Reldesemtiv

CK-2127107

CAS 1345410-31-2

UNII-4S0HBYW6QE, 4S0HBYW6QE

MW 384.4 g/mol, MF C₁₉H₁₈F₂N₆O

1-[2-({[trans-3-fluoro-1-(3-fluoropyridin-2-yl)cyclobutyl]methyl}amino)pyrimidin-5-yl]-1H-pyrrole-3- carboxamide

1-[2-[[3-fluoro-1-(3-fluoropyridin-2-yl)cyclobutyl]methylamino]pyrimidin-5-yl]pyrrole-3-carboxamide

Reldesemtiv, also known as CK-2127107, is a skeletal muscle troponin activator (FSTA) and is a potential treatment for people living with debilitating diseases and conditions associated with neuromuscular or non-neuromuscular dysfunction, muscular weakness, and/or muscle fatigue such as SMA, COPD, and ALS.

Cytokinetics , in collaboration with  Astellas , is developing reldesemtiv, the lead from a program of selective fast skeletal muscle troponin activators, in an oral suspension formulation, for the treatment of indications associated with neuromuscular dysfunction, including spinal muscular atrophy and amyotrophic lateral sclerosis.

• Originator Cytokinetics
• Developer Astellas Pharma; Cytokinetics
• Class Pyridines; Pyrimidines; Pyrroles; Small molecules
• Mechanism of Action Troponin stimulants

• Orphan Drug Status Yes – Spinal muscular atrophy

• Phase II Amyotrophic lateral sclerosis; Chronic obstructive pulmonary disease; Spinal muscular atrophy
• Suspended Muscle fatigue
• No development reported Muscular atrophy

• 05 May 2019 Safety and efficacy data from the phase II FORTITUDE-ALS trial in Amyotrophic lateral sclerosis presented at the American Academy of Neurology Annual Meeting (AAN-2019)
• 07 Mar 2019 Cytokinetics completes the phase III FORTITUDE-ALS trial for Amyotrophic lateral sclerosis in USA, Australia, Canada, Spain, Ireland and Netherlands (PO) (NCT03160898)
• 22 Jan 2019 Cytokinetics plans a phase I trial in Healthy volunteers in the first quarter of 2019

Reldesemtiv, a next-generation, orally-available, highly specific small-molecule is being developed by Cytokinetics, in collaboration with Astellas Pharma, for the improvement of skeletal muscle function associated with neuromuscular dysfunction, muscle weakness and/or muscle fatigue in spinal muscular atrophy (SMA), chronic obstructive pulmonary disease (COPD) and amyotrophic lateral sclerosis (ALS). The drug candidate is a fast skeletal muscle troponin activator (FSTA) or troponin stimulant intended to slow the rate of calcium release from the regulatory troponin complex of fast skeletal muscle fibers. Clinical development for ALS, COPD and SMA is underway in the US, Australia, Canada, Ireland, Netherlands and Spain. No recent reports of development had been identified for phase I development for muscular atrophy in the US. Due to lack of of efficacy determined at interim analysis Cytokinetics suspended phase I trial in muscle fatigue in the elderly.

The cytoskeleton of skeletal and cardiac muscle cells is unique compared to that of all other cells. It consists of a nearly crystalline array of closely packed cytoskeletal proteins called the sarcomere. The sarcomere is elegantly organized as an interdigitating array of thin and thick filaments. The thick filaments are composed of myosin, the motor protein responsible for transducing the chemical energy of ATP hydrolysis into force and directed movement. The thin filaments are composed of actin monomers arranged in a helical array. There are four regulatory proteins bound to the actin filaments, which allows the contraction to be modulated by calcium ions. An influx of intracellular calcium initiates muscle contraction; thick and thin filaments slide past each other driven by repetitive interactions of the myosin motor domains with the thin actin filaments.

[0003] Of the thirteen distinct classes of myosin in human cells, the myosin-II class is responsible for contraction of skeletal, cardiac, and smooth muscle. This class of myosin is significantly different in amino acid composition and in overall structure from myosin in the other twelve distinct classes. Myosin-II forms homo-dimers resulting in two globular head domains linked together by a long alpha-helical coiled-coiled tail to form the core of the sarcomere’s thick filament. The globular heads have a catalytic domain where the actin binding and ATPase functions of myosin take place. Once bound to an actin filament, the release of phosphate (cf. ADP-Pi to ADP) signals a change in structural conformation of the catalytic domain that in turn alters the orientation of the light-chain binding lever arm domain that extends from the globular head; this movement is termed the powerstroke. This change in orientation of the myosin head in relationship to actin causes the thick filament of which it is a part to move with respect to the thin actin filament to which it is bound. Un-binding of the globular head from the actin filament (Ca²⁺ regulated) coupled with return of the catalytic domain and light chain to their starting conformation/orientation completes the catalytic cycle, responsible for intracellular movement and muscle contraction.

Tropomyosin and troponin mediate the calcium effect on the interaction on actin and myosin. The troponin complex is comprised of three polypeptide chains: troponin C, which binds calcium ions; troponin I, which binds to actin; and troponin T, which binds to tropomyosin. The skeletal troponin-tropomyosin complex regulates the myosin binding sites extending over several actin units at once.

Troponin, a complex of the three polypeptides described above, is an accessory protein that is closely associated with actin filaments in vertebrate muscle. The troponin complex acts in conjunction with the muscle form of tropomyosin to mediate the

Ca²⁺ dependency of myosin ATPase activity and thereby regulate muscle contraction. The troponin polypeptides T, I, and C, are named for their tropomyosin binding, inhibitory, and calcium binding activities, respectively. Troponin T binds to tropomyosin and is believed to be responsible for positioning the troponin complex on the muscle thin filament. Troponin I binds to actin, and the complex formed by troponins I and T, and tropomyosin inhibits the interaction of actin and myosin. Skeletal troponin C is capable of binding up to four calcium molecules. Studies suggest that when the level of calcium in the muscle is raised, troponin C exposes a binding site for troponin I, recruiting it away from actin. This causes the tropomyosin molecule to shift its position as well, thereby exposing the myosin binding sites on actin and stimulating myosin ATPase activity.

U.S. Patent No. 8962632 discloses l-(2-((((trans)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl)amino)pyrimidin-5-yl)-lH-pyrrole-3-carboxamide, a next-generation fast skeletal muscle troponin activator (FSTA) as a potential treatment for people living with debilitating diseases and conditions associated with neuromuscular or non-neuromuscular dysfunction, muscular weakness, and/or muscle fatigue.

PATENT

WO 2011133888

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011133888&recNum=202&docAn=US2011033614&queryString=&maxRec=57668

PATENT

WO2016039367 ,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016039367&tab=FULLTEXT

claiming the use of a similar compound for treating stress urinary incontinence.

[Chemical formula 1]

PATENT

WO-2019133605

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019133605&tab=PCTDESCRIPTION&_cid=P11-JXY4C3-99085-1

Process for preparing reldesemtiv , a myosin, actin, tropomyosin, troponin C, troponin I, troponin T modulator, useful for treating neuromuscular disorders, muscle wasting, claudication and metabolic syndrome.

Scheme 1

[0091] Scheme 1 illustrates a scheme of synthesizing the compound of Formula (1C).

Scheme 2

[0092] Scheme 2 illustrates an alternative scheme of synthesizing the compound of Formula (1C).

M

TFAA DS, toluene

Et

to

2 

HCI, H₂0

50°C

Scheme 3

[0093] Scheme 3 illustrates a scheme of converting the compound of Formula (1C) to the compound of Formula (II).

H₂

Ni Raney

NH₃

Scheme 4

[0094] Scheme 4 illustrates a scheme of converting the compound of Formula (II) to the compound of Formula (1).

Examples

[0095] To a flask was added N-methylpyrrolidone (30 mL), tert-butyl cyanoacetate (8.08 g) at room temperature. To a resulting solution was added potassium tert-butoxide (7.71 g), l,3-dibromo-2,2-dimethoxy propane (5.00 g) at 0 °C. To another flask, potassium iodide (158 mg), 2,6-di-tert-butyl-p-cresol (42 mg), N-methylpyrrolidone (25 mL) were added at room temperature and then resulting solution was heated to 165 °C. To this solution, previously prepared mixture was added dropwise at 140-165 °C, then stirred for 2 hours at 165 °C. To the reaction mixture, water (65 mL) was added. A resulting solution was extracted with toluene (40 mL, three times) and then combined organic layer was washed with water (20 mL, three times) and 1N NaOH aq. (20 mL). A resulting organic layer was concentrated below 50 °C under reduced pressure to give 3, 3 -dimethoxy cyclobutane- l-carbonitrile (66% yield,

GC assay) as toluene solution. 1H MR (CDCl₃, 400 MHz) d 3.17 (s, 3H), 3.15 (s, 3H), 2.93-2.84 (m, 1H), 2.63-2.57 (m, 2H), 2.52-2.45 (m, 2H).

Example 2 Synthesis of methyl 3,3-dimethoxycyclobutane-l-carboxylate

[0096] A reactor was vacuumed to 0.02 MPa and less and then inerted with nitrogen to atmosphere for three times. MeOH (339.00 kg), 3-oxocyclobutanecarboxylic acid (85.19 kg, 746.6 mol, 1.0 eq.), Amberlyst-l5 ion exchange resin (8.90 kg, 10% w/w), and

trimethoxymethane (196.00 kg, 1847.3 mol, 2.5 eq.) were charged into the reactor and the resulting mixture was heated to 55±5°C and reacted for 6 hours to give methyl 3,3-dimethoxycyclobutane-l-carboxylate solution in MeOH. 1H NMR (CDCl₃, 400 MHz) d 3.70 (s, 3H), 3.17 (s, 3H), 3.15 (s, 3H), 2.94-2.85 (m, 1H), 2.47-2.36 (m, 4H).

Example 3 Synthesis of 3, 3-dimethoxycyclobutane-l -carboxamide

[0097] The methyl 3, 3 -dimethoxy cyclobutane- l-carboxylate solution in MeOH prepared as described in Example 2 was cooled to below 25°C and centrifuged. The filter cake was washed with MeOH(7.00 kg) and the filtrate was pumped to the reactor. The solution was concentrated under vacuum below 55°C until the system had no more than 2 volumes. MeOH

(139.40 kg) was charged to the reactor and the solution was concentrated under vacuum below 55°C until the system had no more than 2 volumes. MeOH (130.00 kg) was charged to the reactor and the solution was concentrated under vacuum below 55°C until the system had no more than 2 volumes. Half of the resulting solution was diluted with MeOH (435.00 kg) and cooled to below 30°C. NH₃ gas (133.80 kg) was injected into the reactor below 35°C for

24 hours. The mixture was stirred at 40±5°C for 72 hours. The resulting solution was

concentrated under vacuum below 50°C until the system had no more than 2 volumes.

MTBE(l8l.OO kg) was charged into the reactor. The resulting solution was concentrated under vacuum below 50°C until the system had no more than 2 volumes. PE (318.00 kg) was charged into the reactor. The resulting mixture was cooled to 5±5°C, stirred for 4 hours at 5±5°C, and centrifuged. The filter cake was washed with PE (42.00 kg) and the wet filter cake was put into a vacuum oven. The filter cake was dried at 30±5°C for at least 8 hours to give 3,3-dimethoxycyclobutane-l-carboxamide as off-white solid (112.63 kg, 94.7% yield). 1H NMR (CDCf, 400 MHz) d 5.76 (bs, 1H), 5.64 (bs, 1H), 3.18 (s, 3H), 3.17 (s, 3H), 2.84-2.76 (m, 1H), 2.45-2.38 (m, 4H).

[0098] A reactor was vacuumed to 0.02 MPa and less and then inerted with nitrogen to atmosphere for three times. Toluene (500.00 kg), 3,3-dimethoxycyclobutane-l-carboxamide (112.54kg, 706.9 mol, 1.0 eq.), and TEA (158.00 kg, 1561.3 mol, 2.20 eq) were charged into the reactor and the resulting mixture was cooled to 0+ 5°C. TFAA (164.00 kg, 781 mol, 1.10 eq.) was added dropwise at 0±5°C. The resulting mixture was stirred for 10 hours at 20±5°C and cooled below 5±5°C. H₂0 (110.00 kg) was charged into the reactor at below 15 °C. The resulting mixture was stirred for 30 minutes and the water phase was separated. The aqueous phase was extracted with toluene (190.00 kg) twice. The organic phases were combined and washed with H₂0 (111.00 kg). H₂0 was removed by azeotrope until the water content was no more than 0.03%. The resulting solution was cooled to below 20°C to give 3,3-dimethoxycyclobutane-l-carbonitrile solution in toluene (492.00 kg with 17.83% assay content, 87.9% yield).

Example 5 Synthesis of l-(3-fluoropyridin-2-yl)-3,3-dimethoxycyclobutane-l-carbonitrile

[0099] A reactor was vacuumed to 0.02 MPa and less and then inerted with nitrogen to atmosphere for three times. The 3,3-dimethoxycyclobutane-l-carbonitrile solution in toluene prepared as described in Example 4 (246.00 kg of a 17.8% solution of 3,3-dimethoxycyclobutane-l-carbonitrile in toluene, 1.05 eq.) and 2-chloro-3-fluoropyridine (39.17 kg, 297.9 mol, 1.00 eq.) were charged into the reactor. The reactor was vacuumed to 0.02 MPa and less and then inerted with nitrogen to atmosphere for three times. The mixture was slowly cooled to -20±5°C. NaHDMS (2M in THF) (165.71 kg, 1.20 eq) was added

dropwise at -20±5°C. The resulting mixture was stirred at -l5±5°C for 1 hour. The mixture was stirred until the content of 2-chloro-3-fluoropyridine is no more than 2% as measured by HPLC. Soft water (16.00 kg) was added dropwise at below 0°C while maintaining the reactor temperature. The resulting solution was transferred to another reactor. Aq. NH₄Cl (10% w/w, 88.60 Kg) was added dropwise at below 0°C while maintaining the reactor temperature. Soft water (112.00 kg) was charged into the reactor and the aqueous phase was separated and collected. The aqueous phase was extracted with ethyl acetate (70.00 kg) and an organic phase was collected. The organic phase was washed with sat. NaCl (106.00 kg) and collected. The above steps were repeated to obtain another batch of organic phase. The two batches of organic phase were concentrated under vacuum below 70°C until the system had no more than 2 volumes. The resulting solution was cooled to below 30°C to give a l-(3-fluoropyridin-2-yl)-3, 3 -dimethoxy cyclobutane- l-carbonitrile solution. 1H NMR (CDC1₃, 400 MHz) d 8.42-8.38 (m, 1H), 7.50-7.45 (m, 1H), 7.38-7.33 (m, 1H), 3.28 (s, 3 H), 3.13 (s, 3H), 3.09-3.05 (m, 4H).

Example 6 Synthesis of I-(3-fluoropyridin-2-yl)-3-oxocyclohutanecarhonitrile

[0100] A reactor was vacuumed to 0.02 MPa and less and then inerted with nitrogen to atmosphere for three times. Water (603.00 kg) was added to the reactor and was stirred.

Concentrated HC1 (157.30 kg) was charged into the reactor at below 35°C. The l-(3-fluoropyridin-2-yl)-3, 3 -dimethoxy cyclobutane- l-carbonitrile solution prepared as described in Example 5 (206.00 kg) was charged into the reactor and the resulting mixture was heated to 50±5°C and reacted for 3 hours at 50±5°C. The mixture was reacted until the content of 1-(3 -fluoropyridin-2-yl)-3, 3 -dimethoxycyclobutane- l-carbonitrile was no more than 2.0% as measured by HPLC. The reaction mixture was cooled to below 30°C and extracted with ethyl acetate (771.00 kg). An aqueous phase was collected and extracted with ethyl acetate (770.00 kg). The organic phases were combined and the combined organic phase was washed with soft water (290.00 kg) and brine (385.30 kg). The organic phase was concentrated under vacuum at below 60°C until the system had no more than 2 volumes. Propan-2-ol (218.00 kg) was charged into the reactor. The organic phase was concentrated under vacuum at below

60°C until the system had no more than 1 volume. PE (191.00 kg) was charged into the reactor at 40±5 °C and the resulting mixture was heated to 60±5 °C and stirred for 1 hour at 60±5 °C. The mixture was then slowly cooled to 5±5 °C and stirred for 5 hours at 5±5 °C. The mixture was centrifuged and the filter cake was washed with PE (48.00 kg) and the wet filter cake was collected. Water (80.00 kg), concentrated HC1 (2.20 kg), propan-2-ol (65.00 kg), and the wet filter cake were charged in this order into a drum. The resulting mixture was stirred for 10 minutes at 20±5 °C. The mixture was centrifuged and the filter cake was washed with a mixture solution containing 18.00 kg of propan-2-ol, 22.50 kg of soft water, and 0.60 kg of concentrated HC1. The filter cake was put into a vacuum oven and dried at 30±5°C for at least 10 hours. The filter cake was dried until the weight did not change to give l-(3-fluoropyridin-2-yl)-3-oxocyclobutanecarbonitrile as off-white solid (77.15 kg, 68.0% yield). 1H NMR (CDCl₃, 400 MHz) d 8.45-8.42 (m, 1H), 7.60-7.54 (m, 1H), 7.47-7.41 (m, 1H), 4.18-4.09 (m, 2H), 4.02-3.94 (m, 2H).

Example 7 Synthesis of I-(3-fhtoropyridin-2-yl)-3-hydroxycyclobulanecarbonilrile

[0101] To a solution of l-(3-fluoropyridin-2-yl)-3-oxocyclobutanecarbonitrile (231 g,

1.22 mol) in a mixture ofDCM (2 L) and MeOH (200 mL) was added NaBH₄ portionwise at -78° C. The reaction mixture was stirred at -78°C. for 1 hour and quenched with a mixture of methanol and water (1 : 1). The organic layer was washed with water (500 mL><3), dried over Na₂S0₄, and concentrated. The residue was purified on silica gel (50% EtO Ac/hexanes) to provide the title compound as an amber oil (185.8 g, 77.5%). Low Resolution Mass

Spectrometry (LRMS) (M+H) m/z 193.2.

Example 8 Synthesis of (ls,3s)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutane-l-carbonitrile

[0102] To a solution of 1 -(3 -fluoropyridin-2-yl)-3 -hydroxy cyclobutanecarbonitrile (185 g, 0.96 mol) in DCM (1 L) was added DAST portionwise at 0-10 °C. Upon the completion of addition, the reaction was refluxed for 6 hours. The reaction was cooled to rt and poured onto sat. NaHCCf solution. The mixture was separated and the organic layer was washed with water, dried over Na₂S0₄, and concentrated. The residue was purified on silica gel (100% DCM) to provide the title compound as a brown oil (116g) in a 8: 1 transxis mixture. The above brown oil (107 g) was dissolved in toluene (110 mL) and hexanes (330mL) at 70 °C. The solution was cooled to 0 °C and stirred at 0 °C overnight. The precipitate was filtered and washed with hexanes to provide the trans isomer as a white solid (87.3 g). LRMS (M+H) m/z 195.1.

Example 9 Synthesis of ((lr,3r)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methanamine

[0103] A mixture of ( 1.v,3.v)-3-fluoro- 1 -(3-fluoropyridin-2-yl)cyclobutane- 1 -carbonitrile (71 g, 0.37 mol) and Raney nickel (~7 g) in 7N ammonia in methanol (700 mL) was charged with hydrogen (60 psi) for 2 days. The reaction was filtered through a celite pad and washed with methanol. The filtrate was concentrated under high vacuum to provide the title compound as a light green oil (70 g, 97.6%). LRMS (M+H) m/z 199.2.

Example 10 Synthesis of t-butyl 5-bromopyrimidin-2-yl((trans-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl) carbamate

[0104] A mixture of ((lr,3r)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methanamine (37.6 g, 190 mmol), 5-bromo-2-fluoropyrimidine (32.0 g, 181 mmol), DIPEA (71 mL, 407 mmol), and NMP (200 mL) was stirred at rt overnight. The reaction mixture was then diluted with EtOAc (1500 mL) and washed with saturated sodium bicarbonate (500 mL). The

organic layer was separated, dried over Na₂S0₄, and concentrated. The resultant solid was dissolved in THF (600 mL), followed by the slow addition of DMAP (14 g, 90 mmol) and Boc₂0 (117.3 g, 542 mmol). The reaction was heated to 60° C. and stirred for 3 h. The reaction mixture was then concentrated and purified by silica gel chromatography

(EtO Ac/hex) to give 59.7 g oft-butyl 5-bromopyrimidin-2-yl((trans-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl)carbamate as a white solid.

Example 11 Synthesis of t-butyl 5-(3-cyano- 1 H -pyrrol- 1 -yl)pyrimidin-2-yl(((lrans)-3-fhtoro-l-(3-fluoropyridin-2-yl)cyclohutyl)methyl)carhamate

[0105] To a solution oft-butyl 5-bromopyrimidin-2-yl((trans-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl) carbamate (1.0 g, 2.8 mmol) in 15 mL of toluene (degassed with nitrogen) was added copper iodide (100 mg, 0.6 mmol), potassium phosphate (1.31 g, 6.2 mmol), trans-N,N’-dimethylcyclohexane-l, 2-diamine (320 mg, 2.2 mmol), and 3-cyanopyrrole (310 mg, 3.6 mmol). The reaction was heated to 100 °C and stirred for 2 h. The reaction was then concentrated and purified by silica gel chromatography (EtOAc/hexanes) to afford 1.1 g of t-butyl 5-(3-cyano-lH-pyrrol-l-yl)pyrimidin-2-yl(((trans)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl)carbamate as a clear oil.

Example 12 Synthesis of l-(2-((((trans)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl)amino)pyrimidin-5-yl)-lH-pyrrole-3-carboxamide

[0106] To a solution oft-butyl 5-(3-cyano-lH-pyrrol-l-yl)pyrimidin-2-yl(((trans)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl)carbamate (1.1 g, 3.1 mmol) in DMSO (10 mL) was added potassium carbonate (1.3 g, 9.3 mmol). The mixture was cooled to 0 °C and hydrogen peroxide (3 mL) was slowly added. The reaction was warmed to rt and stirred for 90 min. The reaction was diluted with EtO Ac (75 mL) and washed three times with brine (50 mL). The organic layer was then dried over Na₂S0₄, filtered, and concentrated to give a crude solid that was purified by silica gel chromatography (10% MeOH/CH₂Cl2) to afford 1.07 g of a white solid compound. This compound was dissolved in 25% TFA/CH2CI2 and stirred for 1 hour. The reaction was then concentrated, dissolved in ethyl acetate (75 mL), and washed three times with saturated potassium carbonate solution. The organic layer was then dried over Na₂S04, filtered, and concentrated to give a crude solid that was triturated with 75% ethyl acetate/hexanes. The resultant slurry was sonicated and filtered to give 500 mg of l-(2-((((trans)-3-fluoro-l-(3-fluoropyridin-2-yl)cyclobutyl)methyl)amino)pyrimidin-5-yl)-lH-pyrrole-3 -carboxamide as a white solid. LRMS (M+H=385).

//////////////CK-2127107, CK 2127107, CK2127107, Reldesemtiv, Cytokinetics,   Astellas, neuromuscular disorders, muscle wasting, claudication, metabolic syndrome, spinal muscular atrophy, amyotrophic lateral sclerosis, Orphan Drug Status, Spinal muscular atrophy, Phase II

C1C(CC1(CNC2=NC=C(C=N2)N3C=CC(=C3)C(=O)N)C4=C(C=CC=N4)F)F