectroscopy.

Table 2. Embodiments of crystalline forms of compound of Formula I

[0131] The compound that was obtained as described in Example 1 was further crystallized by preparing a slurry in DCM (1:3, for example 10 g of compound in 30 ml DCM) that was stirred at 40^oC for 4 hours, and further stirred for 14 hours at 25^oC, then heptane was slowly added (1:2, for example 20 ml of heptane into the compound/DCM slurry/solution) at 25^oC, stirred at 40^oC for 4 hours, cooled to 25^oC and stirred for further 14 hours at 25^oC. Subsequent filtration led to compound of Formula I in the form of an off-white solid, that was identified as a monohydrate, a 1s embodiment.

CLIP

Journal of Medicinal Chemistry (2020), 63(6), 2915-2929

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Clip

https://clinicaltrials.gov/ct2/show/NCT04552197

The purpose of this study is to evaluate: systemic and local gut (rectum and sigmoid colon) exposure to JNJ-64251330, local tissue Pharmacodynamics (PD) using gut (rectum and sigmoid colon) biopsies (Part 1) and the effect of food on the rate and extent of absorption of JNJ-64251330 from oral tablet dosed with or without food (Part 2).

Familial adenomatous polyposis (FAP) is the most common polyposis syndrome. It is an autosomal dominant inherited disorder characterized by the early onset of hundreds to thousands of adenomatous polyps throughout the colon. JNJ-64251330 (lorpucitinib) is an oral, small molecule, potent pan-janus kinase (JAK) inhibitor that blocks phosphorylation of Signal Transducer and Activator of Transcription (STAT) proteins. pSTAT induces transcription of multiple genes involved in the progression of inflammatory disease. JNJ-64251330 has chemical properties that limits the amount of drug in the blood while delivering the drug to the tissues of the gut. Local inhibition of JAK in the gut may present a promising method to treat inflammatory diseases of the intestinal tract, such as FAP. The study consists of 3 phases: screening phase (30 days) a treatment phase (24 weeks), and follow-up visit (up to 30 days after last dose of study drug). The total duration of the study will be up to 32 weeks. Study evaluations will include efficacy via endoscopies, safety (monitoring of adverse events (AE), serious adverse events (SAEs), events of infections including tuberculosis (TB), clinical laboratory blood tests (complete blood count and serum chemistries), vital signs, and concomitant medication review), pharmacokinetics, pharmacodynamic and biomarkers evaluations.

Adenomatous polyposis coli (APC) also known as deleted in polyposis 2.5 (DP2.5) is a protein that in humans is encoded by the APC gene.^[4] The APC protein is a negative regulator that controls beta-catenin concentrations and interacts with E-cadherin, which are involved in cell adhesion. Mutations in the APC gene may result in colorectal cancer.^[5]

APC is classified as a tumor suppressor gene. Tumor suppressor genes prevent the uncontrolled growth of cells that may result in cancerous tumors. The protein made by the APC gene plays a critical role in several cellular processes that determine whether a cell may develop into a tumor. The APC protein helps control how often a cell divides, how it attaches to other cells within a tissue, how the cell polarizes and the morphogenesis of the 3D structures,^[6] or whether a cell moves within or away from tissue. This protein also helps ensure that the chromosome number in cells produced through cell division is correct. The APC protein accomplishes these tasks mainly through association with other proteins, especially those that are involved in cell attachment and signaling. The activity of one protein in particular, beta-catenin, is controlled by the APC protein (see: Wnt signaling pathway). Regulation of beta-catenin prevents genes that stimulate cell division from being turned on too often and prevents cell overgrowth.

The human APC gene is located on the long (q) arm of chromosome 5 in band q22.2 (5q22.2). The APC gene has been shown to contain an internal ribosome entry site. APC orthologs^[7] have also been identified in all mammals for which complete genome data are available.

////////////////JNJ-64251330, JNJ 64251330, LORPUCITINIB, PHASE 1, CANCER, Adenomatous Polyposis Coli

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MEVOCICLIB, SY 1365

September 20, 2021 2:37 am / Leave a comment

MEVOCICLIB,

CAS 1816989-16-8

SY 1365

N-[(1S,3R)-3-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-1-methylcyclohexyl]-5-[[(E)-4-(dimethylamino)but-2-enoyl]amino]pyridine-2-carboxamide

N-((lS,3R)-3-(5-chloro-4-(lH-indol-3-yl)pyrimidin-2-ylamino)-l-methylcvclohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide

HS Tariff Code: 2934.99.9001

Syros

Molecular Weight	587.12
Formula	C₃₁H₃₅ClN₈O₂

OriginatorSyros Pharmaceuticals
ClassAmides; Amines; Antineoplastics; Chlorinated hydrocarbons; Cyclohexanes; Indoles; Pyridines; Pyrimidines; Small molecules
Mechanism of ActionCyclin-dependent kinase-activating kinase inhibitors
DiscontinuedAcute myeloid leukaemia; Breast cancer; Haematological malignancies; Ovarian cancer; Solid tumours

23 Oct 2019Discontinued – Preclinical for Haematological malignancies and Acute myeloid leukaemia in the USA (Parenteral); Phase-I for Solid tumours, Ovarian cancer and Breast cancer in the USA (IV) because data obtained did not support an optimal profile for patients and indicated higher or frequent dosing
07 Dec 2018Pharmacodynamics data from preclinical trials in Breast cancer presented at the 41st Annual San Antonio Breast Cancer Symposium (SABCS-2018)
15 Nov 2018Adverse events, efficacy and pharmacokinetics data from a phase I trial in Solid tumours presented at the 30^thEORTC-NCI-AACR Molecular Targets and Cancer Therapeutics Symposium (EORTC-NCI-AACR-2018)

Clinical Trial	NCT NumberSponsorConditionStart DatePhaseNCT03134638Syros PharmaceuticalsAdvanced Solid Tumors\|Ovarian Cancer\|Breast CancerMay 12, 2017Phase 1

Mevociclib (SY-1365) is a potent and first-in-class selective CDK7 inhibitor, with a K_i of 17.4 nM. Mevociclib exhibits anti-proliferative and apoptotic effects in solid tumor cell lines. Mevociclib possesses anti-tumor activity in hematological and multiple aggressive solid tumors.

Mevociclib, also known as SY-1365, is a CDK7 inhibitor. In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL-XL expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial anti-tumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax.

Syn

WO2015154038

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015154038

Example 16. Synthesis of N-((lS,3R)-3-(5-chloro-4-(lH-indol-3-yl)pyrimidin-2-ylamino)-l-methylcvclohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide (Compound 267).

[251] (+/-) Benzyl tert-butyl ((lS,3R)-l-methylcvclohexane-l,3-diyl)dicarbamate

(+/-)

[252] A solution of (+/-)-(lS,3R) -3-((ieit-¾itoxycarbonyl)amino)–l -raethylcyclohexanecarboxylic acid prepared as in WO2010/148197 (4.00 g, 15.5 mmol) in toluene (Ϊ 55 mL) was treated with Et₃N (2.4 mL, 17.1 mmol) and DPPA (3.68 mL, Ϊ7.1 mmol) and heated at reflux for lh. Benzyl alcohol (8.0 mL, 77.7 mmol) and Et₃N (4.4 mL , 31 .4 mmol) were added to the reaction mixture and the solution was heated at 100 °C for 72h. The mixture was cooled to room temperature and then diluted with EtOAc (300 mL) and H₂0 (300 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3 x 200 mL). The combined organics layers were washed with brine (100 mL), filtered and evaporated to dryness. The residue was purified by Si0₂ chromatography (EtOAc in hexanes, 0 to 50% gradient) and afforded the title compound (3.40 g, 9.38 mmol, 60%) as a white solid.

[253] Benzyl tert-butyl ((lS,3R)-l-methylcvclohexane-l,3-diyl)dicarbamate and benzyl tert- -l-methylcvclohexane-l,3-diyl)dicarbamate

(+/-)

[254] Both enantiomers of (+/-)-Benzyl tert-butyl ((lS,3R)-l-methylcyclohexane-l,3-diyl)dicarbamate (3.40 g, 9.38 mmol) were separated using preparative chiral HPLC (Chiralpak IA, 5 urn, 20×250 mm; hex/MeOH/DCM = 90/5/5) to yield both compounds benzyl tert-butyl ((lS,3R)-l-methylcyclohexane-l,3-diyl)dicarbamate (1.20 g, 3.31 mmol) and benzyl iert-butyl ((lR,3S)-l-methylcyclohexane-l,3-diyl)dicarbamate (1.15 g, 3.17 mmol) as white solids.

255 Benzyl ((lS,3R)-3-amino-l-methylcvclohexyl)carbamate hydrochloride

[256] A solution of benzyl tert-butyl ((lS,3R)-l-methylcyclohexane-l,3-diyl)dicarbamate (700 mg, 1.93 mmol) in DCM (19 mL) was treated with a 4M solution of HCI in dioxane (9.66 mL, 38.6 mmol) and stirred 16h at rt. The mixture was evaporated to dryness and afforded the title compound (577 mg, 1.93 mmol, 100%) as a white solid which was used in the next step without further purification.

[257] (lS,3R)-Benzyl-3-(5-chloro-4-(l-(phenylsulfonyl)-lH ndol-3-yl)pyrimidin-2-ylamino)-1-methylcyclohexylcarbamate

[258] A solution of 3-(2,5-dichloropyrimidin-4-yl)-l-(phenylsulfonyl)-lH-indole (1.02 g, 2.53 mmol), benzyl (( iS,3 )-3- amino- 1 -methylcyclohexyljcarbaniaie hydrochloride (577 mg, 1.93 mmol) and DIPEA (1.15 mL, 6.60 mmol) in NMP (11 mL) was heated at 135 °C (microwave) for 60 min. The cooled mixture was diluted with EtOAc (250 mL), washed with H₂0 (100 mL), brine (100 mL), dried over MgS0₄, filtered and evaporated to dryness. The residue was purified by Si0₂ chromatography (EtOAc in DCM, 0 to 50% gradient) and afforded the title compound (747 mg, 1.19 mmol, 54%) as a yellow foam.

[259] (lS,3R)-N-(5-chloro-4-(l-(phenylsulfonyl)-lH ndol-3-yl)pyrimidin-2-yl)-3-methylcvclohexane-l,3-diamine

[260] A cooled (-78°C) solution of (lS,3R)-benzyl-3-(5-chloro-4-(l-(phenylsulfonyl)-lH-indol-3-yl)pyrimidin-2-ylamino)-l-methylcyclohexylcarbamate (747 mg, 1.19 mmol) in DCM (39 mL) was treated with a 1M solution of BBr₃ in DCM (2.83 mL, 2.83 mmol) and was slowly warmed up to rt. MeOH (10 mL) was added to the mixture was the resulting solution was stirred lh at rt. The resulting mixture was evaporated to dryness. The residue was purified by reverse phase chromatography (C₁₈, H₂0/ACN +0.1% HC0₂H, 0 to 60% gradient) and afforded the title compound (485 mg, 0.978 mmol, 83%) as a yellow solid.

[261] 5-amino-N-( ( lS,3R)-3-( 5-chloro-4-(l-(phenylsulfonyl)-lH-indol-3-yl)pyrimidin-2-ylamino)-l-methylcvclohexyl)picolinamide

[262] A solution of (lR,3S)-N-(5-chloro-4-(l-(phenylsulfonyl)-lH-indol-3-yl)pyrimidin-2-yl)-3-methylcyclohexane-l,3-diamine (75.0 mg, 0.150 mmol) and 5-aminopicolinic acid (25.0 mg, 0.180 mmol) in DMF (5.0 mL) was treated with HBTU (86.0 mg, 0.230 mmol) and DIPEA (79 μί, 0.45 mmol). The resulting mixture was stirred 5h at rt and diluted with MeTHF (50 mL) and saturated NaHC0₃ (50 mL). The layers were separated and the aqueous layer was extracted with MeTHF (2 x 50 mL). The combined organic layers were dried over MgS0₄, filtered and evaporated to dryness. The residue was purified by Si0₂ chromatography (EtOAc in DCM, 0 to 50% gradient) and afforded the title compound (74.0 mg, 0.120 mmol, 79%) as a light yellow oil.

[263] 5-amino-N-((lS,3R)-3-(5-chloro-4-(lH ndol-3-yl)pyrimidin-2-ylamino)-l-methylcyclohexyDpicolinamide

[264] A solution of 5-amino-N-((lS,3R)-3-(5-chloro-4-(l-(phenylsulfonyl)-lH-indol-3-yl)pyrimidin-2-ylamino)-l-methylcyclohexyl)picolinamide (74.0 mg, 0.120 mmol) in 1,4-dioxane (4.0 mL) was treated with a 2M solution of NaOH in H₂0 (960 μί, 4.78 mmol) and heated at 60°C for lh. The cooled mixture was diluted with MeTHF (30 mL) and H₂0 (30 mL). The layers were separated and the aqueous layer was extracted with MeTHF (3 x 30 mL). The combined organic layers were dried over MgS0₄, filtered and evaporated to dryness affording the title compound (57.0 mg, 0.120 mmol, 100%) as a light yellow oil which was used in the next step without further purification.

[265] N-((lS,3R)-3-(5-chloro-4-(lH ndol-3-yl)pyrimidin-2-ylamino)-l-methylcvd^

( ( E)-4-(dimethylamino)but-2-enamido )picolinamide ( Compound 267)

[266] A cooled (-78°C) solution of 5-amino-N-((lS,3R)-3-(5-chloro-4-(lH-indol-3-yl)pyrimidin-2-ylamino)-l-methylcyclohexyl)picolinamide (57.0 mg, 0.120 mmol) and DIPEA (104 0.598 mmol) in THF/NMP (4.0 mL/1.0 mL) was treated with a 54.2 mg/mL solution of (E)-4-bromobut-2-enoyl chloride in DCM (104 μί, 0.598 mmol). The resulting mixture was stirred 4h at -78°C before addition of a 2M solution of dimethylamine in THF (359 μί, 0.717 mmol). The resulting mixture was warmed up to rt and stirred 45min at this temperature before being evaporated to dryness. The residue was purified by reverse phase chromatography (C₁₈, H₂0/ACN +0.1% HC0₂H, 0 to 50% gradient) and afforded the title compound (15.0 mg, 0.026 mmol, 22%) as a white solid after lyophilization. LCMS: Calculated: 587.12; Found (M+H⁺): 587.39. 1H NMR (500 MHz, DMSO) δ 11.84 (s, 1H), 10.54 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H), 8.64 (s, 1H), 8.47 (s, 1H), 8.25 (dd, J = 8.6, 2.4 Hz, 2H), 7.98 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 7.7 Hz, 1H), 7.25 – 7.07 (m, 3H), 6.81 (dt, J = 15.5, 5.8 Hz, 1H), 6.29 (d, J = 15.4 Hz, 1H), 4.23 – 4.08 (m, 1H), 3.08 (dd, J = 5.7, 1.1 Hz, 2H), 2.46 – 2.37 (m, 1H), 2.18 (s, 6H), 2.04 – 1.95 (m, 2H), 1.87 – 1.70 (m, 3H), 1.63 – 1.46 (m, 4H), 1.39 – 1.26 (m, 1H).

Ref

[1]. Hu S, et al. Discovery and characterization of SY-1365, a selective, covalent inhibitor of CDK7. Cancer Res. 2019 May 7.[2]. Shanhu Hu, et al. SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors.

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CN(C)C\C=C\C(=O)Nc1ccc(nc1)C(=O)N[C@]1(C)C[C@@H](CCC1)Nc1ncc(Cl)c(n1)c1c[NH]c2ccccc21

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SY 5609

September 18, 2021 2:12 pm / Leave a comment

[ Fig. 0001] [ Fig. 0002] [ Fig. 0003] [ Fig. 0004]

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016058544&tab=FULLTEXT&_cid=P10-KTPTVL-74436-1

SY 5609

CAS 2519828-12-5

Cancer, solid tumor

PHASE 1

A highly selective and potent oral inhibitor of cyclin-dependent kinase 7 (CDK7) for potential treatment of advanced solid tumors that harbor the Rb pa thway alterations (Syros Pharmaceuticals, Inc., Cambridge, Massachusetts, USA)

SY-5609 is an oral non-covalent CDK7 inhibitor in early clinical development at Syros Pharmaceuticals for the treatment of patients with advanced breast, colorectal, lung or ovarian cancer, or with solid tumors of any histology that harbor Rb pathway alterations.

OriginatorSyros Pharmaceuticals
ClassAntineoplastics; Small molecules
Mechanism of ActionCyclin-dependent kinase-activating kinase inhibitors
Phase IBreast cancer; Solid tumours

05 Aug 2021Roche plans the phase I/Ib INTRINSIC trial in Colorectal cancer (Combination therapy, Metastatic disease) in USA, Canada, Italy, South Korea, Spain and United Kingdom (NCT04929223)
05 Aug 2021Roche and Syros Pharmaceuticals enters into a clinical trial collaboration to evaluate atezolizumab in combination with SY 5609 in a clinical trial
05 Aug 2021Syros Pharmaceuticals plans a phase I trial in Cancer in second half of 2021
NCT04247126
https://clinicaltrials.gov/ct2/show/NCT04247126

At #ESMO21, we will be presenting new preclinical and clinical data on SY-5609, our highly selective and potent oral CDK7 inhibitor. #oncology #biotech Learn more: https://lnkd.in/gqYmWYhb

A Promising Approach for Difficult-to-Treat Cancers

SY-5609 is a highly selective and potent oral inhibitor of the cyclin-dependent kinase 7 (CDK7) in a Phase 1 dose-escalation trial in patients with advanced breast, colorectal, lung, ovarian or pancreatic cancer, or with solid tumors of any histology that harbor Rb pathway alterations.

SY-5609 represents a new approach to treating cancer that we believe has potential in a range of difficult-to-treat cancers. It has shown robust anti-tumor activity, including complete regressions, in preclinical models of breast, colorectal, lung and ovarian cancers at doses below the maximum tolerated dose. In preclinical studies of breast, lung and ovarian cancers, deeper and more sustained responses were associated with the presence of Rb pathway alterations. SY-5609 has also shown substantial anti-tumor activity in combination with fulvestrant in treatment-resistant models of estrogen receptor-positive breast cancer, including those resistant to both fulvestrant and a CDK4/6 inhibitor. Early dose-escalation data demonstrated proof-of-mechanism at tolerable doses.

Syros to Present New Data from Phase 1 Clinical Trial of SY-5609 in Oral Presentation at ESMO Congress 2021SEPTEMBER 13, 2021

Management to Host Conference Call on Monday, September 20, 2021 at 4:00 p.m. ET

CAMBRIDGE, Mass.–(BUSINESS WIRE)– Syros Pharmaceuticals (NASDAQ:SYRS), a leader in the development of medicines that control the expression of genes, today announced that it will present new data from the dose-escalation portion of the Phase 1 clinical trial of SY-5609, its highly selective and potent oral cyclin-dependent kinase 7 (CDK7) inhibitor, at the ESMO Congress 2021, taking place virtually September 16-21, 2021. The oral presentation will include safety, tolerability, and initial clinical activity data for SY-5609 in patients with breast, colorectal, lung, ovarian and pancreatic cancers, as well as in patients with solid tumors of any histology harboring Rb pathway alterations.

In separate poster presentations, Syros will present new preclinical data evaluating the antitumor and pharmacodynamic activity of intermittent dosing regimens for SY-5609 in ovarian cancer models, as well as new preclinical data evaluating antitumor activity of SY-5609 as a single agent and in combination with chemotherapy in KRAS-mutant models.

The abstracts for the two poster presentations are now available online on the ESMO conference website at: https://www.esmo.org/meetings/esmo-congress-2021/abstracts, and the presentations will become available for on-demand viewing starting September 16 at 08:30 CEST (September 16 at 2:30 a.m. ET). The abstract for the oral presentation on the Phase 1 dose-escalation data will remain embargoed until September 17 at 00:05 CEST (September 16 at 6:05 p.m. ET).

Details of the oral presentation are as follows:

Presentation Title: Tolerability and Preliminary Clinical Activity of SY-5609, a Highly Potent and Selective Oral CDK7 Inhibitor, in Patients with Advanced Solid Tumors
Session Date & Time: Monday, September 20, 17:30-18:30 CEST (11:30-12:30 p.m. ET)
Presentation Time: 17:55-18:00 CEST (11:55-12:00 p.m. ET)
Session Title: Mini Oral Session: Developmental Therapeutics
Presenter: Manish Sharma, M.D., START Midwest
Abstract Number: 518MO

Details of the poster presentations are as follows:

Presentation Title: Preclinical Evaluation of Intermittent Dosing Regimens on Antitumor and PD Activity of SY-5609, a Potent and Selective Oral CDK7 Inhibitor, in Ovarian Cancer Xenografts
Abstract Number: 14P
Presentation Title: SY-5609, a Highly Potent and Selective Oral CDK7 inhibitor, Exhibits Robust Antitumor Activity in Preclinical Models of KRAS Mutant Cancers as a Single Agent and in Combination with Chemotherapy
Abstract Number: 13P

Conference Call Information

Syros will host a conference call on Monday, September 20, 2021 at 4:00 p.m. ET to discuss the new clinical and preclinical data for SY-5609, which will be presented at the ESMO Congress 2021.

To access the live conference call, please dial 866-595-4538 (domestic) or 636-812-6496 (international) and refer to conference ID 4648345. A webcast of the call will also be available on the Investors & Media section of the Syros website at www.syros.com. An archived replay of the webcast will be available for approximately 30 days following the conference call.

About Syros Pharmaceuticals

Syros is redefining the power of small molecules to control the expression of genes. Based on its unique ability to elucidate regulatory regions of the genome, Syros aims to develop medicines that provide a profound benefit for patients with diseases that have eluded other genomics-based approaches. Syros is advancing a robust clinical-stage pipeline, including: tamibarotene, a first-in-class oral selective RARα agonist in RARA-positive patients with higher-risk myelodysplastic syndrome and acute myeloid leukemia; SY-2101, a novel oral form of arsenic trioxide in patients with acute promyelocytic leukemia; and SY-5609, a highly selective and potent oral CDK7 inhibitor in patients with select solid tumors. Syros also has multiple preclinical and discovery programs in oncology and monogenic diseases.

View source version on businesswire.com: https://www.businesswire.com/news/home/20210913005090/en/

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020219926&tab=PCTDESCRIPTION&_cid=P10-KTPT14-70600-1

CN(C)C\C=C\C(=O)Nc1ccc(cc1)C(=O)Nc1cccc(c1)Nc1ncc(Cl)c(n1)c1c[NH]c2ccccc21

THZ1; 1604810-83-4; THZ-1; HY-80013

CLIP

SY 1365 MEVOCICLIB, CAS 1816989-16-8

CN(C)C\C=C\C(=O)Nc1ccc(nc1)C(=O)N[C@]1(C)C[C@@H](CCC1)Nc1ncc(Cl)c(n1)c1c[NH]c2ccccc21

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016058544&tab=FULLTEXT&_cid=P10-KTPTVL-74436-1

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=US206490062&tab=PCTDESCRIPTION&_cid=P10-KTPTVL-74436-1

3-fluoro-4-(methylamino)-N-[(1S,3R)-1-methyl-3-[[4-(7-methyl-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]cyclohexyl]benzamide (Compound 130)

3-chloro-4-[[4-(dimethylamino)-3-hydroxy-butanoyl]amino]-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 129)

4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)-1-methylcyclohexyl)benzamide (Compound 128)

4-amino-3-fluoro-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 127)

4-amino-N-((1S,3R)-3-((5-chloro-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 126)

4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indazol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 124)

Example 25 Synthesis of N1-(4-(((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)carbamoyl)phenyl)oxalamide (Compound 113)

Example 24 Synthesis of N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)-4-(4-(dimethylamino)butanamido)benzamide (Compound 105)

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=US319558608&tab=PCTDESCRIPTION&_cid=P10-KTPTVL-74436-1

4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)tricyclo[3.3.1.1^3,7]decanyl)benzamide (Compound 100).

+/−)-4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-5 hydroxycyclohexyl)benzamide (Compound 101)

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide (Compound 102)

(1S,3R)-N-(4-aminophenyl)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexanecarboxamide (Compound 106)

4-amino-N-((1S,3R)-3-(5-cyclopropyl-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide.HCl (Compound 103)

4-amino-N-((1S,3R)-3-(5-chloro-4-(pyridin-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide (Compound 108)

4-amino-N-((1S,3R)-3-(5-cyano-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide (Compound 107)

(+/−)-4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-5-fluorocyclohexyl)benzamide (Compound 110)

4-amino-N-(5-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)bicyclo[3.1.1]heptan-1-yl)benzamide (Compound 104)

4-amino-N4(1R,5S)-5-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-3,3-difluorocyclohexyl)benzamide (Compound 115)

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzenesulfonamide (Compound 109).

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-2-fluorobenzamide (Compound 112)

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-3-fluorobenzamide (Compound 111).

(+/−)-4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-1-methylcyclohexyl)benzamide (Compound 116).

N-((1S,3R)-3-(4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-4-aminobenzamide (Compound 114).

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-2-morpholinobenzamide(Compound 117).

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyridin-2-ylamino)cyclohexyl)benzamide (Compound 118).

3-amino-N-(trans-4-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide.HCl (Compound 119).

(1S,3R)-N1-(R)-1-(4-aminophenyl)-2,2,2-trifluoroethyl)-N3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)cyclohexane-1,3-diamine (Compound 120).

(1S,3R)-N1-(4-aminobenzyl)-N3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)-N1-methylcyclohexane-1,3-diamine.HCl (Compound 122).

4-amino-N-((1S,3R)-3-(5-chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide.HCl (Compound 123).

Synthesis of 5-amino-N-((1S,3R)-3-(5-chloro-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)picolinamide (Compound 125)

Synthesis of N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)-4-(4-(dimethylamino)butanamido)benzamide (Compound 105)

Synthesis of N1-(4-(((1S,3R)-3-)(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)carbamoyl)phenyl)oxalamide (Compound 113)

Synthesis of 4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indazol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 124)

Synthesis of 4-amino-N-((1S,3R)-3-((5-chloro-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 126)

Synthesis of 4-amino-3-fluoro-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 127).

Synthesis of 4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl) pyrimidin-2-yl)amino)-1-methylcyclohexyl)benzamide (Compound 128)

Synthesis of 3-chloro-4-[[4-(dimethylamino)-3 hydroxy-butanoyl]amino]-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 129).

Synthesis of 3-fluoro-4-(methylamino)-N-[(1S,3R)-1-methyl-3-[[4-(7-methyl-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]cyclohexyl]benzamide (Compound 130)

//////////////SY 5609, 2519828-12-5, Cancer, solid tumor, PHASE 1, SYROS

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ABBV 744

March 11, 2020 11:32 am / Leave a comment

ABBV-744 Chemical Structure

ABBV 744

N-Ethyl-4-[2-(4-fluoro-2,6-dimethylphenoxy)-5-(1-hydroxy-1-methylethyl)phenyl]-6,7-dihydro-6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide

1H-Pyrrolo[2,3-c]pyridine-2-carboxamide, N-ethyl-4-[2-(4-fluoro-2,6-dimethylphenoxy)-5-(1-hydroxy-1-methylethyl)phenyl]-6,7-dihydro-6-methyl-7-oxo-

Molecular Weight	491.55
Formula	C₂₈H₃₀FN₃O₄
CAS No.	2138861-99-9

ABBV-744 is a highly BDII-selective BET bromodomain inhibitor, used in the research of inflammatory diseases, cancer, and AIDS.

Acute Myeloid Leukemia (AML)

Phase I, AbbVie is evaluating oral agent ABBV-744 in early clinical trials for the treatment of metastatic castration resistant prostate cancer (CRPC) and for the treatment of relapsed or refractory acute myeloid leukemia (AML).

PATENT

WO 2017177955

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017177955&tab=FULLTEXT

Bromodomains refer to conserved protein structural folds which bind to N-acetylated lysine residues that are found in some proteins. The BET family of bromodomain containing proteins comprises four members (BRD2, BRD3, BRD4 and BRDt) . Each member of the BET family employs two bromodomains to recognize N-acetylated lysine residues typically, but not exclusively those found on transcription factors (Shi, J., et al. Cancer Cell 25 (2) : 210-225 (2014) ) or on the amino-terminal tails of histone proteins. Numbering from the N-terminal end of each BET protein the tandem bromodomains are typically labelled Binding Domain I (BDI) and Binding Domain II (BDII) . These interactions modulate gene expression by recruiting transcription factors to specific genome locations within chromatin. For example, histone-bound BRD4 recruits the transcription factor P-TEFb to promoters, resulting in the expression of a subset of genes involved in cell cycle progression (Yang et al., Mol. Cell. Biol. 28: 967-976 (2008) ) . BRD2 and BRD3 also function as transcriptional regulators of growth promoting genes (LeRoy et al., Mol. Cell 30: 51-60 (2008) ) . BET family members were recently established as being important for the maintenance of several cancer types (Zuber et al., Nature 478: 524-528 (2011) ； Mertz et al； Proc. Nat’l. Acad. Sci. 108: 16669-16674 (2011) ； Delmore et al., Cell 146: 1-14, (2011) ； Dawson et al., Nature 478: 529-533 (2011) ) . BET family members have also been implicated in mediating acute inflammatory responses through the canonical NF-KB pathway (Huang et al., Mol. Cell. Biol. 29: 1375-1387 (2009) ) resulting in the upregulation of genes associated with the production of cytokines (Nicodeme et al., Nature 468: 1119-1123, (2010) ) . Suppression of cytokine induction by BET bromodomain inhibitors has been shown to be an effective approach to treat inflammation-mediated kidney disease in an animal model (Zhang, et al., J. Biol. Chem. 287: 28840-28851 (2012) ) . BRD2 function has been linked to pre-disposition for dyslipidemia or improper regulation of adipogenesis, elevated inflammatory profiles and increased susceptibility to autoimmune diseases (Denis, Discovery Medicine 10: 489-499 (2010) ) . The human immunodeficiency virus utilizes BRD4 to initiate transcription of viral RNA from stably integrated viral DNA (Jang et al., Mol. Cell, 19: 523-534 (2005) ) . BET bromodomain inhibitors have also been shown to reactivate HIV transcription in models of latent T cell infection and latent monocyte infection (Banerjee, et al, J. Leukocyte Biol. doi: 10.1189/jlb. 0312165) . BRDt has an important role in spermatogenesis that is blocked by BET bromodomain inhibitors (Matzuk, et al., Cell 150: 673-684 (2012) ) . Thus, compounds that inhibit the binding of BET family bromodomains to their cognate acetylated lysine proteins are being pursued for the treatment of cancer, inflammatory diseases, kidney diseases, diseases involving metabolism or fat accumulation, and some viral infections, as well as for providing a method for male contraception. Accordingly, there is an ongoing medical need to develop new drugs to treat these indications.

FIDANZE, Steven D., et al. BROMODOMAIN INHIBITORS. WO 2017177955 A1.

////////////ABBV 744, Acute Myeloid Leukemia, AML, Phase 1 , AbbVie

CC(O)(C)C1=CC(C(C2=C3NC(C(NCC)=O)=C2)=CN(C)C3=O)=C(OC4=C(C)C=C(F)C=C4C)C=C1

LNS-8801 (SRR G-1)

February 12, 2020 10:52 am / Leave a comment

LNS-8801 (SRR G-1)

CAS 925419-53-0

Chemokine receptor-like 2 agonist

C₂₁ H₁₈ Br N O_3, 412.28

Ethanone, 1-[(3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-

1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one

1-[(3aS,4R,9bR)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]ethanone
G 1
G 1 (estrogen receptor agonist)

Linnaeus Therapeutics Inc

NOTE 1-((3aR,4S,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one (RSS G-1 or LNS8812)

Novel crystalline forms of an enantiomerically purified LNS-8801 (SRR G-1), or a derivative useful for treating cancer, hearing disorder, depression and myocardial infarction.

Linnaeus Therapeutics is developing LNS-8801, a G-protein coupled estrogen receptor (GPER) agonist, an oral capsule formulation for the treatment of cancer including melanoma, pancreatic ductal adenocarcinoma, non-small cell lung cancer, solid tumor, hematological cancer, advanced cancer and colon carcinoma.

In October 2019, a phase I trial was initiated in patients with locally advanced or metastatic cancer.

PATENT

WO-2020023391

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020023391&tab=PCTDESCRIPTION&_cid=P21-K6IRN7-26108-1

Novel crystalline and amorphous forms of 1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one (here referred to as SRR-G-1 or LNS-8801) and 1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one (RSS G-1 or LNS8812) and its derivative (having chiral purity of >90%; substantially free of its opposite enantiomer; designated as Form A-C) and their co-crystal (eg benzenesulfonic acid) and compositions and combinations comprising them are claimed. Also claimed is their use for treating cancers (disorder expresses GPER), depression, myocardial infarction, osteoporosis, rheumatoid arthritis, insomnia, inflammatory bowel disease, Crohn’s disease, thymic atrophy and hearing disorder and further type-2 diabetes. Further claimed are methods for reducing the likelihood of pregnancy after intercourse, increasing skin pigmentation and skin protection and preventing cancer, reoccurrence of cancer and inhibiting the progression of cancer

Embodiments of the present invention relate to an enantiomerically purified agonist of the G-protein coupled estrogen receptor (GPER), pharmaceutical compositions comprising an enantiomerically purified SRR G-l, or a derivative thereof, and methods of treating disease states and conditions in subjects in need thereof, and methods of treating disease states and conditions mediated through GPER receptors.

[0003] Estrogens mediate multiple complex physiological responses throughout the body. The responses are in turn mediated through the binding of estrogen to receptors. The classical receptors bind steroids, such as estrogen, and are soluble cytoplasmic/nuclear proteins that function as transcription factors. These receptors are known as estrogen receptor alpha and beta (two closely related proteins) that mediate transcriptional activity. GPER is a 7-transmembrane G protein-coupled receptor that also binds to estrogen with high affinity (K_d~6 nM) and mediates rapid cellular responses including cyclic adenosine monophosphate signaling, calcium mobilization and phosphatidylinositol 3,4,5 trisphosphate production.

[0004] Diseases whose development, progression, and or response to therapy, may be influenced by endogenous, and/or pharmacologic activation of GPER signaling include cancer (including the prevention of cancer, prevention of the reoccurrence of cancer, and the inhibition of the progression of cancer; and particularly melanoma, pancreatic, lymphomas, uveal melanoma, non-small cell lung cancer, breast, reproductive and other hormone-dependent cancers, leukemia, colon cancer, prostate, bladder cancer), reproductive (genito-urological) including endometritis, prostatitis, polycystic ovarian syndrome, bladder control, hormone-related disorders, hearing disorders, cardiovascular conditions including hot flashes and profuse sweating, hypertension, stroke, obesity, diabetes, osteoporosis, hematologic diseases, vascular diseases or conditions such as venous thrombosis, atherosclerosis, among numerous others and disorders of the central and peripheral nervous system, including depression, insomnia, anxiety, neuropathy, multiple sclerosis, neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, as well as inflammatory bowel disease, Crohn’s disease, coeliac (celiac) disease and related disorders of the intestine.

Embodiments of the present invention encompass compounds comprising enantiomerically purified G-l and methods of use in the treatment of diseases. G-l is a racemic mixture of the enantiomers l-((3aS,4R,9bR)-4-(6-bromobenzo[if][l,3]dioxol-5-yl)-3a.4.5.9b-tetrahydro-3H-cyclopenta|c |quinolin-8-yl)ethan- 1 -one (henceforth referred to as “SRR G-l” or “LNS8801”) and l-((3aR,4S,9bS)-4-(6-bromobenzo[if][l,3]dioxol-5-yl)-3a.4.5.9b-tetrahydro-3H-cyclopenta|c |quinolin-8-yl)ethan- 1 -one (henceforth referred to as “RSS G-l” or“LNS8812”).

Enantiomerically purified G-l has been purified in favor of its l-((3aS,4R,9bR)-4-(6-bromobenzo[ri][l,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-l-one enantiomer over the corresponding l-((3aR,4S,9bS)-4-(6-bromobenzo[ri][l,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-l-one enantiomer. Unless specifically described, SRR Gl, or a derivative thereof includes, any physical form, including an amorphous form or any crystalline solid forms such as A, B, C or combinations thereof.

Experimental Section

Scheme 1

[0166] A synthesis of G-l is described in Org. Biomol Client., 2010,8, 2252-2259, which is hereby incorporated by reference, and depicted in Scheme 1. A catalytic amount of Sc(OTf)₃ (0.492 g, 1.0 mmol) in anhydrous acetonitrile (2.0 cm³) was added to the mixture of 6-bromopiperonal (2.30 g, 10.0 mmol). / aminoacetophenone (1.30 g, 10.0 mmol) and cyclopentadiene (3.30 g, 50.0 mmol) in acetonitrile (25 cm³). The reaction mixture was stirred at ambient temperature (~23 ^°C) for 2.0 h. The volatiles were removed in vacuo. The residue was purified by preparative silica gel column chromatography using ethyl acetate-hexanes (10 : 90) to provide G-l (4.03 g, 98%, dr. = 94 : 6) as a white solid. The minor diastereomer was substantially removed by recrystallization to yield a racemic mixure of SRR G-l and RSS G-l.

Example 1: Isolation of the SRR G-l and RSS G-l Enantiomers

[0167] Starting with a highly purified sample of G-l, (±)l-(4-(6-bromobenzo[ri][l,3]dioxol-5-yl)-(3aS*,4R*,9bR*)-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-l-one, (99.4% purity) purchased from Tocris Bioscience, the material was dissolved in 90: l0:0. l(v/v/v) methyl tert-butyl ether / ethanol / diethyl amine and subjected to preparative chromatography using a column packed with Chrialpak 1A resin. Elution was conducted with 90: l0:0. l(v/v/v) methyl tert-butyl ether / ethanol / diethyl amine and the fractions corresponding to each enantiomer were collected and concentered to a solid. The early eluting enantiomer was determined to be the SRR G-l enantiomer by single crystal x-ray structural analysis.

Example 2: SRR G-l Polymorph Screen

[0168] Starting with SRR G-l prepared according to Example 1, a polymorph screening study was conducted analyzing the solids isolated from slurry of the solid, or from fast and slow evaporation and cooling of solutions (Table 1). Two crystal forms were identified, an anhydrous form designated Form A and mono dichloromethane solvate designated Form B. On exposure to elevated temperature the Form B crystal form desolvates to form the Form C crystal form. Amorphous material was generated from purified SRR G-l by two different methods; quick evaporating a diethyl ether solution of SRR G-l or rotary evaporating from a solution of a dichloromethane solution of SRR G-l.

Table 1

PAPER

Anti-Cancer Agents in Medicinal Chemistry (2019), 19(6), 760-771.

/////////RSS G-1, LNS8812, phase I, Locally advanced, metastatic cancer, LNS-8801, SRR G-1, Linnaeus Therapeutics

CC(=O)c1cc4c(cc1)N[C@@H](c2cc3OCOc3cc2Br)[C@H]5CC=C[C@@H]45

CC-90010

February 11, 2020 8:52 am / Leave a comment

CC-90010

C₂₁ H₂₁ N O₄ S, 383.46

CAS 1706738-98-8

1(2H)-Isoquinolinone, 4-[2-(cyclopropylmethoxy)-5-(methylsulfonyl)phenyl]-2-methyl-

4-[2-(Cyclopropylmethoxy)-5-(methylsulfonyl)phenyl]-2-methyl-1(2H)-isoquinolinone
4-[2-(Cyclopropylmethoxy)-5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one
4-[2-(Cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one

Quanticel Pharmaceuticals Inc, Michael John Bennett Juan Manuel Betancort Amogh Boloor Stephen W. Kaldor Jeffrey Alan Stafford James Marvin Veal

Celgene (now a wholly owned subsidiary of Bristol-Myers Squibb ) , following its acquisition of Quanticel , is developing CC-90010, an oral inhibitor of BET (bromodomain and extraterminal) proteins, for the potential treatment of solid tumors and non-Hodgkin’s lymphoma. In August 2019, a phase I trial for diffuse astrocytoma, grade III anaplastic astrocytoma and recurrent glioblastoma was planned

PATENT

WO2018075796 claiming solid composition comprising a bromodomain inhibitor, preferably 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one in crystalline form A.

PATENT

WO2015058160 (compound 89, page 103).

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=9B64008287A0D105A68DDF31141C7419.wapp1nA?docId=WO2015058160&tab=PCTDESCRIPTION

Example 89: 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-l-one

Step 1 : 2-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoquinolin-l-one

[00344] A suspension of 4-bromo-2-methylisoquinolin-l-one (100 mg, 0.42 mmol), bis(pinacolato)diboron (214 mg, 0.84 mmol), Pd(dppf)Cl₂ (31 mg, 0.04 mmol) and potassium acetate (104 mg, 1.05 mmol) in dioxane (2 mL) under nitrogen was warmed up to 90 °C for 135 minutes. It was then cooled down to room temperature and diluted with ethyl acetate (8 mL). The mixture was washed with aqueous saturated solution of NaHC0₃ (8 mL) and brine (8 mL). The organic phase was separated, dried over Na₂S0₄, filtered and concentrated under reduced pressure. The residue was purifed by normal phase column chromatography (10-90% EtOAc/Hexanes) to give the title compound (44 mg, 37%). 1H NMR (CDC1₃, 400 MHz) δ 8.43 (d, J = 7.9 Hz, 1 H), 8.40 (dd, J = 8.2 Hz, 0.9 Hz, 1 H), 7.68 (s, 1 H), 7.65 (ddd, J = 8.2, 8.2, 1.1 Hz, 1 H), 7.46 (t, J = 7.5 Hz, 1 H), 3.63 (s, 3H), 1.38 (s, 12H). LCMS (M+H)⁺ 286. Step 2: 4-[2-(cyclopropylmethox -5-methylsulfonylphenyl]-2-methylisoquinolin-l-one

[00345] The title compound was prepared in a manner similar to Example 18, step 3, substituting 2-bromo-l-(cyclopropylmethoxy)-4-methylsulfonylbenzene for 4-bromo-2-methylisoquinolin-l(2H)-one and 2-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoquinolin-l-one for N-benzyl-2-methoxy-5-(tetramethyl-l,3,2-dioxaborolan-2-yl)benzamide. 1H NMR (DMSO-d6, 400 MHz) δ 0.09 (m, 2 H), 0.29 (m, 1H), 0.35 (m, 1H),

0.94 (m, 1H), 3.22 (s, 3H), 3.57 (s, 3H), 3.95 (m, 2H), 7.16 (d, J = 7.9 Hz, 1H), 7.37 (d, J =

8.8 Hz, 1H), 7.53 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.97 (dd, J = 8.8,

2.4 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H). LCMS (M+H)⁺ 384.

[00346] Alternatively, 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-l-one can be prepared as described below.

Step 1 : 2-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoquinolin-l-one

[00347] A mixture of 4-bromo-2-methylisoquinolin-l-one (8.0 g, 33.6 mmol),

bis(pinacolato)diboron (17.1 g, 67.2 mmol), KOAc (6.6 g, 67.2 mmol), Pd₂(dba)3 (3.1 g, 3.36 mmol) and X-Phos (1.6 g, 3.36 mmol) in anhydrous dioxane (200 mL) was stirred at 60 °C for 12 h. The reaction mixture was concentrated and the residue was purified by column chromatography on silica gel (PE : EA = 15 : 1) to give the title compound (6.0 g, 62 %) as a solid.

Step 2: 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-l-one

[00348] The title compound from Step 1 (5.0 g, 17.5 mmol), 2-bromo-l-(cyclopropylmethoxy)-4-methylsulfonylbenzene (6.4 g, 21 mmol), K₃PO₄ (9.3 g, 43.9 mmol) and Pd(dppf)Cl₂ (1.4 g, 1.75 mmol) in a dioxane/water (100 mL / 10 mL) mixture were stirred at 60 °C for 12 hrs. The reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (EA : DCM = 1 : 4).

Appropriate fractions were combined and concentrated under reduce pressure. The resultant solid was recrystallized from DCM / MTBE (1 : 1, 50 mL) to give the title compound (4.0 g, 60 %) as a white solid. 1H NMR: (CDC1₃, 400 MHz) δ 8.51 (dd, Ji = 8.0 Hz, J₂ = 0.8 Hz, 1 H), 7.98 (dd, Ji = 8.4 Hz, J₂ = 2.4 Hz, 1 H), 7.86 (d, J = 2.4 Hz, 1 H), 7.53 (m, 2 H), 7.16 (d, J = 7.6 Hz, 1 H), 7.10 (m, 2 H), 3.88 (m, 2 H), 3.66 (s, 3 H), 3.09 (s, 3 H), 1.02-0.98 (m, 1 H), 0.44-0.38 (m, 2 H), 0.11-0.09 (m, 2 H). LCMS: 384.1 (M+H)⁺

Patent

WO-2020023438

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020023438&tab=PCTDESCRIPTION&_cid=P10-K6HCMJ-20465-1

A process for preparing bromodomain inhibitor, particularly 4-[2(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one (having HPLC purity of 99%; compound 1; (hereafter referred to as C-90010)) and its hydrates, solvates, prodrugs and salts comprising the reaction of a substituted 4-(methylsulfonyl)phenol compound with a quinoline derivative, followed by purification is claimed. Also claimed are novel intermediates of CC-90010 and their processes for preparation. Further claimed are novel crystalline form of CC-90010. CC-90010 is known and disclosed to be a bromodomain containing protein inhibitor, useful for treating cancer.

Scheme 10: Synthesis of Compound 1

[0090] Acetonitrile (1.6L) was charged to a mixture of Compound 2 (156.7g, 460 mmol), Compound 3 (lOOg, 420 mmol) and potassium phosphate tribasic (223g, l.OSmol). Agitation

was begun and water (400mL) charged to the batch. The system was vacuum purged three times with nitrogen and charged with Pd(PPh3)2Cl2 (2.9g, 4 mmol) and the system vacuum purged three times with nitrogen. The batch was heated to about 65 to about 75 °C (or any temperature in between and including these two values) and contents stirred for at least about 16 hours until reaction was complete by HPLC analysis. The batch was cooled to about 60 to about 70 °C (or any temperature in between and including these two values), agitation halted and the mixture allowed to settle. The bottom aqueous layer was removed. Water (150mL) and acetonitrile (700mL) were charged at about 60 to about 70°C (or any temperature in between and including these two values). Ecosorb C-941 (15g) and Celite (lOg) were charged to the reaction vessel at about 60 to about 70°C (or any temperature in between and including these two values). After lh, the mixture was filtered to remove solids. The solids were washed twice each with 18% water in acetonitrile (500 mL) at about 60 to about 70°C (or any temperature in between and including these two values). The filtrates were combined and concentrated under atmospheric pressure to a final volume of 1.5L. The batch was cooled to about 60 to about 65°C (or any temperature in between and including these two values) and seeded with Compound 1 (1 g). After lh, water (500 mL) was charged over at least 1 hour at about 60 to about 65°C (or any temperature in between and including these two values). The slurry was cooled to about 15 to about 25°C (or any temperature in between and including these two values) over 4 hours. The product was collected by suction filtration. The wet cake was washed with 45% water in acetonitrile (500mL) twice. The product was dried under vacuum at about 40°C with nitrogen purge. Yield: 139g of 1.

[0091] The above procedure for coupling Compound 3 and Compound 2 to produce

Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used.

The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying. Catalysts: Different metal and ligand combination could be used. Examples of metal/ligand combinations can be found in Maluenda, Irene; Navarro, Oscar, Molecules, 2015, 20, 7528. Various catalysts can be including: XPhos-3G (cas# 1445085-55-1); cataCXium® A Pd 3G (CAS# 1651823-59-4); PdCk(DtBPF) (CAS# 95408-45-0); SPhos 3G (Cas# 1445085-82-4); AmPhos 3G (Cas# 1820817-64-8); PCy₃ 3G (Cas# 1445086-12-3); Pd PEPPSI IPent Cas#l 158652-41-5);

Pd(PPh₃)2Cb (Cas# 13965-03-2). Examples of catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.

Table 4: Catalyst screen summary

VI. Purification of Compound 1 fCC-900101 bv crystallization from formic acid and water

[0092] Described herein are methods of purifying Compound 1 by crystallization from formic acid and water. Also described are methods for obtaining three different polymorphs of Compound 1, including the most stable form, Form 1 and two metastable forms, Form 4

The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying. Catalysts: Different metal and ligand combination could be used. Examples of metal/ligand combinations can be found in Maluenda, Irene; Navarro, Oscar, Molecules, 2015, 20, 7528. Various catalysts can be including: XPhos-3G (cas# 1445085-55-1); cataCXium® A Pd 3G (CAS# 1651823-59-4); PdCh(DtBPF) (CAS# 95408-45-0); SPhos 3G (Cas# 1445085-82-4); AmPhos 3G (Cas# 1820817-64-8); PCy₃ 3G (Cas# 1445086-12-3); Pd PEPPSI IPent Cas#l 158652-41-5);

Pd(PPh3)2Cl2 (Cas# 13965-03-2). Examples of catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.

Table 4: Catalyst screen summary

VI. Purification of Compound 1 (CC-90010! bv crystallization from formic acid and water

33 -a

and Form 5. Supporting data (XRPD, DSC, photomicroscopy) for all three forms is provided in the examples below.

[0093] The stmcture of Compound 1 (CC-90010) is shown below:

Example 1: Synthesis of Compound 1

[0217] Synthesis of compound 1 was accomplished according to Scheme 1 below. Referring to Scheme 1, synthesis commenced with bromination of starting material 4-(methylsulfonyl)phenol 4, to produce compound 5. Compound 5 was O-alkylated with (bromomethyl)cyclopropane to produce compound 6. Boronate Compound 2 was then formed by borylation of Compound 6 with Pd catalyst and bis(pinacolato)diboron to produce transient Compound 7, which was subsequenctly treated with diethanolamine (DBA) to afford cross-coupling partner Compound 2. Cross-coupling partner Compound 3 was formed in one pot starting from commercially available Compound 8. Compound 8 was N-methylated and brominated to afford Compound 3. Compounds 2 and 3 were cross-coupled (Norio, M. and Suzuki, A., Chem. Rev., 95(7), 2457-2483 (1995)) to afford the target compound 1.

Scheme 1: Synthesis of compound 1

1.1: Bromination of 4

[0218] The bromination of Compound 4 to produce Compound 5 itself is simple, however stopping at the mono-brominated Compound 5 was challenging. The bis-brominated Compound 5-a (see Scheme 2 below) is a particularly pernicious impurity as it couples downstream to form a di ffi cult-to-purge impurity.

Scheme 2: Bromination of Compound 4

[0219] The key to high purity with reasonable yield was to exploit the solubility differences of the starting material Compound 4 (46 mg/ml at 20 °C) and the product Compound 5 (8 mg/ml) in CH₂CI2. These solubility differences are summarized in Table 3 below.

[0220] This solubility difference is exploited by performing the reaction at a high

concentration to drive Compound 5 out of solution once formed, thereby minimizing its ability to react further with the brominating reagent to form Compound 5-a diBr. The reaction is seeded with Compound 5 to initiate its crystallization.

[0221] In Fig. 22 (Conversion of Compound 4 to Compound 5: Effect of Sulfuric Acid) it can be seen that in the absence of acid the initial reaction to Compound 5 is rapid, however the conversion plateaus at about 30% Compound 5. The main side product was found to be the impurity Compound 5-a diBr (see Fig. 23: Conversion of Compound 5 and Compound 5-a diBr: No H2SO4). Addition of increasing amounts of sulfuric acid leads to a higher conversion to desired Compound 5.

[0222] Fig. 24 (Compound 4 to Compound 5 Reaction Profile: Portion-wise Addition of NBS, Seeding) depicts further reaction control. The portion-wise addition ofNBS after addition of catalytic sulfuric acid minimizes the temperature rise, and the addition of Compound 5 after an initial NBS charge promotes the reactive crystallization of Compound 5. After about 6 to 7 hours of reaction it can be seen that the major product is Compound 5, with only a small (<5%) of the di-brominated impurity formed. In contrast, in a reaction where Compound 4 and all of the NBS were charged followed by the addition of 4 volumes of methylene chloride, a rapid exotherm resulted and undesired Compound 5-a diBr was found to be the major product.

[0223] Thus, the reaction was run under a high concentration in CH₂CI2 with a portion-wise solid addition of NBS (to control both availability of the electrophile and the exotherm). An end of reaction slurry sample typically showed not more than 5% of the starting material Compound 4 remaining. After filtration the crude cake was washed with cold CH₂CI2 and the OkCk-washed filter cake contained not more than 0.5% by weight dibrominated Compound 5-a. It also contained a large amount of HPLC-silent succinimide.

[0224] The following procedure was carried out: Compound 4 (25g, 145mmol) followed by CH₂CI2 (lOOmL) were added to a reaction vessel and agitated. The batch was adjusted to 17 °C to 23 °C. Sulfuric acid was charged (2.7mL, Slmmol) to the batch maintaining 17 °C to 23°C. The batch was stirred at 17 °C to 23 °C for 10 minutes to 20 minutes. The first portion of A-bromosuccimide (NBS) was charged (6.5g, 36.5 mmol) to the batch at 17 °C to 23°C and stirred for at least 30 min. The second portion of NBS was charged (6.5g, 36.5 mmol) to the batch at 17 °C to 23°C and stirred for at least 30 min. The batch was seeded with

Compound 5 (0.02wt) and stirred for ca. 30 min at 17 °C to 23 °C to induce crystallization.

[0225] The third portion of NBS was charged (6.5g, 36.5 mmol) to the batch at 17 °C to 23 °C and stirred for at least 30 min. NBS (6.5g, 36.5 mmol) was charged to the batch at 17 °C to 23 °C and stirred for at least 30 min. Additional CH₂CI2 was charged (50mL) to the batch while maintaining 17 °C to 23 °C to aid in agitation and transfer for filtration. The batch was stirred at 17 °C to 23 °C until complete by HPLC analysis (~20 – 40 h). The product was collected by suction filtration. The filter cake was slurry washed with CH₂CI2 (3 x 50mL) at 17 °C to 23 °C (target 20 °C). The filter cake was slurry washed with purified water (3.0vol) at 65 °C to 75 °C for 2 to 3 hours. Then, the filter cake was slurry washed with purified water (3 x 1.0 vol, 3 x 1.0 wt) at 17 °C to 23°C. The wet cake was dried under vacuum with nitrogen bleed at 60 °C. Yield: 27g 5 (74% molar) >97% by weight. ¾ NMR (500 MHz, de-DMSO) 8.01 (1H, d, ⁴J = 2.1 Hz, RO-Ar meta- H ), 7.76 (1H, dd, J = 8.6 and ⁴J = 2.1 Hz, RO-Ar meta-H ), 7.14 (1H, d, J = 8.6 Hz, RO-Ar ortho- H), 3.38 (1H, br s, OH), 3.20 (3H, s,

CHJ); MS (ES-) calc. 249/251; found 249/251. Melting point (MP): (DSC) 188 °C.

[0226] The above procedure allowed for the following modifications. Solvents: Alternative solvents could be used. Examples include chlorinated solvents, such as chloroform or 1,2 dichloroethane, and non-chlorinated solvents such as acetonitrile, tetrahydrofuran, or 2- methyltetrahydrofuran. Reaction concentration: The reaction concentration can be varied from about 2X vol to about 20 X vol (with respect to Compound 4). Brominating agents: Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination reagent stoichiometry: Different amounts of the brominating reagent can be used, from about 0.8 equiv to about 1.9 equiv. Bromination reagent addition: The brominating reagent can be added all at once, portion wise in about 2 to about 20 portions, or continuously. The addition times can vary from about 0 to about 72 hours. Temperature: Reaction temperatures from about 0 °C to about 40 °C could be used. Acids: Different acids can be envisioned, including benzenesulfonic acid, para-toluenesulfonic acid, triflic acid, hydrobromic acid, and trifluoroacetic acid. Isolation: Instead of directly filtering the product and washing with methylene chloride and water, at the end of reaction an organic solvent capable of dissolving Compound 5 could be charged, followed by an aqueous workup to remove succinimide, and addition of an antisolvent or solvent exchange to an appropriate solvent to crystallize Compound 4. Drying: A temperature range of about 10 to about 60 °C could be used for drying.

[0227] An alternative process to Compound 5 has also been developed. This process is advantageous in that it does not use a chlorinated solvent, and provides additional controls over the formation of the Compound 5-a dibromo impurity. See Oberhauser, T. J Org. Chem 1997, 62, 4504-4506. The process is as follows. Compound 4 (10 g, 58 mmol) and acetonitrile (100 ml) were charged to the reactor and agitated. The batch was cooled to -20 °C. Triflic acid (CF3SO3H or TfOH, 5.5 mL, 62 mmol) was charged while maintaining a batch temperature of -10 to -25 °C. N-bromosuccinimide was charged (NBS, 11.4 g, 64 mmol), stirred at -10 to -25 °C for 30 minutes, then warmed to 20 °C over 3 to 4 hours. Agitation was continued at 15 °C to 25 °C until reaction completion. If the reaction conversion plateaued before completion, the reaction was cooled to -5 to -15 °C, and additional NBS was added, the amount based off of unreacted starting material, followed by warming to 15 °C to 25 °C and reacting until complete.

[0228] After reaction completion, the batch was warmed to 40 °C to 50 °C and concentrated under reduced pressure to 40 mL. The batch was cooled to -5 °C to -15 °C and the resulting product solids were filtered off. The solids were slurry washed three times, each with 20 mL water, for at least 15 minutes. The final cake was dried at 50 °C to 60 °C under reduced pressure to furnish 10 g of 5 containing less than 0.1% MeCN, 0.07% water, and 0.1% triflic acid (TfOH) by weight.

[0229] Alternatives to the above procedure employing MeCN and TfOH are as follows. Brominating agents: Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination Reagent Stoichiometry: Different amounts of the brominating reagent can be used, from about 0.8 equiv to about 2 equiv. Drying: A temperature range of about 10 °C to about 60 °C could be used for drying.

[0230] The impurity 5-a is was prepared and characterized as follows. 10 g of Compound 4 and sulfuric acid (35 mol%) were dissolved in MeOH (10 vol). The mixture was set to stir at 20 °C to 25 °C for 5-10 min and 2.0 equivalents of NBS were charged in one portion. The resulting yellow mixture was stirred for three days at 20-25 °C. The batch was concentrated under reduced pressure and the resulting solid was slurried in water at 95-100 °C for 3 hours. After a second overnight slurry in CH₂CI2 at room temperature, the batch was filtered and dried to give a white solid 5-a (15.0 g, 78%). ¾ NMR (500 MHz, de-DMSO), 8.05 (2H, s, ArH), 3.40 (1H, br s, HO-Ar), 3.28 (3H, s, CH₃); MS (ES^‘) calc. 327/329/331; found

327/329/331; MP (DSC): 226 °C (onset 221 °C, 102 J/g); lit. 224-226 °C.

1.2: O-alkylation of 5 to produce 6

[0231] Compound 6 was prepared according to Scheme 7 below.

Scheme 7: O-alkylation of 5 to produce 6

[0232] Compound 5 (100 g, 398 mmol) and methyl ethyl ketone (MEK, 700 mL) were charged to the reaction vessel and agitated. Potassium carbonate (K2CO3, 325 mesh 82.56 g, 597 mmol) was then charged to the stirred reaction vessel at 15 °C to 25 °C.

Bromomethylcyclopropane (64.4 mL, 664 mmol) was charged to the reaction vessel over at least 1 hour, maintaining the temperature between 15 °C to 25 °C. MEK (200 mL) was added into the reactor and the reactor heated to 65 to 75 °C. The contents of the reaction vessel were stirred at 65 to 75°C for approximately 10 hours until reaction was complete by HPLC analysis. Water (3.0 vol, 3.0wt) was charged to the vessel maintaining the temperature at 65 to 75 °C. The batch was stirred at 65 to 75 °C. The phases were allowed to separate at 65°C to 75 °C and the lower aqueous phase was removed. Water (300 mL) was charged to the vessel maintaining the temperature at 65 °C to 75 °C. The batch was agitated for at least 10 minutes at 65 to 75 °C. The phases were allowed to separate at 65 °C to 75 °C and the lower aqueous phase was removed. The water wash was repeated once. The temperature was adjusted to 40 to 50°C. The mixture was concentrated to car. 500 mL under reduced pressure. The mixture was distilled under reduced pressure at up to 50 °C with MEK until the water content was <1.0% w/w. n-heptane (500mL) was charged to the vessel maintaining the temperature at 40 to 50 °C. The mixture was continuously distilled under vacuum with n-heptane (300mL), maintaining a 1L volume in the reaction vessel. Compound 6 seeds (0.0 lwt) were added at 40 to 50 °C. The mixture was continuously distilled under reduced pressure at up to 50 °C with n-heptane (300mL) while maintaining 1L volume in the reactor. The batch was cooled to 15 to 25 °C and aged for 2 hours. The product was collected by suction filtration. The filter cake was washed with a solution of 10% MEK in n-heptane (5vol) at 15 to 25°C. The filter cake was dried under reduced pressure at up to 40 °C under vacuum with nitrogen flow to afford 95g of 6. ¹H NMR (500 MHz, de-DMSO) 8.07 (1H, d, ⁴J = 2.2 Hz, ArH), 7.86 (1H, d, J = 8.7 Hz, meta-ArH), 7.29 (1H, d, J = 8.8 Hz, ortho-AiK),

4.04 (2H, d, J = 6.9 Hz, OCH₂CH), 3.21 (3H, s, CH₃), 1.31-1.24 (1H, m, OCH), 0.62- 0.58 (2H, m, 2 x CHCHaHb), 0.40-0.37 (2H, m, 2 x CHC¾Hb); MS (ES⁺) calc. 305/307; found 305/307; MP: (DSC) 93 °C.

[0233] The following modifications of the above reaction, synthesis of 6 from 5, may be employed as well. Solvent: Different solvents could be used, for example acetone, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, acetonitrile, or 2-methyl tetrahydrofuran. Reaction volume: Reaction volumes of 3 to 30 volumes with respect to 3 could be used. Base: Different inorganic bases, such as cesium carbonate or phosphate bases (sodium, potassium, or cesium) could be used. Also, organic bases, such as trimethylamine or diisopropyldiimide could be used. Base particle size: Different particle sizes of potassium carbonate from 325 mesh could be used. Reaction temperature: A lower temperature, such

as 50 °C could be used. A higher temperature, such as about 100 °C could be used. Any temperature above the boiling point of the solvent could be run in a pressure vessel.

Isolation: Different solvent ratios of MEK to n-heptane could be used. Different amounts of residual water can be left. Different amounts of seeds, from 0 to 50% could be used.

Seeding could take place later in the process and/or at a lower temperature. An un-seeded crystallization can be employed. A different isolation temperature, from 0 °C to 50 °C could be used. A different wash could be used, for example a different ratio of MEK to n-heptane. A different antisolvent from n-heptane could be used, such as hexane, pentane, or methyl tert-butyl ether. Alternatively, the batch could be solvent exchanged into a solvent where Compound 3 has a solubility of less than 100 mg/ml and isolated from this system. Drying: A temperature range of 10 to 60 °C could be used for drying.

[0234] Compound 10, shown below may also be formed as a result of O-alkylation of unreacted 4 present in product 5, or alternatively from or via a palladium mediated proteodesbromination or proteodesborylation in subsequent chemistry discussed in Example 1.3 below.

[0235] Preparation of methylsulfonylphenyl(cyclopropylmethyl) ether 10: Compound 4 (0.86 g, 5.0 mmol) and K2CO3 (1.04 g, 7.5 mmol) were slurried in acetone (17 mL, 20 vols). Cyclopropylmethyl bromide (0.73 mL, 7.5 mmol) was added in several small portions over ~1 minute and the reaction mixture heated to 50 °C for 48 hours, then cooled to 25 °C. Water (5.0 mL) was added with stirring and the acetone was evaporated on a rotary evaporator from which a fine white solid formed which was filtered off and returned to a vessel as a damp paste. A 1 : 1 mixture of MeOH/ water (8 mL) was added and heated to 40 °C with stirring. After 1 hour, the white solid was filtered off. Some residual solid was washed out with fresh water that was also rinsed through the cake, which was then isolated and left to air dry over the two days to give a dense white solid 10 (1.00 g, 88%). ¾ NMR (500 MHz, CDCb) 7.85

(2H, d, J = 8.8 Hz, RO-Ar ortho-H), 7.00 (2H, d, J = 8.8 Hz, RO-Ar meta- H), 3.87 (2H, d, J = 7.0 Hz, OCH₂CH), 3.02 (3H, s, CHs), 1.34-1.23 (1H, m, OCH₂CH), 0.72-0.60 (2H, m, 2 x CHCH_flHb), 0.42-0.31 (2H, m, 2 x CHCH^.

1.3: Synthesis and Isolation Coupling Partner Boronic Ester 2

[0236] The final bond forming step to Compound 1 is a Suzuki-Miyaura coupling between Compounds 2 and 3, as shown in Scheme 3 below (Norio, M. and Suzuki, A., Chem. Rev., 95(7), 2457-2483 (1995)). Early studies demonstrated that the boronic ester of the isoquinolinone Compound 3-a had poor physical attributes and solid phase stability (Kaila, N. et al., J. Med Chem., 57: 1299-1322 (2014)). The pinacolatoboronate of the O-alkyl phenol, Compound 7, had acceptable solid phase stability and could be isolated via crystallization.

Scheme 3: Suzuki-Miyaura coupling between 2 and 3

[0237] Process robustness studies for the isolation of Compound 7, however, indicated that Compound 7 has poor solution stability, decomposing primarily to the proteodeborylated compound 10, as shown in Scheme 4 below. This was particularly problematic as the isolation process involved a solvent exchange from 2-MeTHF (2-methyl tetrahydrofuran) to iPrOAc (isopropyl acetate), which is not a fast unit operation on scale.

Scheme 4: Modification of 7

[0238] A search for a more stable boronic ester was undertaken. Early attempts targeted making N-methyliminodiacetic acid (MID A) boronate Compound 2-a (E. Gilis and M. Burke,“Multi step Synthesis of Complex Boronic Acids from Simple MIDA Boronates,” J Am. Chem. Soc., 750(43): 14084-14085 (2008)), however, all attempts resulted in product decomposition. Applicant then turned to a relatively obscure boronate formed by the addition of diethanolamine to Compound 7 (Bonin et al., Tetrahedron Lett., 52: 1132-1135 (2011)). Addition of diethanolamine to a solution of Compound 7 led to rapid ester formation and concomitant crystallization of Compound 2.

[0239] The discovery of boronic ester Compound 2 allowed for a simple, fast, high-yielding, high-purity process comprising the following procedure. Tetrahydrofuran (THF, 1500mL) was charged to a flask containing Compound 6 (100g, 328 mmol), bis(pinacolato)diboron (90.7g, 357 mmol) and cesium acetate (CsOAc, 158g, 822 mmol). The system was vacuum purged three times with nitrogen. Pd(PPh3)2Cl2 (13.8g, 20 mmol) was charged to the reaction and the system was vacuum purged three times with nitrogen. The reaction was then heated to 55 to 65°C.

[0240] The batch was stirred for approximately 8 hours until reaction was complete by HPLC analysis. The batch was cooled to 15 to 25 °C (target 20 °C ) and charged with silica gel (20g) and Ecosorb C-941 (20g). After lh, the mixture was filtered to remove solid. The residual solids were washed twice, each with THF (300mL). The filtrate and washes were combined. In a separate vessel, diethanolamine (34.5mL, 360 mmol) was dissolved in THF (250 mL). The diethanolamine solution in THF (25mL) was then charged to the batch. After 10 minutes, the batch was seeded with 2 (1 g) and aged for 1 to 2 hours. The remaining of the diethanolamine solution in THF was charged to the batch over at least 2 hours and the slurry was stirred for at least 2 hours. The product 2 was collected by suction filtration. The wet cake was washed thrice with THF (200mL). The material was dried under vacuum at 40 °C with nitrogen purge yielding 94.6g of 2.

[0241] The reaction to synthesize Compound 2 from Compound 6 described above may be modified as follows. Solvent: Different solvents from THF could be used, such as 1,4 dioxane or 2-methyltetrahydrofuran. Reaction volume: The reaction volume can be varied from 4 to 50 volumes with respect to compound 2. Catalyst and base: Different palladium catalyst and bases can be used for the borylation. Examples can be found in Chow et al., RSC Adv., 3 : 12518-12539 (2013). Borylation reaction temperature: Reaction temperatures from room temperature (20 °C) to solvent reflux can be used. Carbon/ Silica treatment:

The treatment can be performed without silica gel. The process can be performed without a carbon treatment. Different carbon sources from Ecosorb C-941 can be used. Different amounts of silica, from 0.01X to IX weight equivalents, can be used. Different amounts of Ecosorb C-941, from 0.01X to IX weight equivalents, can be used. Crystallization: A different addition rate of diethanolamine can be used. Different amounts of diethanolamine, from 1.0 to 3.0 molar equivalents can be used. A different cake wash with more or less THF can be used. Different amount of seeds from 0.0001X wt to 50X wt can be used.

Alternatively, the process can be unseeded. Drying: A temperature range of 10 °C to 60 °C could be used for drying.

[0242] The subsequent Suzuki-Miyaura coupling between Compounds 2 and 3 also proceeded well, providing over 20 kg of crude compound 1 with an average molar yield of 80% and LCAP of 99.7%.

1.4: Synthesis of Coupling Partner 3

[0243] Cross-coupling partner 3 was prepared by two different processes corresponding to Schemes 8 and 9 shown below.

Scheme 8: Process A for preparation of 3

[0244] According to Process A, Compound 9 (100g, 628 mmol) was dissolved in acetonitrile (450 mL) at room temperature. In a separate vessel, N-bromosuccinimide (NBS, 112g, 628 mmol) was suspended in acetonitrile (1 L). Compound 9 in acetonitrile was charged to the NBS slurry over at least 45 minutes. The contents of the reaction vessel were warmed to 45 °C to 55 °C and the batch stirred until the reaction was complete by HPLC analysis. The batch was cooled to 35 °C to 45 °C and ensured dissolution. Norit SX plus carbon (lOg) was charged to the mixture and the reaction mixture adjusted to 55 °C to 60 °C. The mixture was stirred at 55 °C to 60 °C for about lh and the mixture filtered at 55 °C to 60 °C to remove solids. The solids were washed with acetonitrile (500mL) at 55 °C to 60 °C. The volume of the combined filtrate was reduced to 900 mL by distilling off acetonitrile under reduced pressure. The batch with Compound 3 (lg) and stirred at 35 °C to 45 °C for at least 60 minutes. The contents of the reaction vessel were cooled to 15 °C to 25 °C over at least 1 hour. Water (2000 mL) was charged to the reaction vessel over at least 90 minutes and the slurry aged for at least 60 minutes. The product was collected by suction filtration. The cake was washed with a premixed 5% solution of acetonitrile in water (300mL). The wet cake was dried under vacuum at 40 °C with nitrogen purge. Yield: 120g of 3.

[0245] The above procedure, Process A for this synthesis of 3, may be practiced with alternative reagents and conditions as follows. Solvents: Alternative solvents could be used. Examples include chlorinated solvents, such as methylene chloride, chloroform or 1,2 dichloroethane, and non-chlorinated solvents such as tetrahydrofuran, or 2-methyltetrahydrofuran. Reaction concentration: The reaction concentration can be varied from 2X vol to 40 X vol (with respect to Compound 9). Brominating agents: Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination reagent Stoichiometry: Different amounts of the brominating reagent can be used, from 0.8 equiv to 2 equiv. Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used. The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 °C to 60 °C could be used for drying.

Scheme 9: Process B for preparation of 3

[0246] According to Process B, Compound 3 can be formed starting from 8 via non-isolated compound 9 as follows. Compound 8 (80 g, 55 mmol), cesium carbonate (CS2CO3, 215 g, 66 mmol), and acetonitrile (800 mL) were charged to the reactor. The temperature was adjusted from 15 to 25 °C and iodomethane charged to the reactor (Mel, 86 g, 0.61 mol) while maintaining a batch temperature below 25 °C. The batch was heated to 40 °C and agitated for 10 hours to form Compound 9. The batch was cooled to 25 °C, filtered into a fresh reactor to remove solids, and the solids washed twice with acetonitrile. The combined organic layers were concentrated via atmospheric distillation to about 320 mL.

[0247] In a separate reactor N-bromosuccinimide (NBS, 98.1 g, 0.55 mol) was charged to acetonitrile (800 mL) and agitated. The batch containing Compound 9 was transferred to the NBS solution while maintaining a batch temperature of 15 to 25 °C. The batch was heated to 45 to 55 °C and agitated for at least 4 hours to allow for reaction completion to Compound 3. Upon reaction completion, Norit SX Plus activated carbon (8 g) was charged, and agitated at 45 to 55 °C for one hour. The batch was filtered into a fresh vessel, the Norit SX plus cake was washed with 400 ml of 45 to 55 °C acetonitrile. The acetonitrile layers were combined, cooled to 35 to 45 °C, and distilled under reduced pressure to 720 mL. The batch was adjusted to a temperature of 40 °C, charged with Compound 3 seeds (0.8 g), agitated for one hour, cooled to 15 to 25 °C over at least on hour, then charged with water (1600 mL) over at least two hours. The mixture was agitated for an additional one to two hours, filtered, the cake washed with a premixed 5% solution of acetonitrile in water (240 mL). The wet cake was dried under vacuum at 40°C with nitrogen purge. Yield: 52 g of 3.

[0248] Process B to synthesize Compound 3, described above, may be modified as follows. Solvents: Alternative solvents could be used. Examples include chlorinated solvents, such as methylene chloride, chloroform or 1,2 dichloroethane, and non-chlorinated solvents such as tetrahydrofuran, or 2-methyltetrahydrofuran. Reaction concentration: The reaction concentration can be varied from 2X vol to 40 X vol (with respect to Compound 8).

Alkylating reagent: Alternative methylating reagents to methyl iodide can be used such as dimethylsulfate. Alkylating reagent stoichiometry: 1 to 10 molar equivalents of methyl iodide may be used. Base: Different inorganic bases, such as potassium carbonate or phosphate bases (sodium, potassium, or cesium) could be used. Brominating agents:

Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination reagent stoichiometry: Different amounts of the brominating reagent can be used, from 0.8 equiv to 2 equiv. Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used. Seeding levels from 0.0001% to 50% can be used. The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying.

1.5: Cross-coupling of 2 and 3 to Produce Target Compound 1

[0249] 1 is synthesized by Suzuki cross-coupling of 3 and 2 according to Scheme 10 and as described below.

Scheme 10: Synthesis of 1

[0250] Acetonitrile (1.6L) was charged to a mixture of Compound 2 (156.7g, 460 mmol), Compovmd 3 (lOOg, 420 mmol) and potassium phosphate tribasic (223 g, l.OSmol). Agitation was begun and water (400mL) charged to the batch. The system was vacuum purged three times with nitrogen and charged with Pd(PPh3)2Cl2 (2.9g, 4 mmol) and the system vacuum

purged three times with nitrogen. The batch was heated to 65 to 75°C and contents stirred for at least 16 hours until reaction was complete by HPLC analysis. The batch was cooled to 60 to 70°C, agitation halted and the mixture allowed to settle. The bottom aqueous layer was removed. Water (150mL) and acetonitrile (700mL) were charged at 60 to 70°C. Ecosorb C-941 (15g) and Celite (lOg) were charged to the reaction vessel at 60 to 70°C. After lh, the mixture was filtered to remove solids. The solids were washed twice each with 18% water in acetonitrile (500 mL) at 60 to 70°C. The filtrates were combined and concentrated under atmospheric pressure to a final volume of 1.5L. The batch was cooled to 60 to 65°C and seeded with Compound 1 (1 g). After lh, water (500 mL) was charged over at least 1 hour at 60 to 65°C. The slurry was cooled to 15 to 25°C over 4 hours. The product was collected by suction filtration. The wet cake was washed with 45% water in acetonitrile (500mL) twice. The product was dried under vacuum at 40°C with nitrogen purge. Yield: 139g of 1.

[0251] The above procedure for coupling Compound 3 and Compound 2 to produce

Compound 1 may be modified in any of the ways that follow. Reaction solvents: Different reaction solvents from acetonitrile can be used, including tetrahydrofuran, 2-methyl tetrahydrofuran, toluene, and isopropanol. Boronic ester: Different boronic esters from Compound 2 can be used, including pinacolato ester compound 7, and the free boronic acid of Compound 2. Examples of boronic esters can be found in Lennox, Alister, J.J., Lloyd-Jones, Guy C. Chem. Soc. Rev., 2014, 43, 412. Carbon treatment: Different carbon treatments from Ecosorb C-941 could be used. Different amounts of carbon, from 0.01 to 0.5X weight can be used. The carbon can be eliminated. Different amounts of Celite, from 0.01 to 0.5X weight can be used. Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used. The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying. Catalysts: Different metal and ligand combination could be used. Examples of metal/ligand combinations can be found in Maluenda, Irene; Navarro, Oscar, Molecules, 2015, 20, 7528. Various catalysts can be including: XPhos-3G (cas# 1445085-55-1);

cataCXium® A Pd 3G (CAS# 1651823-59-4); PdCk(DtBPF) (CAS# 95408-45-0); SPhos 3G (Cas# 1445085-82-4); AmPhos 3G (Cas# 1820817-64-8); PCy₃ 3G (Cas# 1445086-12-3); Pd PEPPSI IPent Cas#l 158652-41-5); Pd(PPh₃)2Cl2 (Cas# 13965-03-2). Examples of

catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.

Table 4: Catalyst screen summary

1.6: Crystallization of 1

[0252] The final isolation of Compound 1 requires a polish filtration. For this, the batch must be completely soluble. Unfortunately, Compound 1 has low solubility in almost all

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Class 3 and common Class 2 (e.g. THF, MeCN) solvents (ICH

Harmonized Guideline“Impurities: Guideline for Residual Solvents Q3C(R6)” October 20, 2016). A reasonable solubility was obtained in a warm MeCN-water mix, but this is not an optimal system (requires a heated filtration, MeCN has a residual solvent limit of only 410 ppm). Additional solvents with reasonable solubility (>50 mg/ml) include N-methyl-2- pyrrolidone (NMP) and dimethylacetamide (DMAc); but the development of isolations from these solvents required large volumes and raised residual solvent limit concerns (530 ppm or less for NMT and 1090 ppm or less for DMAc).

catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.

Table 4: Catalyst screen summary

1.6: Crystallization of 1

[0252] The final isolation of Compoxmd 1 requires a polish filtration. For this, the batch must be completely soluble. Unfortunately, Compound 1 has low solubility in almost all

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Class 3 and common Class 2 (e.g. THF, MeCN) solvents (ICH

[0253] Formic acid is one ICH Class 3 solvent in which Compound 1 is highly soluble, having a solubility greater than 250 mg/ml at 20 °C. The solubility curve of Compound 1 in formic acid-Water is quite steep (see Figure 7), which enables a volumetrically efficient process.

[0254] Initial attempts to recrystallize crude Compound 1 involved dissolving in formic acid, polish filtering, and charging polish filtered water to about 20% supersaturation, followed by seeding with the thermodynamically most stable form (Form 1), followed by slow addition of water to the final solvent ratio, filtration, washing, and drying. Applicant observed that during the initial water charge, if the batch self-seeded it formed a thick slurry. X-ray diffraction (XRD), differential scanning calorimetry (DSC), and photomicroscopy demonstrated that a metastable form was produced. Once seeded with Form 1, the batch converted to the desired form (Form 1) prior to the addition of the remaining water. This process worked well during multiple lab runs, consistently delivering the desired form and purity with about 85% yield.

[0255] Unfortunately, upon scale-up, the batch did not convert to Form 1 after seeding. Additional water was charged and the batch began to convert to the desired form (mix of Form 1 and the metastable form by X-ray powder diffraction (XRPD)). When additional water was charged, the XRPD indicated only the metastable form. After a few hours with no change, Applicant continued the water charge to the final solvent ratio, during which time the batch eventually converted to Form 1. This process is summarized in Figure 8.

[0256] It was subsequently found by closer analysis of the plant and laboratory retains that a new metastable form was formed during scale up, with a similar, but different XRPD pattern. This form (metastable B) could be reproduced in the laboratory, but only when the batch has a high formic acid:water ratio and is seeded with Form 1. Without Form 1 seeds, metastable A is the kinetic form. Both metastable forms converted to Form 1 with additional water and/or upon drying, leading Applicant to believe that the metastable forms are formic acid solvates. These findings are summarized in Figure 9.

[0257] While there is little risk in not being able to control the final form, there is a risk of forming a difficult-to-stir slurry which can lead to processing issues. The crystallization procedure was therefore modified to keep a constant formic acid-water ratio. This was performed by charging 2.4X wt. formic acid and 1.75X wt. water (final solvent composition)

to the crystallizer with 0.03X wt. Form 1 seeds, and performing a simultaneous addition of Compound 1 in 6. IX wt. formic acid and 4.4X wt. water. The batch filtered easily and was washed with formic acid/water, then water, and dried under reduced pressure to yield 8.9 kg of Compound 1 (92% yield) with 99.85% LCAP and N.D. formic acid.

Example 2: Exemplary high throughput experimentation reaction

[0258] The following procedure is an exemplary high throughput experimentation reaction.

[0259] An overview of the reaction is shown below in Scheme 5:

Scheme 5: Reaction conditions tested for cross-coupling reaction of 2 and 3

[0260] Pd catalysts were dosed into the 24-well reactor vial as solutions (100 pL of 0.01 M solution in tetrahydrofuran (THF) or dichloroethane (DCE) depending upon the solubility of the ligand). Plates of these ligands are typically dosed in advance of the reaction, the solvent is removed by evacuation in an evaporative centrifuge and plates are stored in the glovebox. The catalysts screened in the coupling are the following: XPhos, SPhos, CataCXium A, APhos, P(Cy)3, PEPPSI-IPent. For the first five ligands, these were initially screened as the Buchwald Pd G2/G3 precatalysts.

[0261] To the plates was then added a stock solution of Compound 3 (10 pmol) and Compound 2 (12 pmol) dissolved in the following solvents: dimethylformamide (DMF),

tetrahydrofuran (THF), butanol (/r-BuOH), and toluene. The base was then added as a stock solution (30 mmol) in 20 mL of water.

[0262] A heatmap summarizing catalyst performance is shown in Figs. 10A and 10B. High performance liquid chromatography (HPLC) yields for this screening span from <5% up to -85%. Larger circles indicate higher yield. Lighter circles indicate higher cleanliness.

[0263] A similarly designed screening of base and solvent also indicate that a range of alcoholic solvents (methanol, ethanol, propanol, 2-butanol, 2-propanol, and /-amyl alcohol) are also all viable in this coupling chemistry. Bases such as potassium phosphate, potassium carbonate, potassium acetate, and potassium hydroxide were all successful in achieving the coupling. Fig. 10B shows a heatmap with HPLC yields ranging from -50 – 95%. Larger, darker circles indicate higher yield.

[0264] This chemistry from microvial screening has been scaled to a laboratory process. To a 3 -necked jacketed 250 mL flask equipped with overhead stirring, nitrogen inlet, and thermocouple was added Compound 3 (1.0 eq, 4.00 grams), Compound 2 (1.2 eq, 1.71 x wt), potassium carbonate (3.0 eq, 1.74 x wt). The reactor was inerted three times and then degassed 2-propanol (24 x vol.) followed by degassed water (6 x vol) was then added.

Stirring was then initiated at 300 rpms. The reactor was then stirred and blanketed with nitrogen for 1 hour. The catalyst was then added (0.01 eq, 0.028 x wt) and stirring continued (300 rpms) and the reactor was heated into the Tj = 65 °C.

[0265] After 2 hours, with full conversion confirmed analytically, trioctylphosphine (0.1 eq, 0.16 x wt) dosed, and reaction mixture allowed to cool slowly to room temperature hours.

The reaction mixture was then filtered, washed with 2-propanol (4 x vol), 2-propanol: water (4: 1, 4 x vol), and then with water (4 x vol). Note: If 2 is dimer present in cake, an additional ethyl acetate (EtOAc) wash (4 x vol) can be added for purging. The cake was then transferred to a vacuum oven to dry overnight at 40 °C, -40 cm Hg, under nitrogen flow. After transfer to a bottle, 6.03 grams of 1 were isolated, 98.6% assay, 91% overall yield.

Scheme 6: Alternative reagents and solvents for cross-coupling

[0266] Based on the previously delineated results, it was expected that a variety of monodentate (PPI13 [triphenylphosphine], PBu3 [tributylphosphine], etc) and bidentate phosphines (dppf [1,1 ‘-bis(diphenylphosphino)ferrocene], BINAP [2,2 -bis(diphenylphosphino)- 1 , 1 -binaphthyl], Xantphos [4,5-bis(diphenylphosphino)-9,9-dimethylxanthene], dppe [l,2-bis(diphenylphosphino)ethane], etc) ligated to any number of Pd sources (Pd halides, Pd(H) precatalyts, Pd(0) sources) could reasonably be employed to arrive at the Compound 1 crude material. A range of organic solvents ranging from non-polar (heptane, benzene), protic (alcohols), polar aprotic (dimethylsulfoxide, dimethylformamide, dimethylacetamide, acetonitrile) as well as a variety of esters and ketones (acetone, 2-butanone, ethylacetate) should also serve as effective solvents for this reactivity. Finally, inorganic bases of varying strength (phosphates, carbonates, acetates, etc) along with organic variants such as triethylamine, l,8-diazabicyclo(5.4.0)undec-7-ene, and others in a wide pKa range are viable as stoichiometric basic additives.

Example 3: Exemplary Compound 5 process

[0267] The purpose of this example was to describe an exemplary process for making Compound 5.

[0268] Charge 4 (lOg, 58mmol) and acetonitrile (lOOmL) to a reaction vessel and start the stirrer. Adjust the batch to -18 °C to -22 °C (target -20 °C). Charge triflic acid (5.5mL, 62mmol) to the batch maintaining -10 °C to -25 °C (target -20 °C). Stir the batch at -10 °C to -25 °C (target -20 °C) for 10 to 20 minutes. Charge NBS (11.38g, 64mmol) to the batch at -10 °C to -25 °C (target -20 °C) and stir for ca. 30 min at -10 °C to -25 °C (target -20 °C). Warm the batch to 20 °C over 3-4 hours (reaction will occur when internal temp is between 5 °C and 15 °C). Stir the batch at 15 °C to 25 °C (target 20 °C) for approximately 1 hour and sample for reaction completion.

[0269] If Compound 4 relative to Compound 5 is more than 5%:

[0270] Cool the bath to -5 °C to -15 °C (target -10 °C) (cooling below 0 °C to ensure selectivity). Charge NBS to the batch according to the follow formula: Mass of NBS = (% Compound 4 x lOg). Warm the batch to 20 °C over 1-2 hours. Stir the batch at 15 °C to 25 °C (target 20 °C) for approximately 1 hour and check reaction for completion. Proceed to next line.

[0271] If Compound 4 relative to Compound 5 is less than 5%:

[0272] Warm the batch to 40 °C to 50 °C (target 48 °C). Concentrate the batch under reduced pressure to a final volume of ~40mL. Cool the batch to -15 °C to -5 °C (target -10 °C) and stir for ca. lh. Filter the batch by suction filtration. Slurry wash the filter cake with purified water (3 x 20mL) at 15 °C to 25 °C (target 20 °C) for 10 to 15 minutes each wash. Remove a sample of the filter cake for analysis by ¾ NMR. Continue washing cake until the residual succimide is below 1.0%mol% relative to 5. Dry the filter cake at up to 60°C under vacuum and nitrogen purge. Analyse the 5 by HPLC analysis (97%w/w to 99%w/w). Expected yield: 60-85% theory (90-110% w/w).

Example 4: Purification of Compound 1 (CC-90010) by crystallization from formic acid and water.

[0273] This example describes a method for the purification of Compound 1 by

crystallization from formic acid and water. Also detailed are methods for obtaining three different polymorphs of Compound 1, including the most stable form, Form 1.

[0274] Figure 11 shows ^XH NMR of Compound 1 (CC-90010). Solvent: d6DMSO; and Figure 12 shows microscopy of Compound 1 (CC-90010) Form I. Figure 13 shows XRPD of Compound 1 (CC-90010) Form I, with peak information detailed in Table 6:

PATENT

US 20190008852

WO 2018081475

US 20180042914

WO 2016172618

WO 2015058160

/////////CC-90010, solid tumors , non-Hodgkin’s lymphoma, PHASE 1, CANCER, QUANTICEL

CS(=O)(=O)c4cc(C1=CN(C)C(=O)c2ccccc12)c(OCC3CC3)cc4

AK 3280

August 16, 2019 10:07 am / 1 Comment on AK 3280

AK-3280

AK 3280; GDC3280; RG 6069

C₁₉ H₁₅ F₃ N₄ O_2, 388.34

CAS 1799412-33-1

4H-Benzimidazol-4-one, 1,5-dihydro-1-methyl-7-(1-methyl-1H-pyrazol-4-yl)-5-[4-(trifluoromethoxy)phenyl]-

Ci8Hi₄N50₂F₃, mass 389.3 g/mol),

ROCHE,

Ark Biosciences , under license from Roche , is developing AK-3280, an antifibrotic agent, for the potential oral treatment of IPF. In July 2018, Ark intended to further clinical development of the drug, for IPF. In June 2019, a phase I trial was planned in Sweden.

Originator Genentech
Mechanism of Action Undefined mechanism

Phase I Interstitial lung diseases

19 Jun 2019Ark Biosciences plans a phase I trial for Idiopathic pulmonary fibrosis (In volunteers) in Sweden (PO, Tablet), in August 2019 , (NCT03990688)
28 Sep 2018GDC 3280 is still in phase I trials for Interstitial lung diseases (Genentech pipeline, September 2018)
28 Jun 2018No recent reports of development identified for phase-I development in Fibrosis(In volunteers) in United Kingdom (PO)

Introduction

GDC 3280 (also known as RG 6069), an orally administered drug, is being developed by Genentech, for the treatment of interstitial lung diseases. Early stage clinical development is underway in the UK.

Company Agreements

In September 2018, Genentech licensed exclusive worldwide development and commercialisation rights of GDC 3280 to Ark Biosciences, for the treatment of idiopathic pulmonary fibrosis

Key Development Milestones

As at September 2018, GDC 3280 is still in phase I development for interstitial lung disease (Genentech pipeline, September 2018).

In December 2015, Genentech completed a phase I trial that evaluated the safety, pharmacokinetics and tolerability of GDC 3280 in healthy volunteers, compared with placebo (GB29751; EudraCT2015-000560-33; NCT02471859). The randomised, double-blind, single and multiple oral dose trial was initiated in June 2015 and enrolled eight volunteers in the UK.

PATENT

WO-2019152863

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019152863&tab=PCTDESCRIPTION&_cid=P12-JZDLP2-41289-1

Novel crystalline salt forms of 1-methyl-7-(1-methyl-lH-pyrazol-4-yl)-5-(4-(trifluoromethoxy)phenyl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one (compound I; presumed to be AK-3280 ), processes for their preparation and compositions comprising them are claimed.

Compound I is an orally available small molecule having the structure:

[0004] Compound I has therapeutic value in several different indications that display fibrotic pathophysiology, including idiopathic pulmonary fibrosis (IPF).

[0005] Idiopathic pulmonary fibrosis is a disease of unknown etiology that occurs mainly in middle-aged and elderly patients, which is characterized by progressive fibrosis of the lung, leading to pulmonary insufficiency and death. Because fibrosis has long been considered to be a clinically irreversible process, treatments have traditionally been focused on managing the symptoms and complications, with little hope of significantly slowing progression of the condition. For many years, mainstay treatments have been typically anti inflammatory, immunosuppressive, and anti-oxidant agents. The effectiveness of these therapies in the treatment of IPF and other fibrotic conditions appears to be minimal and variable, and their side effects are often poorly tolerated by patients.

[0006] New treatment options have only recently become available. Both pirfenidone and nintedanib have been approved for use in the treatment of IPF. Current research efforts to develop new anti-fibrotic agents are targeting multiple mechanisms proposed to be linked to the underlying molecular pathogenic processes. This changing landscape has raised hopes and expectations for what might be achievable with new single agents or combination therapies targeting additional pathways.

Preparation of Compound I and its salts

[0045] A synthesis of Compound I and its tosylate salt is shown in the scheme below:

[0046] l-methyl-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one (5) was synthesized in 4 steps, including a copper-catalyzed coupling reaction e.g., a Goldberg-Ullmann coupling reaction. In another aspect of the invention, intermediate (5) is synthesized using any transition metal-catalyzed coupling reaction. The skilled chemist would know that intermediate (5) could be synthesized from intermediate (4) and compounds

of the general formula: OCF3 , wherein the leaving group“LG” includes but is not limited to halogen, tosylate, mesylate, triflate, etc.

[0047] Compound I was synthesized in 6 steps, using a transition metal cross-coupling reaction, e.g., a Suzuki reaction. In another aspect of the invention, Compound I is synthesized using any cross -coupling reaction. Compound I is synthesized from intermediate 6 containing any leaving group. For example, the skilled chemist would use compounds of

the general formula:
, wherein the leaving group“LG” includes but is not limited to halogen, tosylate, mesylate, triflate, etc.

An alternative synthesis of Compound I and its salts is shown in the scheme below:

Example 13 – Synthesis of Compound I Tosylate Salt

[00183] A process for the formation of mono- and di-tosylate salts of Compound I was developed and a batch was performed to successfully produce the mono-tosylate salt.

Step 1 : Synthesis of2-chloro-N-methyl-3-nitropyridin-4-amine

[00184] A reactor was charged with 2,4-dichloro-3-nitropyridine and 3.0 volumes of DMF. The solution was stirred at 20-25 °C until a clear solution was obtained. The solution was then cooled to 0-5 °C, and 2.1 equivalents of 40% methylamine in water were slowly added over at least 2 hours at 0-5 °C. The reaction mixture was stirred for at least 2 hours at 0-5 °C until conversion to the product was 95% (as measured by HPLC). The reaction mixture was diluted by slowly adding 10 volumes of water over at least 30 minutes at 0-5 °C. The obtained suspension was stirred for at least 60 minutes at 0-5 °C. The precipitate was collected by filtration, and the filter cake was rinsed via the reactor with 10 volumes of water at 0-5 °C. The damp filter cake was then dried in a flow of dry nitrogen to yield 2-chloro-A-methyl-3-nitropyridin-4-amine in 78% yield.

Step 2: Synthesis of 2-chloro-N⁴ -methylpyridine-3, 4-diamine

[00185] A reactor was charged with catalyst [2% Pt on charcoal, 59 %wt. water] (0.0004 equivalents Pt), damp 2-chloro-/V-methyl-3-nitropyridin-4-amine from step 1 and 9.4 volumes of THF. The solution was stirred, and then the suspension was transferred from the glass-reactor to an autoclave. The line was rinsed with 1.2 volumes of THF into the autoclave, and the autoclave was purged with nitrogen for 15 minutes at 50 rpm, followed by hydrogen for 15 minutes at 150 rpm. The autoclave was closed, and the hydrogen pressure was adjusted to 2 bar at 20-30 °C. The reaction mixture was stirred for 4-8 hours at 2 bar and 20-30 °C.

[00186] Next, the autoclave was released to atmospheric pressure and purged with nitrogen for at least 15 minutes. Conversion to the product was verified by HPLC, and then the catalyst was removed by filtration. The filtered catalyst was rinsed with 1.3 volumes of THF and the filtrates were combined. The combined filtrates were charged to a second reactor via a particle filter, and the line was rinsed with 0.5 volumes of THF. The solution was concentrated to a final volume of 2.5 volumes by distillation under reduced pressure at 40-45 °C.

[00187] The solution was then diluted with 10 volumes of THF in portions while concentrating the solution to a final volume of 2.5 volumes by distillation under reduced pressure at 45-50 °C. The reactor was purged with nitrogen to atmospheric pressure, and 5.0 volumes of heptane were added to the residue at 40-50 °C. The reaction mixture was cooled over 2 hours to 20-25 °C, and stirring was continued for 1 hour. The reaction mixture was then further cooled to 0-5 °C over 1 hour, and stirring was continued for 1 hour. The precipitated product was collected by filtration, rinsed via the reactor with 5.0 volumes of heptane, and the damp filter cake was dried in a vacuum drying oven at max. 40 °C until loss on drying was < 2 % weight, giving 2-chloro-/V⁴-methylpyridine-3, 4-diamine in 85% yield.

Step 3 : Synthesis of -inelhyl- 1 ,5-dihvdro-4H-iinidazoi4,5-c h yridin-4-one

[00188] A reactor was charged with 2-chloro-/V⁴-methylpyridine-3, 4-diamine and 4 volumes of formic acid. The reaction mixture was heated to smooth reflux within one hour, and reflux was maintained for 6 hours. The reaction mixture was then cooled to

approximately 60 °C, and conversion to the product was verified by HPLC.

[00189] The reaction mixture was then concentrated by distillation under reduced pressure at 60-80 °C to a final volume of 2 volumes. The temperature of the solution was adjusted to 60 °C, maintaining the temperature above 50 °C to avoid precipitation.

[00190] Next, a second reactor was charged with 10 volumes of acetone, and heated to gentle reflux. The product solution from the first reactor was slowly transferred to the acetone in the second reactor over 20 minutes, and the line was rinsed with approximately 0.05 volumes of formic acid. Reflux of the obtained suspension was maintained for 15 minutes. The slurry was cooled to 0 °C within 1 hour, and stirring was continued for 1 hour at that temperature. The precipitate was collected by filtration, and the filter cake was rinsed via the reactor with 3.7 volumes of cold acetone at 0-10 °C. The filter cake was dried in a flow of dry nitrogen or in a vacuum drying oven at 50 °C until loss on drying was < 2% of weight, giving 1 -methyl- 1 ,5-dihydiO-4/7-imidazo[4,5-c]pyndin-4-onc in 95% yield.

Step 4: Synthesis of l-methyl-5-(4-(trifluoromethoxy)phenyl)-J5-dihvdro-4H-imidaz.o[4,5-c]pyridin-4-one

[00191] A first reactor (Reactor A) was charged with 1 -methyl- 1 ,5-dihydro-4/7-imidazo[4,5-c]pyridin-4-one (1.0 mol equivalent), Cu(0Ac)₂ H₂0 (0.1 mol equivalents), and K₂C0₃ (1.1 mol equivalents). The reactor was closed and the atmosphere replaced with nitrogen.

[00192] Next, l-bromo-4-(trifluoromethoxy)benzene (1.5 mol equivalents) and N-methylpyrrolidinone (5.4 volume equivalents) were added, whereupon a suspension was formed. The suspension was stirred until the temperature had fallen again to approximately 20-25 °C and gas evolution had slowed. The reaction mixture was heated to approximately 130-150 °C at which time a blue/green color was observed, changing to dark brown after some time. The reaction was stirred at 130-150 °C for at least 40 hours. Stirring times of 40 hours up to 72 hours were required to reach an acceptable level of conversion. In general, higher reaction temperatures supported faster conversion.

[00193] Next, the reaction mixture was cooled to approximately 20-30 °C, and 25% aqueous NH₃ (0.7 volume equivalents) was added, followed by water (3.5 volume equivalents). The resulting suspension was transferred into a second reactor (Reactor B). Additional water was added (18.1 volume equivalents) to the reaction mixture via Reactor A, followed by n-heptane (3.2 volume equivalents). The resulting suspension was cooled to approximately 0-5 °C, and stirred for approximately 2 hours.

[00194] The suspension was filtered, and the filter cake was washed with water (9.7 volume equivalents). The filter cake was then dissolved in dichloromethane (14.1 volume equivalents) and transferred back into reactor B. To this solution was added water (5.7 volume equivalents) via the filter, followed by 25% aq. NH₃(1.6 volume equivalents). The mixture was stirred for approximately 1 hour at approximately 15-25 °C.

[00195] Next, the layers were separated, and dichloromethane was added (3.6 volume equivalents) to the aqueous layer. The biphasic mixture was stirred at approximately 15-25 °C for approximately 20-30 minutes. The layers were separated over a period of at least 1 hour, and to the combined organic layers was added a solution of NH₄Cl (2.5 mol equivalents) in water (7.0 volume equivalents). The biphasic mixture was stirred at approximately 15-25 °C for about 20-30 minutes, then the layers were separated over the course of 1 hour.

[00196] The lower organic layer was filtered through a particle filter and diluted with toluene (7.1 volume equivalents) via the filter. The organic layer was concentrated under ambient pressure at approximately 80 °C, until no further liquid was seen to evaporate and a precipitate began to form. Toluene was added (16.6 volume equivalents), then concentrated in vacuo, followed by addition of more toluene (7.1 volume equivalents) and again concentrated in vacuo. The suspension was cooled to approximately 0-5 °C, stirred for approximately 2 hours, and filtered. The filter cake was washed with toluene (2.9 volume equivalents), and dried in vacuo at approximately 50 °C until the loss on drying was 0.5% of the weight to give l-methyl-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-47/-imidazo[4,5-c]pyridin-4-one as a beige-colored solid in 83.1% yield.

Step 5 : Synthesis of 7-bromo- 1 -methyl-5-(4-( trifluoromethoxy Iphenyl )- l,5- 4H-

imidaz.o[4,5-clpyridin-4-one

[00197] A first reactor (Reactor A) was charged with water (1.8 volume equivalents) and cooled to approximately 0-5 °C, to which was slowly added 96% sulfuric acid (14 mol. equivalents) at approximately 0-20 °C. The temperature of the solution was adjusted to approximately 0-5 °C, and l -mcthyl-5-(4-(tnfluoromcthoxy)phcnyl)-l ,5-dihydro-4/7-imidazo[4,5-c]pyridin-4-one (1.0 mol equivalent) was added in 3-4 portions at approximately 0-5 °C. The temperature of the mixture was adjusted to approximately 0-5 °C, and N-bromosuccinimide (1.0 mol equivalents) was slowly added in 3-4 portions, while maintaining the temperature at approximately 0-5 °C.

[00198] The reaction mixture was stirred for about 1 hour at approximately 0-5 °C, and then for an additional 4-16 hours at approximately 0-22 °C. Conversion to the product was confirmed by HPLC, then the reaction mixture was cooled to approximately 0-5 °C.

[00199] A second reactor (Reactor B) was charged with water (42.7 volume equivalents) and cooled to approximately 0-5 °C. The reaction mixture from Reactor A was transferred into the pre-cooled water in Reactor B at a temperature below 30 °C over 2 hours. The reaction was rinsed with water (1.6 volume equivalents), and 50% aqueous sodium hydroxide (25 mol. equivalents) was carefully added at approximately 0-30 °C over about 2 hours until the pH reached 2-5.

[00200] Next, MTBE (6.5 volume equivalents) was added at approximately 0-20 °C, and the mixture was stirred for about 5 minutes. Additional 50% aqueous sodium hydroxide (2 mol. equivalents) was added at approximately 0-30 °C until the pH of the solution was in the range of 10-14. The reaction was stirred for at least 1.5 hours at approximately 15-25 °C, and then the layers were allowed to separate over a period of at least 1 hour. The suspension was filtered, taking care to capture the product, which accumulated at the interface of the aqueous and organic layers. The filter cake was washed with MTBE (1.7 volume equivalents), water (3.0 volume equivalents), and then MTBE again (3.0 volume equivalents). The product was dried in vacuo at below 50 °C until the loss on drying was < 1% of the weight, giving 7-bromo-l-methyl-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-47/-imidazo[4,5-c]pyridin-4-one as a pale beige-colored solid in 97.6% yield.

Step 6: Synthesis of 1 -methyl-7 -( 1 -methyl-lH-pyraz.ol-4-yl )-5-(4-( trifluoromethoxy )pheml )-J5-dihvdro-4H-imidaz.o[4,5-c]pyridin-4-one (Compound /)

[00201] A reactor was charged with 7-bromo-l-methyl-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-4//-imidazo[4,5-c]pyridin-4-one (1.0 mol equivalents), ( 1 -methyl- 1 //-pyrazol-4-yl)boronic acid pinacol ester (l-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l//-pyrazole, 1.6 mol equivalents), Pd[Ph₃]₄ (0.025 mol equivalents, and K₂C0₃ (2.0 mol equivalents), to which were added acetonitrile (10.0 volume equivalents) and water (3.0 volume equivalents). The reaction mixture was stirred for approximately 10-20 minutes at about 20-25 °C to form a suspension.

[00202] The mixture was heated to slight reflux, whereupon a biphasic, yellow solution formed. The mixture was stirred at slight reflux for at least 10 hours. The reaction mixture was cooled to between 30-50 °C, then passed through a particle filter. The filter was washed with acetonitrile (2.6 volume equivalents), the filtrates were combined, and the solution was concentrated to a final volume of approximately 120 mL (4.8 volume equivalents) under reduced pressure at below 60 °C.

[00203] To the resulting suspension was added water (1.9 volume equivalents), methanol (26 mL, 1.0 volume equivalents), and dichloromethane (14.8 volume equivalents). The mixture was warmed to about 30-35 °C and stirred until two clear layers were observed. The layers were allowed to separate without stirring at about 30-35 °C, and additional dichloromethane (3.7 volume equivalents) was added to the aqueous layer. The mixture was warmed to approximately 30-35 °C and stirred for about 5 minutes, and then the layers were allowed to separate at approximately 30-35 °C.

[00204] To the combined organic layers was added water (1.9 volume equivalents), and the mixture was warmed to approximately 30-35 °C and stirred for about 5 minutes. The layers were separated at approximately 30-35 °C. Charcoal was added to the combined organic layers and stirred for 30-60 minutes at approximately 30-35 °C. The charcoal was removed by filtration, and the filter was washed with dichloromethane (39 mL, 1.6 volume equivalents).

[00205] The solution was concentrated to approximately 4.0 volume equivalents at ambient pressure and at below 50 °C, then diluted with methanol (5.0 volume equivalents). The solution was again concentrated to approximately 4.0 volume equivalents at ambient pressure and below 60 °C, diluted with methanol (5.0 volume equivalents), and concentrated to a final volume of approximately 3.0 volume equivalents under reduced pressure below 60 °C.

[00206] To the resulting suspension was added methanol (2.9 volume equivalents), and the suspension was warmed to approximately 45-55 °C and stirred for about 1 hour. The suspension was cooled to approximately 0-5 °C within approximately 1 hour, stirred for 1 hour at approximately 0-5 °C, and then filtered. The filter cake was washed with cold methanol (pre-cooled to approximately 0-10 °C, 2.9 volume equivalents), and the product was dried under a stream of nitrogen and in vacuo at below 60 °C until the loss on drying was < 1% by weight, giving Compound I (l-methyl-7-(l-methyl-l -pyrazol-4-yl)-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-4//-imidazo[4,5-c]pyridin-4-one) as a white solid in 88.5% yield.

Step 7: Recrystallization of 1 -methyl-7 -(1 -methyl- lH-pyraz.ol-4-yl)-5-( 4-(trifluoromethoxy)phenyl)-J5-dihvdro-4H-imidaz.o[4,5-c]pyridin-4-one (Compound /)

[00207] A reactor was charged with crude l-methyl-7-(l -methyl- l//-pyrazol-4-yl)-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-47/-imidazo[4,5-c]pyridin-4-one from step 6, and to this was added glacial acetic acid (1.5 volume equivalents). The suspension was warmed to approximately 50-60 °C and stirred until a clear solution was obtained, approximately 10-20 minutes. The warm solution was passed through a particle filter into a second reactor.

[00208] To this solution was added ethanol (10.0 volume equivalents) at approximately 45-55 °C over 2 hours. The suspension was stirred for approximately 30 minutes at approximately 45-55 °C, then cooled to approximately 0-5 °C over about 4 hours. The suspension was then stirred for approximately 4-16 hours at about 0-5 °C.

[00209] Next, the suspension was filtered and the filter cake was washed with cold isopropanol (4.2 volume equivalents) at approximately 0-20 °C. The product was dried under a nitrogen stream and in vacuo at below 60 °C until the loss on drying was < 1% by weight, giving Compound I ( 1 – mcthyl-7-( 1 -methyl- 1 /7-pyrazol-4-yl)-5-(4-(tnfluoromcthoxy)phcnyl)-l,5-dihydro-47/-imidazo[4,5-c]pyridin-4-one) as a white solid in 93.0% yield.

Step 8 : Synthesis of 1 -methyl-7 -( 1 -methyl- 1 H-pyrazol-4-yl )-5-(4-( trifluoromethoxy )phenyl )- 1 ,5-dihvdro-4H-imidaz.oi 4,5-clpyridin-4-one, mono – mono -tosylate

salt)

[00210] A reactor was charged with Compound I ( 1 -mcthyl-7-( 1 -methyl- 1 /7-pyrazol-4-yl)-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-4//-imidazo[4,5-c]pyridin-4-one, 1.00 mol equivalent), para-toluenesulfonic acid monohydrate (1.05 mol equivalents), acetone (6.75 volume equivalents), and water (0.75 volume equivalents). The mixture was stirred at 15-25 °C until a clear solution formed, and then this solution was filtered through a particle filter into a second reactor.

[00211] The filter was washed with acetone (2.5 volume equivalents), and to the combined filtrates was added MTBE (7.5 volume equivalents) at 15-25 °C and Compound I mono-tosylate seeding crystals (0.001 mol equivalents).

[00212] The resulting suspension was stirred at 15-25 °C for approximately 30-60 minutes, and MTBE was added (22.5 volume equivalents) at 15-25 °C during a period of

approximately 30 minutes. Stirring was continued at 15-25 °C for approximately 30-60 minutes, and then the suspension was filtered. The filter was washed with MTBE (2.5 volume equivalents), and the material was dried in vacuo at below 55 °C to give Compound I mono-tosylate salt (l-methyl-7-(l-methyl-l//-pyrazol-4-yl)-5-(4-(trifluoromethoxy)phenyl)-l,5-dihydro-47/-imidazo[4,5-c]pyridin-4-one, mono-tosylate salt) as a white, crystalline solid in 93% yield.

PATENT

WO2018102323 ,

claiming use of a specific compound, orally administered, in combination with food (eg low, medium or high fat meal) for treating fibrotic, inflammatory or autoimmune disorders eg idiopathic pulmonary fibrosis IPF, assigned to Genentech Inc ,

References

Roche licenses IPF candidate to Ark Biosciences. Internet-Doc 2019;.

Available from: URL: https://scrip.pharmaintelligence.informa.com/deals/201820364
Roche Q3 2018. Internet-Doc 2018;.

Available from: URL: https://www.roche.com/dam/jcr:f9cad8fc-8655-4692-9a85-efbe1cf7a59b/en/irp181017.pdf
A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Ascending, Single- and Multiple-Oral-Dose, Safety, Tolerability, and Pharmacokinetic Study of GDC-3280 in Healthy Subjects

ctiprofile

// AK-3280, AK 3280, AK3280, GDC 3280, RG 6069, PHASE 1, Idiopathic pulmonary fibrosis

SY-008

July 29, 2019 10:09 am / Leave a comment

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

SY-008

CAS 1878218-66-6

FREE FORM 1480443-32-0

SGLT1 inhibitor (type 2 diabetes),

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

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

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

MF H₅₀ N₄ O₆ . C₂ H₄ O₂

MW 58.8 g/mol,C₃₅H₅₄N₄O₈

Originator Eli Lilly

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

Phase I Diabetes mellitus

28 Aug 2018 No recent reports of development identified for phase-I development in Diabetes-mellitus in Singapore (PO)
24 Jun 2018 Biomarkers information updated
12 Mar 2018 Phase-I clinical trials in Diabetes mellitus (In volunteers) in China (PO) (NCT03462589)
Eli Lilly is developing SY 008, a sodium glucose transporter 1 (SGLT1) inhibitor, for the treatment of diabetes mellitus. The approach of inhibiting SGLT1 could be promising because it acts independently of the beta cell and could be effective in both early and advanced stages of diabetes. Reducing both glucose and insulin may improve the metabolic state and potentially the health of beta cells, without causing weight gain or hypoglycaemia. Clinical development is underway in Singapore and China.

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

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

PATENT

WO 2013169546

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

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

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

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

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

Preparation 1

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

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

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

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

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

Preparation 2

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

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

-Cl-

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

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

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

Preparation 3

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

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

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

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

Preparation 4

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

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

Preparation 5

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

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

Preparation 6

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

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

Preparation 7

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

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

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

Preparation 8

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

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

Example 1

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

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

Preparation 9

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

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

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

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

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

Preparation 10

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

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

Preparation 11

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

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

Preparation 12

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

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

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

Preparation 13

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

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

Preparation 14

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

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

Preparation 15

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

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

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

Preparation 16

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

dihydrochloride.

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

First alternative synthesis of Example 1

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

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

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

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

Example 2

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

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

O H

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

Example 3

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

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

Example 1 Preparation 17

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

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

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

Preparation 18

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

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

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

Preparation 19

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

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

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

Second alternative Synthesis of Example 1

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

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

PATENT

WO 2015069541

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

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

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

BETA-D- GLUCOPYRANOSIDE ACETATE

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

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

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

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

or hydrate thereof.

Preparation 1

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

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

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

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

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

Preparation 2

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

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

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

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

Preparation 3

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

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

3-ol.

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

Preparation 4

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

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

Preparation 5

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

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

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

Preparation 6

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

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

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

Preparation 7

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

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

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

Preparation 8

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

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

Preparation 9

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

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

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

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

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

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

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

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

Preparation 11

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

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

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

Preparation 12a

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

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

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

Preparation 13

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

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

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

Scheme 3

Preparation 14

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

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

Preparation 15

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

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

Preparation 16

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

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

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

Preparation 17

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

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

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

Preparation 12b

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

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

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

Preparation 18

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

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

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

Example 1

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

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

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

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

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

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

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

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

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

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

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

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

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

PATENT

CN105705509

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

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

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

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

PATENT

WO-2019141209

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

Diabetes is a group of lifelong metabolic diseases characterized by multiple causes of chronic hyperglycemia. Long-term increase in blood glucose can cause damage to large blood vessels and microvessels and endanger the heart, brain, kidney, peripheral nerves, eyes, feet and so on. According to the statistics of the World Health Organization, there are more than 100 complications of diabetes, which is the most common complication, and the incidence rate is also on the rise. The kidney plays a very important role in the body’s sugar metabolism. Glucose does not pass through the lipid bilayer of the cell membrane in the body, and must rely on the glucose transporter on the cell membrane. Sodium-coupled glucose co-transporters (SGLTs) are one of the transporters known to be responsible for the uptake of carbohydrates such as glucose. More specifically, SGLT1 is responsible for transporting glucose across the brush border membrane of the small intestine. Inhibition of SGLT1 results in a decrease in glucose absorption in the small intestine and can therefore be used in the treatment of diabetes.

Ellerelli has developed a novel SGLTs inhibitor for alternative drugs and treatments for diabetes. CN105705509 discloses the SGLTs inhibitor-pyrazole compound, which has the structure shown in the following formula (1):

It is well known for drug production process has strict requirements, the purity of pharmaceutical active ingredients will directly affect the safety and effectiveness of drug quality. Simplified synthetic route optimization, and strictly control the purity of the intermediates has a very important role in improving drug production, quality control and optimization of the dosage form development.

CN105705509 discloses a method for synthesizing a compound of the formula (1), wherein the intermediate compound 2-{(3E)-4-[3-methyl4-({5-(propyl-2-yl)) is obtained by the step B in Scheme 4. -3-[(2,3,4,6-tetra-acetyl-β-D-glucopyranosyl)oxy]-1H-pyrazol-4-yl}methyl)phenyl]but-3- Tert-butyl-1-enyl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (Compound obtained in Preparation Example 12b) was obtained as a yellow foam, yield 68.6%, purity 94 %, this step involves silica gel column purification, low production efficiency, high cost, and poor quality controllability; the intermediate 2-{(3E)-4-[3-methyl 4-({5- (prop-2-yl)-3-[(2,3,4,6-tetra-acetyl-β-D-glucopyranosyl)oxy)-1H-pyrazol-4-yl}methyl) Phenyl]but-3-en-1-yl}-2,9-diazaspiro[5.5]undecane (Compound obtained in Preparation Example 18) as a yellow solid with a purity of 95.0%; The resulting intermediate compounds were all of low purity. Moreover, CN105705509 produces a compound of formula (1) having a purity of 96.7% as described in the publications of the publications 0141 and 0142. The resulting final compound is not of high purity and is not conducive to subsequent drug preparation.

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

Example 1

626 g of the compound of the formula (16), 6 L of acetonitrile, 840 g of cesium carbonate and 1770 g of 2,3,4,6-tetra-O-pivaloyl-α-D-glucosyl bromide (formula (17) The compound is sequentially added to the reaction vessel, heated to 40 ° C to 45 ° C, and reacted for 4 to 5 hours, then cooled to 20 to 25 ° C, filtered, and the obtained solid is rinsed once with acetonitrile; the filter cake is dissolved with 8 L of ethyl acetate and 10 L of water. After the liquid separation, the organic phase was concentrated to about 3 L, 10 L of acetonitrile was added, and the mixture was stirred for 12 h to precipitate a solid, which was filtered. The filter cake was rinsed with acetonitrile and dried under vacuum at 60 ° C for 24 h to give white crystals, 652 g of compound of formula (9c). The yield was 61%, the HPLC purity was 98.52%, and the melting point was 180.0-182.1 °C. ¹ H NMR (400 MHz, MeOD) (see Figure 1): δ 7.10 (s, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.39 (d, J=15.6,1H), 6.19-6.12 (m,1H), 5.59 (d, J=8.4 Hz, 1H), 5.40-5.35 (t, J=9.6 Hz, 1H), 5.17-5.06 (m, 2H) , 4.18-4.14 (dd, J = 12.4 Hz, 4.4 Hz, 1H), 4.10-4.06 (dd, J = 12.4 Hz, 1.6 Hz, 1H), 3.92-3.89 (dd, J = 10 Hz, 2.4 Hz, 1H) , 3.64-3.54 (dd, J=20 Hz, 16.8 Hz, 2H), 3.31-3.30 (m, 4H), 2.86-2.79 (m, 1H), 2.37-2.29 (m, 11H), 1.63-1.38 (m, 17H), 1.15-1.05 (m, 42H). MS (m/z): 1035.7 (M+H).

640 g of the compound of the formula (9c) and 6.4 L of ethyl acetate were successively added to the reaction vessel, and the temperature was lowered to 15 ° C to 20 ° C. 1176 g of p-toluenesulfonic acid monohydrate was added in portions for 2 to 3 hours; after the reaction was over, 3.5 L of a 9% potassium hydroxide aqueous solution was added, and the mixture was stirred for 10 minutes, and the aqueous phase was discarded. The organic phase was washed successively with 3.5 L of 9% and 3.5 L of 3% aqueous potassium hydroxide and concentrated to 2.5 L. 21L of n-heptane was added to the residue, and the mixture was stirred for 12 hours; filtered, and the filter cake was rinsed with n-heptane; the filter cake was dried under vacuum at 60 ° C for 24 h to obtain white crystals, p-toluene of the compound of formula (10c). The sulfonate salt was 550 g, the yield was 80%, the purity was 97.59%, and the melting point was 168.0-169.2 °C. ¹ H NMR (400 MHz, MeOD) (see Figure 2): δ 7.72 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.10 (s, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.39 (d, J = 15.6, 1H), 6.19-6.12 (m, 1H), 5.60 (d, J = 8.0 Hz, 1H) ), 5.41-5.37 (t, J = 9.6 Hz, 1H), 5.17-5.06 (m, 2H), 4.18-4.14 (dd, J = 12.4 Hz, 4.0 Hz, 1H), 4.10-4.07 (d, J = 11.6Hz, 1H), 3.94-3.91 (dd, J=7.2Hz, 2.8Hz, 1H), 3.64-3.54 (dd, J=20.0Hz, 16.8Hz, 2H), 3.31-3.30 (m, 4H), 2.86 -2.79 (m, 1H), 2.49-2.29 (m, 14H), 1.78-1.44 (m, 8H), 1.15-1.05 (m, 42H). MS (m/z): 935.7 (M+H).

82.6 g of potassium hydroxide, 5.5 L of absolute ethanol and 550 g of the p-toluenesulfonate of the compound of the formula (10c) were sequentially added to the reaction vessel, and stirred at 45 to 50 ° C for about 4 hours. The temperature was lowered to 20 to 25 ° C, filtered, and the solid was rinsed with ethanol. The filtrate and the eluent were combined, and 65 g of acetic acid was added thereto, followed by stirring for 15 min. The reaction solution was concentrated under reduced pressure to about 1.5 L, and then 52 g of acetic acid was added. After stirring for 20 min, 4.5 L of ethyl acetate containing 3% water and 160 mL of purified water were added dropwise. After the dropwise addition, continue stirring for 3 to 4 hours. Filter and filter cake was rinsed with ethyl acetate containing 3% water. The solid was transferred to a reaction kettle, 500 mL of water was added and stirred for 18 h. After filtration, the filter cake was washed successively with water and an ethanol/ethyl acetate mixed solvent. The filter cake was dried under vacuum at 35 to 40 ° C for 4 hours to obtain a white solid, 245 g of compound of formula (1), yield 75%, purity 99.55%. ¹ H NMR (400 MHz, MeOD) (see Figure 3): δ 7.11 (s, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.39 (d, J=16.0,1H), 6.20-6.13 (dt, J=15.6 Hz, 6.8 Hz, 1H), 5.03-5.01 (m, 1H), 3.83 (d, J=11.2, 1H), 3.71-3.59 (m, 3H), 3.35-3.30 (m, 4H), 3.09-3.06 (t, J = 6 Hz, 4H), 2.87-2.77 (m, 1H), 2.49-2.31 (m, 6H), 2.30 (s, 3H), 2.26(s, 2H), 1.90 (s, 3H), 1.78 (m, 2H), 1.68 (m, 2H), 1.65 (m, 2H), 1.44-1.43 (m, 2H), 1.13 (d, J = 6.8 Hz, 3H), 1.11 (d, J = 6.8 Hz, 3H), MS (m/z): 599.5 (M+H).

Example 2

5.00 kg of the maleate salt of the compound of the formula (16), 40 L of tetrahydrofuran, 5.47 kg of potassium phosphate and 11.67 kg of 2,3,4,6-tetra-O-pivaloyl-α-D-glucosyl bromide The compound (formula (17)) is sequentially added to the reaction vessel, heated to 40 to 45 ° C, and reacted for 4 to 5 hours, then cooled to 15 to 25 ° C, filtered, and the solid was rinsed once with tetrahydrofuran. The filter cake was dissolved in 36 L of ethyl acetate and 20 L of water and then separated. The organic phase was concentrated to ca. 18 L, 64 L acetonitrile was added and stirred for 15 h. Filtration, the filter cake was rinsed with acetonitrile, and dried under vacuum at 60 ° C for 24 h to give white crystals of the compound of formula (9c), 4.50 kg, yield 57%, HPLC purity 99.19%.

4.45 kg of the compound of the formula (9c) and 45 L of butyl acetate were sequentially added to the reaction vessel, and the temperature was lowered to 15 ° C to 20 ° C. 4.13 kg of methanesulfonic acid was added in portions and the reaction was carried out for 2 to 3 hours. 22 L of a 9% aqueous potassium hydroxide solution was added, stirred for 10 min, and the liquid phase was discarded. The organic phase was washed successively with 10 L of 9%, 4.5 L of 10% and 2 L of 2.5% aqueous potassium hydroxide and concentrated to 15 L. 68 L of n-heptane was added to the residue, and the mixture was stirred for further 12 h. Filtered and the filter cake was rinsed once with n-heptane. The solid was dried under vacuum at 60 ° C for 24 h to obtain white crystals. The methanesulfonic acid salt of the compound of formula (10c) was 4.37 kg, yield 99%, purity 97.94%.

0.73 kg of potassium hydroxide, 43 L of methanol and 4.30 kg of the compound of the formula (10c) were sequentially added to the reaction vessel, and stirred at 45 to 50 ° C for 4 hours. The temperature was lowered to 20 to 25 ° C, filtered, and 0.56 kg of acetic acid was added to the filtrate, and the mixture was stirred for 15 minutes. The reaction solution was concentrated to about 15 L under reduced pressure, and 0.40 g of acetic acid was added. After stirring for 10 min, 39 L of 3% water in ethyl acetate and 1.3 L of purified water were added dropwise. After the dropwise addition, stirring was continued for about 2 hours. Filter and filter cake was rinsed once with ethyl acetate containing 3% water. The solid was transferred to a reaction kettle, and 3.5 L of water was added and stirred for 18 h. After filtration, the filter cake was washed successively with water and an ethanol/ethyl acetate mixed solvent. The cake was vacuum dried at 35 to 40 ° C to give a white solid. Compound (1) (1), 1.84 g, yield 67%, purity 99.65%.

Patent ID	Title	Submitted Date	Granted Date
US9573970	4–5-(PROPAN-2-YL)-1H-PYRAZOL-3-YL BETA-D GLUCOPYRANOSIDE ACETATE	2014-10-30	2016-07-28

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

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

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

CC-90009

July 22, 2019 1:27 pm / Leave a comment

2-(4-Chlorophenyl)-N-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-2,2-difluoroacetamide.png

CC-90009

CC-90009-AML-001

CAS 1860875-51-9

461.8 g/mol, C₂₂H₁₈ClF₂N₃O₄

2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

4-Chloro-N-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1-oxo-1H-isoindol-5-yl]methyl]-α,α-difluorobenzeneacetamide
Benzeneacetamide, 4-chloro-N-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1-oxo-1H-isoindol-5-yl]methyl]-α,α-difluoro-

Phase 1 Clinical, Acute myelogenous leukemia, Protein cereblon modulator

Useful for treating chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia or acute myeloid leukemia.

Celgene is developing CC-90009, a cereblon E3 ligase modulator, for treating AML; in January 2019, data from a phase I trial were expected later that year.

0iginator Celgene Corporation
Class Antineoplastics
Mechanism of Action CRBN protein modulators; Ubiquitin protein ligase complex modulators

Phase I Acute myeloid leukaemia

28 Mar 2019 No recent reports of development identified for clinical-Phase-Unknown development in Acute-myeloid-leukaemia in USA (IV)
01 Sep 2016 Phase-I clinical trials in Acute myeloid leukaemia (Second-line therapy or greater) in Canada (IV) (NCT02848001)
04 Aug 2016 Celgene plans a phase I trial for Acute Myeloid Leukaemia in USA and Canada (NCT02848001)

In September 2016, Celgene initiated a phase I dose-finding trial of CC 90009 in patients with relapsed or refractory acute myeloid leukaemia (NCT02848001; CC-90009-AML-001). The open-label study intends to enrol 60 patients in the US and Canada

CC-90009 is a cereblon modulator. CC-90009 specifically binds to CRBN, thereby affecting the activity of the ubiquitin E3 ligase complex. This leads to the ubiquitination of certain substrate proteins and induces the proteasome-mediated degradation of certain transcription factors, including Ikaros (IKZF1) and Aiolos (IKZF3), which are transcriptional repressors in T-cells. This reduces the levels of these transcription factors, and modulates the activity of the immune system, which may include the activation of T-lymphocytes. .

Development Overview

cereblon modulator CC-90009A modulator of cereblon (CRBN), which is part of the cullin 4-RING E3 ubiquitin ligase complex (CRL4-CRBN E3 ubiquitin ligase; CUL4-CRBN E3 ubiquitin ligase), with potential immunomodulating and pro-apoptotic activities. Upon administration, CC-90009 specifically binds to CRBN, thereby affecting the activity of the ubiquitin E3 ligase complex. This leads to the ubiquitination of certain substrate proteins and induces the proteasome-mediated degradation of certain transcription factors, including Ikaros (IKZF1) and Aiolos (IKZF3), which are transcriptional repressors in T-cells. This reduces the levels of these transcription factors, and modulates the activity of the immune system, which may include the activation of T-lymphocytes. In addition, this downregulates the expression of other proteins, including interferon regulatory factor 4 (IRF4) and c-myc, which plays a key role in the proliferation of certain cancer cell types. CRBN, the substrate recognition component of the E3 ubiquitin ligase complex, plays a key role in the ubiquitination of certain proteins. Check for active clinical trials using this agent. (NCI Thesaurus)

WO 2017120446,

PATENT

WO2016007848

US 20170348298

WO 2017120415

WO 2017120446

WO 2017120437

PATENT

WO2017214014

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017214014&tab=PCTDESCRIPTION

Provided herein are methods of treating, preventing, managing, and/or ameliorating a hematologic malignancy with 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide or a stereoisomer or a mixture of

stereoisomers, an isotopologue, pharmaceutically acceptable salt, tautomer, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. Further provided is a compound for use in methods of treating, preventing, managing, and/or ameliorating a hematologic malignancy, wherein the compound is 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide or a stereoisomer or a mixture of stereoisomers, an isotopologue, pharmaceutically acceptable salt, tautomer, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

The term Compound 1 refers to”2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide” having the structure:

and its stereoisomers or mixture of stereoisomers, isotopologues, pharmaceutically acceptable salts, tautomers, solvates, hydrates, co-crystals, clathrates, or polymorphs thereof. In certain embodiments, Compound 1 refers to 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide and its tautomers. In certain embodiments, Compound 1 refers to a polymorph of 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-l-

oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide. In certain embodiments, Compound 1 refers to polymorph Form C of 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide. In one embodiment, the stereoisomer is an enantiomer.

PATENT

WO-2019136016

Novel isotopologs of the compound presumed to be CC-90009 , processes for their preparation and compositions comprising them are claimed.

Patent ID	Title	Submitted Date
US2017199193	METHODS FOR TREATING CANCER AND THE USE OF BIOMARKERS AS A PREDICTOR OF CLINICAL SENSITIVITY TO THERAPIES	2017-01-06
US2018224435	METHODS FOR MEASURING SMALL MOLECULE AFFINITY TO CEREBLON	2018-02-02
US2018353496	FORMULATIONS OF 2-(4-CHLOROPHENYL)-N-((2-(2,6-DIOXOPIPERIDIN-3-YL)-1-OXOISOINDOLIN-5-YL)METHYL)-2,2-DIFLUOROACETAMIDE	2018-07-19
US2017196847	FORMULATIONS OF 2-(4-CHLOROPHENYL)-N-((2-(2,6-DIOXOPIPERIDIN-3-YL)-1-OXOISOINDOLIN-5-YL)METHYL)-2,2-DIFLUOROACETAMIDE	2017-01-06
US2017348298	TREATMENT OF A HEMATOLOGIC MALIGNANCY WITH 2-(4-CHLOROPHENYL)-N-((2-(2,6-DIOXOPIPERIDIN-3-YL)-1-OXOISOINDOLIN-5-YL)METHYL)-2,2-DIFLUOROACETAMIDE	2017-06-05

Patent ID	Title	Submitted Date	Granted Date
US2018221361	ANTIPROLIFERATIVE COMPOUNDS AND METHODS OF USE THEREOF	2018-04-09
US9968596	Antiproliferative compounds and methods of use thereof	2017-10-02	2018-05-15
US2017197934	SOLID FORMS OF 2-(4-CHLOROPHENYL)-N-((2-(2,6-DIOXOPIPERIDIN-3-YL)-1-OXOISOINDOLIN-5-YL)METHYL)-2,2-DIFLUOROACETAMIDE, AND THEIR PHARMACEUTICAL COMPOSITIONS AND USES	2017-01-06
US9499514	ANTIPROLIFERATIVE COMPOUNDS AND METHODS OF USE THEREOF	2015-07-09	2016-01-14
US9808451	ANTIPROLIFERATIVE COMPOUNDS AND METHODS OF USE THEREOF	2016-09-23

////////CC-90009 , CC 90009 , CC90009, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, phase I, CANCER, CC-90009-AML-001

Clc1ccc(cc1)C(F)(F)C(=O)NCc2ccc3C(=O)N(Cc3c2)C4CCC(=O)NC4=O

	Bonkasaurus on Infigratinib phosphate
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A Promising Approach for Difficult-to-Treat Cancers

3-chloro-4-[[4-(dimethylamino)-3-hydroxy-butanoyl]amino]-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 129)

4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)-1-methylcyclohexyl)benzamide (Compound 128)

4-amino-3-fluoro-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 127)

4-amino-N-((1S,3R)-3-((5-chloro-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 126)

4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indazol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 124)

Example 25 Synthesis of N1-(4-(((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)carbamoyl)phenyl)oxalamide (Compound 113)

Example 24 Synthesis of N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)-4-(4-(dimethylamino)butanamido)benzamide (Compound 105)

4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)tricyclo[3.3.1.13,7]decanyl)benzamide (Compound 100).

+/−)-4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-5 hydroxycyclohexyl)benzamide (Compound 101)

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide (Compound 102)

(1S,3R)-N-(4-aminophenyl)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexanecarboxamide (Compound 106)

4-amino-N-((1S,3R)-3-(5-cyclopropyl-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide.HCl (Compound 103)

4-amino-N-((1S,3R)-3-(5-chloro-4-(pyridin-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide (Compound 108)

4-amino-N-((1S,3R)-3-(5-cyano-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide (Compound 107)

(+/−)-4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-5-fluorocyclohexyl)benzamide (Compound 110)

4-amino-N-(5-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)bicyclo[3.1.1]heptan-1-yl)benzamide (Compound 104)

4-amino-N4(1R,5S)-5-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-3,3-difluorocyclohexyl)benzamide (Compound 115)

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzenesulfonamide (Compound 109).

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-2-fluorobenzamide (Compound 112)

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-3-fluorobenzamide (Compound 111).

(+/−)-4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)-1-methylcyclohexyl)benzamide (Compound 116).

N-((1S,3R)-3-(4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-4-aminobenzamide (Compound 114).

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)-2-morpholinobenzamide(Compound 117).

4-amino-N-((1S,3R)-3-(5-chloro-4-(1H-indol-3-yl)pyridin-2-ylamino)cyclohexyl)benzamide (Compound 118).

3-amino-N-(trans-4-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide.HCl (Compound 119).

(1S,3R)-N1-(R)-1-(4-aminophenyl)-2,2,2-trifluoroethyl)-N3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)cyclohexane-1,3-diamine (Compound 120).

(1S,3R)-N1-(4-aminobenzyl)-N3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)-N1-methylcyclohexane-1,3-diamine.HCl (Compound 122).

4-amino-N-((1S,3R)-3-(5-chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-ylamino)cyclohexyl)benzamide.HCl (Compound 123).

Synthesis of 5-amino-N-((1S,3R)-3-(5-chloro-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-ylamino)cyclohexyl)picolinamide (Compound 125)

Synthesis of N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)-4-(4-(dimethylamino)butanamido)benzamide (Compound 105)

Synthesis of N1-(4-(((1S,3R)-3-)(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)carbamoyl)phenyl)oxalamide (Compound 113)

Synthesis of 4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indazol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 124)

Synthesis of 4-amino-N-((1S,3R)-3-((5-chloro-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)benzamide (Compound 126)

Synthesis of 4-amino-3-fluoro-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 127).

Synthesis of 4-amino-N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl) pyrimidin-2-yl)amino)-1-methylcyclohexyl)benzamide (Compound 128)

Synthesis of 3-chloro-4-[[4-(dimethylamino)-3 hydroxy-butanoyl]amino]-N-[(1S,3R)-3-[[4-(1H-indazol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-1-methyl-cyclohexyl]benzamide (Compound 129).

Synthesis of 3-fluoro-4-(methylamino)-N-[(1S,3R)-1-methyl-3-[[4-(7-methyl-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]cyclohexyl]benzamide (Compound 130)

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Introduction

Company Agreements

Key Development Milestones

References

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Development Overview

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4-amino-N-(3-(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-ylamino)tricyclo[3.3.1.1^3,7]decanyl)benzamide (Compound 100).