Tazemetostat

Current developer:  Epizyme, Inc., Cambridge, MA 02139.

EPZ-6438 (Tazemetostat)
CAS: 1403254-99-8

HBR 1467052-75-0

タゼメトスタット臭化水素酸塩

Current developer:  Epizyme, Inc., Cambridge, MA 02139.

EPZ-6438 (Tazemetostat)
CAS: 1403254-99-8

HBR

Chemical Formula: C34H44N4O4
Exact Mass: 572.33626

USFDA APPROVED 23/1/2020 AS HBR SALT, TAZVERIK, EPIZYME

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide
SIMLES: O=C(C1=CC(C2=CC=C(CN3CCOCC3)C=C2)=CC(N(CC)C4CCOCC4)=C1C)NCC5=C(C)C=C(C)NC5=O

(1,1′-Biphenyl)-3-carboxamide, N-((1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(4-morpholinylmethyl)-

N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(oxan-4-yl)amino)-4-methyl-4′-((morpholin-4-yl)methyl)(1,1′-biphenyl)-3-carboxamide

UNII-Q40W93WPE1

Tazemetostat, sold under the brand name Tazverik, is a medication used for the treatment of adults and adolescents aged 16 years and older with metastatic (when cancer cells spread to other parts of the body) or locally advanced (when cancer has grown outside the organ it started in, but has not yet spread to distant parts of the body) epithelioid sarcoma not eligible for complete resection (surgically removing all of a tissue, structure, or organ).^[1]

Tazemetostat is a cancer drug that acts as a potent selective EZH2 inhibitor.^[2]

Tazemetostat blocks activity of the EZH2 methyltransferase, which may help keep the cancer cells from growing.^[1] Most cases of epithelioid sarcoma begin in the soft tissue under the skin of an extremity, though it can start in other areas of the body.^[1] Surgical removal is considered the main treatment when the cancer is localized to one area of the body.^[1] Chemotherapy or radiation may also be given.^[1] However, there is a high likelihood for local and regional spread of the disease even with treatment and approximately 50% of patients have metastatic disease at the time of diagnosis.^[1] Metastatic disease is considered life-threatening to the patient.^[1]

The most common side effects are pain, fatigue, nausea, decreased appetite, vomiting and constipation.^[1] People taking tazemetostat are at increased risk of developing secondary malignancies including: T-cell lymphoblastic lymphoma (a type of blood cancer that affects the lymphatic system usually found in the lymph nodes), myelodysplastic syndrome (a disorder resulting from poorly formed or dysfunctional blood cells) and acute myeloid leukemia (a cancer of the blood and bone marrow).^[1]

According to the NCI Drug Dictionary, “tazemetostat is an orally available, small molecule selective and S-adenosyl methionine (SAM) competitive inhibitor of histone methyl transferase EZH2, with potential antineoplastic activity. Upon oral administration, tazemetostat selectively inhibits the activity of both wild-type and mutated forms of EZH2. Inhibition of EZH2 specifically prevents the methylation of histone H3 lysine 27 (H3K27). This decrease in histone methylation alters gene expression patterns associated with cancer pathways and results in decreased tumor cell proliferation in EZH2 mutated cancer cells. EZH2, which belongs to the class of histone methyltransferases (HMTs), is overexpressed or mutated in a variety of cancer cells and plays a key role in tumor cell proliferation.”^[3]

History

The U.S. Food and Drug Administration (FDA) approved tazemetostat in January 2020,^[1] based on the results of a clinical trial (NCT02601950) enrolling 62 subjects with metastatic or locally advanced epithelioid sarcoma.^[1][4] During the clinical trial, subjects received 800 milligrams (mg) of tazemetostat twice a day until the disease progressed or the subject reached an unacceptable level of toxicity.^[1][4] Tumor response assessments were performed every eight weeks during the clinical trial.^[1] The trial measured how many subjects experienced complete or partial shrinkage (by a certain amount) of their tumors during treatment (overall response rate).^[1] The overall response rate was 15%, with 1.6% of subjects having a complete response and 13% having a partial response.^[1] Of the nine subjects that had a response, six (67%) subjects had a response lasting six months or longer.^[1]

The trial was conducted at 22 sites in France, United Kingdom, Taiwan, Italy, Canada, Belgium, and the United States.^[4]

The FDA granted the application for tazemetostat accelerated approval and orphan drug designation.^[1] The FDA granted the approval of Tazverik to Epizyme Inc.^[1]

PATENT

PRODUCT PAT

US 8410088 EXP 21/1/2034

WO 2012142504

US 9090562 EXP 13/4/32

SEE  Proceedings of the National Academy of Sciences of the United States of America (2013), 110(19), 7922-7927, S7922/1-S7922/5….http://www.pnas.org/content/110/19/7922.abstract

http://www.epizyme.com/wp-content/uploads/2014/11/Ribrag-ENA-FINAL.pdf

Tazemetostat, also known as EPZ-6438,  is a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity. EPZ-6438 induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells.

Treatment of xenograft-bearing mice with EPZ-6438 leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation.

These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers. EPZ-6438 is currently in clinical trials.

Epizyme, Inc., Eisai R&D Management Co.Ltd.

Epizyme is developing tazemetostat, a lead from several small molecule EZH2 inhibitors, for treating cancer (phase 1 clinical, as of April 2015). Japanese licensee Eisai was developing the program for the potential oral treatment of cancers, including non-Hodgkin’s lymphoma; however, in March 2015, Epizyme regained worldwide, ex-Japan, rights to the program.

It appeared that Eisai was planning to investigate the program in Japan .

WO-2015057859 From, Eisai Research Institute; Epizyme Inc, indicates Novel crystalline polymorphic form C of tazemetostat, useful for treating an EZH2-mediated cancer, including non-Hodgkin’s lymphoma and breast cancer.

see WO2013155317, claiming novel hydrobromide salt of tazemetostat.

PREDICT

………………………………….

PATENT

WO 2012142504

http://www.google.com/patents/WO2012142504A1?cl=en

Example 44: Synthesis of N-((4,6-dimethyl-2-oxo-l ,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(moφholinomethyl)-[l , – biphenyl]-3-carboxamide

Compound 44

[Step 1 : Synthesis of 5-brom -2-methyl-3-nitrobenzoic acid

To stirred solution of 2-methyl-3-nitrobenzoic acid ( 100 g, 552 mmol) in cone. H₂S0₄ (400 mL), 1 ,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol) was added in a portion wise manner at room temperature and the reaction mixture was then stirred at room temperature for 5 h. The reaction mixture was poured onto ice cold water, the precipitated solid was filtered off, washed with water and dried under vacuum to afford the desired compound as a solid ( 140 g, 98%). The isolated compound was taken directly into the next step. Ή NMR (DMSO-4$, 400 MHz) δ 8.31 (s, 1 H), 8.17 (s, 1 H), 2.43 (s, 3H).

Step 2: Synthesis of methyl -bromo-2-methyl-3-nitrobenzoate

To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1 105 mmol) in DMF (2.8L) at room temperature was added sodium carbonate (468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60 °C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate ( 1 L X 3). The combined filtrate was washed with water (3L X 5) and the aqueous phase was back extracted with ethyl acetate (1L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (290g, 97% yield). The isolated compound was taken directly into the next step. Ή NMR (CDC1₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (290 g,

1058 mmol) in ethanol (1 .5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5L water). The resulting mixture was stirred at 80°C to which iron powder (472 g, 8451 mmol) was added in a portion wise manner. The resulting reaction mixture was heated at 80 °C for 12 h. Upon completion as determined by TLC, the reaction mixture was hot filtered over celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5L). The combined filtrate was concentrated in-vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2L) and extracted with ethyl acetate (5L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (220 g, 85%). The compound was taken directly into the next step. Ή NMR (CDC1₃, 400 MHz) δ 7.37 (s, 1 H), 6.92 (s, 1 H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Step 4: Synthesis of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate

To a stirred solution of methyl 3-amino-5-bromo-2-methylbenzoate (15 g, 61 .5 mmol) and dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) in dichloroethane (300 mL) was added acetic acid (22 g, 369 mmol) and the reaction mixture stirred at room temperature for 15 minutes, then the reaction mixture was cooled to 0°C and sodium triacetoxyborohydnde (39 g, 184 mmol) was added. The reaction mixture was stirred overnight at room temperature. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH of 7-8 was obtained. The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a solid ( 14 g, 69%). ‘H NMR (DMSO-<fc, 400 MHz) δ 7.01 (s, 1 H), 6.98 (s, 1 H), 5.00 (d, 1 H, J=7.6 Hz), 3.84-3.87 (m, 2H), 3.79 (s, 31 1), 3.54-3.56 (m_f 1 H), 3.43 (L 21 1, J 12 Hz), 2.14 (s. 31 1). 1 . 1 – 1 .84 (m_: 211). 1 .47- 1 .55 (m, 2H).

Step 5: Synthesis of methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2- methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g, 85.2 mmol) and acetic acid ( 15.3 g, 256 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0 °C and sodium

triacetoxyborohydnde (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100- 200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%). Ή NMR (DMSO-cfo 400 MHz) δ 7.62 (s, 1 H), 7.52 (s, 1 H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1 H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1 .37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Step 6: Synthesis of 5-bromo-N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-3 -(ethyl (tetrahydro-2H-pyra -4-yl) amino)-2-methylbenzamide

To a stirred solution of 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2- methylbenzoate (14 g, 39.4 mmol) in ethanol ( 100 mL) was added aqueous NaOH (2.36 g, 59.2 mmol in 25mL water) and the resulting mixture was stirred at 60 °C for 1 h. Upon completion of the reaction as determined by TLC, the solvent was removed under reduced pressure and the residue obtained was acidified with IN HC1 until a pH 7 was obtained and then aqueous citric acid solution was added until a pH 5-6 was obtained. The aqueous layer was extracted with 10% MeOH in DCM (200mL X 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the respective acid (14 g, 100%).

The above acid (14 g, 40.9 mmol) was then dissolved in DMSO (70 mL) and 3- (amino methyl)-4, 6-dimethylpyridin-2( l H)-one ( 12.4 g, 81 .9 mmol) was added to it. The reaction mixture was stirred at room temperature for 15 minutes, then PYBOP (31.9 g, 61.4 mmol) was added and stirring was continued for overnight at room temperature. Upon completion of the reaction as determined by TLC, the reaction mixture was poured onto ice- cold water (700 mL), stirred for 30 minutes and the precipitated solid was collected by filtration, washed with water (500 mL) and air dried. The solid obtained was stirred with acetonitrile (75mL X 2), filtered and air dried. The solid obtained was again stirred with 5% MeOH in DCM ( l OOmL), filtered and dried completely under vacuum to afford the title compound as a solid ( 14 g, 74 %). Ή NMR (DMSO- ₆, 400 MHz) δ 1 1.47 (s, 1 H), 8.23 (t, 1 H), 7.30 (s, 1 H), 7.08 (s, 1 H), 5.85 (s, 1 H), 4.23 (d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=l 0.4 Hz), 3.20-3.26 (m, 2H), 3.00-3.07 (m, I H), 2.91 -2.96 (m, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H), 1.58-1.60 (m, 2H), 1.45-1.50 (m, 2H), 0.78 (t, 3H, J=6.8 Hz).

Step 7: Synthesis of N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-5- (ethyl (tetrahydro-2H-pyran-4-yl) amino)-4-methyl-4′-(morpholinomethyl)-[l , l ‘-biphenyl]-3- carboxamide

TITLE COMPD

To a stirred solution of 5-bromo-N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzamide (14 g, 29.5 mmol) in dioxane/ water mixture (70 mL/ 14 mL) was added 4-(4-(4, 4, 5, 5-tetramethyl- l , 3, 2- dioxaborolan-2-yl) benzyl) morpholine (13.4 g, 44.2 mmol) followed by addition of Na₂C0₃ (1 1 .2 g, 106.1 mmol). The solution was purged with argon for 15 minutes and then Pd (PPh₃)₄ (3.40 g, 2.94 mmol) was added and the solution was again purged with argon for a further 10 min. The reaction mixture was heated at 100°C for 4 h. After completion (monitored by TLC), the reaction mixture was diluted with water and extracted with 10% MeOH/DCM.

The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100- 200 mesh silica gel) eluting with methanol: DCM to the title compound as a solid (12 g, 71 %).

Analytical Data: LCMS: 573.35 (M + 1 )⁺; HPLC: 99.5% (@ 254 nm) (R,;3.999; Method: Column: YMC ODS-A 1 50 mm x 4.6 mm x 5 μ; Mobile Phase: A; 0.05% TFA in water/ B; 0.05% TFA in acetonitrile; Inj. Vol : 10 μΐ, Col. Temp.: 30 °C; Flow rate: 1 .4 mL/min.;

Gradient: 5% B to 95% B in 8 min, Hold for 1 .5 min, 9.51 -12 min 5% B);

Ή NMR (DMSO-i ₆, 400 MHz) 5 1 1 .46 (s, I H), 8. 19 (t, 1 H), 7.57 (d, 2H, J=7.2 Hz), 7.36-7.39 (m, 3H), 7.21 (s, I H), 5.85 (s, I H), 4.28 (d, 2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57 (bs, 4H), 3.48 (s, 2H), 3.24 (t, 2H, J=10.8Hz), 3.07-3.09 (m, 2H), 3.01 (m, I H), 2.36 (m, 4H), 2.24 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 1 .64-1 .67 (m, 2H), 1 .51 – 1 .53 (m, 2H), 0.83 (t, 3H, J=6.4 Hz).

TRIHYDROCHLORIDE

Step 8: Synthesis of N-((4,6-dimethyl-2-oxo-l ,2-dihydropyridin-3-yl)methyl)-5- (ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[ 1 , 1 ‘-biphenyl]-3- carboxamide trihydrochloride

N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-5-(ethyl (tetrahydro- 21 l-pyran-4-yl) amino)-4-methyI-4′-(niorpholinornethyl)-[ 1 , 1 ‘-biphenyl]-3-carboxamide ( 12 g, 21.0 mmol) was dissolved in methanolic HC1 (200 mL) and stirred at room temperature for 3 h. After three hours of stirring, the reaction mixture was concentrated under reduced pressure. The solid obtained was stirred with ether ( l OOmL X 2) to afford the desired salt as a solid ( 1 1 g, 77 %).

Analytical Data of the tri-HCl salt: LCMS: 573.40 (M + 1 )⁺; HPLC: 99.1 % (@ 254 nm) (R,;3.961 ; Method: Column: YMC ODS-A 150 mm x 4.6 mm x 5 μ; Mobile Phase: A; 0.05% TFA in water/ B; 0.05% TFA in acetonitrile; Inj. Vol: 10 pL, Col. Temp.: 30 °C; Flow rate: 1.4 mL/min.; Gradient: 5% B to 95% B in 8 min, Hold for 1.5 min, 9.51 -12 min 5% B);

Ή NMR (D₂0 400 MHz) δ 7.92 (bs, I H,) 7.80 (s, I H), 7.77 (d, 2H, J=8 Hz), 7.63 (s, I H), 7.61 (s, I H), 6.30 (s, I H), 4.48 (s, 2H), 4.42 (s, 2H), 4.09-4.1 1 (m, 4H), 3.95-3.97 (m, 2H), 3.77 (t, 3H, J=10.4 Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H), 2.35 (s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1 .76 (m, 2H), 1 .04 (t, 3H, J=6.8 Hz).

…………………………………………

PATENT

WO2013155317

 http://www.google.com/patents/WO2013155317A1?cl=en

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl] -3-carboxamide hydrobromide:

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-5-(ethyl

(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’-biphenyl]-3- carboxamide hydrobromide:

As used herein, “Compound I” refers to N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl]-3-carboxamide. The hydrobromide of Compound I can be used to inhibit the histone methyltransferase activity of EZH2, either in a subject or in vitro. The hydrobromide of Compound I can also be used to treat cancer in a subject in need thereof.

Scheme 1

……………………………………..Compound I                                       Compound I – HBr

HPLC

HPLC was conducted on an Agilent 1200 HPLC quaternary pump, low pressure mixing, with an in-line degasser. Analytical method conditions: 8 μΐ_^ sample (20 mg of ER-581982-06 diluted with 50 mL of a methanol to provide approximately 0.4 mg/mL solution) was injected onto a Agilent Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 3.5 um), Chromatography conditions: mobile phase A, water with 5mM ammonium formate; mobile phase B, 5 mM ammonium formate in 50/45/5 acetonitrile/methanol/water; flow rate, 1.5 ml/min.; gradient: isocratic at 10% B from 0 to 3 min; linear increase to 70% B from 3 to 7 min; isocratic at 70% B from 7 to 12 min; linear increase to 100% B from 12 to 15 min isocratic at 100% B from 15 to 20 min;

column temperature, 35 °C; detection, UV 230 nm. Approximate retention time of Compound I = 10.7 min.

Synthesis of Polymorph A

5-bromo-2-methyl-3-nitrobenzoic acid stirred solution of 2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) in cone. H₂S0₄ (400 mL), l,3-dibromo-5,5-dimethyl-2,4- imidazolidinedione (88 g, 308 mmol) was added in a portion wise manner at room temperature and the reaction mixture was then stirred at room temperature for 5 h. The reaction mixture was poured onto ice cold water, the precipitated solid was filtered off, washed with water and dried under vacuum to afford the desired compound as a solid (140 g, 98%). The isolated compound was taken directly into the next step. 1H NMR (DMSO-J₆, 400 MHz) δ 8.31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).

Methyl 5-bromo-2-methyl-3-nitrobenzoate To a stirred solution of 5-bromo-2- methyl-3-nitrobenzoic acid (285 g, 1105 mmol) in DMF (2.8L) at room temperature was added sodium carbonate (468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60 °C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate (1L X 3). The combined filtrate was washed with water (3L X 5) and the aqueous phase was back extracted with ethyl acetate (1L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (290g, 97% yield). The isolated compound was taken directly into the next step. 1H NMR (CDC1₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Methyl 3-amino-5-bromo-2-methylbenzoate (1) To a stirred solution of methyl 5- bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol (1.5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5L water). The resulting mixture was stirred at 80°C to which iron powder (472 g, 8451 mmol) was added in a portion wise manner. The resulting reaction mixture was heated at 80 °C for 12 h. Upon completion as determined by TLC, the reaction mixture was hot filtered over celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5L). The combined filtrate was concentrated in- vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2L) and extracted with ethyl acetate (5L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (220 g, 85%). The compound was taken directly into the next step. 1H

NMR (CDCI₃, 400 MHz) δ 7.37 (s, 1H), 6.92 (s, 1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (2) A reactor was charged with methyl 3-amino-5-bromo-2-methylbenzoate (455.8 g, 1.87 mol), 1,2- Dichloroethane (4.56 L), and acetic acid (535 ml, 9.34 mol). To the mixture were added dihydro-2H-pyran-4(3H)-one (280 g, 2.80 mol) and sodium triacetoxyborohydride (594 g, 2.80 mol) maintaining the internal temperature below 40 °C. The mixture was stirred at 25 °C for 2.5 h and then the reaction was quenched with a solution of sodium hydroxide (448 g, 11.20 mol) in water (5.61 L). After stirring for 20 minutes at ambient temperature, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (3.65 L). The organic layers were combined, washed with brine (1.5 L), and concentrated under vacuum.

The residue was treated with ethyl acetate (1.8 L) and heated to 65-70 °C. The mixture was stirred at 65-70 °C for 15 minutes to give a clear solution and then treated with n-heptane (7.3 L) maintaining the temperature between 60-70 °C. Once the heptane was completely added to the solution, the mixture was held at 65-70 °C for 15 minutes and then allowed to cool to 18- 22 °C over 3 h. The resulting suspension was stirred at 18-22 °C for 4 h, cooled to 0-5 °C over 1 h, and held at 0-5 °C for 2 h. The precipitate was filtered, washed twice with n-heptane (1.4 L), and dried under vacuum to give the title compound (540 g, 88%). The XRPD pattern of this compound is shown in Figure 17.

Methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate (3)

To a stirred solution of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g, 85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0 °C and sodium triacetoxyborohydride (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%). 1H NMR DMSO-d₆, 400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1.37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-

[l,l’-biphenyl]-3-carboxylate (4): A mixture of methyl 5-bromo-3-(ethyl(tetrahydro-2H-pyran- 4-yl)amino)-2-methylbenzoate (580 g, 1.63 mol), 4-(4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan- 2-yl)benzyl)morpholine (592 g, 1.95 mol), 1,4-dioxane (3.86 L), sodium carbonate (618 g, 5.83 mol), and water (771 ml) was degassed by bubbling nitrogen through the mixture at 20 °C for 20 minutes and treated with tetrakis(triphenylphosphine)palladium(0) (14.11 g, 12.21 mmol). The resulting mixture was degassed for an additional 20 minutes and then heated to 87-89 °C for 17 h. After cooling to 20 °C, the mixture was diluted with ethyl acetate (5.80 L) and a solution of (R)-2-Amino-3-mercaptopropionic acid (232 g) in water (2.320 L). After stirring for 1 h at 20 °C, the organic layer was separated and washed again with a solution of (R)-2-Amino-3- mercaptopropionic acid (232 g) in water (2.320 L). The aqueous layers were combined and extracted with ethyl acetate (5.80 L). The organic layers were combined, washed with a solution of sodium hydroxide (93 g) in water (2.32 L), and concentrated under vacuum at 35 °C to give the title compound as an orange oil (1.21 kg, 164% yield).

5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl]-3-carboxylic acid (5): Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′- (morpholinomethyl)-[l,l’-biphenyl]-3-carboxylate (69.0 g, 152.5 mmol) (based on the theoretical yield from the previous step) was suspended in ethanol (380 mL) and treated with a solution of sodium hydroxide (24.84 g, 621.0 mmol) in water (207 mL). The mixture was stirred at 40°C for 18 h. After cooling to 0-5 °C, the mixture was neutralized to pH 6.5 with 1 N hydrochloric acid (580 mL) maintaining the temperature below 25 °C. Then, the mixture was extracted twice with a mixture of dichloromethane (690 mL) and methanol (69.0 mL). The organic layers were combined and concentrated under vacuum to give a crude product as a yellow solid (127g).

The crude product was dissolved in 2-methyltetrahydrofuran (656 mL) at 70 °C and then treated with IPA (828 mL). The mixture was allowed to cool to rt over 3-4 h and then stirred overnight at rt. The precipitate was filtered, washed twice with IPA (207 mL), and dried under vacuum to give the title compound as an off white solid (53.54 g, 80%). The XRPD pattern of this compound is shown in Figure 9.

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H- pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’-biphenyl]-3-carboxamide

(Compound I): A mixture of 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′- (morpholinomethyl)-[l,l’-biphenyl]-3-carboxylic acid (540 g, 1.23 mol) and 3-(aminomethyl)- 4,6-dimethyl-dihydro-pyridin-2(lH)-one hydrochloride (279 g, 1.48 mol) was suspended in DMSO (2.70 L) and treated with triethylamine (223 ml, 1.60 mol). The mixture was stirred at 25 °C for 30 min and treated with EDC-HC1 (354 g, 1.85 mol) and HOBT hydrate (283 g, 1.85 mol). The reaction mixture was stirred at rt for 16 h. After addition of triethylamine (292 ml, 2.09 mol), the mixture was cooled to 15 °C, diluted with water (10.1 L) maintaining the temperature below 30 °C, and stirred at 19-25 °C for 4 h. The resulting precipitate was filtered, washed twice with water (2.70 L), and dried under vacuum to give a crude product (695 g, wt-wt analysis = 78%).

For the further purification of the product, recrystallization was conducted. A crude product (20.00 g, 34.92 mmol) was suspended in a mixture of ethanol (190 ml) and water (10.00 ml) and heated to 75°C until a clear solution was obtained. The solution was allowed to cool to rt overnight. The precipitate was filtered, washed twice with a mixture of ethanol (30.0 ml) and water (30.0 ml), and dried under vacuum at 35 °C to give the title compound as an off white solid (14.0 g, 70% recovery from the crude and 90% yield based on wt-wt assay).

4-((3′-(((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[l,l’-biphenyl]-4-yl)methyl)morpholin- 4-ium bromide (Polymorph A): A crude N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)am

biphenyl]-3-carboxamide (595 g, 464 g based on wt-wt assay, 810.3 mmol) was suspended in ethanol (3.33 L). After heating to 70 °C, the mixture was treated with 48% aqueous HBr (97 ml, 850.8 mmol) and stirred at 70 °C for 30 min. The resulting orange-red solution was treated with ethyl acetate (3.33 L) maintaining the temperature above 60 °C. The mixture was slowly cooled to rt over 18 h. The mixture was cooled to 0 °C over 1 h and stirred at that temperature for 5.5 h. The resulting precipitate was filtered, washed twice with ethyl acetate (1.39 L), and dried under vacuum to give the title compound as an off white solid (515 g, 97% yield).

Recrystallization of Polymorph A: 4-((3′-(((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[l,l’-biphenyl]-4- yl)methyl)morpholin-4-ium bromide (0.50 g, 0.77 mmol; 95.6% pure by HPLC) was suspended in ethanol (3.0 mL) and heated to 80 °C until a clear solution was obtained. To the solution was added MTBE (5.0 mL) slowly. The resulting solution was allowed to cool to 18-22 °C over 3 h and stirred at 18-22 °C for 15 h. The precipitate was filtered, washed twice with MTBE (2 mL) and dried under vacuum to give 0.45 g of the title compound (89% recovery, 96.6% pure by HPLC).

Compound I is protonated at the nitrogen of the morpholino substituent, providing a monohydrobromide of Compound I having the following structure:

This particular monohydrobromide can be referred to as “4-((3′-(((4,6-dimethyl-2-oxo- l,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′- methyl-[l, -biphenyl]-4-yl)methyl)morpholin-4-ium bromide.” Figure 11 depicts the X-ray crystal structure of this particular salt form.

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see

WO-2015057859

Eisai Research Institute; Epizyme Inc

Novel crystalline polymorphic form C of tazemetostat, useful for treating an EZH2-mediated cancer, including non-Hodgkin’s lymphoma and breast cancer.

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PAPER

RSC Advances (2015), 5(33), 25967-25978

http://pubs.rsc.org/en/content/articlelanding/2015/ra/c5ra02365c#!divAbstract

DOI: 10.1039/C5RA02365C

The histone lysine methyltransferase EZH2 has been implicated as a key component in cancer aggressiveness, metastasis and poor prognosis. This study discovered a new class of hexahydroisoquinolin derivatives as EZH2 inhibitors. A structure–activity relationship study showed that the steric hindrance was important to the activity for EZH2. A preliminary optimization study led to the discovery of several potent compounds with low nanomolar to sub-nanomolar potency for EZH2. Biological evaluation indicated that SKLB1049 was a highly potent with improved solubility compared to EPZ6438, SAM-competitive, and cell-active EZH2 inhibitor that decreased global H3K27me3 in SU-DHL-6 and Pfeiffer lymphoma cells in a concentration- and time-dependent manner. Further study indicated that SKLB1049 caused cell arrest in G₀/G₁ phase. These compounds would be useful as chemical tools to further explore the biology of EZH2 and provided us with a start point to develop new EZH2 inhibitors.

1: Knutson SK, Warholic NM, Johnston LD, Klaus CR, Wigle TJ, Iwanowicz D, Littlefield BA, Porter-Scott M, Smith JJ, Moyer MP, Copeland RA, Pollock RM, Kuntz KW, Raimondi A, Keilhack H. Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas. PLoS One. 2014 Dec 10;9(12):e111840. doi: 10.1371/journal.pone.0111840. eCollection 2014. PubMed PMID: 25493630; PubMed  Central PMCID: PMC4262195.

2: Knutson SK, Kawano S, Minoshima Y, Warholic NM, Huang KC, Xiao Y, Kadowaki T,  Uesugi M, Kuznetsov G, Kumar N, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Waters NJ, Smith JJ, Porter-Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Uenaka T, Pollock RM, Kuntz KW, Yokoi A, Keilhack H. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol Cancer Ther. 2014 Apr;13(4):842-54. doi: 10.1158/1535-7163.MCT-13-0773. Epub 2014 Feb 21. PubMed PMID: 24563539

3. Inhibitors of human histone methyltransferase EZH2, and methods of use thereof for treating cancer. By Kuntz, Kevin W.; Knutson, Sarah K.; Wigle, Timothy James Nelson . From U.S. Pat. Appl. Publ. (2013), US 20130040906 A1 20130214.

4. Aryl-or heteroaryl-substituted benzamide compounds as anticancer agents and their preparation By Kuntz, Kevin Wayne; Chesworth, Richard; Duncan, Kenneth William; Keilhack, Heike; Warholic, Natalie; Klaus, Christine; Zheng, Wanjun; Seki, Masashi; Shirotori, Syuji; Kawano, Satoshi From PCT Int. Appl. (2012), WO 2012142504 A1 20121018.

5: Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Porter Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Pollock RM, Kuntz KW, Keilhack H. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7922-7. doi: 10.1073/pnas.1303800110. Epub 2013 Apr 25. PubMed PMID: 23620515; PubMed Central PMCID: PMC3651445.

References

External links

• “Tazemetostat”. Drug Information Portal. U.S. National Library of Medicine.

Tazemetostat

Clinical data
Trade names	Tazverik
Other names	EPZ-6438
AHFS/Drugs.com	Monograph
MedlinePlus	a620018
License data	US DailyMed: Tazemetostat US FDA: Tazverik
Legal status
Legal status	US: ℞-only
Identifiers
IUPAC name[show]
CAS Number	1403254-99-8
PubChem CID	66558664
DrugBank	DB12887
ChemSpider	30208713
UNII	Q40W93WPE1
KEGG	D11485 D11444
ChEMBL	ChEMBL3414621
Chemical and physical data
Formula	C34H44N4O4
Molar mass	572.750 g·mol−1
3D model (JSmol)	Interactive image
SMILES[hide] CCN(C1CCOCC1)C2=CC(=CC(=C2C)C(=O)NCC3=C(C=C(NC3=O)C)C)C4=CC=C(C=C4)CN5CCOCC5
InChI[hide] InChI=1S/C34H44N4O4/c1-5-38(29-10-14-41-15-11-29)32-20-28(27-8-6-26(7-9-27)22-37-12-16-42-17-13-37)19-30(25(32)4)33(39)35-21-31-23(2)18-24(3)36-34(31)40/h6-9,18-20,29H,5,10-17,21-22H2,1-4H3,(H,35,39)(H,36,40) Key:NSQSAUGJQHDYNO-UHFFFAOYSA-N

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