DEUTERATED FEZOLINETANT

Abstract

Deuterated fezolinetant (R)-(4-fluorophenyl)-(8-methyl-3-(3-(methyl-d3)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]trizolo[4,3-a]pyrazin-7(8H)-yl)methanone:

##STR00001##

or a pharmaceutically acceptable salt or solvate thereof, as an NK-3 antagonist. Also, methods of modulating NK-3 receptor activity including administering an effective amount of the compound or pharmaceutically acceptable salt or solvate thereof. Additionally, a process for manufacturing the compound or pharmaceutically acceptable salt or solvate thereof.

Claims

1-8. (canceled)

9. (R)-(4-fluorophenyl)-(8-methyl-3-(3-(methyl-d3)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]trizolo[4,3-a]pyrazin-7(8H)-yl)methanone or a pharmaceutically acceptable salt or solvate thereof.

10. The (R)-(4-fluorophenyl)-(8-methyl-3-(3-(methyl-d3)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]trizolo[4,3-a]pyrazin-7(8H)-yl)methanone or a pharmaceutically acceptable salt or solvate thereof according to claim 9, formulated in a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

11. A method for modulating NK-3 receptor activity in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of (R)-(4-fluorophenyl)-(8-methyl-3-(3-(methyl-d3)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]trizolo[4,3-a]pyrazin-7(8H)-yl)methanone or a pharmaceutically acceptable salt or solvate thereof.

12. The method according to claim 11, for treating and/or preventing a disease selected from depression, anxiety, psychosis, schizophrenia, psychotic disorders, bipolar disorders, cognitive disorders, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), pain, convulsion, obesity, inflammatory diseases including irritable bowel syndrome (IBS) and inflammatory bowel disorders, emesis, pre-eclampsia, airway related diseases including chronic obstructive pulmonary disease, asthma, airway hyperresponsiveness, bronchoconstriction and cough, urinary incontinence, reproduction disorders, contraception and sex hormone-dependent diseases including but not limited to benign prostatic hyperplasia (BPH), prostatic hyperplasia, metastatic prostatic carcinoma, testicular cancer, breast cancer, ovarian cancer, androgen dependent acne, male pattern baldness, endometriosis, abnormal puberty, uterine fibrosis, uterine fibroid tumor, uterine leiomyoma, hormone-dependent cancers, hyperandrogenism, hirsutism, virilization, polycystic ovary syndrome (PCOS), premenstrual dysphoric disease (PMDD), HAIR-AN syndrome (hyperandrogenism, insulin resistance and acanthosis nigricans), ovarian hyperthecosis (HAIR-AN with hyperplasia of luteinized theca cells in ovarian stroma), other manifestations of high intraovarian androgen concentrations (e.g. follicular maturation arrest, atresia, anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility), androgen-producing tumor (virilizing ovarian tumor or virilizing adrenal tumor), menorrhagia and adenomyosis.

13. The method according to claim 11, for treating and/or preventing hot flashes.

14. The method according to claim 11, for lowering the circulating LH levels.

15. A process of manufacturing (R)-(4-fluorophenyl)-(8-methyl-3-(3-(methyl-d3)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]trizolo[4,3-a]pyrazin-7(8H)-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of: a) reacting a compound of Formula (i) ##STR00016## wherein: PG represents a suitable protecting group; with a compound of Formula (ii) ##STR00017## so as to obtain a compound of Formula (iii) ##STR00018## wherein PG is as defined above; b) deprotecting compound of Formula (iii) with a suitable deprotection agent to afford compound of Formula (iv) ##STR00019## c) N-acylating compound of Formula (iv), with a compound of Formula (v) ##STR00020## leading to (R)-(4-fluorophenyl)-(8-methyl-3-(3-(methyl-d3)-1,2,4-thiadiazol-5-yl)-5,6-dihydro-[1,2,4]trizolo[4,3-a]pyrazin-7(8H)-yl)methanone.

16. The process according to claim 15, wherein the protecting group PG is selected from 2,4-dimethoxybenzyl (DMB), 4-methoxybenzyl (PMB), tert-butoxycarbonyl (Boc), allyl, diphenyl-phosphiramide (DPP) and 2-trimethylsilylethanesulfonyl (SES).

17. The process according to claim 15, wherein the protecting group PG is 2,4-dimethoxybenzyl (DMB).

Description

BRIEF DESCRIPTION OF THE DRAWING

[0117] FIG. 1 is an X-ray crystal structure of deuterated fezolinetant with thermal displacement ellipsoids drawn at the 50% probability level.

EXAMPLES

Chemistry Examples

[0118] All reported temperatures are expressed in degrees Celsius ( C.); all reactions were carried out at room temperature (rt) unless otherwise stated.

[0119] All reactions were followed by thin layer chromatography (TLC) analysis (TLC plates, silica gel 60 F.sub.254, Merck) was used to monitor reactions, establish silica-gel flash chromatography conditions. All other TLC developing agents/visualization techniques, experimental set-up or purification procedures that were used in this invention, when not described in specific details, are assumed to be known to those conversant in the art and are described in such standard reference manuals as: i) Gordon, A. J.; Ford, R. A. The Chemist's CompanionA Handbook of Practical Data, Techniques, and References, Wiley: New York, 1972; ii) Vogel's Textbook of Practical Organic Chemistry, Pearson Prentice Hall: London, 1989.

[0120] The following abbreviations are used:

[0121] CCSC: chlorocarbonylsulfenyl chloride

[0122] Cpd: Compound,

[0123] DCM: Dichloromethane,

[0124] equiv.: Equivalent(s),

[0125] EtOH: Ethanol,

[0126] g: Gram(s),

[0127] h: Hour(s),

[0128] mg: Milligram(s),

[0129] mL: Milliliter(s),

[0130] mmol: Millimole(s),

[0131] min: Minute(s),

[0132] RT: Room temperature,

[0133] R.sub.t: retention time

[0134] TFA: Trifluoroacetic acid,

[0135] TLC: Thin layer chromatography.

[0136] The intermediates and compounds described below were named using ChemBioDraw Ultra version 12.0 (PerkinElmer).

[0137] Synthetic Scheme

[0138] Deuterated fezolinetant may be synthesized using the methodology described in the following schemes (Part A and Part B):

[0139] Part A: Preparation of Deuterated Key Intermediate (ii)

##STR00014##

[0140] Part B: Synthesis of Deuterated Fezolinetant Using Intermediate (ii)

##STR00015##

[0141] Synthesis of deuterated fezolinetant was performed through key intermediate (ii). Part A corresponds to the synthesis of intermediate (ii). Part B leads to deuterated fezolinetant (d3-fezolinetant), using intermediate (ii), using procedures adapted from WO2014/154895.

[0142] Experimental Details

[0143] Part AStep 1): Formation of d.sub.3-Acetamide (b)

[0144] To d.sub.3-acetic acid (a) (10 g, 1 equiv.) in DCM (100 mL) CDI (25.3 g, 1 equiv.) was added and the resultant mixture stirred at RT for 30 min, thereupon ammonia gas was bubbled through the reaction mixture for 40 min at 0-5 C. Thereafter the bubbling was stopped, the mixture was filtered and the filtrate was evaporated under reduced pressure to give 30.95 g crude product that was purified using flash chromatography on silica to furnish 6.65 g (yield: 73%) deuterated acetamide (b) was obtained (GC (column RTX-1301 30 m0.32 mm0.5 m) R.sub.t 7.4 min, 98%).

[0145] Part AStep 2): Ring Closure Leading to Compound (c)

[0146] d.sub.3-Acetamide (b) (3.3 g, 1 equiv.) and chlorocarbonylsulfenyl chloride (CCSC) (8.4 g, 1.2 equiv.) were combined in 1,2-dichloroethane (63 mL), and refluxed for 4.5 h. CCSC can be prepared as per the procedure described in Adeppa et al. (Synth. Commun., 2012, Vol. 42, pp. 714-721). The volatiles were then removed to obtain 6.60 g (102% yield) oxathiazolone (c) product as a yellow oil. The product was analyzed by GC (R.sub.t=7.8 min, 97%). .sup.13C NMR (CDCl.sub.3): 16.0, 158.7, 174.4 ppm.

[0147] Part AStep 3): Formation of Compound (d)

[0148] To oxathiazolone (c) (6.6 g, 1 equiv) in m-xylene (231 mL) methyl cyanoformate (14.70 g, 3.2 equiv.) was added. The mixture was stirred at 130 C. for 19 h and thereafter the volatiles removed under reduced pressure at 50 C. to obtain 4.53 g brown oil (yield: 51%). The product (d) was analyzed by GC(R.sub.t=11.8 min, 81%) and mass spectrometry (M+H=162).

[0149] Part AStep 4): Formation of Intermediate (ii)

[0150] The ester (d) obtained above (3.65 g, 1 equiv.) was dissolved in ethanol (45 mL). The undissolved material was filtered off then hydrazine hydrate (2.3 mL, 1.15 equiv. 55 .sup.w/.sub.w % in H.sub.2O) was added to the stirred solution. Thick suspension formed in minutes, the suspension was stirred for 45 min, filtered and washed with EtOH (3 mL) to furnish intermediate (ii) a pale yellow solid (2.43 g, 55% yield). Mass spectrometry (M+H=162, M+Na=184); .sup.1H NMR (d.sub.6-DMSO): 4.79 ppm (br s, 2H), 10.55 ppm (br s, 1H); .sup.13C NMR (d.sub.6-DMSO): 17.4 ppm, 155.6 ppm, 173.4 ppm, 183.0 ppm.

[0151] Part BStep a): Formation of Compound (iii)

[0152] Intermediate (i) was prepared as described in WO2014/154895.

[0153] Intermediate (ii) (490 mg, 3.04 mmol) and compound (i) (1.0 g (87 mol % 1.3 content), 2.97 mmol) were taken up in MeOH and the reaction mixture was stirred at a temperature ranging from 55 C. to 70 C. for a period of time ranging from 6 hours to 8 hours. The reaction was deemed complete by TLC. The reaction mixture was evaporated and the crude product was purified by flash chromatography on silica in DCM: MeOH eluent to afford 1.13 g (97% yield) of compound (iii) as a yellow oil. .sup.1H NMR (CDCl3): (ppm) 7.26 (d, 1H), 6.48-6.49 (2H), 4.50 (m, 1H), 4.30 (m, 1H), 4.09 (m, 1H), 3.94 (d, 1H), 3.80 (s, 6H), 3.61 (d, 1H), 3.22 (m, 1H), 2.75 (m, 1H), 1.72 (d, 3H); Mass spectrometry (M+H=390, 2M+Na=801). Chiral LC (column: Chiralpak IC, 2504.6 mmeluent: MTBE/MeOH/DEA 98/2/0.1) 99.84%.

[0154] Part BStep b): Deprotection Leading to Compound (iv)

[0155] Intermediate (iii) prepared above (1.05 g, 2.7 mmol) was dissolved in DCM and washed with aq. NaOH. The organic phase was dried, then TFA (1.56 mL, 2.3 g, 7.5 equiv.) was added at RT. The resulting solution was stirred at RT for 2 h. The reaction was monitored by TLC. After completion of the reaction water was added to the reaction mixture, and the precipitate filtered and washed with water. The phases were separated, the pH of the aq. phase was adjusted to pH 13 by addition of 20% aq. NaOH. NaCl was then added to the aqueous solution that was then extracted with DCM. The organic phase was evaporated under reduced pressure to give 504 mg of compound (iv) (78% yield). .sup.1H NMR (d.sub.6-DMSO): (ppm) 4.42 (m, 1H), 4.10 (m, 2H), 3.0 (m, 1H), 2.82 (m, 1H), 1.46 (d, 3H). .sup.13C NMR (d.sub.6-DMSO): (ppm) 174.8, 173.4, 156.2, 145.0, 48.1, 45.7, 40.7, 19.1. Mass spectrometry (M+H=240, 2M+Na=501).

[0156] Part BStep c): Acylation and Recrystallization to Form Deuterated Fezolinetant

[0157] Intermediate (iv) (450 mg, 1.88 mmol) was dissolved in DCM, then sat. aq. NaHCO.sub.3 was added and the mixture was stirred for 30 min. To this mixture 4-fluorobenzoyl chloride (v) (220 L, 1 equiv.) was added dropwise at RT. The reaction was stirred for a period of time ranging from about 20 min to overnight at RT and reaction progress monitored by TLC. After completion the phases were separated, the organic phase was washed with water, dried over MgSO.sub.4, filtered and evaporated under reduced pressure to give 745 mg crude d.sub.3-fezolinetant (110% yield). The crude product was purified by flash chromatography using MeOH:DCM together with a second batch, then crystallized (EtOH/H.sub.2O) before final analysis. .sup.1H NMR (d.sub.6-DMSO): (ppm) 7.60 (m, 2H), 7.33 (m, 2H), 5.73 (m, 1H), 4.68 (dd, 1H), 4.31 (m, 1H), 4.06 (m, 1H), 3.65 (m, 1H), 1.61 (d, 3H). .sup.13C NMR (d.sub.6-DMSO): (ppm) 174.4, 173.5, 168.7, 163.7, 161.8, 154.1, 144.9, 131.6, 129.5, 115.5, 44.7, 18.7. Isotopic purity based on an intense molecular ion observed at m/z=362.2 Da is estimated as approximately 100% isotopic purity. Chiral purity (LC) (column: Chiralpak IC, 2504.6 mmeluent: n-hexane/EtOH/DEA 80/20/0.1)>99.9%. A single crystal X-ray structure of the deuterated fezolinetant final product was obtained (FIG. 1) that confirmed the structure of the compound as well as the stereochemistry.

BIOLOGY EXAMPLES

[0158] Functional Assay

[0159] Aequorin Assay with Human NK-3 Receptor.

[0160] Changes in intracellular calcium levels are a recognized indicator of G protein-coupled receptor activity. The efficacy of compounds of the invention to inhibit NKA-mediated NK-3 receptor activation was assessed by an in vitro Aequorin functional assay. Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. In the presence of the cofactor coelenterazine, apoaequorin emits a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.

[0161] Antagonist Testing.

[0162] The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC.sub.80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.

[0163] Inhibition curves are obtained for compounds of the invention and the concentrations of compounds which inhibit 50% of reference agonist response (IC.sub.80) were determined (see results in table 1 below). The IC.sub.50 values shown in table 1 indicate that deuterated fezolinetant is a potent NK-3 antagonist compounds.

[0164] Competitive Binding Assays

[0165] The affinity of compounds of the invention for the human NK-3 receptor was determined by measuring the ability of compounds of the invention to competitively and reversibly displace a well-characterized NK-3 radioligand in a concentration-dependent manner.

[0166] .sup.3H-SB222200 Binding Competition Assay with Human NK-3 Receptor.

[0167] The ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist .sup.3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK-3 receptor. The membranes were incubated with 5 nM .sup.3H-SB222200 (ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl.sub.2 1 mM/MgCl.sub.2 5 mM/BSA 0.5%/Saponin 10 g/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of .sup.3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentration that displaced 50% of bound radioligand (IC.sub.80) were determined by linear regression analysis and then the apparent inhibition constant (K.sub.i) values were calculated by the following equation: K.sub.i=IC.sub.50/(1+[L]/K.sub.d) where [L] is the concentration of free radioligand and K.sub.d is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973) (see results in table 1 below).

[0168] Table 1 shows biological results obtained using the .sup.3H-SB222200 binding competition assay with compounds of the invention. These results indicate deuterated fezolinetant displays potent affinity for the human NK-3 receptor and that deuterated fezolinetant retains the biological activity of fezolinetant.

TABLE-US-00001 TABLE 1 Functional assay: Aequorin assay Competitive with human NK-3 receptor binding assay with hNK-3 AEQ human NK-3 receptor Cpd (antagonist IC.sub.50, nM) hNK-3 (K.sub.i, nM) Deuterated 13 17 fezolinetant fezolinetant 18 23

[0169] CYP 450 Profilling Assay

[0170] P450-Glo Screening assay (Promega) are used to evaluate the potential of the compounds of the invention to inhibit cytochrome P450 isoforms (CYP 1A2# V9770, 2C9# V9790, 2C19# V9880, 2D6# V9890, 3A4# V9910). These assays employ luminogenic CYP450 probe substrates that are derivatives of beetle luciferin, a substrate for luciferase enzymes. The derivatives are converted by P450s cytochrome to luciferin, which in turn reacts with luciferase to produce an amount of light that is directly proportional to the activity of the P450.

[0171] P450-Glo assays are performed in two steps, the P450-Glo substrates are first converted by cytochrome P450 enzyme to a luciferin product which is then detected as a luminescent signal from a luciferase reaction. To perform the assay, the cytochrome P450 mixture with cytochrome P450 enzyme and a P450-Glo substrate is prepared at pH 7.4 in a P04 buffer at the optimal concentration for each cytochrome P450 isoform. The compounds of the invention (Dose response curve from 100 M to 30 nM) are added to the mixture in duplicates. Luciferin-Free water+0.1% DMSO is used as negative control and known inhibitor as positive control. The reactions are initiated by adding the NADPH regeneration system and are performed at 37 C. Luciferin detection reagent is added to stop cytochrome P450 activity and initiate the D-luciferin detection reaction. The IC50 value (compound concentration required to inhibit cytochrome activity by 50%) of the compound of the invention were then determined.

[0172] Table 2 shows CYP P450 Inhibition Profile obtained with compound of the invention. When tested in the above-described assay, the deuterated fezolinetant surprisingly displays a better CYP profile on CYP2C9 and 2C19 compare to fezolinetant, indicating no or a very low CYP P450 inhibition on all five Cytochrome P450 isoforms.

TABLE-US-00002 TABLE 2 Cytochrome Deuterated fezolinetant fezolinetant P450 isoform IC.sub.50 M IC.sub.50 M CYP1A2 100 100 CYP2C9 100 47.95 CYP2C19 89.92 41.98 CYP2D6 100 100 CYP3A4 92.08 90.44

[0173] Evaluation of Gonadotropins Following Oral Dosing of Deuterated Fezolinetant and Fezolinetant in Castrate Monkey

[0174] The evaluation of efficacy for agents modulating the hypothalamic-pituitary-gonadal (HPG) axis may be performed by evaluating luteinizing hormone (LH) in castrate monkeys, as established in the literature (for example, Fraser et al., Endocrinol., 2015, Vol. 156, pp. 4214-4225; Struthers et al., Endocrinol., 207, Vol. 148, pp. 857-867).

[0175] Sexually mature, male, cynomolgus monkeys (Macaca fascicularis; N=4, age: 4-5 years, body weight range between 4.41-5.69 kg) were castrated and allowed to recover for >6 months prior to this experiment. Monkeys were group housed and maintained on a 12 h alternating light and dark cycle on a standard laboratory chow diet supplemented with fruit. Water was provided ad libitum. A pilot study was performed where monkeys were treated with vehicle (0.5% methylcellulose/water) and blood samples were collected at the time intervals specified below. This is the response to vehicle. Animals were given a one-week recovery period prior to re-testing, in all cases. The test articles, deuterated fezolinetant and fezolinetant, were similarly formulated in 0.5% methylcellulose/water and administered by oral gavage at 1, 3, and 10 mg/kg (dose volume=5 mL/kg in all cases). Dosing occurred at 08h00. Blood samples were collected by venepuncture at: 0 (pre-dose), 0.5, 1, 1.5, 2.5, 5, 8, 12, 24, 32 and 48 h post-dose. Samples were collected into centrifuge tubes containing K.sub.2EDTA and centrifuged at 2500 g for 15 min. The plasma was decanted off the samples into vials designated for pharmacokinetic analysis whereas the serum was collected for LH pharmacokinetic analysis, respectively. All sample vials were immediately frozen and stored at 20 C. until assay.

[0176] Serum LH was measured using the Leydig cell method (Wickings et al., J. Reprod. Fert., 1979, Vol. 57, pp. 497-504) according to a method previously qualified with established acceptance criteria for calibration of LH and testosterone. Briefly, for each run, 2-3 male mice were sacrificed, testes collected and a Leydig cell suspension prepared according to published methods. Treatment of Leydig cells with LH will elicit the secretion of testosterone and the latter is measured by ELISA. For each run, a standard (monkey LH standards, Scripps) curve for LH was constructed by plotting the log (LH concentration) against the square root of the testosterone produced, which gave a linear plot over the dose range. The formula of this standard curve was then used for the quantification of LH concentration in unknown samples (eg. collected from the in vivo testing) by interpolation.

[0177] Pharmacokinetic (PK) analyses were performed on plasma samples collected at coincident time intervals as for the LH analyses. Liquid chromatography coupled with tandem mass spectrometry method (LC-MS/MS) was established to quantify fezolinetant and deuterated fezolinetant concentrations in cynomolgus monkey plasma samples (determinations validated by comparison against an internal standard). PK parameters were calculated using non-compartmental analysis (Phoenix WinNonlin, version 6.1). The linear log trapezoidal algorithm, weighting 1/Y*Y was used for parameters calculation. Mean PK parameters were calculated from individual animals in each treatment group. Concentrations below the lower limit of quantification (LLOQ) were excluded for the calculation of PK parameters.