WO2023240205A1 - Deuterated compounds - Google Patents

Abstract

The present disclosure provides deuterated compounds and their use as T-type calcium channel blockers in the treatment or prevention of various diseases or disorders associated with calcium T channels.

Description

Deuterated Compounds

TECHNICAL FIELD

The present disclosure relates to deuterated compounds and their use as T-type calcium channel blockers in the treatment or prevention of various diseases or disorders associated with calcium T channels.

BACKGROUND

Intracellular calcium concentrations control important life processes such as signal transduction pathways, hormones and neurotransmitter release, muscular contraction, gene expression and cell division. Control of calcium influx across the cellular membrane is in part regulated by a family of transmembrane proteins tenned voltage-gated calcium channels (VOCs). They are activated by changes in electrical potential difference across the membrane and have been further classified into different subtypes based on biophysical and pharmacological considerations: Cavl.x (L-type for Long-lasting), Cav2.x (N-, P/Q- and R-types; N for Neuronal, P for Purkinje cells, Q (after P) and R for Remaining or Resistant) and Cav3.x (T-type for Transient). The L, N, P and Q-type channels activate at more positive potentials (high voltage activated) and display diverse kinetics and voltage-dependent properties. The T-type class (or “low voltage-activated”) is characterized by fast inactivation (transient) and small conductance (tiny) and is composed of three members due to the different main pore-forming al subunits: Cav3.1 (al G), Cav3.2 (al H) and Cav3.3 (al I).

The compounds of the present disclosure are calcium T channel blockers and therefore useful for the prevention or treatment of diseases or disorders where calcium T channels are involved.

SUMMARY

The present application provides, inter alia, a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein the constituent members are defined herein.

The present disclosure further provides pharmaceutical compositions comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The present disclosure further provides methods of blocking a T-type calcium channel, comprising contacting the T-type calcium channel with a compound described herein, or a pharmaceutically acceptable salt thereof.

The present disclosure further provides methods of blocking a T-type calcium channel in a patient, comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt thereof.

The present disclosure further provides methods of treating a disease or disorder associated with a T-type calcium channel in a patient, comprising administering to the patient a compound described herein, or a pharmaceutically acceptable salt thereof.

The present disclosure further provides compounds described herein, or a pharmaceutically acceptable salt thereof, for use in any of the methods described herein.

The present disclosure further provides uses of a compound described herein, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for use in any of the methods described herein. DESCRIPTION OF DRAWINGS

FIG. 1 shows recovery of total radioactivity in incubations of Compound 2 with liver microsomes and hepatocytes of various species.

FIG. 2 shows proposed metabolic pathways of Compound 1. The aglycon M29 was only detected after enzymatic cleavage of the corresponding glucuronic acid conjugate M4.

FIG. 3 shows cross-species comparison of Compound 2 metabolic profdes after 60 minutes incubation with liver microsomes. All values are expressed as percent of total chromatogram radioactivity and rounded to two significant figures. Empty cells indicate the absence of a metabolite.

FIG. 4 shows radiochromatogram following 60 minutes incubation of Compound 2 with human liver microsomes.

FIG. 5 shows radiochromatogram following 60 minutes incubation of Compound 2 with Wistar rat liver microsomes.

FIG. 6 shows radiochromatogram following 60 minutes incubation of Compound 2 with Beagle dog liver microsomes.

FIG. 7 shows radiochromatogram following 60 minutes incubation of Compound 2 with cynomolgus monkey liver microsomes.

FIG. 8 shows radiochromatogram following 60 minutes incubation of Compound 2 with CD-I mouse liver microsomes.

FIG. 9 shows radiochromatogram following 60 minutes incubation of Compound 2 with NZW rabbit liver microsomes.

FIGs. 10A-10C show radiochromatogram following 60 minutes incubation of Compound 2 with liver microsomes of human (FIG. 10A), rat (FIG. 10B), and mouse (FIG. 10C) in the absence of NADPH.

FIG. 11 shows radiochromatogram following 60 minutes incubation of Compound 2 in the absence of liver microsomes.

FIG. 12 shows cross-species comparison of Compound 2 metabolic profiles in incubations with hepatocytes. All values are expressed in percent of total chromatogram radioactivity and rounded to two significant figures. Empty cells indicate the absence of a metabolite.

FIGs. 13A-13B show radiochromatogram following 4 h (FIG. 13A) and 24 h (FIG. 13B) incubation of Compound 2 with ready-plated human hepatocytes (batch 1).

FIGs. 14A-14B show radiochromatogram following 4 h (FIG. 14A) and 24 h (FIG. 14B) incubation of Compound 2 with ready-plated human hepatocytes (batch 2).

FIGs. 15A-15B show radiochromatogram following 4 h (FIG. 15 A) and 24 h (FIG. 15B) incubation of Compound 2 with cryopreserved human hepatocytes (batch 3).

FIGs. 16A-16B show radiochromatogram following incubation of Compound 2 with fresh human hepatocytes (batch 4) in the absence (FIG. 16A) or presence (FIG. 16B) of P-glucuronidase.

FIG. 17 shows radiochromatogram following 24 h incubation of Compound 2 with Wistar rat hepatocytes.

FIG. 18 shows radiochromatogram following 24 h incubation of Compound 2 with Beagle dog hepatocytes.

FIG. 19 shows radiochromatogram following 24 h incubation of Compound 2 with cynomolgus monkey hepatocytes.

FIG. 20 shows radiochromatogram following 4 h incubation of Compound 2 with CD-I mouse hepatocytes.

FIG. 21 shows radiochromatogram following 6 h incubation of Compound 2 with NZW rabbit hepatocytes.

FIG. 22 shows radiochromatogram following 24 h incubation of Compound 2 in the absence of hepatocytes.

FIG. 23 shows stability of Compound 2 in plasma. All values are expressed in percent of total chromatogram radioactivity and rounded to two significant figures; n.d.: not determined. Empty cells: 0.

FIG. 24 shows stability of Compound 2 in blood at 37 °C. All values are expressed in percent of total chromatogram radioactivity; n.d.: not determined. Empty cells: 0. FTG. 25A-25D show representative radiochromatograms following 6 h incubation of Compound 2 with rat plasma at 37 °C (FIG. 25 A), 4 h incubation with rat plasma and 0.1 % DCV at 37 °C (FIG. 25B), 4 h incubation with rat plasma and 0.2 mM BNPP at 37 °C (FIG. 25C), and 4 h incubation with rat plasma at 4 °C (FIG. 25D).

FIGs. 26A-26B show representative radiochromatograms following incubation of Compound 2 with blood of human (FIG. 26A) and rat (FIG. 26B) at 37°C.

DETAILED DESCRIPTION

Compound 1 (i.e., N-(l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl)-2-(4-(l- (trifluoromethyl)cyclopropyl)phenyl)acetamide) is a selective and orally available, brainpenetrating T-type calcium channel blocker in development for the prevention or treatment of epilepsy (see e.g., U.S. Patent Nos.: 9,932,314; 10,065,929; 10,246,426; 10,899,695; 11,059,803; and 11,213,517, the disclosures of which are each incorporated herein by reference in their entireties), such as epileptic encephalopathy with continuous spike-and-wave during sleep, and essential tremor.

Compound 1

The present application provides deuterated analogs of Compound 1, and pharmaceutically acceptable salts thereof. Substitution with heavier isotopes, such as deuterium, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances, (see e.g., A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more metabolism sites may afford one or more of the therapeutic advantages. In some embodiments, the present application provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁵, R¹⁷, and R¹⁸ are each independently selected from hydrogen and deuterium; and wherein at least one R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ is deuterium.

In some embodiments, one to eighteen of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are deuterium, for example, one to sixteen, one to fourteen, one to twelve, one to ten, one to eight, one to six, one to four, or one to two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are deuterium .

In some embodiments, one to six of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are deuterium. In some embodiments, two to six of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R^1(J, R^u, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are deuterium. In some embodiments, two to four of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are deuterium. In some embodiments, four to six of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are deuterium.

In some embodiments, one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ is deuterium. In some embodiments, two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium. In some embodiments, three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium. In some embodiments, four of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹³, R¹⁶, R¹⁷, and R¹⁸ are each deuterium. In some embodiments, five of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium. In some embodiments, six of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,

R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹³, R¹⁶, R¹⁷, and R¹⁸ are each deuterium. In some embodiments, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

In some embodiments, the compound of Formula I is a compound of Formula II:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is a compound of Formula III:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is a compound of Formula IV:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is a compound of Formula V:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is selected from:

or a pharmaceutically acceptable salt thereof.

The compounds disclosed and described herein allow atoms at each position of the compound independently to have: 1) an isotopic distribution for a chemical element in proportional amounts to those usually found in nature or 2) an isotopic distribution in proportional amounts different to those usually found in nature unless the context clearly dictates otherwise. A particular chemical element has an atomic number defined by the number of protons within the atom’s nucleus. Each atomic number identifies a specific element, but not the isotope; an atom of a given element may have a wide range in its number of neutrons. The number of both protons and neutrons in the nucleus is the atom's mass number, and each isotope of a given element has a different mass number. A compound wherein one or more atoms have an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature is commonly referred to as being an isotopically-labeled compound. Each chemical element as represented in a compound structure may include any isotopic distribution of said element. For example, in a compound structure a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be an isotopic distribution of hydrogen, including but not limited to protium (¹H) and deuterium (²H) in proportional amounts to those usually found in nature and in proportional amounts different to those usually found in nature. Thus, reference herein to a compound encompasses all potential isotopic distributions for each atom unless the context clearly dictates otherwise. Examples of isotopes include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, chlorine, bromine, and iodine. As one of skill in the art would appreciate, any of the compounds as disclosed and described herein may include radioactive isotopes. Accordingly, also contemplated is use of compounds as disclosed and described herein, wherein one or more atoms have an isotopic distribution different to those usually found in nature, such as having ²H or ³H in greater proportion, or ^UC, ¹³C, or ¹⁴C in greater proportion than found in nature. By way of general example, and without limitation, isotopes of hydrogen include protium ( ¹ H), deuterium (²H), and tritium (³H). Isotopes of carbon include carbon-11 (^UC), carbon-12 (¹²C), carbon-13 (¹³C), and carbon-14 (¹⁴C). Isotopes of nitrogen include nitrogen-13 (¹³N), nitrogen-14 (¹⁴N) and nitrogen- 15 (¹⁵N). Isotopes of oxygen include oxygen- 14 (¹⁴O), oxygen- 15 (¹⁵O), oxygen- 16 (¹⁶O), oxygen- 17 (¹⁷O), and oxygen- 18 (¹⁸O). Isotope of fluorine include fluorine-17 (^{1 Z}F), fluorine-18 (¹⁸F) and fluorine-19 (¹⁹F). Isotopes of phosphorous include phosphorus-31 (³¹P), phosphorus-32 (³²P), phosphorus-33 (³³P), phosphorus-34 (³⁴P), phosphorus-35 (³⁵P) and phosphorus-36 (³⁶P). Isotopes of sulfur include sulfur-32 (³²S), sulfur-33 (³³S), sulfur-34 (³⁴S), sulfur-35 (³⁵S), sulfur-36 (³⁶S) and sulfur-38 (³⁸S). Isotopes of chlorine include chlorine-35 (³⁵C1), chlorine-36 (³⁶C1) and chlorine-37 (³⁷C1). Isotopes of bromine include bromine-75 (⁷⁵Br), bromine-76 (⁷⁶Br), bromine-77 (⁷⁷Br), bromine-79 (⁷⁹Br), bromine-81 (⁸¹Br) and bromine-82 (⁸²Br). Isotopes of iodine include iodine-123 (¹²³I), iodine-124 (¹²⁴I), iodine-125 (¹²⁵I), iodine-131 (¹³¹I) and iodine-135 (¹³⁵I). In some embodiments, atoms at every position of the compound have an isotopic distribution for each chemical element in proportional amounts to those usually found in nature. In some embodiments, an atom in one position of the compound has an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature (remainder atoms having an isotopic distribution for a chemical element in proportional amounts to those usually found in nature). In some embodiments, atoms in at least two positions of the compound independently have an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature (remainder atoms having an isotopic distribution for a chemical element in proportional amounts to those usually found in nature). In some embodiments, atoms in at least three positions of the compound independently have an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature (remainder atoms having an isotopic distribution for a chemical element in proportional amounts to those usually found in nature). In some embodiments, atoms in at least four positions of the compound independently have an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature (remainder atoms having an isotopic distribution for a chemical element in proportional amounts to those usually found in nature). In some embodiments, atoms in at least five positions of the compound independently have an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature (remainder atoms having an isotopic distribution for a chemical element in proportional amounts to those usually found in nature). In some embodiments, atoms in at least six positions of the compound independently have an isotopic distribution for a chemical element in proportional amounts different to those usually found in nature (remainder atoms having an isotopic distribution for a chemical element in proportional amounts to those usually found in nature).

Certain compounds, for example those having incorporated radioactive isotopes such as ³H and ¹⁴C, are also useful in drug or substrate tissue distribution assays. Tritium (³H) and carbon-14 (¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Compounds with isotopes such as deuterium (²H) in proportional amounts greater than usually found in nature may afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Isotopically-labeled compounds can generally be prepared by performing procedures routinely practiced in the chemical art. Methods are readily available to measure such isotope perturbations or enrichments, such as, mass spectrometry, and for isotopes that are radio-isotopes additional methods are available, such as, radio-detectors used in connection with HPLC or GC.

As used herein, “isotopic variant” means a compound that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such a compound. In certain embodiments, an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, protium (¹H), deuterium (²H), tritium (³H), carbon-11 (ⁿC), carbon-12 (¹²C), carbon-13 (¹³C), carbon-14 (¹⁴C), nitrogen- 13 (¹³N), nitrogen- 14 (¹⁴N), nitrogen- 15 (¹⁵N), oxygen- 14 (¹⁴O), oxygen- 15 (¹⁵O), oxygen- 16 (¹⁶O), oxygen- 17 (¹⁷O), oxygen- 18 (¹⁸O), fluorine- 17 (¹⁷F), fluorine- 18 (¹⁸F), phosphorus-31 (³¹P), phosphorus-32 (³²P), phosphorus-33 (³³P), sulfur-32 (³²S), sulfur-33 (³³S), sulfur-34 (³⁴S), sulfur-35 (³⁵S), sulfur-36 (³⁶S), chlorine-35 (³⁵C1), chlorine-36 (³⁶C1), chlorine-37 (³⁷C1), bromine-79 (⁷⁹Br), bromine-81 (⁸¹Br), iodine-123 (¹²³I), iodine-125 (¹²⁵I), iodine-127 (¹²⁷I), iodine-129 (¹²⁹I), and iodine-131 (¹³¹I). In certain embodiments, an “isotopic variant” of a compound is in a stable form, that is, non-radioactive. In certain embodiments, an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (^XH), deuterium (²H), carbon-12 (¹²C), carbon-13 (¹³C), nitrogen-14 (¹⁴N), nitrogen-15 (¹⁵N), oxygen- 16 (¹⁶O), oxygen- 17 (¹⁷O), and oxygen- 18 (¹⁸O). In certain embodiments, an “isotopic variant” of a compound is in an unstable form, that is, radioactive. In certain embodiments, an “isotopic variant” of a compound of the disclosure contains unnatural proportions of one or more isotopes, including, but not limited to, tritium f'H), carbon-11 (ⁿC), carbon-14 (¹⁴C), nitrogen-13 (¹³N), oxygen-14 (¹⁴O), and oxygen-15 (¹⁵O). It will be understood that, in a compound as provided herein, any hydrogen can include ²H as the major isotopic form, as example, or any carbon include be ¹³C as the major isotopic form, as example, or any nitrogen can include ¹⁵N as the major isotopic form, as example, and any oxygen can include ¹⁸O as the major isotopic form, as example. In certain embodiments, an “isotopic variant” of a compound contains an unnatural proportion of deuterium (²H).

With regard to the compounds provided herein, when a particular atomic position is designated as having deuterium or “D” or “d”, it is understood that the abundance of deuterium at that position is substantially greater than the natural abundance of deuterium, which is about 0.015%. A position designated as having deuterium typically has a minimum isotopic enrichment factor of, in certain embodiments, at least 3500 (52.5% deuterium incorporation), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation) at each designated deuterium position.

Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.

The present disclosure further provides synthetic methods for incorporating radioisotopes into compounds of the disclosure. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and an ordinary skill in the art will readily recognize the methods applicable for the compounds of disclosure.

Methods of Use

The present disclosure also relates to a method for the prevention or treatment of a disease or disorder mentioned herein comprising administering to a subject a compound of Formula I (e.g., a therapeutically effective amount of a compound of Formula I), or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is administered in an amount of between about 1 mg and about 1000 mg per day, for example, between about 5 mg and about 500 mg per day, about 25 mg and about 400 mg per day, or about 50 mg and about 200 mg per day. Whenever the word “between” is used to describe a numerical range, it is to be understood that the end points of the indicated range are explicitly included in the range. For example: if a temperature range is described to be between 40 °C and 80 °C, this means that the end points 40 °C and 80 °C are included in the range; or if a variable is defined as being an integer between 1 and 4, this means that the variable is the integer 1, 2, 3, or 4.

Unless used regarding temperatures, the term “about” (or alternatively the term “around”) placed before a numerical value “X” refers in the current application to an interval extending from X minus 10% of X to X plus 10% of X, and preferably to an interval extending from X minus 5% of X to X plus 5% of X. In the particular case of temperatures, the term “about” placed before a temperature “Y” refers in the current application to an interval extending from the temperature Y minus 10 °C to Y plus 10 °C, and preferably to an interval extending from Y minus 5 °C to Y plus 5 °C.

For avoidance of any doubt, if compounds are described as useful for the prevention or treatment of certain diseases, such compounds are likewise suitable for use in the preparation of a medicament for the prevention or treatment of said diseases.

The compounds provided herein are useful, for example, for the prevention or treatment of diseases or disorders where calcium T channels are involved. Example diseases or disorders where calcium T channels are involved include, but are not limited to:

• epilepsy (e.g. absence epilepsy, childhood absence and other forms of idiopathic generalized epilepsies, epileptic encephalopathy with continuous spike-and-wave during sleep, and temporal lobe epilepsy);

• sleep disorders and sleep disturbances;

• pain (e.g., inflammatory pain, neuropathic pain, peripheral pain, and chronic pain associated with peripheral axonal injury);

• neurological diseases and disorders (e.g., essential tremors, Parkinson’s disease, schizophrenia, depression, anxiety, psychosis, neurodegenerative disorders, autism, and drug addiction); • cardiovascular diseases and disorders (e.g., hypertension, cardiac arrhythmias, atrial fibrillation, congenital heart failure, and heart block);

• cancer;

• diabetes and diabetic neuropathy; and

• infertility and sexual dysfunction.

In some embodiments, the disease or disorder is selected from epilepsy, neurological disease and disorders, and pain. In some embodiments, the disease or disorder is epilepsy or pain. In some embodiments, the disease or disorder is neurological disease and disorders

In some embodiments, the disease or disorder is epilepsy. In some embodiments, the epilepsy is selected from epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS), and childhood absence epilepsy. In some embodiments, the epilepsy is epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS). In some embodiments, the epilepsy is childhood absence epilepsy.

The term “epilepsy” describes recurrent unprovoked seizures wherein the term “seizure” refers to an excessive and/or hypersynchronous electrical neuronal activity. Different types of “epilepsy” can be found, for example, Berg et al., Epilepsia, 2010; 51(4): 676-685, the disclosure of which is incorporated herein by reference in its entirety. The term “epilepsy” as used herein preferably refers to absence epilepsy, childhood absence and other forms of idiopathic generalized epilepsies, temporal lobe epilepsy.

The term “pain” preferably refers to inflammatory pain, neuropathic pain, peripheral pain, or chronic pain associated with peripheral axonal injury.

The term “neurological diseases and disorders” preferably refers to essential tremors, Parkinson’s disease, schizophrenia, depression, anxiety, psychosis, neurodegenerative disorders, autism, or drug addiction. In some embodiments, the neurological diseases and disorders is essential tremor.

The term “cardiovascular diseases and disorders” preferably refers to hypertension, cardiac arrhythmias, atrial fibrillation, congenital heart failure, or heart block. The compounds described herein are also useful in a method of reducing the concentration of calcium in a neuronal cell, and wherein said reduction in calcium is achieved by blocking the calcium T-channel present in such neuronal cell. The compounds provide herein are also useful in a method of decreasing burst firing discharges in a neuronal cell and wherein said decrease of burst firing is achieved by blocking the calcium T-channel. In some embodiments, the method provided herein comprise administering a compound provided herein (i.e., a compound of any of Formulas I-V), or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds provided herein may be metabolized by one or more cytochrome P450 isoforms. Examples of cytochrome P450 isoforms in a subject (e.g., a mammalian subject), include, but are not limited to, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2G1, CYP2J2, CYP2R1, CYP2S1, CYP3A4, CYP3A5, CYP3A5P1, CYP3A5P2, CYP3A7, CYP4A11, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F1, CYP4F12, CYP4X1, CYP4Z1, CYP5A1, CYP7A1, CYP7B1, CYP8A1, CYP8B1, CYP11A1, CYP11B1, CYP11B2, CYP17, CYP19, CYP21, CYP24, CYP26A1, CYP26B1, CYP27A1, CYP27B1, CYP39, CYP46, and CYP51.

As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system.

As used herein, the term “patient” or “subject” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent such as an amount of any of the solid forms or salts thereof as disclosed herein that elicits the biological or medicinal response in a tissue, system, animal, subject, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. An appropriate “effective” amount in any individual case may be determined using techniques known to a person skilled in the art.

The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients or carriers are generally safe, non-toxic and neither biologically nor otherwise undesirable and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use. In some embodiments, each component is “pharmaceutically acceptable” as defined herein. See, e g., Remington: The Science and Practice of Pharmacy, 21st ed; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.

As used herein, the term “treating” or “treatment” refers to inhibiting the disease; for example, inhibiting a disease, condition or disorder in a subject who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology) or ameliorating the disease; for example, ameliorating a disease, condition or disorder in a subject who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.

In some embodiments, the compounds of the disclosure are useful in preventing or reducing the risk of developing any of the diseases referred to herein; e.g., preventing or reducing the risk of developing a disease, condition or disorder in a subject who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiply dependent form). Conversely, various features of the disclosure which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

Combination Therapies

The compounds provided herein can also be used, for example, in one or more methods and/or uses described herein in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an antiepileptic therapeutic agent, or a pharmaceutically acceptable salt thereof. Examples of additional therapeutic agents useful in combination with the compounds provided herein can be found, for example, in U.S. Patent No.: 11,213,517, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the additional therapeutic agent is selected from 6-(2,3- dichlorophenyl)-l,2,4-triazine-3,5-diamine, (S)-2-(2-oxopyrrolidin-l-yl)butanamide, and 2-propylpentanoic acid, or a pharmaceutically acceptable salt of any of the aforementioned. In some embodiments, the additional therapeutic agent is 6-(2,3- dichlorophenyl)-l,2,4-triazine-3,5-diamine, or a pharmaceutically acceptable salt thereof. In some embodiments, the additional therapeutic agent is (S)-2-(2-oxopyrrolidin-l- yl)butanamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the additional therapeutic agent is 2-propylpentanoic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds provided herein and the additional therapeutic agents are comprised in a single pharmaceutical composition. In some embodiments, the compound provided herein and the additional therapeutic agents are comprised in separated pharmaceutical compositions (e.g., two or more pharmaceutical compositions). In some embodiments, the compounds provided herein and the additional therapeutic agents are administered simultaneously. In some embodiments, the compound provided herein and the additional therapeutic agents are administered sequentially.

In some embodiments, the combination exhibits synergistic effect. In some embodiments, the combination exhibits additive effect.

Pharmaceutical Formulations

The production of the pharmaceutical compositions can be effected in a manner which will be familiar to any person skilled in the art (see e.g., Remington, The Science and Practice of Pharmacy 21st Edition (2005), Part 5, “Pharmaceutical Manufacturing” [published by Lippincott Williams & Wilkins]) by bringing the described compounds of formula (I), or their pharmaceutically acceptable salts, optionally in combination with other therapeutically valuable substances, into a galenical administration form together with suitable, non-toxic, inert, therapeutically compatible solid or liquid carrier materials and, if desired, usual pharmaceutical adjuvants.

The compounds provided herein, and pharmaceutically acceptable salts thereof, can be used as medicaments, e.g., in the form of pharmaceutical compositions for enteral (e.g., oral) or parenteral administration, including topical application or inhalation.

EXAMPLES

The compounds of the present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.

Example 1. Synthesis

Compounds according to the present disclosure may be prepared from commercially available or well-known starting materials according to the methods described herein or by analogous methods. The compounds obtained may al so be converted into salts, especially pharmaceutically acceptable salts thereof in a manner known per se.

General remarks: All solvents and reagents are used as obtained from commercial sources unless otherwise indicated. Temperatures are indicated in degrees Celsius (°C). Unless otherwise indicated, the reactions take place at room temperature (rt) under an argon or nitrogen atmosphere and are run in a flame dried round-bottomed flask equipped with a magnetic stir bar. In mixtures, relations of parts of solvent or eluent or reagent mixtures in liquid form are given as volume relations (v/v), unless indicated otherwise. Characterization methods used:

LC-MS

UPLC/MS analyses are performed on Acquity UPLC setup. The column temperature is 40°C

The LC retention times are obtained using the following elution conditions:

LC-MS: Analytical UPLC on a Agilent Zorbax RRHD SB-Aq (2.1x50mm, 1 ,8um); detection at 210 nm and MS; Gradient of water/ 0.04% TFA (A) and MeCN (B). The eluent flow rate was 0.8 mL/min and the characteristics of the eluting mixture proportion in function of the time t from start of the elution are summarized in the table below (a linear gradient being used between two consecutive time points):

Preparative LC-MS methods used:

Preparative HPLC/MS purifications are performed on a Gilson HPLC system, equipped with a Gilson 215 autosampler, Gilson 333/334 pumps, Finnigan AQA MS detector system, and a Dionex UV detector, using a Waters Xbridge Cl 8 or an Waters Atlantis column, with a linear gradient of water/formic acid 0.02% (A) and MeCN (B) (acidic conditions) or water/ammonia 0.02% (A) and MeCN (B) (basic conditions). Combiflash

Flash column chromatography was performed using a combiflash from Teledyne ISCO. NMR

¹ H-NVIR spectra were recorded at rt with a Brucker NMR 500 spectrometer ¹ H (500 MHz) equipped with a Barker's DCH cryoprobe. Chemical shifts are reported in ppm downfield from tetramethyl silane using residual solvent signals as internal reference. The multiplicity is described as singulet s, doublet d, triplet t, quadruplet q, hextet h, or multiplet m. Broad signals are indicated as br. Abbreviations aq. aqueous ca. circa comb. combined

DCM di chloromethane

DIPEA N,N-diisopropylethylamine

DMAC dimethylacetamide

DMSO dimethylsulfoxide

DPPF 1 , 1 '-Bis(diphenylphosphino)ferrocene eq. equivalent

EtOAc ethyl acetate

EtOH ethanol

HATU l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium

3 -oxide hexafluorophosphate Hept heptane

HPLC high performance liquid chromatography

LC liquid chromatography

LG leaving group

MeCN acetonitrile

MS mass spectroscopy

NaOAc sodium acetate

NEts tri ethyl amine NMR nuclear magnetic resonance org. organic

Pd2dba3 tris(dibenzylideneacetone)dipalladium(0) prep. preparative rt room temperature sat. saturated soln. solution

T3P propylphosphonic anhydride tR retention time

The preparation of compound 1’ (N-(l-((5-cyanopyridin-2-yl)methyl)-lH- pyrazol-3-yl-4-d)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide) is described in Scheme 1. Nitropyrazole 2’ is alkylated with bromide 3’ in presence of a base such as K₂CO₃ and tetrabutylammonium bromide in a solvent such as acetone to give the nitroderivative 4’. Compound 4’ is reduced to the aminopyrazole 5’ following a Bechamp protocol (Fe, aq. NH4CI in EtOH). The amine 5’ undergoes an amide coupling with the known acid 6’ (O. Bezemjon et al., J. Med Chem 2017,60, 9769-9789) using an activating agent such as HATU in presence of a base such as DIPEA to give amide 7’. The bromopyrazole 7’ is converted into the deuterated analog 1’ using a catalyst such as Pd(OH)2 in a presence of a base such as NaOAc in a solvent such as EtOAc or Pd/C in a presence of a base such as NEt3 in a deuterated solvent like D3COD under a D2- atmosphere.

Scheme 1: Synthesis of compounds 1’ and 8’

Compound 8’ (N-(l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl-5-d)-2-(4-(l- (trifluoromethyl)cyclopropyl)phenyl)acetamide) is prepared in a similar manner as compound 1’, starting from the nitropyrazole 9’ (see Scheme 1).

The preparation of compound 10’ (N-(l-((5-cyanopyridin-2-yl)methyl-d2)-lH- pyrazol-3-yl)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide) is described in Scheme 2. Methyl 5 -bromopicolinate 11’ is reduced with sodiumborodeuteride in a solvent such as CD3OD to yield alcohol 12’. Bromopyridine 12’ undergoes a cyanation using ZnCNi in the presence of a catalyst such as Pd₂dba₃ and a ligand such as DPPF in a solvent such as DMAC to give nitrile 13’. Alcohol 13’ is activated e.g. as a mesylate and reacted with nitropyrazole 14’ in the presence of a base to give compound 15’, which undergoes a Bechamp reaction to afford amine 16’. Amide coupling between amine 16’ and acid 6’ in the presence of coupling agent such as HATU and a base such as DIPEA yields compound 10’.

Scheme 2: Synthesis of compound 10’

The synthesis of compound 17’ (N-(l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol- 3-yl)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide-2,2-d2) is described in Scheme 3. The carboxylic acid 6’ is deuterated following a known procedure (see e.g. W02020203610) to give deuterated phenylacetic acid 18’. Amide coupling between the known aminopyrazole 19’ (O. Bezen^on et al., Med. Chem. 2017,«5d, 9769-9789) and the acid 18’ using an activating reagent such as HATU or T3P and a base such as DIPEA yields compound 17’.

Scheme 3: Synthesis of compound 17’

The synthesis of compound 20’ (N-(l -((5-cyanopyridin-2-yl)methyl)-lH-pyrazol- 3-yl-4,5-d2)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide) is described in

Scheme 4. Nitropyrazole 21’ is alkylated with the bromide 3’ in the presence of a base such as K₂CO₃ to give the nitroderivative 22’. Deuteration and concomitant nitroreduction of the dibromoderivative 22’ using a palladium catalyst in the presence of a base under D₂-atmosphere affords deuterated amine 23’. A final amide coupling between amine 23’ and the acid 6’ using an activating agent such as HATU in the presence of a base such as DIPEA yields compound 20’.

Scheme 4: Synthesis of compound 20’ Compound 24’ (N-(l -((5-cyanopyridin-2-yl)methyl-d2)-lH-pyrazol-3-yl-4,5-d2)-

2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide) is prepared as presented in Scheme 5. Alcohol 13’ is activated e.g. as a mesylate and used to alkylate nitropyrazole 21’ in the presence of a base to give compound 25’. Deuteration and concomitant nitroreduction of the dibromoderivative 25’ yields the amine 26’ which undergoes an amide coupling with acid 6’ in the presence of an activating agent such as HATU and a base such as DIPEA to give the final compound 24’.

Scheme 5: Synthesis of compound 24’ As depicted in Scheme 6, deuterated compounds 27’ (N-(l-((5-cyanopyridin-2- yl)methyl-d2)-lH-pyrazol-3-yl)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide- 2,2-d2) or 28’ (N-(l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl-4,5-d2)-2-(4-(l- (trifluoromethyl)cyclopropyl)phenyl)acetamide-2,2-d2) are prepared by amide coupling between carboxylic acid 18’ and the amines 16’ or 23’ using a coupling activating agent such as HATU in the presence of a base such as DIPEA.

Scheme 6: Synthesis of compounds 27’ and 28’

The following compounds were prepared using the methods described above. Compound 1’: JV-(l-((5-cyanopyridin-2-yl)methyl)-TH-pyrazol-3-yl-4-d)-2-(4-(l- (trifluoromethyl)cyclopropyl)phenyl)acetamide

Step 1: 6-((4-bromo-3-nitro-lH-pyrazol-l-yl)methyl)nicotinonitrile (4’)

At rt under argon, 4-bromo-3 -nitro pyrazole (2’) (100 mg, 0.521 mmol, 1.0 eq.), 6-bromomethyl-nicotinonitrile (3’) (103 mg, 0.521 mmol, 1.0 eq.) and tetrabutylammonium bromide (33.6 mg, 0.104 mmol, 0.2 eq) were taken up in acetone (3 mL), then K2CO3 (360 mg, 2.6 mmol, 5.0 eq.) was added and the dark brown mixture was stirred overnight at rt. The suspension was fdtered and washed with acetone. The filtrate was concentrated under reduced pressure. The residue was purified by Combiflash (column: 40 g, flow: 40 mL/min, Heptane to Heptane + EtOAc 4:6) to give a brown oil that solidified upon standing. LC-MS (1): tR = 0.76min; [M+H]⁺: 307.81. 'H NMR (500 MHz, CDCh) 8: 8.89 (d, J= 1.4 Hz, 1 H), 8.04 (dd, J= 8.1, 2.1 Hz, 1 H), 7.81 (s, 1 H), 7.45 (d, J= 8.1 Hz, 1 H), 5.54 (s, 2 H).

Step 2: 6-((3-amino-4-bromo-lH-pyrazol-l-yl)methyl)nicotinonitrile (S’)

To a suspension at rt of 6-((4-bromo-3-nitro-lH-pyrazol-l- yl)methyl)nicotinonitrile (4’) (2.09 g, 6.77 mmol, 1 eq.) in EtOH/water 2: l (70 mL), iron powder, ca. 70 mesh (1.87 g, 33.9 mmol, 5 eq.) and ammonium chloride (1.81 g, 33.9 mmol, 5 eq.) were added. The suspension was heated to 75 °C and stirred at 75 °C overnight. The suspension was filtered over Celite and the Celite rinsed with EtOH. The filtrate was concentrated under reduced pressure. The residue was taken up in water and extracted with DCM (4 x). The comb. org. phases were dried over NaiSCh, concentrated under reduced pressure to give a light orange solid. LC-MS (1): tR = 0.60min; [M+H]⁺: 278.04. 'H NMR (500 MHz, CDCh) 8: 8.85 (dd, J =2.0, 0.7 Hz, 1 H), 7.94 (dd, J= 8.2, 2.0 Hz, 1 H), 7.38 (s, 1 H), 7.15-7.17 (m, 1 H), 5.28 (s, 2 H), 3.81 (s, 2 H).

Step 3: N-(4-bromo-l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl)-2-(4-(l-

( trifluoromethyl)cyclopropyl)phenyl)acetamide (7’)

To a solution at rt of 2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetic acid (6’) (Patent WO2015/186056) (507 mg, 2.08 mmol, 1.05 eq.) in MeCN (13 mL), DIPEA (0.745 mL, 4.35 mmol, 2.20 eq.) and HATU (790 mg, 2.08 mmol, 1.051 eq) were added. The yellow solution was stirred at rt for 5 min, then 6-((3-amino-4-bromo-lH-pyrazol-l- yl)methyl)nicotinonitrile (5’) (550 mg, 1.98 mmol, 1.00 eq.) was added. The yellow solution was stirred at rt for 5 h. The mixture was concentrated in vacuo. The residue was purified by prep. HPLC (column : Waters XBridge, 30x50 mm, 10 um, UV/MS, basic conditions). LC-MS (1): t_R = 0.89min; [M+H]⁺: 503.71. ’H NMR (500 MHz, CDCh) 8: 8.84 (s, 1 H), 7.94-7.95 (m, 1 H), 7.49-7.55 (m, 3 H), 7.36-7.37 (m, 2 H), 7.23 (d, J= 8.1 Hz, 1 H), 6.98 (s, 1 H), 5.42 (s, 2 H), 3.80 (s, 2 H), 1.39 (s, 2 H), 1.04 (s, 2 H).

Step 4: N-( I -( (5-cyariopyridin-2-yl)methyl)-lH-pyrazol-3-yl-4-d)-2-(4-( 1 - (trifluoromethyl)cyclopropyl)phenyl)acetamide (1 ’)

To a mixture under N2 of N-(4-bromo-l -((5-cyanopyridin-2-yl)methyl)-lH- pyrazol-3-yl)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide (7’) (50 mg, 0.1 mmol, 1 eq.) and EhN (70 pL, 0.5 mmol, 5 eq.) in D3COD (2 mL), palladium on activated charcoal (10% Pd basis, 5 mg) was added. The flask was evacuated and backfilled with D2 (3x). The mixture was stirred at rt under a D2 atmosphere for 1.5 hour. The mixture was filtered through an HPLC filter (WHATMAN). The filtrate was concentrated in vacuo. The residue was purified by prep. HPLC (column: Water X- Bridge, 30x75 mm, 10 um, UV/MS, basic conditions). LC-MS (1): tR = 0.95min; [M+H]⁺: 426.98. ’H NMR (500 MHz, DMSO) 8: 10.72 (s, 1 H), 8.99 (dd, J= 2.1, 0.7 Hz, 1 H), 8.28 (dd, J= 8.2, 2.1 Hz, 1 H), 7.79 (s, 1 H), 7.39 (m, 2 H), 7.32 (m, 2 H), 7.18- 7.19 (m, 1 H), 5.44 (s, 2 H), 3.58 (s, 2 H), 1.31 (m, 2 H), 1.09 (s, 2 H). Compound 8’: N-(l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl-5-d)-2-(4-(l- (trifluoromethyl)cyclopropyl)phenyl)acetamide

Step 1: 6-( (5-bromo-3-nitro-lH-pyrazol-l-yl)methyl)nicotinonitrile

To a suspension of 5-bromo-3-nitro-lH-pyrazole (9’) (566 mg, 2.95 mmol, 1.0 eq.) and K2CO3 (2.04 g, 14.7 mmol, 5.0 eq.) in MeCN (20 mL), tetrabutylammonium bromide (194 mg, 0.59 mmol, 0.2 eq.) and 6-bromomethyl-nicotinonitrile (3’) (612 mg, 2.95 mmol, 1.0 eq.) were added in sequence. The mixture was stirred at 60 °C for 18 hours. The mixture was allowed to cool to rt and concentrated in vacuo. The residue was partitioned between water (50 mL) and EtOAc (50 mL). The layers were separated. The aq. phase was extracted with EtOAc (2 x 50 mL). The comb. org. phases were dried over MgSO4 and concentrated in vacuo. The residue was purified by Combiflash (column: 40 g, flow: 40 mL/min, Heptane to Hept/EtOAc 4:6) to yield a brown solid. LC-MS (1): tR = 0.82min; [M+H]⁺: 307.90. 'H NMR (500 MHz, DMSO) 8: 8.98 (dd, J = 2.0, 0.7 Hz, 1 H), 8.38 (dd, J= 8.2, 2.0 Hz, 1 H), 7.57 (dd, J= 8.2, 0.7 Hz, 1 H), 7.48 (s, 1 H), 5.81 (s, 2 H).

Step 2: 6-( (3-amino-5-bromo-lH-pyrazol-l-yl)methyl)nicotinonitrile

To a solution of 6-((5-bromo-3-nitro-lH-pyrazol-l-yl)methyl)nicotinonitrile (369 mg, 1.2 mmol, 1 eq.) in EtOH/water 2:1 (12 mL), iron powder ca. 70 mesh (338 mg, 5.99 mmol, 5 eq.) and ammonium chloride (320 mg, 5.99 mmol, 5 eq.) were added. The mixture was stirred at 75 °C for 18 hours. The mixture was allowed to cool to rt and filtered over Celite. The celite was rinsed with EtOH. The filtrate was concentrated in vacuo. The residue was taken up in water (20 mL) and extracted with DCM (3 x 25 mL). The comb. org. phases were dried over MgSCU and concentrated in vacuo to give an orange solid. The product was used without further purification. LC-MS (1): tR = 0.63min; [M+H]⁺: 278.01. ^XH NMR (500 MHz, DMSO) 8: 8.99 (dd, J = 2.2, 0.7 Hz, 1 H), 8.30 (dd, J= 8.2, 2.2 Hz, 1 H), 7.09 (dd, J= 8.2, 0.7 Hz, 1 H), 5.68 (s, 1 H), 5.28 (s, 2 H), 4.93 (s, 2 H).

Step 3: N-(5-bromo-l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl)-2-(4-(l-

(trifluoromethyl)cyclopropyl)phenyl)acetamide To a solution of 2-(4-(l -(trifluoromethyl)cyclopropyl)phenyl)acetic acid (6’) (Patent WO2015/186056) (128 mg, 0.525 mmol, 1.05 eq.) in MeCN (4 mL), DIPEA (0.188 mL, 1.1 mmol, 2.20 eq.) and HATU (200 mg, 0.525 mmol, 1.05 eq.) were added. The yellow solution was stirred at rt for 5 minutes, then 6-((3-amino-5-bromo-lH- pyrazol-l-yl)methyl)nicotinonitrile (139 mg, 0.5 mmol, 1.00 eq.) was added and the solution was stirred at rt for 20 hours. The mixture was purified by prep. HPLC (column: Water X-Bridge, 30x75 mm, 10 um, UV/MS, basic conditions). LC-MS (1): tR = 1.02min; [M+H]⁺: 503.80. 'HNMR (500 MHz, DMSO) 8: 10.88 (s, 1 H), 8.99 (dd, J= 22, 0.8 Hz, 1 H), 8.31 (dd, J= 8.2, 2.2 Hz, 1 H), 7.40 (m, 2 H), 7.31 (m, 2 H), 7.27 (dd, J = 8.2, 0.5 Hz, 1 H), 6.67 (s, 1 H), 5.49 (s, 2 H), 3.60 (s, 2 H), 1.32 (m, 2 H), 1.10 (s, 2 H).

Step 4: N-( 1 -( (5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl-5-d)-2-(4-( 1 - ( trifluoromethyl)cyclopropyl)phenyl)acetamide (8’)

To a mixture under N2 of N-(5-bromo-l-((5-cyanopyridin-2-yl)methyl)-lH- pyrazol-3-yl)-2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetamide (95 mg, 0.188 mmol, 1 eq.) and EtN (0.131 mL, 0.942 mmol, 5 eq.) in D3COD (3 mL), palladium on activated charcoal (10% Pd basis, 10 mg) was added. The flask was evacuated and backfilled with D2 (3x). The mixture was stirred at rt under a D2 atmosphere for 2 hours. The mixture was filtered through an HPLC filter (WHATMAN). The filtrate was concentrated in vacuo The residue was purified by prep. HPLC (column: Water X- Bridge, 30x75 mm, 10 um, UV/MS, basic conditions). LC-MS (1): tR = 0.95min; [M+H]⁺: 426.99. ^XHNMR (500 MHz, DMSO) 6: 10.72 (s, 1 H), 8.99 (dd, J= 22, 0.8 Hz, 1 H), 8.28 (dd, J= 8.2, 2.2 Hz, 1 H), 7.39 (m, 2 H), 7.32 (m, 2 H), 7.18-7.19 (m, 1 H), 6.51 (s, 1 H), 5.44 (s, 2 H), 3.58 (s, 2 H), 1.31 (d, J= 1.7 Hz, 2 H), 1.09 (s, 2 H).

Compound 17’ : N-(l-((5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl)-2-(4-(l-

(trifluoromethyl)cyclopropyl)phenyl)acetamide-2,2-d2

Stepl: 2-(4-(l-(trifluoromethyl)cyclopropyl)phenyl)acetic-2,2-d2 acid (18’)

To a turbid solution of 2-(4-( 1 -(trifluoromethyl)cyclopropyl)phenyl)acetic acid (6’) (244 mg, 1 mmol, 1 eq.) in D2O (2 mL), sodium deuteroxide 40 wt. % in D2O (3 mL) was added. The mixture was stirred at 100 °C for 20 hours. The reaction mixture was allowed to cool to rt and diluted with D2O (5 mL). The aq. layer was washed with F.tiO (1 x 10 mL) then acidified with 6N aq. HC1 soln, until pH~l. The aq. layer was extracted with DCM (3 x 10 mL). The comb. org. phases were dried over MgSCL and concentrated in vacuo to give a pale yellow solid. The product was used without further purification. LC-MS (1): t_R = 0.88min; no ionization. 'H NMR (500 MHz, DMSO) 8: 12.36 (br s, 1 H), 7.40 (d, J = 8.1 Hz, 2 H), 7.28 (m, 2 H), 1.33 (m, 2 H), 1.11 (s, 2 H).

Step 2: N-( I -( (5-cyanopyridin-2-yl)methyl)-lH-pyrazol-3-yl)-2-(4-( I - (trifluoromethyl)cyclopropyl)phenyl)acetamide-2,2-d2 (18’)

To an ice cooled solution of 6-((3-amino-lH-pyrazol-l-yl)methyl)nicotinonitrile (19’) (Patent WO2015/186056) (19.9 mg, 0.1 mmol, 1 eq.) and 2-(4-(l- (trifluoromethyl)cyclopropyl)phenyl)acetic-2,2-d2 acid (18’) (24.6 mg, 0.1 mmol, 1 eq.) in EtOAc (1 mL), T3P (50% solution in EtOAc) (0.119 mL, 0.2 mmol, 2 eq.) and pyridine (25 pL, 0.3 mmol, 3 eq.) were added. The mixture was kept at 4 °C for 18 hours. The mixture was diluted with EtOAc (25 mL) and washed with 0.1M aq. HC1 soln. (25 mL), sat. aq. NaHCOs (25 mL), sat. aq. NaCl soln. (25 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by prep. HPLC (column: Water X- Bridge, 30x75 mm, 10 um, UV/MS, basic conditions). The fractions containing product were concentrated in vacuo. The residue was diluted with sat. aq. NaHCOs soln. (10 mL) and extracted with DCM (3 x 10 mL). The comb. org. phases were dried over MgSO4 and concentrated in vacuo. LC-MS (1): t_R = 0.95min; [M+H]⁺: 428.03. 'HNMR (500 MHz, DMSO) 8: 10.71 (s, 1 H), 8.99 (dd, J= 22, 0.8 Hz, 1 H), 8.28 (dd, J= 8.2, 2.2 Hz, 1 H), 7.79 (d, J = 23 Hz, 1 H), 7.39 (m, 2 H), 7.31-7.33 (m, 2 H), 7.18-7.20 (m, 1 H), 6.51 (d, J= 2.3 Hz, 1 H), 5.44 (s, 2 H), 1.32 (m, 2 H), 1.09 (s, 2 H).

Example 2: In vitro Methods - Measurement of calcium channel flux by means of FLIPR assays.

HEK293 cells recombinantly expressing either voltage-dependent T-type calcium channel subunit alpha- 1G (Cav3.2) or voltage-dependent L-type calcium channel subunit alpha-lC (Cavl.2) are assayed for calcium flux using the calcium indicator dye Calcium 6 (Molecular Devices) and FLIPR technology (Fluorometric Imaging Plate Reader, Molecular Devices) (Xie X, Van Deusen AL, Vitko I, Babu DA, Davies LA, Huynh N, Cheng H, Yang N, Barrett PQ, Perez-Reyes E. Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches. Assay and Drug Development Technologies 2007, 5(2), 191 - 203). The HEK293 cells recombinantly expressing Cav3.2 are maintained in DMEM growth medium Gibco) supplemented with 10 % Fetal Bovine Serum (FBS), 100 U/ml penicilin (Gibco), 100 pg/ml streptomycin (Gibco) and 1 mg/ml G418 (Gibco). HEK293 cells recombinantly expressing Cavl .2 are maintained in MEM (Gibco), 10% FBS (Gibco); 2mM L-Glutamine (Gibco); 1% Pen/strep; 400 pg/ml G418; 10 pg/ml Zeocin. Cells are washed once with PBS, then dissociated in 0.25 % trypsin/EDTA (Gibco) and seeded into PureCoat Amine coated 384-well black (Corning), clear bottom plates at a density of 20,000 cells/well, in culture medium for the HEK-hCav3.2 cell line, and without antibiotics forthe HEK-hCavl.2 cell line. The seeded plates are incubated overnight at 37°C, 5% CO2.

Immediately prior to performing the assay, medium is removed, and cells are treated for 1 hour at 37°C with 50 pl/well of loading buffer containing the Calcium 6 dye prepared in assay buffer: HBSS IX (137 mM NaCl; 5.4 mM KCl; 0.25 mM Na2HPC>4; 1.3 mM CaCl₂; 0.4 mM MgSCU; 0.5 mM MgCh; 0.4 mM KH2PO4), 0.375 g/L NaHCCh, 20 mM Hepes,l% FBS (Gibco), pH 7.4. After the Ih incubation, cells are allowed to rest at RT in the dark for 30 minutes.

For the Cav3.1 assay, the cells are loaded in presence of Probenecid (AATBioquest; 2.5 mM final concentration in loading buffer) for 1 hour at 37°C. Then, the loading buffer is discarded, and cells are kept in 50 pl/well of assay buffer (HBSS IX; 0.375 g/L NaHCCL; 20 mM Hepes; 1 % FBS; pH 7.4) for 30 min at RT in the dark.

Stock solutions of test compounds are prepared to a concentration of 10 mM in DMSO. For the Cav3.2 assay, serial dilutions of the compounds are prepared in TEAC buffer (100 mM tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCh; 5 mM KC1; 1 mM MgCE; 1 % FBS; pH 7.2), for the Cavl .2 assay serial dilutions are prepared in assay buffer (HBSS IX; 0.375 g/L NaHCCh; 20 mM Hepes; 1 % FBS; pH 7.4). Test compounds are added to the cells to give a 3-fold dilution range from 10 pM to 0 05 nM The compounds are incubated with the cells for 3 minutes and Ca²⁺ entry is stimulated by adding either CaCh to a final concentration of 10 mM (Cav3.2 assay) or by adding KC1 to a final concentration of 75 mM (Cavl.2 assay). The kinetics of fluorescence increase are recorded for every well and the area under the fluorescence trace for every compound concentration is used to generate inhibition curves using non-linear regression sigmoidal concentration-response curve analysis with in-house software. IC50 values are calculated and represent the compound concentration required to inhibit 50% of the signal that is obtained in the presence of vehicle instead of test compound. Antagonistic activities (IC50 values) have been measured for the for the Cav3.1- and the Cav3.3 -channel.

In the following table, ICso-values for the Cav3.X-channel are presented for certain compounds.

Example 3. Metabolic Profiles of the T-Type Calcium Channel Blocker, Compound 2, with Blood, Plasma, and Liver Preparations From Mouse, Rat, Rabbit, Dog, Monkey, and Human

Summary

The metabolism of Compound 2 was investigated using liver microsomes and hepatocytes of man and a set of animal species used or considered to be used in preclinical safety testing. The in vitro metabolic profile of Compound 2 with human liver preparations was characterized by the formation of five metabolites, i.e., M1-M5. It was found that Compound 2 undergoes three primary metabolic pathways: oxidative dealkylation of the pyrazole ring to form Ml, hydrolysis of the amide bond to form M2, and hydroxylation to yield M29. The aglycon M29 was never observed directly, but was instead observed as M4, the product of subsequent glucuronidation. Also, Ml was the product of aP450/FMO- dependent reaction and undergoes further conjugation with pentose to yield the humanspecific metabolite, M3. Hydrolysis to M2 was shown to be catalyzed by microsomal enzymes other than cytochrome P450s. M5 is a cysteine conjugate of 3-aminopyrazole.

Rat and cynomolgus monkey together covered four of the five human metabolites, i.e., Ml, M2, M4 and M5, observed in vitro and therefore appear as the most suitable animal species for preclinical safety testing from a metabolism perspective.

Compound 2 was hydrolyzed to M2 in plasma of rat, monkey and mouse whereas no degradation was observed in human and rabbit plasma. M2 was also formed in blood from human, rat and monkey, but not in blood from rabbit and mouse.

Background

The metabolic profile of Compound 1 and Compound 2 have been determined in vitro. Compound 2 is the ¹⁴C-labelled analogue of Compound 1 bearing the radiolabel in the pyrazole moiety of the molecule.

Compound 1 Compound 2

The ¹⁴C labeled Compound 2 was used as a tool compound. One of skill in the art will recognize that the deuterated compounds can also be used instead of Compound 2 for any of the assays described herein.

Study Objective

The objective of this in vitro study was the determination and comparison of Compound 2 metabolic profiles using liver microsomes, hepatocytes, blood, and plasma, from a number of animal species envisaged for toxicity testing, as well as of man. For this purpose, the number and proportion of metabolites generated following incubation of the ¹⁴C-labelled analogue, Compound 2, at a single concentration of 10 pM with liver preparations from CD-I mouse, Wistar rat, NZW rabbit, Beagle dog, cynomolgus monkey, and man were determined using HPLC coupled with 14C-radiodetection. The stability in plasma and blood of all species used in the safety evaluation was investigated as Compound 2 hydrolyzed to metabolite M2. Data on plasma and blood stability were generated in support of the plasma protein binding and blood partitioning studies.

Chemicals

¹⁴C-radiolabelled compound, Compound 2, was obtained from Quotient Bioresearch UK Ltd (Cardiff, UK). The compound was supplied as a solution in ethanol at a concentration of 0.5 mCi/mL with a radiochemical purity of >99.9 % and a specific activity of 55 mCi/mmol. Non-labelled Compound 2 (i.e , Compound 1) was supplied as a powder with a purity of 99.2 %. Dichlorvos-Pestanal, bis-(4-nitrophenyl)-phosphate, p- nitrophenyl acetate, glucose-6- phosphate (di sodium salt) and NADP were purchased from Sigma (Buchs, Switzerland). Glucose-6-phosphate dehydrogenase was supplied by Roche Diagnostics (Mannheim, Germany). The liquid scintillation cocktail for HPLC analysis, Optiflow Safe 2, was purchased from Berthold Technologies GmbH (Regensdorf, Switzerland). Leibovitz's L-15 and William’s E media were supplied from Life Technologies (Basel, Switzerland). The liquid scintillation cocktail for determination of total radioactivity, Irga Safe plus, was purchased from Perkin Elmer (Zurich, Switzerland). All other chemicals and solvents used throughout this study were of highest commercially available quality.

For the preparation of the Compound 2 working solution, aliquots of the stock solution in ethanol with an initial radioactive concentration of 0.5 mCi/mL were diluted in acetonitrile/water (1:1, v/v) to a final concentration of 1 mM. This working solution was stored at -20 °C.

HPLC/MS Equipment and Data Acquisition

The HPLC/MS system for the recording of metabolic profiles and mass spectra consisted of two Shimadzu pumps LC-30AD (Shimadzu, Reinach, Switzerland) equipped with a Shimadzu membrane degasser DGU-30A5, a Shimadzu diode array detector SPD- M20A, a Shimadzu column oven CTO-20A, and a Shimadzu autosampler model SIL- 20 AC. Radio detection was performed by a Berthold radioflow detector LB513 with a 200 pL liquid cell Z-200-6M, a LB5036 pump for supplementing liquid scintillation cocktail at 3 mL/min (Berthold AG, Regensdorf, Switzerland). Detection of mass data was performed by a LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The acquisition and analysis of radiochemical and mass data were done using the RadioStar (version 5.0.12.4, Berthold AG, Regensdorf, Switzerland) and Xcalibur software packages (version 2.2 SP1.48, Thermo Fisher Scientific, Waltham, MA, USA). As used herein, the disclosed m/z values refer to the singly protonated [M+H]⁺. The following MS equipment and parameters were used:

LTQ XL (Thermo Fisher

Mass Spectrometer Scientific, Waltham, MA, USA)

Ionization H-ESI+ (positive ionization mode)

H-ESI Voltage 3.0 kV Capillary Temperature 275 °C Sheath Gas 40 psi Auxiliary Gas 10 psi Capillary 35 V

HPLC Method

The chromatographic separation of Compound 2 and its metabolites was achieved on a GL Sciences InertSustain Cl 8 column (3 pm, 250 x 4.6 mm ID) at 30 °C. The total HPLC flow rate was set to 1 mL/min and split prior to 14C-radiochemical- and mass determination in a ratio of 8/2, respectively. Mobile phases consisted of 20 mM ammonium formate adjusted to pH 8.5 with ammonium hydroxide (phase A), and methanol (phase B). Serial UV-detection in a wavelength range of 190-500 nm, preliminary mass analytic and 14C-radiochemical detection were performed. The applied gradient for the separation of Compound 2 and its metabolites is described below.

Using these chromatographic conditions, Compound 2 had a retention time of 73.6 min. The variability in retention time in the different chromatograms did not exceed 0.5 min over the total run time of 90 min.

NADPH-Regenerating System

The NADPH-regenerating system used for the liver microsomal incubations was prepared as a 10-fold concentrated stock solution and kept at -20 °C. It consisted of 11 mM NADP, 100 mM glucose-6-phosphate and 50 mM magnesium chloride in 0.1 M phosphate buffer (pH 7.4). 20 lU/mL of glucose-6-phosphate dehydrogenase was added before use.

Liver Microsomes

Pooled liver microsomes were obtained. An overview on the enzyme specification in these preparations is provided in Table 1.

Table 1.

Hepatocytes

Cryopreserved human hepatocytes (batch 3) were provided by Celsis (Neuss, Germany), while NZW rabbit and CD-I mouse cells were provided by Biopredic International (Rennes, France). Freshly prepared and ready-plated cultures of Beagle dog, cynomolgus monkey and human hepatocytes (batches 1, 2 and 4) were obtained from Primacyt (Schwerin, Germany) and Biopredic International (Rennes, France), and used immediately after receipt. Hepatocytes from Wistar rat were prepared at Actelion Pharmaceutical Ltd following the standard two-step collagenase perfusion method (see e.g.. Seglen & Fossa, Exp. Cell Res. 1978, 116: 199-206). Cell viabilities were measured after hepatocyte preparation using a Vi-cell viability analyzer (Beckman Coulter, Nyon, Switzerland) for the determination of cell number and initial cell viability with a trypan blue dye exclusion test. Hepatocytes with viabilities below 70% were rejected for use. With the exception of rabbit hepatocytes in suspension, hepatocytes were used in a ready- plated 24-well format. The viabilities of cryopreserved and fresh cells in these experiments ranged from 76%-92%. The metabolic capacity of the liver cells was verified using in-process controls. Table 2 provide an overview of the characteristics of the hepatocyte batches used in this study.

Table 2.

(1) fresh ready-plated human hepatocytes; (2) cryopreserved human hepatocytes.

Plasma and Blood

Pooled frozen human plasma was obtained from Bioreclamation IVT (Brussels, Belgium), whereas pooled frozen plasma from Wistar rat, cynomolgus monkey, CD-I mouse and NZW rabbit was provided by Harlan (Itingen, Switzerland). Cynomolgus monkey blood was obtained from Covance (Neuss, Germany), while blood from human, rat, mouse and rabbit was obtained from Actelion Pharmaceutical Ltd (Allschwil, Switzerland). Blood was collected by venipuncture from the large arm vein of healthy volunteers not taking aspirin. Characteristics of plasma and blood used in this study are summarized in Table 3.

Table 3.

Incubations with Liver Microsomes

Incubations of Compound 2 with liver microsomes of all species were performed at a single substrate concentration of 10 pM in 2 mL amber reaction vials. In a total volume of 200 pL, a 2.0 pL-aliquot of the Compound 2 stock solution was added to 100 mM phosphate buffer (pH 7.4) containing the liver microsomes at a protein concentration of 1 mg/mL for human, rabbit, rat, monkey and mouse liver microsomes, and at 3 mg/mL for dog liver microsomes. The mixture was incubated at 37 °C in an Eppendorf thermomixer and agitation at 650 rpm. The organic solvent concentration in all incubations was kept <1 % (v/v). The reaction was initiated by addition of 20 pL of prewarmed NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated either immediately after the start of the reaction (control) or after 60 minutes, by addition of 200 pL of ice-cold acetonitrile. Samples were centrifuged at 20'800 g and 10 °C for 5 min. Prior to HPLC analysis, a 100 pL-aliquot of the supernatant was mixed with 300 pL mobile phase A in order to mimic initial HPLC conditions. Control experiments were performed in the absence of either the NADPH- regenerating system or the liver microsomes under otherwise identical conditions. Tn these controls, the volumes of both co-factors were replaced by 100 mM phosphate buffer (pH 7.4).

Cell Culture Media for Hepatocytes

Freshly prepared hepatocytes were cultured in William’s E medium supplemented with 10 % fetal bovine serum and 4 pg/mL bovine insulin. Cryopreserved cells were thawed and the supplied medium was replaced by culture medium. Incubations with Compound 2 were performed using a culture medium additionally fortified with 0.48 pg/mL hydrocortisone and 400 pM L-glutamine. Neither the culture nor the incubation media contained phenol red or antibiotics.

Incubations with Plated Hepatocytes

Freshly isolated and collagenase-perfused rat liver was kept in Leibovitz's medium and was mechanically dissociated using sterile pipette tips. To facilitate liver cell isolation from tissue, the cell suspension was transferred through a nylon mesh cell strainer with a pore size of 70 pm into a sterile 50 mb centrifugation tube. The cell suspension was centrifuged at 50 g and 4 °C for 4 min, the cell pellet re-suspended in Leibovitz’s medium, followed by purification on a Percoll cushion (15%) and another centrifugation step at 50 g and 4 °C for 4 min. After centrifugation, cells were resuspended in 1 mL culture medium. An aliquot of the cell suspension was submitted to a Vi-Cell viability analyzer (Beckman Coulter, Nyon, Switzerland) for the determination of cell number and initial cell viability, by a trypan blue dye exclusion test. Viabilities are summarized in Table 2. The cell suspension was then adjusted with culture medium at a nominal density of 5 x 105 viable cells/mL. 400 pL-aliquots of this suspension were dispensed into collagen-coated 24-well plates and incubated at 37 °C for a period of about 3 h in a humidified atmosphere containing 5 % CO2.

Primary cultures of human, monkey and dog hepatocytes were purchased in a ready-plated format and cultured in culture medium for 0.5 h prior to use for incubations. Cryopreserved hepatocytes from man, rabbit and mouse were quickly thawed at 37 °C until visible ice was gone, and purified on a Percoll cushion (15 %) at 50 g for 4 min at 4 °C. The cell pellet was re-suspended in 1 mL culture medium and cell viabilities determined as described above. The cell suspension was then adjusted with culture medium at a nominal density of 5 x 105 viable cells/mL. 400 pL-aliquots of this suspension were dispensed into collagen-coated 24-well plates and incubated at 37 °C for a period of about 3 h in a humidified atmosphere containing 5% CO2 to allow cell attachment.

At the end of the pre-incub ati on period, the medium was removed from each well and replaced by 200 pL of pre-warmed (37 °C) incubation medium containing Compound 2 at a final concentration of 10 pM. Triplicate wells were sampled after 0, 4 and 24 h of incubation by addition of 200 pL of ice-cold acetonitrile. The entire well content was transferred into 2 mL amber reaction vials. Samples were stored frozen at - 20 °C pending analysis. Prior to HPLC analysis, samples were thawed at 37 °C, vortex- mixed and centrifuged at 20'800 g and 10 °C for 5 min. A 100 pL-aliquot of the supernatant was mixed with 300 pL mobile phase A in order to mimic initial HPLC conditions and submitted to HPLC analysis.

Incubations with Hepatocytes in Suspension

Cryopreserved hepatocytes from NZW rabbit were thawed at 37 °C, and purified on a Percoll cushion (15%) at 50 g for 4 min at 4 °C. The resulting cell pellet was resuspended in 1 mL culture medium and cell viabilities determined as described above. The cell suspension was adjusted with incubation medium at a nominal density of 1 x 10⁶ viable cells/mL. 500 pL-aliquots of this cell suspension were dispensed into 2 mL amber reaction vials and placed in an Eppendorf thermomixer at 37 °C and 850 rpm to keep the cells in suspension. A 5 pL-aliquot of the Compound 2 stock solution was added to reach a final concentration of 10 pM. After pre-defined incubation periods of 0, 2 and 6 h, the reaction was stopped by the addition of 500 pL of ice-cold acetonitrile. Samples were stored frozen at -20 °C pending analysis. Prior to HPLC analysis, samples were centrifuged at 20'800 g and 10 °C for 5 min, and a 100 pL-aliquot of the supernatant was mixed with 300 pL mobile phase A in order to mimic initial HPLC conditions.

Enzymatic Cleavage of Glucuronic Acid Conjugates

The samples of Compound 2 after 22 h incubation with human hepatocyte batch 4 were treated with β -glucuronidase to identify potential glucuronic acid conjugates. For this purpose, triplicate wells of an incubation plate were supplemented with 10 pL of a 140 lU/mL β-glucuronidase solution. The mixtures were incubated for further 2 h at 37 °C in a humidified atmosphere containing 5 % carbon dioxide. The reaction was stopped by addition of 200 pL acetonitrile, followed by centrifugation at 20'800 g and 10 °C for 5 min. Prior to HPLC analysis, samples were centrifuged at 20'800 g and 10 °C for 5 min, and a 100 pL-aliquot of the supernatant was mixed with 300 pL mobile phase A in order to mimic initial HPLC conditions. Control experiments in the absence of P-glucuronidase were run in parallel under otherwise identical conditions.

Incubation with Plasma and Blood

Plasma and blood of cynomolgus monkey, Wistar rat, CD-I mouse, New Zealand White rabbit and man were fortified with Compound 2 at a final concentration of 10 pM in a total reaction volume of 1 mL. After an incubation time of 0.5, 1 and 2 h for blood, or 2, 4 and 6 h for plasma, 200 pL-aliquots of the incubation mixture were mixed with 600 pL of a 8:2 (v/v) mixture of acetonitrile and methanol to lyse blood cells and precipitate proteins. The samples were centrifuged at 20'800 g for 10 min at 10 °C and 100 pL aliquots of the supernatants were mixed with 300 pL mobile phase A in order to mimic initial HPLC conditions. Control experiments to assess hydrolase activity in plasma and blood using p -nitrophenyl acetate as a substrate for esterase enzymes were performed simultaneously under identical incubation and sample work-up conditions.

Additional experiments were performed to assess whether the hydrolysis of Compound 2 could be suppressed at 4 °C or by addition of hydrolase inhibitors. The effect of hydrolase inhibitors was tested by pre-incubating 0.1 % (v/v) DCV or 0.2 mM BNPP with Wistar rat, CD-I mouse, and cynomolgus monkey plasma at 37 °C for about 5 minutes prior to the addition of Compound 2. Incubations and sample work-up followed the procedure outlined above.

Determination of Recoveries

Recoveries were determined as the ratio of total radioactivity before and after centrifugation of the quenched incubation mixtures. For this purpose, triplicate 10 pL aliquots of each incubation were mixed with 4 mL of IRGA SafePlus liquid scintillation cocktail and submitted for liquid scintillation counting using a Tricarb 2300 TR liquid scintillation analyzer (Perkin Elmer, Zurich, Switzerland) with luminescence correction and on-line quenching correction by means of an internal standard. Results of the recovery determinations are summarized in FIG. 1. Mean recoveries were in excess of 97% and 84% for liver microsomal and hepatocyte incubations, respectively.

Results and Discussion

Metabolite Identification

Preliminary structural elucidation of Compound 2 metabolites was performed. The 24 h incubation of Compound 2 with cryopreserved human liver cells (batch 3) was selected for further LC-MS investigations as it contained all metabolites observed with human liver preparations. A scheme outlining the metabolic pathways of metabolites is shown in FIG. 2.

Compound 2 undergoes three primary metabolic pathways: oxidative dealkylation of the pyrazole ring to Ml and hydrolysis of the amide bond to yield M2. M4 is a secondary metabolite and the product of hydroxylation in the pyrazole amide moiety followed by glucuronidation. M29, the aglycon of M4, (i.e., the primary hydroxylation product of the pyrazole moiety) was only detected after treatment with β-glucuronidase. Mass spectrometry data indicate that M29 was not present in incubations with Compound 2 with liver microsomes or hepatocytes. The exact chemical structure of M4 and its precursor is yet unknown. Conjugation of Ml with a pentose gives the phase II metabolite M3. M5 is a cysteine conjugate of 3-aminopyrazole. Experiments with Liver Microsomes

Compound 2 was incubated at a single substrate concentration of 10 pM for up to 60 minutes with liver microsomes of CD-I mouse, Wistar rat, NZW rabbit, Beagle dog, cynomolgus monkey and man at microsomal protein concentrations of 1 or 3 mg/mL. Control experiments in the absence of liver microsomes or the NADPH-regenerating system were performed in order to demonstrate that metabolite formation was indeed P450/FMO-dependent. FIG. 3 gives an overview on the number of metabolites formed and their individual relative contributions. The radiochromatograms of these incubations including controls are presented in FIGs. 4-11.

The metabolism of Compound 2 was slow in all the species. Incubation with human liver microsomes led to the formation of a single product, the dealkylated Ml. Ml was consistently observed with liver microsomes of all animal species. Metabolite M2 was additionally observed with rat and mouse liver microsomes, while M5 was selectively formed with rat liver microsomes. M2 was also observed in incubations with rat and mouse liver microsomes in the absence of the NADPH-regenerating system (FIG. 10) indicating that M2 is formed by microsomal proteins other than P450s. No turnover was observed in control incubations without liver microsomes (FIG. 11).

Experiments with Hepatocytes

Incubation of Compound 2 with hepatocytes from CD-I mouse, Wistar rat, NZW rabbit, Beagle dog, cynomolgus monkey and man were performed at a single substrate concentration of 10 pM for up to 24 h. FIG. 12 gives an overview on the number of metabolites formed in the different incubations together with their individual relative contributions. The respective radiochromatograms including the control without cells are presented in FIGs. 13-22.

The metabolism of Compound 2 with human hepatocytes was investigated using four different batches of human liver cells (FIGs. 13-16). Up to five products, designated M1-M5, were observed. All five metabolites were seen following 24 h-incubation of Compound 2 with batches 3 and 4, none individually exceeding 21 % and with an overall turnover of 32% and 52%, respectively. Only Ml was observed with human hepatocyte batches 1 , 2 and 3 after an incubation time of 4 h. Metabolites M2 and M3 were detected in batch 1 after 24 h-incubation, accounting for 9.2% and 3.1%, respectively. Metabolites Ml and M2 were detected in batch 2 after 24 h-incubation representing 3.1% and 10%, respectively. Control experiments in the absence of liver cells confirmed that all five products were indeed Compound 2 metabolites (FIG. 22).

Compound 2 yielded five metabolites with rat hepatocytes (FIG. 17) after 24 h incubation and a turnover of 88%. M2, M4 and M5, representing 40%, 2.1% and 33% of total radioactivity, respectively, were common to human hepatocytes. The two non-human metabolites M9 and MIO were detected in addition.

After 24 h incubation, dog and mouse hepatocytes (FIGs. 18 and 20) yielded metabolites Ml and M2, accounting for 4.2% and 8.5% in dog liver cells, respectively, while 12% and 8.0% were observed in mouse liver cells.

Incubation with monkey hepatocytes (FIG. 19) resulted in the formation of seven metabolites with a turnover of 55% after 24 h. Ml and M8 were the major products accounting for 17% and 12%, respectively, while all others were present in a range from 2.2%-7.1%.

Rabbit hepatocytes (FIG. 21) resulted in the formation of M2 accounting for 4.0% after 6 h of incubation.

Enzymatic Cleavage of Glucuronic Acid Conjugates

The presence of glucuronic acid conjugates was investigated using a fresh hepatocyte sample from human batch 4. This sample was incubated for 2 h with P- glucuronidase. Control experiments in the absence of β-glucuronidase were performed under otherwise identical conditions. The corresponding radiochromatograms are shown in FIG. 16 and the results are summarized in FIG. 12.

Only metabolite M4 disappeared after P-glucuronidase treatment. Mass spectrometry data revealed a molecular mass of m/z 444, i.e., a hydroxylation product M29 co-eluting with parent Compound 2. This aglycon M29 was not detected in any other in vitro incubation. Stability of Compound 2 in Plasma and Blood

Compound 2 was incubated with plasma of Wistar rat, CD-I mouse, NZW rabbit, cynomolgus monkey and man at 37 °C for 2, 4 and 6 hours, as well as with blood of the same species for 0.5, 1 and 2 hours. The rate constant k of the hydrolysis to M2 was determined from the slope of log concentration versus time plot.

Additional experiments using hydrolase inhibitors and lower temperatures were performed to investigate whether Compound 2 hydrolysis in plasma can be suppressed. The relative proportions of Compound 2 and hydrolysis product M2 are summarized in FIGs. 23-24, while representative radiochromatograms are depicted in FIGs. 25-26.

Significant species differences were observed in the susceptibility to hydrolysis of Compound 2 in plasma and blood. Hydrolysis of Compound 2 was observed in plasma of rat, monkey and mouse, whereas no hydrolysis was evident in plasma of man and rabbit. The order of the enzymatic hydrolysis rates was rat > monkey > mouse, as reflected by the degradation rate constants of 0.10, 0.04 and 0.01 h'¹, respectively. Control incubations at 37 °C in 100 mM phosphate buffer (pH 7.4) under otherwise identical conditions showed no hydrolysis of Compound 2. Species differences in plasma hydrolase expression and activity were described in Bahar et al., J. Pharm. Sci. 2012, 101 :3979-88.

The hydrolytic activity of rat, monkey and mouse plasma was fully suppressed at 4 °C, or by addition 0.1% of the esterase inhibitor dichlorvos (DCV). In contrast, addition of bis (4-nitrophenyl) phosphate (BNPP) did not completely suppress the hydrolytic activity in plasma of rat and monkey.

In blood, hydrolysis of Compound 2 was observed in rat, monkey and man, whereas Compound 2 was stable in blood of mouse and rabbit. The order of enzymatic hydrolysis rates was rat > human > monkey, with degradation rate constants of 0.17, 0.11 and 0.07 h'¹, respectively. Hydrolysis rates were not affected by the choice of anticoagulant, as there was no difference in human blood treated with heparin or EDTA. /?-Nitrophenyl acetate was used as positive control for hydrolase activity in plasma and blood. In sum, Compound 2 was hydrolyzed to M2 in plasma of rat, monkey and mouse whereas no degradation was observed in human and rabbit plasma. M2 was also formed in blood from human, rat, and monkey, but not in blood from rabbit and mouse. Comparison of Hepatocytes and Microsomal Incubations

Metabolic profiles of microsomal and hepatocytes incubations were compared to assign metabolites to phase I or phase II metabolism. The primary metabolite Ml, observed in liver microsomes from all species was also detected in hepatocyte incubations of human, dog, monkey and mouse. Since its formation was dependent on NADPH, it is most likely a product of a P450/FMO-catalyzed reaction. The hydrolysis product M2, observed in hepatocytes of all species was only observed in microsomes of rat and mouse. Its formation in the absence of NADPH suggests a P450-independent hydrolysis. Metabolites M3 and M4 are products of phase II metabolism and were only present in hepatocyte incubations.

Coverage of Human Metabolites formed by liver preparations

Overall, human liver preparations yielded five metabolites: M1-M5. Ml, M2, M4 and M5 were observed in the rat. The same four metabolites were observed in cynomolgus monkey, while only Ml and M2 were observed in the dog. The pentose conjugate M3 was not observed in liver microsomes or hepatocytes of any animal species and thus appears to be specific to man.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patent, patent applications, and publications, cited in the present application is incorporated herein by reference in its entirety.

Claims

WHAT TS CLAIMED IS:

1 . A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each independently selected from hydrogen and deuterium; and wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ is deuterium.

2. The compound of claim 1 , wherein one of R¹, R², R³, R⁴, R⁵, R⁶, R^z, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ is deuterium.

3. The compound of claim 1, wherein two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

4. The compound of claim 1, wherein three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

5. The compound of claim 1, wherein four of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

6. The compound of claim 1, wherein five of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

7. The compound of claim 1, wherein six of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

8. The compound of claim 1, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each deuterium.

9. The compound of claim 1, which is a compound of Formula II:

or a pharmaceutically acceptable salt thereof.

10. The compound of claim 1, which is a compound of Formula III:

or a pharmaceutically acceptable salt thereof.

11. The compound of claim 1, which is selected from:

or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition, comprising a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

13. A method of blocking a T-type calcium channel, comprising contacting a T-type calcium channel with a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

14. A method of treating a disease or disorder selected from epilepsy, a sleep disorder, a sleep disturbance, pain, a neurological disorder, a cardiovascular disorders, cancer, diabetes, diabetic neuropathy, infertility, and sexual dysfunction, in a subject, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

15. The method of claim 14, wherein the disease or disorder is epilepsy.

16. The method of claim 14 or 15, wherein the epilepsy is selected from epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS), childhood absence epilepsy, and essential tremor.