HK1236524A1 - Inhibiting the transient receptor potential a1 ion channel - Google Patents

Description

Inhibition of transient receptor potential A1 ion channel

Requirement of priority

This application claims priority to U.S. provisional application No. 61/983,223 filed on 23/4 of 2014 and U.S. provisional application No. 61/987,272 filed on 1/5 of 2014, which are incorporated herein by reference in their entireties.

Technical Field

The present invention relates to compounds of formula (I), or a pharmaceutically acceptable salt, pharmaceutical preparation or pharmaceutical composition thereof, and their use for the treatment of pain, inflammatory diseases, neurological diseases, skin diseases, pulmonary diseases and cough, and for inhibiting the transient receptor potential a1 ion channel (TRPA 1).

Background

The transient receptor potential a1 (referred to herein as "TRPA 1") is a nonselective cation channel involved in human pain perception. TRPA1 is found in sensory neurons and functions as a detector to help correlate detection of toxic chemicals, tissue damage and inflammation with pain. Activation of TRPA1 is thought to cause pain by inducing inflammation of pain-responsive neurons and driving central sensitization in the spinal cord. TRPA1 stimulation may also increase inflammation of sensory neurons, leading to the release of pro-inflammatory neuropeptides such as NK-A, P substance and CGRP, which induce vasodilation and help recruit immune cells. Endogenous reactive compounds produced during various inflammatory processes activate TRPA1, including 4-hydroxynonenal released during liposome peroxidation; cyclopentane prostaglandins synthesized by COX enzymes; hydrogen peroxide produced by oxidative stress. Activation of TRPA1 also sensitizes TRPA1 to cold. Furthermore, acquired functional mutations in TRPA1 cause familial paroxysmal pain syndromes; patients with this disease have episodic pain, which may be caused by cold. Thus, TRPA1 is believed to play a role in pain, cold allodynia, and inflammatory pain related to nerve injury.

Compounds that inhibit the TRPA1 ion channel are useful, for example, in the treatment of diseases that can be ameliorated, eliminated or prevented by the inhibition of the TRPA1 ion channel. For example, pharmaceutical compositions that inhibit TRPA1 may be used to treat pain. Inhibition of TRPA1 (e.g., by gene excision and chemical antagonism) has been shown to result in reduced pain behavior in mice and rats. Knockout mice lacking functional TRPA1 have reduced pain responses to TRPA1 activators including AITC, formalin, acrolein, 4-hydroxynonenal, and, in addition, greatly reduced thermal and mechanical hypersensitivity in response to the inflammatory mediator bradykinin (e.g., Kwan, k.y. et al Neuron 2006, 50, 277, 289; Bautista, d.m. et al Cell 2006, 124, 1269-. In an animal pain model, inflammatory and nerve injury-induced cold hyperalgesia was prevented and reversed by gene-specific antisense down-regulation of TRPA1 expression (see, e.g., Obata, K. et al, J Clin Invest (2005)115, 2393-. TRPA1 inhibitor compounds are effective in a variety of rodent pain models. TRPA1 inhibitors have been shown to reduce mechanical hypersensitivity and cold allodynia after complete freund' S adjuvant induction of inflammation without altering normal cold sensation in natural animals, and also to improve function in the rat mono-iodoacetate osteoarthritis model (see, e.g., Materazzi, S et al, Eur JPhysiol (2012), 463(4): 561-9; weii H et al, Anesthesiology 2012, 117(1): 137-48; Koivisto, a et al, Pharmacol Res (2012), 65(1): 149-58). TRPA1 inhibitor compounds have demonstrated reduced pain behavior in rodents injected with AITC (mustard oil), formalin, cinnamaldehyde, acrolein, and other TRPA1 activators. TRPA1 inhibitor compounds have also been shown to be effective in rodent models for post-operative pain, (see, e.g., Wei et al, Anesthesiology (2012), 117(1): 137-48); chemotherapy-induced peripheral neuropathy (see, e.g., Trevisan, et al, Cancer Res (2013)73(10):3120-31) and painful diabetic neuropathy (see, e.g., Koivisto, et al, Pharmacol Res (2011)65: 149-.

Disclosure of Invention

The compounds described herein are useful for treating diseases in which inhibition of the TRPA1 ion channel would be beneficial, for example, treating pain. In some embodiments, the compounds described herein have superior properties relative to other compounds in the art that inhibit TRPA 1. For example, in some embodiments, the compounds described herein inhibit the TRPA1 ion channel without raising serum biomarkers of hepatotoxicity. In some embodiments, a compound described herein, e.g., a compound of formula (I), has favorable aqueous solubility relative to other compounds in the art that inhibit TRPA1 (including compounds having aqueous solubility suitable for formulation into pharmaceutical compositions for intravenous administration).

Described herein are compounds of formula (I) and pharmaceutically acceptable salts thereof:

wherein the variables are as described herein, e.g., in the detailed description section below.

Also described herein are purified pharmaceutical preparations and pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The compounds and compositions described herein are useful for treating a variety of diseases in a subject. For example, described herein are methods of treatment, such as methods of treating a TRPA 1-mediated disease in a subject, comprising administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Also described herein are methods of treating pain in a subject comprising administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Exemplary types of pain include neuropathic pain, e.g., painful diabetic neuropathy, chemotherapy-induced peripheral neuropathy, lower back pain, trigeminal neuralgia, post-herpetic neuralgia, sciatica, and complex regional pain syndrome; inflammatory pain, e.g., pain derived from rheumatoid arthritis, osteoarthritis, temporomandibular disease; PDN or CIPN; visceral pain, e.g., from pancreatitis, inflammatory bowel disease, colitis, crohn's disease, endometriosis, pelvic pain, and angina; pain selected from the group consisting of: cancer pain, burn pain, oral pain, crush and injury-induced pain, incision pain, bone pain, sickle cell disease pain, fibromyalgia, and musculoskeletal pain; or pain from hyperalgesia or allodynia.

In some embodiments, the method comprises treating an inflammatory disease in a subject, the method comprising administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In some embodiments, the method comprises treating a neuropathy in a subject, the method comprising administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the neuropathy results from diabetes, chemical injury, chemotherapy, and or trauma.

In some embodiments, the method comprises treating a skin disorder in a subject, the method comprising administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Exemplary skin disorders include atopic dermatitis, acute pruritus, psoriasis, urticaria, eczema, pompholyx, mouth ulcers, and diaper rash.

In some embodiments, the method comprises treating a pulmonary disease in a subject, the method comprising administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Exemplary pulmonary diseases include obstructive diseases such as chronic obstructive pulmonary disease. Other exemplary pulmonary diseases include asthma and cough.

In addition, the compounds described herein, e.g., compounds of formula (I), can be used to prepare pharmaceutical compositions formulated for oral administration. In some embodiments, the compounds described herein can be formulated as compositions for intravenous administration. In embodiments, a compound or composition described herein can be used to treat pain.

The compounds described herein, e.g., compounds of formula (I), can include molecules having one or more chiral centers. For example, unless otherwise stated, the compositions of formula (I) may comprise different amounts of stereoisomers of formulae (Ia), (Ib), (IIa) and (IIb). In one embodiment, the composition comprising a compound of formula (Ia) or (IIa) preferably comprises a therapeutically effective amount of a compound having a stereochemical structure as shown in formula (Ia) or (IIa) (e.g., enantiomeric or diastereomeric excess of a particular isomer of formula (Ia) or (IIa) relative to the corresponding stereoisomer of formula (Ib) or (IIb)). In one embodiment, a composition comprising a compound of formula (I) comprises a therapeutically effective amount of a compound having a stereochemical structure shown in formula (Ib) or (IIb) (e.g., enantiomeric or diastereomeric excess of a particular isomer of formula (Ib) or (IIb) relative to the corresponding stereoisomer of formula (Ia)).

Furthermore, the compounds of formula (I) may comprise one or more isotopes of said atoms present in formula (I). For example, the compound of formula (I) may comprise: those in which H (or hydrogen) is replaced by any isotopic form of hydrogen, including¹H、²H or D (deuterium) and³h (tritium); those in which C is replaced by any isotopic form of carbon, including¹²C、¹³C and¹⁴c; those in which O is replaced by any isotopic form of oxygen, including¹⁶O、¹⁷O and¹⁸o; those in which N is replaced by any isotopic form of nitrogen, including¹³N、¹⁴N and¹⁵n; those in which P is replaced by any isotopic form of phosphorus, including³¹P and³²p; those in which S is replaced by any isotopic form of sulfur, including³²S and³⁵s; those in which F is replaced by any isotopic form of fluorine, saidIsotopic forms include¹⁹F and¹⁸f; and the like. In one embodiment, the compounds represented by formula (I) include isomers of the atoms present therein in their naturally occurring abundance.

Brief Description of Drawings

Figure 1 is a graph depicting the X-ray powder diffraction (XRPD) pattern of the solid crystalline form of compound 2 (form a) after slurry treatment in ethanol.

Figure 2 is a graph depicting the X-ray powder diffraction pattern of the anhydrous solid crystalline form of compound 2 (form B) after slurry treatment in 97% ethanol/3% water and drying under vacuum (-80 ℃ for 1 day).

Fig. 3 is a graph depicting the results of Differential Scanning Calorimetry (DSC) analysis of an anhydrous solid crystalline form of compound 2 (form B).

Figure 4 is a graph depicting the results of thermogravimetric analysis (TGA) of the anhydrous solid crystalline form of compound 2 (form B).

Fig. 5 is a graph depicting the results of a Dynamic Vapor Sorption (DVS) analysis of the anhydrous solid crystalline form of compound 2 (form B).

FIG. 6 depicts micronization into d₉₀A spectrum of overlapping results of XRPD analysis of an anhydrous solid crystalline form of compound 2 (form B) with values before (light grey curve) and after (dark grey curve) less than 10 microns.

Figure 7 is a graph depicting the effect of oral administration of different doses of compound 2 in a model of CFA-induced cold hyperalgesia in rats.

Figure 8 is a graph depicting the duration of formalin-mediated pain behavior following oral administration of compound 2. Compound 2 was administered 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or 24 hours prior to formalin injection to assess the persistence of the benefits of treatment.

Figure 9 is a graph depicting an exemplary profile of the CYP450 response phenotype for compound 2 (also referred to herein as example 2).

Figure 10 is a graph depicting the solubility of micronized formulations of compound 2 at pH ranges from 2.00 to 8.00.

Figure 11 is a graph depicting plasma levels of compound 2 (i.e., example 2) in a rat, dog, or monkey model following administration of a 10mg/kg oral dose.

Figure 12 is a graph depicting a comparison of the pharmacokinetic profiles of compound 2 (i.e., example 2) in capsule and suspension formulations in fed and fasted monkeys.

Figure 13 is a graph depicting the analgesic effect observed with low dose oral administration of compound 2 (i.e., example 2) and control (compound a, i.e., comparative a) in a CFA model.

Fig. 14 is a graph depicting the dose response observed following oral administration of compound 2 (i.e., example 2) in a formalin model.

Figure 15 is a graph depicting the efficacy observed with intravenous administration of different doses of compound 1 (i.e., example 1) in the formalin model.

Figure 16 is a graph depicting the change in pulmonary resistance (early and late asthmatic response) in sheep stimulated with allergen following administration of compound 2.

Figure 17 is a graph depicting the effect of compound 2 (i.e., example 2) on airway hyperresponsiveness measurements in a sheep model of allergic asthma.

Figure 18 is a graph depicting serum biomarkers of hepatotoxicity in beagle dogs before and after receiving a once daily oral dose of compound 2 for 5 days.

Figure 19 is a graph depicting the change in serum biomarkers of hepatotoxicity between control and compound 2 (orally administered) in beagle dogs on day 5 receiving a once daily oral dose of compound 2 for 5 days.

Fig. 20 is a graph depicting the change in hepatotoxic serum biomarkers in SD rats (Sprague-dawley rat) after receiving a once daily oral dose of compound 2 for 28 days.

Fig. 21 is a graph depicting the change in serum biomarkers of hepatotoxicity between controls and compound 2 (orally administered) in day 28 SD rats that received a once daily oral dose of compound 2 for 28 days.

Figure 22 is a graph depicting serum biomarkers of hepatotoxicity in cynomolgus monkeys after receiving a once daily oral dose of compound 2 for 28 days.

Figure 23 is a graph depicting the serum biomarker change in hepatotoxicity between control and compound 2 (orally administered) in cynomolgus monkeys on day 28 receiving a once daily oral dose of compound 2 for 28 days.

Detailed Description

Definition of

The invention is not limited in its application to the details of the methods and compositions described herein. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

"about" and "approximately" shall generally refer to an acceptable degree of error in the measured quantity given the nature or accuracy of the measurement. Exemplary degrees of error are within 20 percent (%) of a given value or range of values, typically within 10%, and more typically within 5%.

As described herein, an amount of a compound or combination effective to treat a disease (e.g., a disease described herein), a "therapeutically effective amount," an effective amount, "or an" effective course of treatment "refers to an amount of a compound or combination that, upon single or multiple dose administration to a subject, is effective to treat the subject or cure, ameliorate, alleviate, or improve a subject with a disease (e.g., a disease described herein), which exceeds that expected in the absence of such treatment.

The term "pharmaceutically acceptable" as used herein refers to a compound or carrier (e.g., excipient) that can be administered to a subject with a compound described herein (e.g., a compound of formula (I)), and which does not destroy its pharmacological activity and is non-toxic when administered at a dose sufficient to deliver a therapeutic amount of the compound.

As noted above, certain embodiments of the compounds of the present invention may comprise basic functional groups, such as amino or alkylamino, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this respect the term "pharmaceutically acceptable salts" means the relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting the purified compounds of the invention in their free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative Salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, and the like (see, e.g., Berge et al (1977)' "Pharmaceutical Salts", J.pharm.Sci.66: 1-19).

In other cases, the compounds of the present invention may contain one or more acidic functional groups and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances means the relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the purified compound in its free acid form with a suitable base, such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

The term "treating" or "treatment," as used herein, refers to applying or administering a compound to a subject, alone or in combination with other drugs, e.g., a subject having a disease (e.g., a disease described herein), symptoms of a disease, or a predisposition to a disease, with the goal of curing, healing, alleviating, relieving, altering, repairing, ameliorating, promoting, or affecting the disease.

As used herein, the term "subject" is intended to include both human and non-human animals. Exemplary human subjects include human subjects having a disease, such as the diseases described herein. The term "non-human animal" in the present invention includes all vertebrates, e.g., non-mammals (e.g., chickens, amphibians, reptiles) and mammals, e.g., non-human primates, domestic and/or agricultural animals, e.g., sheep, dogs, cats, cows, pigs, etc.

The terms "antagonist" and "inhibitor" are used interchangeably and refer to an agent that decreases or inhibits a biological activity, such as inhibiting the activity of an ion channel, such as TRPA 1. TRPA1 inhibitors include inhibitors having any combination of the structural and/or functional properties disclosed herein.

In connection with methods of inhibition or treatment, for example, an "effective amount" of a TRPA1 antagonist refers to the amount of antagonist in a formulation that, when administered as part of a desired dosage regimen, results in a desired clinical or functional outcome. Without being bound by theory, an effective amount of a TRPA1 antagonist for use in the methods of the present invention includes an amount of a TRPA1 antagonist effective to reduce one or more in vitro or in vivo functions of the TRPA1 channel. Exemplary functions include, but are not limited to, membrane polarization (e.g., antagonists can prevent depolarization of the cell), ion flux, ion concentration in the cell, outward current, and inward current. Compounds that antagonize TRPA1 function include compounds that antagonize the in vitro or in vivo functional activity of TRPA 1. The ability of a compound to inhibit TRPA1 function in an in vitro assay serves as a reasonable surrogate for the activity of the compound, as a particular functional activity is only readily observed in that assay. In certain embodiments, an effective amount is an amount sufficient to inhibit TRPA 1-mediated current flow and/or an amount sufficient to inhibit TRPA 1-mediated ion flow.

The term "hydrate," as used herein, refers to a compound formed by the binding of water to a parent compound.

The term "preventing" when used in reference to a disease, such as a local recurrence (e.g., pain), a disease such as cancer, a complex syndrome such as heart failure or any other medical condition, is well understood in the art and includes compositions that reduce the frequency of onset of symptoms of, or delay the onset of symptoms of, a medical condition in a subject relative to a subject not receiving the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancer growth in a patient population receiving prophylactic treatment compared to a non-treated control population, and/or delaying the appearance of detectable cancer growth in a treated population compared to a non-treated control population, e.g., by a statistically and/or clinically significant amount. Preventing infection includes, for example, reducing the number of diagnoses of infection in the treated population compared to the untreated control population and/or delaying the onset of symptoms of infection in the treated population compared to the untreated control population. Preventing pain includes, for example, reducing the level of pain perception experienced by the subject in the treated population compared to the untreated control population or alternatively delaying pain perception.

The term "prodrug" is intended to encompass compounds that are converted under physiological conditions to the therapeutically active agents of the present invention. A common method of making prodrugs consists in making them include selected moieties that hydrolyze under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by the enzymatic activity of the host animal.

The term "solvate" as used herein means a compound formed by solvation (e.g., a compound formed by binding of solvent molecules to solute molecules or ions).

The terms "TRPA 1", "TRPA 1 protein" and "TRPA 1 channel" may be used interchangeably in the context of the present application. These terms are intended to encompass the SEQ ID NO: 1. SEQ ID NO: 3 or SEQ ID NO: 5 or an equivalent polypeptide or functional biologically active fragment thereof (e.g., a polypeptide). In certain embodiments, the term is intended to encompass SEQ ID NO: 1. SEQ ID NO: 3 or SEQ ID NO: 5, a polypeptide consisting or consisting essentially of same. TRPA1 includes polypeptides that retain TRPA1 function and comprise: (i) SEQ ID NO: 1. SEQ ID NO: 3 or SEQ ID NO: 5, all or part of the amino acid sequence set forth in seq id no; (ii) SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; (iii) and SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 amino acid sequences that are at least 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical; and (iv) functional fragments thereof. The polypeptides of the invention also include SEQ id no: 1. SEQ ID NO: 3 or SEQ ID NO: 5, such as homologues () and paralogs (paralogs).

The "enantiomeric excess" or "% enantiomeric excess" of a composition can be calculated using the equation shown below. In the examples shown below, the compositions comprise 90% of one enantiomer, e.g., the S enantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.

ee＝(90-10)/100＝80％。

Thus, a composition comprising 90% of one enantiomer and 10% of the other enantiomer is claimed to have an enantiomeric excess of 80%.

The "diastereomeric excess" or "% diastereomeric excess" of a composition can be calculated using the equation shown below. In the examples shown below, the compositions comprise 90% of one diastereomer, and 10% of the other enantiomer.

de＝(90-10)/100＝80％。

Thus, a composition comprising 90% of one diastereomer and 10% of the other enantiomer is claimed to have a diastereomer excess of 80%.

Chemical definition

In various places in the specification, substituents of the compounds of the present invention are disclosed in groups or ranges. It is specifically contemplated that the invention includes each and every individual subcombination of these groups and ranges. For example, the term "C_1-6Alkyl "means in particular that methyl, ethyl, C are disclosed separately₃Alkyl radical, C₄Alkyl radical, C₅Alkyl and C₆An alkyl group.

For compounds of the invention in which a variable occurs more than once, each variable may be a different moiety selected from the Markush group defining the variable. For example, when a structure is described as having two R groups present on the same compound at the same time; the two R groups may represent different moieties selected from the Markush group defined for R.

It is also to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for clarity, described in the context of separate embodiments, may also be provided separately or in any suitable subcombination.

As used herein, "alkyl" by itself or as part of another substituent, unless otherwise stated, refers to a straight or branched chain and may have an optionally specified plurality of carbon atoms (i.e., C)₁-C₆Meaning 1 to 6 carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, e.g., homologs and isomers of n-pentyl, n-hexyl, and the like.

As described herein, "AAlkyl "refers to divalent alkyl radicals, e.g., -CH₂-、-CH₂CH₂-、-CH₂CH₂CH₂-、-CH₂CH₂CH₂CH₂-、-CH₂CH₂CH₂CH₂CH₂-and-CH₂CH₂CH₂CH₂CH₂CH₂-.

As used herein, "alkenyl" may be a straight or branched hydrocarbon chain, containing at least one double bond, and having from 2 to 6 carbon atoms (i.e., C)₂-C₆Alkenyl). Examples of alkenyl groups include, but are not limited to, groups such as vinyl, prop-1-enyl (i.e., allyl), but-1-enyl, pent-1, 4-dienyl, and the like.

As described herein, "alkoxy" may be a straight or branched chain alkoxy group having 1 to 6 carbon atoms (i.e., C)₁-C₆Alkoxy groups). Examples of alkoxy groups include, but are not limited to, groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, tert-butyloxy, pentyloxy, hexyloxy, and the like.

As used herein, "alkynyl" can be a straight or branched hydrocarbon chain, containing at least one triple bond, having from 2 to 6 carbon atoms (i.e., C)₂-C₆Alkynyl). Examples of alkynyl groups include, but are not limited to, groups such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

As used herein, "amino" or "amine" refers to-NH₂A group.

As used herein, "aryl" refers to a polyunsaturated aromatic hydrocarbon moiety that can be a single ring or multiple rings (e.g., 1 to 2 rings) that are fused together or linked covalently, having 6 to 12 carbon atoms (i.e., C)₆-C₁₂Aryl). Non-limiting examples of aryl groups include phenyl, 1-naphthyl, 2-naphthyl, and 4-biphenyl.

As used herein, "cycloalkyl" refers to a monocyclic or polycyclic group, which is a cyclic groupContains only carbon and hydrogen and may be saturated or partially unsaturated. Cycloalkyl includes groups having 3 to 10 ring atoms (i.e., C)₃-C₁₀Cycloalkyl groups). Examples of cycloalkyl groups include, but are not limited to, groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl (cycloseptyl), cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like.

As used herein, "halo" or "halogen", by itself or as part of another substituent, refers to a fluorine, chlorine, bromine, or iodine atom, unless otherwise indicated. The term "halo" by itself or as part of another substituent means fluoro, chloro, bromo, or iodo.

As described herein, "haloalkyl" and "haloalkoxy" can include alkyl and alkoxy structures substituted with one or more halo groups or combinations thereof. For example, the terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy, respectively, where the halogen is fluorine.

As described herein, "heteroalkyl" may include optionally substituted alkyl groups having one or more backbone chain atoms selected from atoms other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, or combinations thereof. Can be given a range of values, e.g. C₁-C₆Heteroalkyl, which refers to the number of carbons in the chain, includes from 1 to 6 carbon atoms in this example. For example, -CH₂OCH₂CH₃The radical being referred to as "C₃"Heteroalkyl group". The attachment to the remainder of the molecule may be through a heteroatom or carbon in the heteroalkyl chain.

As used herein, "heteroaryl" refers to a 5-to 14-membered aromatic group (e.g., C)₂-C₁₃Heteroaryl) which comprises one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic or bicyclic ring system. Bivalent radicals derived from monovalent heteroaryl radicals (the name of which ends in the "-radical", by removing one hydrogen atom from the atom having the free valence) are named by adding the "-subunit" to the name of the corresponding monovalent radical, e.g. havingThe pyridyl group having two points of attachment is a pyridylene group. An N-containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic group in which at least one backbone atom of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused or non-fused. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl group is attached to the remainder of the molecule through any atom of the ring. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolinyl, isoquinolinyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuryl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2, 4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like.

As used herein, "heterocyclyl" or "heterocycloalkyl" can be a stable 3-to 18-membered non-aromatic mono-, di-, or tricyclic heterocyclic group containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Examples of heterocycloalkyl include, but are not limited to, groups such as dioxolanyl, thieno [1,3] dithiacyclohexyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl (4-piperidonyl), azetidinyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl (quinuclidinyl), thiazolidinyl, tetrahydrofuranyl, trithianyl (thianyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxo-thiomorpholinyl, 1, 1-dioxo-thiomorpholinyl, and the like.

As used herein, "hydroxy" or "hydroxy" refers to-OH.

As used herein, "cyano" refers to — CN.

As used herein, "nitro" refers to–NO₂。

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic rings of organic compounds, aromatic and nonaromatic substituents (e.g., alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which may be further substituted on its own), and halogen, carbonyl (e.g., aldehyde, ketone, ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester, thiocarboxylate, or thiocarbamate), amino (e.g., -N (R) s^b)(R^c) Wherein each R is^bAnd R^cIndependently is H or C₁-C₆Alkyl), cyano, nitro, -SO₂N(R^b)(R^c)，–SOR^dAnd S (O)₂R^dWherein each R is^b、R^cAnd R^dIndependently is H or C₁-C₆An alkyl group. Exemplary substituents include, for example, those described above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of the present invention, a heteroatom, such as nitrogen, may have a hydrogen substituent and/or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatom. The present invention is not limited in any way by the permissible substituents of organic compounds.

It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is according to the allowed valencies of the substituting atoms and substituents and that the substitution results in a stable compound, e.g., that no spontaneous transformation, such as by rearrangement, cyclization, elimination, etc., occurs.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of abbreviations used by those of ordinary skill in Organic Chemistry appears in the first edition of the Journal of Organic Chemistry, each volume; this List is typically presented in the form of a table, entitled Standard List of Abbrevicions. The abbreviations contained in the list and all abbreviations used by those of ordinary skill in organic chemistry are incorporated herein by reference.

Equivalents of the above compounds of interest include compounds that are otherwise comparable and have the same general characteristics (e.g., ability to inhibit TRPA1 activity) in which simple variations of one or more substituents are made that do not adversely affect the potency of the compound. In general, the compounds of the present invention may be prepared by methods exemplified in general reaction schemes, e.g., as described below or modifications thereof, using readily available starting materials, reagents and conventional synthetic procedures. In these reactions, it is also possible to use variants which are known per se but are not mentioned here.

For the purposes of the present invention, the chemical elements are identified according to the CAS version of the periodic Table of the elements, Handbook of chemistry and Physics, 67th Ed., 1986-87 (encloser). In addition, for the purposes of the present invention, the term "hydrocarbon" is intended to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic branched and unbranched carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds which may be substituted or unsubstituted.

Compound (I)

Described herein are compounds useful for treating diseases in which inhibition of TRPA1 would be beneficial. These diseases are described herein.

The compounds include compounds of formula (I)

Wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

In some embodiments, R¹Is C₁-C₆Alkyl radicals, e.g., -CH₃. In some embodiments, R¹Is H.

In some embodiments, R²Is H or C₁-C₆Alkyl radicals, e.g., -CH₃，–CD₃or-CHF₂.

In some embodiments, each R is¹And R²Independently is C₁-C₆Alkyl radicals, e.g., -CH₃. In some embodiments, each R is¹And R²Independently is-CH₃And R is³Is H.

In some embodiments, R³Is H. In some embodiments, R³Is C₁-C₆Alkyl radicals, e.g., -CH₃。

In some embodiments, R¹And R²And R³Each independently is C₁-C₆Alkyl radicals, e.g., -CH₃。

In some embodiments, the compound of formula (I) is a compound of formula (Ia):

in some embodiments, R¹And R²And R³Each independently is C₁-C₆Alkyl radicals, e.g., -CH₃。

In some embodiments, the compound of formula (I) of claim 1 is a compound of formula (Ib):

in some embodiments, R¹And R²And R³Each independently is C₁-C₆Alkyl radicals, e.g., -CH₃。

In some embodiments, R⁴Is a heterocyclic group, for example, a 4 to 8-membered ring. In some embodiments, the heterocyclic group is attached through a nitrogen atom. In some embodiments, R⁴Is a substituted heterocyclic group. In some embodiments, R⁴Selected from the following groups:

in some embodiments, R⁴Selected from the following groups:

and m is 1.

In some embodiments, R⁴Selected from the following groups:

in some embodiments, R⁴Selected from the following groups:

and m is 1.

In some embodiments, R⁴Selected from the following groups:

in some embodiments, m is 0. In some embodiments, m is 1.

In some embodiments, R⁶Is alkyl, haloalkyl or cyano, e.g. alkyl or haloalkyl, such as-CF₃。

In some embodiments, R⁴Selected from the following groups:

in some embodiments, the compound of formula (I) is formula (II):

wherein:

n is an integer of 0 to 4; and is

m is an integer from 0 to 4.

In some embodiments, the compound of formula (I) is of formula (IIa):

wherein:

n is an integer of 0 to 4; and is

m is an integer from 0 to 4.

In some embodiments, the compound of formula (I) is of formula (IIb):

wherein:

n is an integer of 0 to 4; and is

m is an integer from 0 to 4.

In some embodiments, the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) has a melting point greater than or equal to about 100 ℃. In some embodiments, the compound of formula (I) has a melting point greater than or equal to about 125 ℃, about 150 ℃, about 175 ℃, or about 180 ℃. In some embodiments, the compound of formula (I) has a melting point in the range of about 180 ℃ to about 205 ℃. In some embodiments, the compound of formula (I) has a melting point in the range of about 190 ℃ to about 200 ℃. In some embodiments, the compound of formula (I) has a melting point in the range of about 190 ℃ to about 196 ℃.

In some embodiments, a solid crystalline form of the compound of formula (I) is prepared by slurry treatment with a suitable solvent (e.g., ethanol, water, or a combination thereof). In some embodiments, a solid crystalline form (e.g., an anhydrous solid crystalline form) of the compound of formula (I) is prepared by slurry treatment with a suitable solvent (e.g., ethanol, water, or a combination thereof) followed by additional treatment (e.g., vacuum treatment, e.g., -80 ℃ for 1 day).

In some embodiments, a solid crystalline form of the compound of formula (I) (e.g., prepared by slurry treatment with a suitable solvent, such as ethanol, water, or a combination thereof, and optionally followed by additional treatment, such as vacuum treatment, e.g., at-80 ℃ for 1 day) has a melting point greater than or equal to about 100 ℃. In some embodiments, the solid crystalline form of the compound of formula (I) has a melting point greater than or equal to about 125 ℃, about 150 ℃, about 175 ℃, or about 180 ℃. In some embodiments, the solid crystalline form of the compound of formula (I) has a melting point in the range of about 180 ℃ to about 205 ℃. In some embodiments, the solid crystalline form of the compound of formula (I) has a melting point in the range of about 190 ℃ to about 200 ℃. In some embodiments, the solid crystalline form of the compound of formula (I) has a melting point in the range of about 190 ℃ to about 196 ℃.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof, which is referred to as compound 2, example 2, or the compound of example 2 herein.

In some embodiments, a solid crystalline form of compound 2 (e.g., form a) is prepared by slurry treatment with a suitable solvent (e.g., ethanol, water, or a combination thereof). In some embodiments, the solid crystalline form of compound 2 (e.g., form a) has an X-ray powder diffraction pattern comprising characteristic peaks, expressed in 2 Θ, at one or more of the following angles: about 7.67 °, about 12.52 °, about 13.49 °, and about 19.31 °. In some embodiments, the solid crystalline form of compound 2 (e.g., form a) has characteristic peaks as shown in figure 1.

In some embodiments, a solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) is prepared by slurry treatment with a suitable solvent (e.g., ethanol, water, or a combination thereof) followed by additional treatment (e.g., vacuum treatment, e.g., -80 ℃ for 1 day). In some embodiments, the solid crystalline form of compound 2 (e.g., the anhydrous solid crystalline form of compound 2, e.g., form B) has an X-ray powder diffraction pattern comprising characteristic peaks, expressed in 2 Θ, at one or more of the following angles: about 9.78 °, about 12.98 °, about 19.20 °, and about 19.67 °. In some embodiments, the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has characteristic peaks as shown in figure 2.

In some embodiments, the solid crystalline form of compound 2 (e.g., the anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point greater than or equal to about 100 ℃. In some embodiments, the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point greater than or equal to about 125 ℃, about 150 ℃, about 175 ℃, or about 180 ℃. In some embodiments, the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point in the range of about 180 ℃ to about 205 ℃. In some embodiments, the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point in the range of about 190 ℃ to about 200 ℃. In some embodiments, the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point in the range of about 190 ℃ to about 196 ℃. In some embodiments, the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a differential scanning calorimetry curve as shown in figure 3.

In some embodiments, a solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) is prepared by slurry treatment with a suitable solvent (e.g., ethanol, water, or a combination thereof) followed by additional treatment (e.g., vacuum treatment, e.g., -80 ℃ for 1 day), wherein the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point greater than or equal to about 150 ℃ and an X-ray powder diffraction pattern includes characteristic peaks at one or more of the following angles, expressed in 2 Θ: about 9.78 °, about 12.98 °, about 19.20 °, and about 19.67 °. In some embodiments, a solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) is prepared by slurry treatment with a suitable solvent (e.g., ethanol, water, or a combination thereof) followed by additional treatment (e.g., vacuum treatment, e.g., -80 ℃ for 1 day), wherein the solid crystalline form of compound 2 (e.g., an anhydrous solid crystalline form of compound 2, e.g., form B) has a melting point in the range of 185 ℃ to about 205 ℃ and an X-ray powder diffraction pattern includes characteristic peaks at one or more of the following angles, expressed in 2 Θ: about 9.78 °, about 12.98 °, about 19.20 °, and about 19.67 °.

Certain embodiments of the present invention include purified pharmaceutical formulations comprising a compound of formula (I). In some embodiments, the pharmaceutical formulation comprises a diastereomer in an excess of greater than or equal to about 55% (e.g., about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 99.5%) over the diastereomer of the other diastereomer. In some embodiments, the pharmaceutical formulation comprises a diastereomer in an excess of greater than or equal to about 95% over the diastereomer of the other diastereomer. In some embodiments, the pharmaceutical formulation comprises a diastereomer in greater than or equal to about 99% enantiomeric excess of the diastereomer of another diastereomer.

In some embodiments, the pharmaceutical formulation comprises less than or equal to about 10% moisture content (e.g., water content). In some embodiments, the pharmaceutical composition comprises less than or equal to about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01%, or about 0.001% moisture content (e.g., water content). In some embodiments, the pharmaceutical formulation is substantially free of moisture (e.g., water).

In some embodiments, the pharmaceutical formulation comprises a compound of formula (I), wherein the compound is:

or a pharmaceutically acceptable salt thereof, which is referred to as compound 2, example 2, or the compound of example 2 herein.

In some embodiments, the pharmaceutical formulation comprises a compound of formula (I), wherein the compound is compound 2, or a pharmaceutically acceptable salt thereof, and the formulation has a diastereomeric excess of compound 2 of greater than or equal to about 99%. In some embodiments, the pharmaceutical formulation comprises a compound of formula (I), wherein the compound is compound 2, or a pharmaceutically acceptable salt thereof, and the formulation has a moisture content (e.g., water content) of less than or equal to about 0.1%. In some embodiments, the pharmaceutical formulation comprises a compound of formula (I), wherein the compound is compound 2, or a pharmaceutically acceptable salt thereof, and the formulation has a diastereomeric excess of compound 2 greater than or equal to about 99% and a moisture content (e.g., water content) of less than or equal to about 0.1%.

In some embodiments, the pharmaceutical formulation comprises a solid crystalline form of compound 2 (e.g., form a) having an X-ray powder diffraction pattern comprising characteristic peaks, expressed in 2 Θ, at one or more of the following angles: about 7.67 °, about 12.52 °, about 13.49 °, and about 19.31 °, and the formulation has a diastereomeric excess of compound 2 greater than or equal to about 99% and a moisture content (e.g., water content) of less than or equal to about 0.1%.

In some embodiments, the pharmaceutical formulation comprises a solid crystalline form of compound 2 (e.g., form B) having a melting point in the range of 185 ℃ to about 205 ℃ and an X-ray powder diffraction pattern comprising characteristic peaks, expressed in 2 Θ, at one or more of the following angles: about 9.78 °, about 12.98 °, about 19.20 °, and about 19.67 °, and the formulation has a diastereomeric excess of compound 2 greater than or equal to about 99% and a moisture content (e.g., water content) of less than or equal to about 0.1%.

Compounds of formula (I) include molecules having water solubility suitable for oral or parenteral (e.g., intravenous) administration, resulting in or enabling their use in the treatment of diseases described herein, e.g., the treatment of pain. In some embodiments, the compound is formulated in a composition suitable for oral administration. The potency of a compound of formula (I) described herein to inhibit the TRPA1 ion channel was measured using the method of example 33. Table 14 discloses the potency of exemplary compounds to inhibit TRPA1 in vitro (as measured by the method of example 33).

Preferred compounds of formula (I) include TRPA1 ion channel inhibiting compounds and having an IC₅₀Compounds with values (obtained by the method of example 33) of less than about 100nM (preferably, less than about 75nM, more preferably less than about 25 nM).

The compounds of formula (I) inhibit the TRPA1 ion channel. In some embodiments, the compounds of formula (I) may be administered as part of an oral or parenteral (e.g., intravenous) pharmaceutical composition to treat a disease (e.g., pain) described herein in a therapeutically effective manner.

Certain compounds of the present invention may exist in specific geometric or stereoisomeric forms, and the present invention encompasses all such compounds, including cis-and trans-isomers, R-and S-enantiomers, diastereomers, (D) -isomers, (L) -isomers, racemic mixtures thereof, and other mixtures thereof, which are also within the scope of the present invention. For example, if a chiral center is present in the molecule, the invention includes racemic mixtures, enantiomerically enriched mixtures, and substantially enantiomerically or diastereomerically pure compounds. The composition may comprise, for example, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% of a single enantiomer or diastereomer. Additional asymmetric carbon atoms may be present on substituents such as alkyl groups. All such isomers and mixtures thereof are contemplated to be included in the present invention.

The compounds described herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, radioactive isotopes may be used, such as, for example, tritium (f) ((r))³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) A radiolabeled compound. All isotopic variations of the compounds of the present invention, whether nonradioactive or not, are intended to be encompassed within the scope of the present invention. For example, deuterated compounds and incorporation¹³All compounds of C are included within the scope of the present invention.

Certain compounds disclosed herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent for the applications for which the invention is concerned and are intended to be within the scope of the invention.

Pharmaceutical composition

Pharmaceutical compositions comprising a compound described herein, e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof, are useful for treating or ameliorating a disease described herein, e.g., a disease responsive to inhibition of TRPA1 ion channels in a subject (e.g., humans and animals).

The amount and concentration of the compound of formula (I) in the pharmaceutical composition and the amount of the pharmaceutical composition to be administered to a subject can be selected based on clinically relevant factors such as medically relevant characteristics of the subject (e.g., age, body weight, sex, other medical conditions, etc.), solubility of the compound in the pharmaceutical composition, potency and activity of the compound, and the mode of administration of the pharmaceutical composition. For more information on the route of administration and dosage regimen, the reader is referred to CompressedMedicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press1990 Vol.5, Chapter 25.3.

Although the compounds disclosed herein can be administered alone, it is preferred that the compounds be administered as a pharmaceutical formulation, wherein the compound is combined with one or more pharmaceutically acceptable excipients or carriers. The compounds disclosed herein may be formulated for administration in any convenient manner for human or veterinary medicine. In certain embodiments, the compounds included in the pharmaceutical formulations may be active per se, or may be, for example, prodrugs that are capable of being converted to the active compound in a physiological environment.

The phrase "pharmaceutically acceptable" is employed 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, or other problem or complication, commensurate with a reasonable benefit/risk level.

Examples of pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) a ringer's solution; (19) ethanol; (20) a phosphate buffer solution; (21) cyclodextrins such asAnd (22) other non-toxic compatible materials used in pharmaceutical formulations.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butyl Hydroxyanisole (BHA), Butyl Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Solid dosage forms (e.g., capsules, tablets, pills, dragees, powders, granules, etc.) may comprise one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) humectants, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) a colorant.

Liquid dosage forms may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

In addition to the active compounds, ointments, pastes, creams and gels may contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can also contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The formulations may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, the amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%, by 100%.

Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as lozenges, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide sustained or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose, other polymer matrices, liposomes and/or microspheres in varying proportions to provide the desired release characteristics. For example, they may be sterilized by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately prior to use. These compositions may also optionally comprise opacifying agents and may be such compositions that they release the active ingredient(s) only, or preferably, in certain parts of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. If appropriate, one or more of the above-mentioned excipients can also be used for microencapsulating the active ingredient.

Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers or boosters.

The formulations disclosed herein can be delivered by a device. Exemplary devices include, but are not limited to, catheters, wires, stents, or other intraluminal devices. Other exemplary delivery devices also include patches, bandages, dental trays, or dental devices. A further advantage of transdermal patches is that the compounds of the present invention are delivered to the body in a controlled manner. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be used to increase the amount of compound that flows into the skin. The rate of such flow can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, ophthalmic ointments, drops, solutions, and the like are also contemplated as being within the scope of the present invention.

In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions with poorly water soluble crystalline or amorphous materials. The rate of absorption of the drug may then depend on its dissolution rate, which in turn depends on the crystal size and crystalline form. Alternatively, parenteral administration of a drug may be delayed in absorption by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are prepared by forming microencapsule matrices of the subject compounds in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Long acting injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

When the compounds of the present invention are administered as medicaments to humans and animals, they may be administered per se or as a pharmaceutical composition comprising, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of an active ingredient together with a pharmaceutically acceptable carrier.

The formulation may be administered topically, orally, transdermally, rectally, vaginally, parenterally, intranasally, intrapulmonary, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intracardially, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsulally, subarachnoid, intraspinally, intrasternally, or by inhalation.

One embodiment is an antitussive composition for oral administration comprising an IC at 1 micromolar or less₅₀Such antitussive compositions may include one or more additional active agents for treating cough, allergy or asthma symptoms selected from antihistamines, 5-lipoxygenase inhibitors, leukotriene inhibitors, H3 inhibitors, β -adrenergic receptor agonists, xanthine derivatives, α -adrenergic receptor agonists, mast cell stabilizers, expectorants, NK1, NK2, and NK3 tachykinin receptor antagonists.

Another embodiment is a metered dose aerosol dispenser comprising an aerosol pharmaceutical composition for pulmonary or nasal delivery comprising an IC at 1 micromolar or less₅₀An agent that inhibits TRPA 1-mediated currents. For example, it may be a metered dose inhalerDry powder inhalers or air-jet nebulizers.

Dosage form

The actual dosage level of the active ingredient in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient.

The selected dosage level will depend upon a variety of factors well known in the medical arts, including the activity of the particular compound or ester, salt or amide thereof employed in the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or substances used in conjunction with the particular compound employed, the age, sex, body weight, condition, general health and prior medical history of the patient being treated.

A clinician or veterinarian of ordinary skill in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, a clinician or veterinarian can start using a compound of the invention in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such effective dosages will generally depend on the factors described above. Typically, intravenous, intracerebroventricular, intrathecal and subcutaneous dosages of the compounds described herein for a subject range from about 0.0001 to about 100mg per kilogram body weight per day. For example, the dose may be 1-50, 1-25 or 5-10 mg/kg. Typically, the oral dosage of a compound described herein for a subject ranges from about 1 to about 1,000 mg/day (e.g., about 5 to about 500 mg/day).

If desired, an effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses divided into appropriate intervals throughout the day, optionally in unit dosage form.

Method of treatment

The compounds described herein are useful for treating or preventing the diseases described herein. For example, provided herein are compounds having TRPA1 inhibitory activity for use in preventing, treating or ameliorating symptoms of a disease or disorder associated with TRPA 1. A compound of formula (I), or a pharmaceutical composition comprising one or more compounds of formula (I), can be administered to treat a disorder, condition, or disease described herein, such as a disease that can be treated by inhibition of TRPA 1. For example, pharmaceutical compositions comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, are useful as perioperative analgesics, e.g., in the management of mild to moderate acute post-operative pain and as an adjunct opioid analgesic in the management of moderate to severe acute pain. Pharmaceutical compositions comprising a therapeutically effective dose of a compound of formula (I) can be administered to a patient in a clinically safe and effective manner to treat pain, including one or more separate administrations of a pharmaceutical composition comprising a compound of formula (I). Other exemplary methods include treating Peripheral Diabetic Neuropathy (PDN) and chemotherapy-induced peripheral neuropathy (CIPN). For example, a pharmaceutical composition comprising a therapeutically effective dose of a compound of formula (I) or a pharmaceutically acceptable salt thereof can be administered (e.g., intravenously) to a subject in need thereof multiple times per day (e.g., BID) over a course of 1 or more days to treat pain in the subject. Pharmaceutical compositions comprising compounds of formula (I) may also be useful in treating or ameliorating pulmonary diseases, such as obstructive diseases, e.g., Chronic Obstructive Pulmonary Disease (COPD), asthma (e.g., cold-induced asthma, exercise-induced asthma, allergy-induced asthma, and occupational asthma), and cough.

A therapeutically effective amount of a compound of formula (I) can be readily determined by the skilled person for the treatment of diseases associated with TRPA1 receptor modulation based on the test results given below. Generally, a suitable daily dose of a compound of the invention is the amount of the compound that will produce the lowest dose of therapeutic effect. The effective dose will generally depend on a variety of factors. Typically, the compounds of the invention are administered to a patient orally, sublingually, rectally, intravenously, topically, transdermally, by inhalation, and intracerebroventricularly in a dosage range of about 0.0001 to about 100 mg/kg of body weight per day. For example, the dose may be 1-50mg/kg, 1-25mg/kg or 5-10 mg/kg. For example, a therapeutically effective dose is contemplated to be from about 0.001mg/kg to about 50mg/kg/kg of body weight of the patient to be treated, more preferably from about 0.01mg/kg to about 10mg/kg/kg of body weight of the patient to be treated. The therapeutically effective dose may conveniently be administered in two or more sub-dose forms at appropriate intervals throughout the day. The sub-doses may be formulated in unit dosage forms, for example, each containing from about 0.1 to about 1000mg, more particularly from about 1 to about 500mg, of the active ingredient per unit dosage form.

The exact dose and frequency of administration will depend upon the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient and other drugs which the patient may take, as is well known to those skilled in the art. Furthermore, the "therapeutically effective amount" may be decreased or increased depending on the response of the patient being treated and/or depending on the evaluation of the physician prescribing the compounds of the instant invention. The effective daily dosage ranges stated above are therefore only to be regarded as guidance. A physician or veterinarian of ordinary skill in the art can readily determine the effective amount to prescribe the desired pharmaceutical composition.

Exemplary diseases suitable for treatment with the compounds or compositions described herein are provided below.

Pain (due to cold or dampness)

The compounds of formula (I) for modulating TRPA1 may be suitable for use in the formulation of analgesic medicaments for the treatment and/or prevention of pain in mammals, especially humans. Endogenous activators of TRPA1 are produced in a number of pathological conditions, including tissue injury, inflammation, and metabolic stress. The compounds and pharmaceutical compositions of the present invention can be administered to treat pain, including neuropathic pain, caused by activation of TRPA 1. Associated neuropathic pain conditions include, but are not limited to, painful diabetic neuropathy, chemotherapy-induced peripheral neuropathy, lower back pain, trigeminal neuralgia, postherpetic neuralgia, sciatica, and complex regional pain syndrome.

The compositions and methods provided herein are also useful in connection with the treatment of inflammation and inflammatory pain. These diseases include rheumatoid arthritis, osteoarthritis, temporomandibular disorders (temporomandibular disorders). In some embodiments, the compositions and methods provided herein can be used to treat headache, e.g., migraine.

The disclosed compounds are also useful in the treatment of visceral pain and inflammation. Related diseases include pancreatitis, inflammatory bowel disease, colitis, crohn's disease, endometriosis, pelvic pain, and angina.

Other exemplary pain indications for which the compounds disclosed herein may be used include temporomandibular disease, cancer pain (resulting from underlying disease or from treatment), burn pain, oral pain treated by cancer, crush and injury induced pain, incisional pain, bone pain, sickle cell disease pain, fibromyalgia, and musculoskeletal pain. TRPA1 has been shown to be responsible for Cancer-related pain (see, e.g., Trevisan et al, Cancer Res (2013)73(10): 3120-3131); post-operative pain (see, e.g., Wei et al, analytics (2012)117: 137-); pathological Pain (see, e.g., Chen et al, Pain (2011)152: 2549-; pain associated with chemical injury (see, e.g., Macpherson et al, JNeurosci (2007)27(42):11412-11415) plays a role.

Hyperalgesia (e.g., mechanical hyperalgesia, cold hyperalgesia) or increased sensitivity to pain (e.g., acute, chronic). Multiple chemosensitivity is a chemical exposure-related disease with multiple organ symptoms, including respiratory symptoms and headache.

Allodynia (e.g., cutaneous allodynia, e.g., head, external to the head) is pain caused by a stimulus that does not normally cause pain, e.g., temperature or a physical stimulus, and is distinct from hyperalgesia, which generally refers to an extreme, exaggerated response to a normally painful stimulus.

Migraine headache

Compounds of formula (I) that are useful for modulating TRPA1 may be suitable for use in the formulation of medicaments for the treatment and/or prevention of migraine in mammals, particularly humans. Exposure to activators of TRPA1 has been shown to cause migraine in susceptible persons. These activators include, but are not limited to, cymenone, nitroglycerin, cigarette smoke, and formaldehyde. Thus, the TRPA1 antagonists of the present invention represent obvious potential therapeutic agents for the treatment of chronic and acute migraine.

Inflammatory diseases and disorders

The compositions and methods provided herein can also be used in connection with the treatment of inflammatory diseases. These diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases such as multiple sclerosis, and immune system diseases. TRPA1 has been shown to play a role in pancreatic pain and inflammation (see, e.g., Schwartz et al, Gastroenterology (2011)140(4): 1283-1291).

Peripheral neuropathies, such as diabetic neuropathy (e.g., painful diabetic neuropathy) are specific disorders that involve neuronal and inflammatory components. Without being bound by mechanical theory, TRPA1 antagonists of the present invention may be useful in the treatment of peripheral neuropathies including, but not limited to, diabetic neuropathy. In addition to use in treating peripheral neuropathy (e.g., reducing inflammation), the inhibitors of the inventive subject matter may also be used to reduce pain associated with peripheral neuropathy. TRPA1 has been shown to play a role in neuropathies and neuropathic pain (see, e.g., Wei et al, Anesthesiology (2009)111: 147-54; Koivisto et al, Pharmacol Res (2011)65: 149-.

Neurogenic inflammation typically occurs when neuronal hyperexcitability leads to the release of peptides that cause inflammation. These peptides include substance P and CGRP. Blockade of TRPA1 may reduce neuronal activity and thus may block neurogenic inflammation. For example, neurogenic inflammation in the respiratory tract can lead to asthma and allergic rhinitis symptoms, and neurogenic inflammation in the dura mater can also mediate migraine.

Pancreatitis

Pancreatitis is an inflammation of the pancreas. The pancreas is the large gland in the back of the stomach and is near the duodenum. Generally, digestive enzymes do not become active until they reach the small intestine where they begin to digest food. However, if these enzymes become active inside the pancreas, they begin to "digest" the pancreas itself. TRPA1 has been shown to play a role in pancreatic pain and inflammation (see, e.g., Schwartz et al, Gastroenterology (2011)140(4): 1283-1291.).

Acute pancreatitis is often, although not exclusively, caused by gallstones or alcohol abuse. Acute pancreatitis usually begins with pain in the upper abdomen, which can last for several days. The pain can be severe and can become persistent. The pain may be localized to the abdomen or may reach the back and other areas. Sometimes and for some patients, the pain is sudden and intense. At other times or for other patients, the pain begins to be mild and worsens after a meal. People with acute pancreatitis often look and feel extremely painful. Other symptoms may include abdominal swelling and tenderness, nausea, vomiting, fever, and rapid pulse. Severe cases of acute pancreatitis can lead to dehydration and hypotension and may even lead to organ failure, internal bleeding or death.

During the onset of acute pancreatitis, the blood levels of amylase and lipase are typically increased at least 3-fold. Blood levels of glucose, calcium, magnesium, sodium, potassium and bicarbonate may also change.

Current treatments depend on the severity of the episode. Generally, the treatments are designed to support important bodily functions, control pain, and prevent complications. Although acute pancreatitis typically resolves within a few days, it is often desirable to control pain during an attack. The compounds disclosed herein may be used to alleviate pain associated with acute pancreatitis.

If damage to the pancreas persists, chronic pancreatitis can develop. Chronic pancreatitis occurs when digestive enzymes attack and destroy the pancreas and nearby tissues, resulting in scarring and pain. Chronic pancreatitis can be caused by alcoholism or blocked, damaged or narrowed pancreatic ducts. In addition, genetic factors appear to affect the disease, and in some cases there is no identifiable cause (so-called idiopathic pancreatitis).

Most people with chronic pancreatitis have abdominal pain. Pain can be exacerbated when eating or drinking water, spreading to the back or becoming persistent and potentially disabling. Other symptoms include nausea, vomiting, weight loss, and fatty stools.

Pain relief is the first step in the treatment of chronic pancreatitis. Once pain is controlled, it is contemplated to implement a high carbohydrate and low fat diet program. Pancreatic enzymes can be used to help compensate for reduced enzymes produced from damaged pancreas. Sometimes insulin or other medication is required to control blood glucose.

Although drug therapy is commonly used to control pain, surgery may be necessary to relieve pain. Surgery is necessary to evacuate the enlarged pancreatic duct, or even to remove a portion of the severely damaged pancreas.

Chronic pancreatitis is often accompanied by pain. For example, pain is present in about 75% of alcoholic chronic pancreatitis patients, 50% of late-onset idiopathic chronic pancreatitis patients and 100% of early-onset idiopathic chronic pancreatitis patients (DiMagno, Gastroenterology (1999)116(5): 1252-1257).

A small number of patients with pain have lesions that are easily identified and they are relatively easily treatable surgically or endoscopically. In other patients, pain is generally thought to be due to different causes, including increased pancreatic pressure, ischemia, and fibrosis. However, without being bound by theory, these phenomena may not be the root cause of pain. Pain may arise from damage to the perineurium and subsequent exposure of the nerve to a background of neuronal sensitization induced by mediators and products of inflammation.

Given the importance of effective control of pain in patients with chronic pancreatitis, other therapies for treating painful symptoms are important and useful. The compounds disclosed herein may be used to control pain associated with chronic pancreatitis; they may be used alone or as part of an overall treatment plan for managing patients with chronic pancreatitis. For example, the compounds may be administered with pancreatin and/or insulin as part of a treatment regimen designed to control patients with chronic pancreatitis.

Not only are cancer treatments painful, they can even be toxic to healthy tissue.

Certain chemotherapeutic agents can produce painful neuropathy. Thus, the compounds disclosed herein may represent potentially important therapeutic agents for the treatment of pain and/or inflammation associated with cancer therapy leading to neuropathy.

The primary function of prostaglandins is to protect the gastric mucosa. Among such functions is the modulation of intracellular calcium levels in human gastric cells that play a critical role in cell proliferation. Thus, inhibition of prostaglandins by non-steroidal anti-inflammatory drugs (NSAIDs) inhibits calcium influx in stomach cells (Kokoska et al (1998) surgery (Stlouis)124 (2): 429-437). The most effective NSAIDs at relieving inflammation also produce the most gastrointestinal damage (Canadian Family physicians, 1 month 5 days 1998, p.101). Thus, the ability to independently modulate calcium channels in specific cell types may help mitigate such side effects of anti-inflammatory therapies. Additionally or alternatively, administration of a TRPA1 inhibitory compound of the present invention may be combined with an NSAID, thereby promoting pain relief using a reduced dose of the NSAID.

TRPA1 may mediate persistent nociception in chronic pancreatitis; and may involve a shift from acute inflammation to chronic inflammation and hyperalgesia in pancreatitis. TRPA1 may also mediate irritation and fever in, for example, the nasal and oral mucosa and respiratory tract.

Neurological diseases

Because TRPA1 overactivity can lead to toxic calcium loading, TRPA1 antagonists can also be useful in preventing neuropathy associated with: diabetes, chemical injury, chemotherapy, drugs such as statins, HIV/AIDS, Fabry's disease, vitamin deficiencies, hereditary polyneuropathy such as CTM disease (Marie-Charcot Tooth disease), and trauma. Peripheral neurodegenerative diseases such as amyotrophic lateral sclerosis may also be suitable for treatment with TRPA1 antagonists.

Pulmonary disease and cough

The compositions and methods provided herein may also be used in connection with the treatment of pulmonary diseases, including, but not limited to, asthma (including exercise-induced asthma, atopic asthma, allergic asthma), Chronic Obstructive Pulmonary Disease (COPD), emphysema, cystic fibrosis, bronchiectasis, bronchiolitis, allergic bronchopulmonary aspergillosis, obstructive bronchiolitis (popcorn worker's lung), diseases due to chemical exposure including exposure to diacetyl, formaldehyde and other irritants. These diseases also include tuberculosis, obstructive pulmonary diseases including asbestosis, radiation fibrosis, hypersensitivity pneumonitis, infant respiratory distress syndrome, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonitis sarcoidosis, eosinophilic pneumonia, lymphangioleiomyomatosis, pulmonary langerhans cell histiocytosis, and alveolar proteinosis; respiratory tract infections, including upper respiratory tract infections (e.g., common cold, sinusitis, tonsillitis, pharyngitis, and laryngitis) and lower respiratory tract infections (e.g., pneumonia); tumors of the respiratory system (malignant) (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell undifferentiated carcinoma, carcinoid, mesothelioma, metastatic cancer of the lung, metastatic germ cell cancer, metastatic renal cell carcinoma) or benign (e.g., lung hamartoma, congenital malformations such as lung isolation and Congenital Cystic Adenomatous Malformations (CCAM)); pleural cavity diseases (e.g., empyema and mesothelioma); and pulmonary vascular diseases, for example, pulmonary embolism such as thromboembolism, and air pocket embolism (iatrogenic), pulmonary arterial hypertension, pulmonary edema, pulmonary hemorrhage, inflammation, and damage to capillaries in the lung (leading to bleeding into the alveoli). Other treatable diseases include diseases that affect respiratory mechanics (e.g., obstructive sleep apnea, central sleep apnea, guillain-barren syndrome (guillian-barren), and myasthenia gravis).

The compounds of the invention are also useful in the treatment, reduction or prevention of cough (with or without sputum production), cough associated with asthma, cough associated with influenza, hemoptysis (hemoptysis), cough of unknown etiology, allergy-induced cough, and cough due to chemical exposure.

Skin diseases

Various itch causing agents activate TRPA1 directly or TRPA1 by activating receptors coupled downstream of TRPA 1. The compositions and methods provided herein can also be used to treat aspects associated with pruritus. Indications include, but are not limited to, conditions caused by exposure to exogenous chemicals, such as contact dermatitis, poison ivy, itching due to cancer (including lymphoma), itching caused by drugs such as chloroquine, itching caused by reactive drug metabolites or itching caused by dry skin.

Other exemplary indications include atopic dermatitis, psoriasis, urticaria, eczema, pompholyx, mouth ulcers, diaper rash.

Itching (pruritus)

Itching, or acute pruritus, although an important protective function by, for example, warning of harmful agents in the environment, can also be a debilitating condition, which is associated with, for example, a variety of skin, systemic and nervous system disorders. Some forms of pruritus are mediated by histamine signaling and are therefore susceptible to treatment with, for example, antihistamines. However, most pathophysiological pruritic conditions are not sensitive to antihistamine treatment. The compounds and pharmaceutical compositions of the present invention can be administered to treat pruritus.

Atopic Dermatitis (AD) is a chronic pruritus and inflammatory disease of the skin. Patients with severe AD may develop asthma and allergic rhinitis, also known as an atopic course of disease. Rashes and itching may be associated with atopic diseases. Chronic pruritus, for example, in AD and psoriasis; including pathophysiological marks such as large scratches, extensive epidermal hyperplasia resulting from, for example, eczema, renal failure, cirrhosis, neurological diseases, some cancers.

Allergic contact dermatitis is a common skin disorder associated with inflammation and persistent itching.

The methods disclosed herein can inhibit skin edema, keratinocyte hyperplasia, nerve growth, leukocyte infiltration, and antihistamine-resistant scratching behavior. The methods disclosed herein can inhibit allergic reactions to, for example, exogenous stimuli, e.g., haptens, oxazolones, urushiol (e.g., from poison ivy).

Disease and injury models

Compounds that antagonize TRPA1 function are useful in the prevention and treatment of any of the injuries, diseases, disorders, or conditions described above. In addition to in vitro testing of the activity of these compounds, their efficacy can be readily tested in one or more animal models. There are a number of animal models for studying pain. Various models use different agents or procedures in order to model pain caused by injury, disease or other conditions (see, e.g., Blackburn-Munro (2004) Trends in Pharmacol Sci (2004)25: 299-.

Exemplary behavioral tests for studying chronic pain include spontaneous pain, allodynia and hyperalgesia tests. The literature is as above. To assess spontaneous pain, posture, gait, signs of nociception (e.g., licking the paw, over-modification, over-probing behavior, protection of damaged body parts, and self-mutilation) can be observed. To determine the pain caused, behavioral responses can be examined after exposure to heat (e.g., a thermal injury model).

Exemplary animal models of pain include, but are not limited to, the Trevisan model and the models described in the Koivisto literature, including streptozotocin-induced painful diabetic neuropathy, bortezomib (bortezomib) -induced peripheral neuropathy, and oxaliplatin-induced peripheral neuropathy; chung model, retained nerve injury model (the specific nerveinjury model), carrageenan-induced hyperalgesia model, freund's complete adjuvant-induced hyperalgesia model, thermal injury model, formalin model and Bennett model.

In the Trevisan literature, the chemotherapy-induced peripheral neuropathy model involves the induction of the CIPN phenotype in mice treated with bortezomib or oxaliplatin (Trevisan et al, Cancer Res (2013) 73: 3120-. Treatment of animals with TRPA1 inhibitors can be assessed using any of a variety of pain tests, such as the Von Frey hair test, hot plate test, cold stimuli, chemical hyperalgesia, or rotarod test.

Models of Peripheral Diabetic Neuropathy (PDN) in the Koivisto literature include the induction of Diabetes (DM) in rats with streptozotocin and the evaluation of the plantar trpA1 agonist-induced axonal reflex (Koivisto et al, Pharmacol Res (2011)65: 149-. The reduction of DM-induced attenuation of skin axonal reflex can be assessed for treatment with compounds that inhibit TRPA 1.

The Chung model of neuropathic Pain (without inflammation) involves ligation of one or more spinal nerves (see, e.g., Chung et al Methods Mol Med (2004) 99: 35-45; Kim and Chung, Pain (1992) 50: 355- & 363). Ligation of spinal nerves results in a variety of behavioral changes in animals, including thermal hyperalgesia, cold allodynia, and persistent pain. Compounds that antagonize TRPA1 can be administered to ligated animals to assess whether they reduce these ligation-induced behavioral changes compared to that observed in the absence of the compound.

Degelatinum-induced hyperalgesia and Freund's complete adjuvant (CFA) -induced hyperalgesia are models of inflammatory pain (see, e.g., Walker et al J Pharmacol Exp Ther (2003)304: 56-62; McGaraughty et al BrJ Pharmacol (2003)140: 1381-. Compounds that antagonize TRPA1 can be administered to carrageenan-or CFA-stimulated animals to assess whether they reduce cold, mechanical, or thermal hypersensitivity, as compared to that observed in the absence of the compound. In addition, the ability of compounds to antagonize TRPA1 function to reduce cold and/or mechanical hypersensitivity may also be assessed in these models. In general, the carrageenan-induced hyperalgesia model is believed to mimic acute inflammatory pain while the CFA model is believed to mimic chronic pain and chronic inflammatory pain.

Exemplary models of inflammatory pain include a rat model of intraplantar injection of bradykinin. Briefly, animals were evaluated for baseline heat sensitivity using a Hargreave instrument. The TRPA1 blocker is then administered systemically. Bradykinin was then injected into the paw and hyperalgesia occurred. Thermal escape latencies were then determined at various time points over the next few hours (Chuang et al, 2001; Vale et al, 2004).

Inflammation is often an important contributor to pain. As such, it is useful to identify compounds that act as anti-inflammatory agents. Many compounds that reduce neural activity also prevent neurogenic inflammation. For direct measurement of inflammation, rat paw volumes can be evaluated using an plethysmometer. After taking baseline measurements, carrageenan can be injected into the paw and the volume can be monitored over hours in animals treated with vehicle or drug. Drugs that reduce paw swelling are considered anti-inflammatory.

Migraine is associated with significant pain and failure to perform normal work. There are several models of migraine, including the rat neurogenic inflammation model (see, e.g., Buzzi et al Br J Pharmacol (1990)99: 202-.

The Bennett model uses chronic ischemia of the paw to mimic chronic pain (see, e.g., Xanthos et al JPan (2004) 5: S1). This provides an animal model of chronic pain, including post-operative pain, complex regional pain syndrome and reflex sympathetic neurotrophic disorder. Chronic ischemia induces behavioral changes in animals, including hyperalgesia to mechanical stimuli, sensitivity to cold, painful behavior (e.g., shaking, licking and/or paw loving), and hyperalgesia. A compound that antagonizes TRPA1 can be administered to stimulated animals in order to assess whether they reduce any or all of these behaviors as compared to when the compound is not present. Similar experiments can be performed in a model of thermal injury or UV-burn to simulate post-operative pain.

Other models of neuropathic pain include central pain models based on spinal cord injury. Chronic pain is produced by inducing spinal cord injury, for example, by dropping a heavy object onto the surgically exposed area of the spinal cord (e.g., a drop weight model). Spinal cord injury can also be induced by: crushing or compressing the spinal cord, by delivering the neurotoxin, using photochemistry or by cutting into halves.

Other models of neuropathic pain include peripheral nerve injury models. Exemplary models include, but are not limited to, a neuroma model, a Bennett model, a Seltzer model, a Chung model (ligated at L5 or L5/L6), a sciatic nerve freezing denervation model, a lower tail trunk excision model, and a sciatic nerve inflammatory neuritis model. The literature is as above.

An exemplary model of neuropathic pain associated with a particular disease may also be utilized. Diabetes and herpes zoster are two diseases commonly associated with neuropathic pain. Even after the onset of acute herpes zoster, some patients continue to suffer from post-herpetic neuralgia and experience persistent pain that persists for years. Neuropathic pain and/or post-herpetic neuralgia caused by herpes zoster can be studied in a post-herpetic neuralgia model (PHN). Diabetic neuropathy can be studied in a mouse model of diabetes as well as in a chemically induced model of diabetic neuropathy.

As noted above, cancer pain can be of any of a variety of causes and a number of animal models exist for testing cancer pain involving, for example, chemotherapeutic agents or tumor infiltration. Exemplary models of toxin-associated cancer pain include vincristine-induced peripheral neuropathy models, paclitaxel-induced peripheral neuropathy models, and cisplatin-induced peripheral neuropathy models. A typical model of cancer pain caused by tumor infiltration is the cancer invasion pain model (CIP). The literature is as above.

Primary and metastatic bone cancers are associated with megalgia. There are several models of bone cancer pain, including the mouse femoral cancer pain model (FBC), the mouse calcaneus cancer pain model (CBC), and the rat tibial cancer model (TBC). The literature is as above.

Other models of pain are the formalin model. Similar to the carrageenan and CFA models, the formalin model involves injecting the stimulus intradermally or intraperitoneally into the animal. Formalin injection, i.e. 37% formaldehyde solution, is the most commonly used agent for intradermal paw injection (formalin test). Injection of a 0.5-15% solution of formalin (typically about 3.5%) into the dorsal or plantar aspect of the anterior or posterior paw produced a biphasic pain response of increasing and decreasing intensity about 60 minutes after injection. Typical responses include lifting, licking, gnawing, or shaking the paw. These responses are considered painful. The initial phase of the response (also referred to as early phase) lasting 3-5 minutes may be due to direct chemical stimulation of the nociceptors. This is followed by 10-15 minutes during which the animal shows little or no nociceptive behaviour. The second phase of the response (also called late phase) begins about 15-20 minutes after formalin injection and lasts 20-40 minutes, initially with an increase in the number and frequency of nociceptive behaviors, reaches a peak, and then decreases. The intensity of these nociceptive behaviors depends on the concentration of formalin used. The second phase comprises the sensitization phase, during which inflammatory phenomena occur. The two-phase response to formalin injection makes the formalin model a suitable model for studying nociception and acute inflammatory pain. In certain aspects, it may also be a model of neuropathic pain.

In addition to any of the models of chronic pain described above, compounds that antagonize TRPA1 function may also be tested in one or more acute pain models (see, e.g., Valenzano et al (2005) Neuropharmacology 48:658 672). Regardless of the compounds tested in models of chronic pain, acute pain, or both, these studies are generally (although not exclusively) conducted with, for example, mice, rats, or guinea pigs. In addition, compounds can be tested in various cell lines that provide in vitro pain assays. Wang and Wang (2003).

Many individuals seeking treatment for pain suffer from visceral pain. Animal models of visceral Pain include rat models of inflammatory uterine Pain (see, e.g., Wesselmann et al, Pain (1997)73: 309-. The efficacy of TRPA1 compounds may be assessed by writhing, gastrointestinal inflammation, or a decrease in bladder excitability.

To test the efficacy of TRPA1 antagonists in treating cough, an conscious guinea pig cough model experiment can be readily used (see, e.g., Tanaka and Maruyama (2003) J Pharmacol Sci 93: 465-470; McLeod et al (2001) Br J Pharmacol 132: 1175-1178). In short, guinea pigs are used as a useful animal model of cough because unlike other rodents such as mice and rats, guinea pigs do cough. In addition, guinea pig coughs appear to resemble human coughs in the posture, behavior, and performance of coughed animals.

To induce cough, conscious guinea pigs are exposed to an inducer, such as citric acid or capsaicin. Animal responses were determined by counting the number of coughs. The effectiveness of a cough suppressant (e.g., a compound that inhibits TRPA1) can be determined by administering the active agent and evaluating the ability of the active agent to reduce the number of coughs resulting from exposure to citric acid, capsaicin, or other similar cough-inducing agent. In this manner, TRPA1 inhibitors useful in the treatment of cough can be readily evaluated and identified.

Other cough models may include an unconscious guinea pig model (see, e.g., rougett et al (2004) Br JPharmacol 141: 1077-. Any of the above models may be applied to other animals capable of coughing. Other exemplary animals capable of coughing include cats and dogs.

The compounds of the invention can be tested in a variety of asthma models. An example is the murine ovalbumin model of asthma (see, e.g., Caperes AI et al, Proc Natl Acad Sci U S A. (2009)106(22): 9099-. In this model, ovalbumin is injected into the abdominal cavity multiple times over a two week period. At some time in the third week, the animals were stimulated for airway hyperresponsiveness with intranasal ovalbumin, and inflammation and inflammatory cytokine production could be measured. The compounds were administered during the stimulation phase of the model. Trpa1 knockout mice can be substituted into the above models as reported by Caceres et al.

The sheep model of conscious allergy is an example of a large animal model of asthma that can be used to assess the effect of compounds on the antigen-induced late phase response of asthma (Abraham wm., Am J RespirCrit Care Med (2000)162(2): 603-11). Briefly, baseline airway reactivity was measured by plethysmograph in conscious sheep, followed by nebulization of ascaris suum extract to induce asthma. After capturing the baseline reading, animals were stimulated with a nebulized dose of ascaris suum. Antigen sensitivity is determined by the decrease in lung airflow resistance from baseline. Once the animal demonstrates antigen-sensitivity, test compounds will be administered and additional lung airflow resistance readings captured to assess airway reactivity changes. Models of horses and beagle dogs are also sometimes used.

Other models may include the Brown Norway rat model of asthma and the C57BL/6J mouse model, as described by Raemdonck et al (Raemdonck K et al, Thorax (2012) Jan; 67(1): 19-25). Briefly, Brown Norway rats and C57BL/6J mice can be sensitized and stimulated with aerosol-delivered ovalbumin. Once sensitivity is confirmed by a decrease in lung function (as measured by whole body plethysmograph readings), the compounds of the present invention may be administered. Visual and audible signs of respiratory distress may also be present, including asthma.

Dermatitis (dermatitis)

There are currently a variety of mouse models of skin disease. For example, Liu et al describe various models of oxazolone and urushiol-induced contact dermatitis (see, e.g., Liu B et al, FASEB J. (2013)27(9): 3549-63). Briefly, Trpa1 knockout mice received topical administration of oxazolone or urushiol to induce a dermatitis and itch response. Epidermal thickness can also be measured by drilling ear holes and stimulated areas compared to untreated ears. In vivo therapeutic compounds can be determined by administering the compound to an animal before or after treatment with oxazolone or urushiol. The scratching behavior was recorded by a camera placed in the upper part of the observation room. An observer blinded to the treatment group recorded the time it took for the animal to scratch during the course of 30 minutes.

An alternative mouse model of dry skin induced itch involves the administration of acetone, ether and water to mice, as reported by Wilson et al (Wilson SR et al, J Neurosci (2013)33(22): 9283-94). In this model, the area to be treated was shaved and the mice received topical acetone and ether twice daily in the area to be observed, e.g. the cheek or back. The in vivo efficacy of a therapeutic compound can be determined by administering the compound to an animal before and after administration of acetone and ether. Scratching behavior was recorded by a video camera for up to 20 minutes and quantified by an observer blinded to the treatment group.

In addition, pruritus can be induced by direct injection of agents that cause pruritus. Examples of such agents are found in Akayimo and Carstens, 2013. Some examples are: chloroquine (Wilson et al, 2011), bile acids, TSLP (Wilson et al, 2013), and IL-31(Cevikbas et al, 2014). Itching, which usually occurred during a defined period, was recorded by an observer blinded to the treatment group.

There are a number of rodent incontinence models. They include incontinence models induced by nerve damage, urinary tract insults, and inflammation. Urethral invasion models include the rat bladder outflow obstruction model (see, e.g., Pandita, RK, anddAndersson KE. J Urol (1999) 162: 943-948). The model of inflammation included the injection of mustard oil into the bladder.

To test the effectiveness of TRPA1 inhibitor compounds in treating incontinence, rats may be administered different concentrations of the compound (e.g., low, medium, and high concentrations) after surgery to effect partial Bladder Outflow Obstruction (BOO). The efficacy of different doses of TRPA1 inhibitory compounds may be compared to a control group given vehicle alone (sham control group). Efficacy can be further compared to rats given a positive control, such as atropine. Atropine is expected to reduce overactive bladder following obstruction of a portion of the bladder outflow tract in the BOO model. Note that when testing compounds in a BOO model, the compounds can be administered directly to the bladder or urethra (e.g., via a catheter) or systemically (e.g., orally, intravenously, intraperitoneally, etc.).

Recently several rat models of pancreatic Pain have been described (Lu, 2003, Anesthesiology 98 (3): 734-. Lu et al induced pancreatitis by systemic delivery of dibutyl tin dichloride (dibutylin dichloride) in rats. Rats showed an increase in the ventral von frey filament-stimulated withdrawal and a decrease in the heat-stimulated withdrawal latency over the 7-day period. The pain state induced in these animals is also characterized by increased levels of substance P in the spinal cord (Lu et al, 2003). To test the efficacy of TRPA1 inhibitors in this model, TRPA1 inhibitors may be administered after or concurrently with the delivery of dibutyltin dichloride. The control group of animals may be administered a vehicle or a known pain relief agent. Pain indicators can be measured. The efficacy of a TRPA1 inhibitor can be evaluated by comparing the pain index observed in animals receiving the TRPA1 inhibitor to the pain index of animals not receiving the TRPA1 inhibitor. In addition, the potency of TRPA1 inhibitors may be compared to the potency of known pain medications.

The efficacy of the Von Frey silk test as a means of determining injury behavior was also demonstrated by inducing pancreatitis with systemic L-arginine administration (Winston et al, 2003). The efficacy of TRPA1 inhibitors may be similarly tested following induction of pancreatitis by systemic L-arginine administration.

Lu et al also describe the direct behavioral determination of pancreatic pain using acute nociceptive stimulation of the pancreas through an internal duct in conscious and freely moving rats. These assays included cag crossing (cage crossing), feeding and hindlimb extension as responses to bradykinin infusion in the pancreas. Intrathecal administration of D-APV (NMDA receptor antagonist) or morphine alone results in partial relief of visceral pain behavior in this model. The combination of both reduced pain behavior to baseline. The efficacy of TRPA1 inhibitors can be similarly tested in this system.

Any of the above animal models may be used to evaluate the efficacy of TRPA1 inhibitors in the treatment of pain associated with pancreatitis. The efficacy was compared to the treatment-free group or the placebo control group. Additionally or alternatively, efficacy may be assessed in comparison to one or more known pain relief drugs.

Examples

General procedure

All reactions are carried out under an inert atmosphere, typically nitrogen. All non-aqueous reactions were carried out using anhydrous solvents. All reactions were stirred using a mechanical stir bar or an overhead mechanical stirrer. All saturated extraction solutions are aqueous (e.g. saturated NH)₄Cl). All desiccants are anhydrous. Drying the organic solution using a desiccant implies that the desiccant is removed from the organic solution by filtration. The chromatography refers to silica gel column chromatography. Preparative Thin Layer Chromatography (TLC) was run on silica gel plates. Concentration of the reaction mixture means concentration under reduced pressure and use of a rotary evaporator. Drying the final product means drying under high vacuum conditions. Sonication means the use of an ultrasonic bath. All of¹H-NMR data were obtained at 400 MHz. Mass spectra were acquired in the positive ion mode and reported as the protonated species MH⁺Unless otherwise indicated.

Abbreviations

DCM dichloromethane

DIC N, N' -diisopropylcarbodiimide

DIPEA N, N' -diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF N, N-dimethylformamide

EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

EA Ethyl acetate

Ether

h hours

HOAc acetic acid

HOAT 1-hydroxy-7-azabenzotriazole

LAH lithium aluminum hydride

MeOH methanol

min for

n-BuLi n-butyllithium

NMP N-methylpyrrolidone

Pd/C Palladium/activated carbon, typically 10% Palladium Supported

PE Petroleum Ether

RT Room temperature

TBAI tetrabutylammonium iodide

TEA Triethylamine

TFA trifluoroacetic acid

TLC thin layer chromatography

THF tetrahydrofuran

Preparation of synthetic intermediates

Preparation of 1(2S) -2- (1, 3-dimethyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid

Step 1(R) -methyl 2- (methylsulfonyloxy) propionate

A solution of methyl (R) -2-hydroxypropionate (30g, 0.28mol) and TEA (80mL, 0.56mol) in DCM (300mL) was cooled to 0 deg.C and methanesulfonyl chloride (33.6mL, 0.42mol) was added dropwise over 1h at 0 deg.C. The mixture was stirred at 10-20 ℃ for 1.5 hours. The resulting mixture was quenched with ice-water (100 mL). The organic layer was separated, washed with water (2 × 50mL) and brine, and Na₂SO₄Dried and concentrated to give the crude product methyl (R) -2- (methylsulfonyloxy) propionate (50g, 95.2%) as a brick-red oil, which was used without purification.

Step 2(2S) -methyl 2- (1, 3-dimethyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionate

To 1, 3-dimethyl-3, 4,5, 7-tetrahydro-1H-purine-2, 6-dione (112g, 0.62mol) and K at 18 deg.C₂CO₃(171g, 1.24mol, 2eq) in DMF (2.2L) was added methyl (R) -2- (methylsulfonyloxy) propionate (226g, 1.24 mol). The mixture was stirred at 18 ℃ overnight; then it is treated with saturated NH₄Cl (2L) quench. The resulting mixture was extracted with DCM (3 × 1L). The combined organic phases were washed with water (5X 500mL) and brine, and Na₂SO₄Dried and concentrated. The residue was poured into DCM and extracted with 6N HCl (2 × 200 mL). The combined aqueous phases were back-extracted with DCM (2 × 50 mL). The combined organic phases are washed with Na₂SO₄Dried and concentrated to give the desired product as a light brown oil (65g, 39.3%), which was used without further purification. MH⁺267.

Step 3(2S) -2- (1, 3-dimethyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid

To a solution of methyl (2S) -2- (1, 3-dimethyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionate (39g, 145mmol) in dioxane (400mL) was added 6N HCl (200 mL). The mixture was refluxed for 3h, cooled to room temperature, and then concentrated to remove dioxane and most of the aqueous phase. The residue was triturated in water (70mL) and filtered. The solid was collected by filtration to give the title product (17.3g, ee: 99%). The filtrate was concentrated to dryness and the residue was purified by chromatography, eluting with DCM/MeOH (40/1 to 15/1) to give more product (3.2g, ee: 95%). The overall yield was 55.1%.¹HNMR(DMSO-d₆)13.28(s，1H)，8.21(s，1H)，5.47(q，J＝7.4Hz，1H)，3.44(s，3H)，3.21(s，3H)，1.76(d，J＝7.4Hz，3H).MH⁺253.

Chiral HPLC details: chiralcel AD column, 250 × 4.6mm, 10 um. Mobile phase: hexane (0.1% TFA)/IPA (0.1% TFA)70/30.

Preparation of 25, 5-dimethylpyrrolidin-2-one hydrochloride

Step 14-methyl-4-nitropentanoate

To a solution of 2-nitropropane (5.06g, 56.84mmol) in dioxane (3mL) was added Triton B (0.55mL, 40% aqueous solution). The reaction was warmed to 70 ℃ and methyl acrylate (4.78g, 55.58mmol) was added dropwise. After the addition, the reaction was heated at 100 ℃ for 4 hours. The reaction is cooled toAt room temperature, 1N HCl (2mL) was added and the resulting mixture partitioned between EA and water. The combined organic layers were washed with brine, washed with Na₂SO₄Dried and concentrated to give the crude product (10g, 100%) as an oil.¹H NMR(CDCl₃)3.68(s，3H)，2.35-2.31(m，2H)，2.27-2.23(m，2H)，1.60(s，6H).

Step 25, 5-dimethylpyrrolidin-2-one

To NiCl₂To a solution of hexahydrate (0.67g, 2.86mmol) in MeOH (30mL) was added NaBH in portions₄(0.33g, 8.57 mmol). The reaction was sonicated for 0.5 hours; methyl 4-methyl-4-nitropentanoate (1.0g, 5.77mmol) was then added dropwise. Add additional NaBH in portions₄(0.66g, 17.14 mmol). The resulting mixture was stirred at room temperature overnight. The mixture was filtered through celite and the filtrate was concentrated to one-quarter volume. The residue was taken up in DCM and saturated NaHCO₃Are distributed among the devices. The organic layer was washed with brine, washed with Na₂SO₄Dried and concentrated to give the crude product (0.35g, 53.7%) as an oil. MH⁺114.

Step 35, 5-dimethylpyrrolidin-2-one hydrochloride

To a suspension of LAH (121mg, 3.18mmol) in THF (8mL) was added 5, 5-dimethylpyrrolidin-2-one (0.3g, 2.65mmol) and the reaction was heated at 60 ℃ overnight. The reaction was cooled to 0 ℃ and carefully quenched with water (0.2mL) followed by 15% NaOH (0.2 mL). The mixture was filtered through celite. Concentrated hydrochloric acid was added to the filtrate. The mixture was concentrated to give the crude product (0.2g, 75.5%) as a white solid, which was used without purification. MH⁺100.

Preparation of 32 '- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

Step 15-bromo-2- (2, 2-dimethylpyrrolidin-1-yl) pyrimidine

5-bromo-2-chloropyrimidine (2.3g, 11.9mmol) and K were reacted at room temperature₂CO₃(6.6g, 47.6mmol) to a solution in DMF (20mL) was added a solution of 2, 2-dimethylpyrrolidine (2.26g, 16.7mmol) in DMF (4 mL). The resulting reaction mixture was stirred at 50 ℃ for 2 days. The reaction was poured into ice water while stirring. The precipitate was collected to give crude 5-bromo-2- (2, 2-dimethylpyrrolidin-1-yl) pyrimidine (2.3g, 76.6%) as a pale yellow solid, which was used in the next step without any further purification. MH⁺256.

Step 22- (2, 2-dimethylpyrrolidin-1-yl) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidine

To a mixture of 5-bromo-2- (2, 2-dimethylpyrrolidin-1-yl) pyrimidine (17.6g, 68.9mmol), bis (pinacolato) diborane (24.5g, 96.5mmol) and KOAc (13.5g, 0.14mol) in 1, 4-dioxane (320mL) was added Pd (PPh)₃)₂Cl₂(2.4g, 3.45 mmol). The mixture was stirred at 80 ℃ for 20 h. The reaction was cooled to room temperature, poured into ice-water, and extracted with EA (4x 200 mL). The combined organic layers were washed with brine, washed with Na₂SO₄Dried and concentrated to give a dark residue. The residue was purified by chromatography, eluting with PE/EA (40:1) to give 2- (2, 2-dimethyl-2-methyl-EA)Pyrrolidin-1-yl) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidine (11.5g, 49% over two steps) as a yellow solid.¹HNMR(DMSO-d₆)8.58(s，2H)，3.69(m，2H)，1.92(m，4H)，1.55(s，6H)，1.33(s，12H).

Step 32 '- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

To a mixture of 2- (2, 2-dimethylpyrrolidin-1-yl) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidine (9.5g, 31mmol) in 1, 4-dioxane (140mL) was added 4-amino-2-chloropyrimidine (4.5g, 34.5mmol) and 2M K₂CO₃(20.4mL, 40.7 mmol). N for the orange mixture₂Degassing; pd (PPh) was then added₃)₄(3.65g, 3.1 mmol). The reaction was stirred at 80 ℃ overnight. The reaction was cooled to room temperature, poured into water and extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with brine, washed with Na₂SO₄Dried and concentrated. The residue was purified by chromatography, using PE: EA (1:1) to give the compound 2'- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-amine (8.96g,>100%) as a yellow solid. MH +271.

Preparation of 4(S) -2- (2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine

Step 1(S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine

Mixing (S) -2-, (Trifluoromethyl) pyrrolidine hydrochloride (40g, 0.23mol), K₂CO₃A mixture of (94.6g, 0.68mol) and 5-bromo-2-chloropyrimidine (48g, 0.25mol) in DMF (200mL) was stirred at 100 ℃ for 24 hours, then N was added₁,N₂Dimethylethane-1, 2-diamine (4mL) and the reaction was stirred for an additional 2h to consume excess 5-bromo-2-chloropyrimidine. The reaction was quenched with water (400mL) and extracted with EA (3 × 500 mL). The combined organic phases were washed with 10% LiCl aqueous solution and Na₂SO₄Dried and concentrated. The residue was purified by chromatography, using PE: EA eluted (50:1) to give (S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (50g, 74%) as a white solid.¹H NMR(DMSO-d₆)8.54(s，2H)，4.90-4.94(m，2H)，3.56-3.58(m，2H)，2.02-2.16(m，4H)。MH⁺296.

Step 2(S) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-ylboronic acid

A solution of (S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (50g, 0.17mol) and triisopropylborate (44.4g, 0.23mol) in THF (400mL) was cooled to-78 deg.C and n-BuLi (105mL, 2.4M in hexanes) was added dropwise. The reaction was stirred at-78 ℃ for 2 h. The reaction was quenched with water (150mL) and allowed to warm to room temperature. The reaction was concentrated to retain the aqueous phase. The aqueous phase was extracted with ether (2 × 50mL) to remove impurities (product in aqueous layer). The pH was adjusted to 5 with 6N HCl, which was then extracted with EA (3x 100 mL). The combined organic phases are washed with Na₂SO₄Dried and concentrated to give (S) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-ylboronic acid (45g, quantitative yield) as an off-white solid. MH⁺262.

Step 3(S) -2- (2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine

To (S) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-ylboronic acid (9.5g, 36.4mmol), 2-chloropyrimidin-4-amine (4.3g, 33.1mmol) and Na₂CO₃(7.0g, 66.2mmol) in dioxane (105mL) and water (35mL) Pd (PPh) was added₃)₄(3.8mg, 3.31 mmoL). The mixture was degassed with nitrogen and then stirred at 110 ℃ for 3 hours. The reaction was cooled and filtered through celite. The filtrate was partitioned between EA (300mL) and water (150 mL). The organic phase was washed with brine (100mL) and Na₂SO₄Dried and concentrated. The residue was purified by chromatography eluting with DCM/MeOH (100: 1 to 80:1 to 70: 1) to give (S) -2- (2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine (8g, 78%) as a white solid.¹H-NMR(CDCl₃)9.16(s，2H)，8.13-8.14(d，J＝10Hz，1H)，6.97(s，2H)，6.34-6.35(d，J＝6Hz，1H)，5.09-5.13(m，1H)，3.67-3.72(m，2H)，2.06-2.21(m，4H).MH⁺311.

Preparation of 5(R) -2- (2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine

The title compound was prepared using the procedure of preparation 4. MH⁺311

Preparation of 6(2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid

Step 1(2S) -methyl 2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionate

To 1-methyl-3, 4,5, 7-tetrahydro-1H-purine-2, 6-dione (6.904g, 41.5mmol) and K at 50 deg.C₂CO₃(5.734g, 41.5mmoL) to a suspension in DMF (150mL) was added methyl (R) -2- (methylsulfonyloxy) propanoate (5.818g, 32.0 mmoL). The reaction was stirred at 50 ℃ overnight and then saturated NH₄Cl (2L) quench. The resulting mixture was extracted with EA (3 × 200 mL). The combined organic phases were washed with water (5 × 500mL) and brine. Na for organic phase₂SO₄Dried and concentrated. The residue was purified by chromatography (0-3% MeOH: DCM) to give the title product as a white solid (1.649g, 20%). MH⁺253.

Step 2(2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid

To a mixture of methyl (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionate (96.9mg, 0.38mmol) in dioxane (3mL) was added 6N HCl (2 mL). The reaction was refluxed for 3h, cooled to room temperature and concentrated to give the product as a white solid (92mg, 100%); MH⁺239.

Preparation of 7(S) -2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid

Step 1(S) -methyl 2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionate:

to a solution of methyl (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionate (600mg, 2.38mmol) in DMF (2ml) at room temperature was added sodium 2-chloro-2, 2-difluoroacetate (508mg, 3.33mmol), followed by Cs₂CO₃(229mg 3.81 mmoL). The reaction was heated at 60 ℃ for 12 hours. The reaction was cooled to room temperature, diluted with cold water and extracted twice with EA. The combined organic layers were washed with brine, over MgSO₄Dried and concentrated. The residue was purified by chromatography with MeOH: DCM (0-3%) was eluted to give methyl (S) -2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionate (164mg, 23%) as a colorless oil. MH⁺303.

Step 2(S) -2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid

To a mixture of methyl (S) -2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionate (64mg, 0.21mmol) in dioxane (1mL) was added 6N HCl (1 mL). The reaction was refluxed for 3h, cooled to room temperature, and then concentrated. The precipitate was collected to give the product as a white solid (61mg, 100%). MH⁺289.

Preparation of 82 '- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

Step 15-bromo-2- (3, 3-difluoroazetidin-1-yl) pyrimidine

5-bromo-2-chloropyrimidine (450mg, 2.3mmol), 3-difluoroazetidine hydrochloride (275.1mg, 2.1mmol), and K were added to a closed tube₂CO₃(589.3mg, 4.3mmol) and DMF (3 mL). The tube was sealed and stirred at 130 ℃ for 2 hours. The reaction was cooled to room temperature and poured into water (4 mL). The solid was collected by filtration and dried to give 5-bromo-2- (3, 3-difluoroazetidin-1-yl) pyrimidine (300mg, 51.4%) as a white solid. MH⁺250.

Step 2(2- (3, 3-Difluoroazetidin-1-yl) pyrimidin-5-yl) boronic acid

To a solution of 5-bromo-2- (3, 3-difluoroazetidin-1-yl) pyrimidine (300mg, 1.2mmol) and triisopropylboronic acid ester (0.4mL, 1.8mmol) in THF (6mL) was added n-BuLi (0.6mL, 2.4M in hexanes, 1.5mmol) dropwise at-78 ℃. The mixture was stirred at-78 ℃ for 2 hours. The reaction was quenched with water and warmed to room temperature. The solvent was concentrated and the residual aqueous layer was extracted with ether (2 × 10 mL). The aqueous layer was adjusted to pH 6 with 1N HCl and extracted with EA (3 × 10 mL). The combined organic layers were washed with brine, washed with Na₂SO₄Dried, and concentrated to give the product (170mg, 65.6%) as a white solid. MH⁺216.

Step 32 '- (3, 3-Difluoroazetidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

(2- (3, 3-Difluoroazetidin-1-yl) pyrimidin-5-yl) boronic acid (170.0mg, 0.8mmol), 4-amino-2-chloropyrimidine (102.4mg, 0.8mmol), Pd (PPh)₃)₂Cl₂(56.2mg, 0.08mmol) and Na₂CO₃A mixture of (167.5mg, 1.6mmol) in 1, 4-dioxane (5mL) and water (1mL) was degassed with nitrogen and stirred at 90 ℃ overnight. Will be describedThe mixture was cooled to room temperature and EA was poured in. The organic phase was separated, washed with water and brine, and Na₂SO₄Dried and concentrated. The residue was dissolved in ether. The insoluble residue was removed by filtration and the filtrate was concentrated to give 2'- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidine]-4-amine (130mg, 62.3%) as a white solid. MH⁺265.

Preparation of 92-chloro-N- (2'- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidinyl ] -4-yl) acetamide

To 2'- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidine at 0 ℃]A solution of (60mg, 0.2mmol) of the (4) -amine in DMF (2mL) was added dropwise 2-chloroacetyl chloride (0.03mL, 0.34 mmol). The reaction was stirred at room temperature for 2h, then it was poured into EA. The organic phase was washed with water and brine, washed with Na₂SO₄Drying and concentrating to obtain 2-chloro-N- (2'- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (50mg, 64.7%) as a yellow solid. MH⁺341.

Preparation of 10(2- (4, 4-difluoropiperidin-1-yl) pyrimidin-5-yl) boronic acid

Step 15-bromo-2- (4, 4-difluoropiperidin-1-yl) pyrimidine

5-bromo-2-chloropyrimidine (633.3mg, 3.3mmol), 4-difluoropiperidine hydrochloride (472.8mg, 3.0mmol), K were added to the closed tube₂CO₃(829.3mg, 6.0mmol) and DMF (4 mL). The tube was sealed and stirred at 130 ℃ for 2 h; then put it inCooled to room temperature and poured into water (5 mL). The solid precipitate was collected and dried to give 5-bromo-2- (4, 4-difluoropiperidin-1-yl) pyrimidine (640mg, 77%) as a white solid. MH⁺278.

Step 2(2- (4, 4-Difluoropiperidin-1-yl) pyrimidin-5-yl) boronic acid

To a solution of 5-bromo-2- (4, 4-difluoropiperidin-1-yl) pyrimidine (640mg, 2.3mmol) and triisopropylboronic acid ester (0.8mL, 3.5mmol) in THF (8mL) was added n-BuLi (2mL, 2.4M in hexanes, 1.5mmol) dropwise at-78 ℃. The mixture was stirred at-78 ℃ for 2 hours. The reaction was quenched with water and allowed to warm to room temperature. The reaction was concentrated and the residual aqueous mixture was extracted with ether (2 × 10 mL). The aqueous phase was adjusted to pH 6 with 1N HCl and extracted with EA (3 × 10 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried, and concentrated to give (2- (4, 4-difluoropiperidin-1-yl) pyrimidin-5-yl) boronic acid (420mg, 74.8%) as a white solid. MH⁺244.

Preparation of 112-chloro-N- (2-chloropyrimidin-4-yl) acetamide

To a mixture of 4-amino-2-chloropyrimidine (2.0g, 15.4mmol) and DMF (25mL) was added 2-chloroacetyl chloride (0.03mL, 0.34mmol) dropwise at 0 ℃. The reaction was stirred at room temperature overnight and then poured into EA. The organic phase was washed with water and brine, washed with Na₂SO₄Dried and concentrated. The residue was triturated with DCM and the solid was collected to give 2-chloro-N- (2-chloropyrimidin-4-yl) acetamide (1.3g, 20.5%) as a yellow solid. MH⁺206.

Preparation of 12N- (2-Chloropyrimidin-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide

2-chloro-N- (2-chloropyrimidin-4-yl) acetamide (1.1g, 5.4mmol), 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (966.3mg, 5.4mmol), K₂CO₃A mixture of (1.1g, 8.1mmol) and TBAI (198.2mg, 0.5mmol) in DMF (20mL) was stirred at 90 ℃ for 10 min. The reaction was cooled to room temperature and then diluted with EA. The resulting mixture was washed with water and saturated NH₄Cl and brine, washed with Na₂SO₄Dried and concentrated. The residue was recrystallized from DCM to give N- (2-chloropyrimidin-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -acetamide (1.3g, 69.4%) as a white solid. MH⁺350.

Preparation of 133-azabicyclo [3.1.0] hexane hydrochloride

Step 13-benzyl-3-azabicyclo [3.1.0] hexane-2, 4-dione

To 3-oxabicyclo [3.1.0]To a mixture of hexane-2, 4-dione (2.3g, 20.5mmol) in AcOH (30mL) was added DMAP (150mg) and benzylamine (2.2mL, 20.5 mmol). The mixture was stirred at 100 ℃ for 40 hours; then cooled to room temperature. The reaction was concentrated and the residue was dissolved in EA. The organic phase was washed with water and brine, washed with Na₂SO₄Dried and concentrated. The residue was purified by chromatography, using PE: EA (8:1 to 5:1) to give 3-benzyl-3-azabicyclo [3.1.0]Hexane-2, 4-dione (3.7g, 89.6%) as a white solid. MH⁺202.

Step 23-benzyl-3-azabicyclo [3.1.0] hexane

To 3-benzyl-3-azabicyclo [3.1.0]To a solution of hexane-2, 4-dione (2.0g, 10.0mmoL) in THF (30mL) was added LAH (1.5g, 40.0mmoL L). The resulting mixture was heated at reflux for 4h, then cooled to 0 ℃. The cold reaction mixture was carefully saturated with NH₄Cl was quenched and then filtered. The filtrate was concentrated to give the title compound (1.5g, 86.7%) as a clear oil. MH⁺174.

Step 33-azabicyclo [3.1.0] hexane hydrochloride

Reacting 3-benzyl-3-azabicyclo [3.1.0]A mixture of hexane (1.3g, 7.5mmol), 10% Pd/C (130mg), and concentrated HCl (0.63mL, 7.5mmol) in MeOH (20mL) was stirred at room temperature under a hydrogen atmosphere (balloon) for 18 h. The reaction was filtered through celite and the filtrate was concentrated to give the title compound (850mg, 95%) as a white solid. MH⁺84

Preparation of 142 '- (3-azabicyclo [3.1.0] hex-3-yl) - [2,5' -bipyrimidine ] -4-amine

Step 13- (5-Bromopyrimidin-2-yl) -3-azabicyclo [3.1.0] hexane

5-bromo-2-chloropyrimidine (671) was added to the closed tube.7mg, 3.5mmol), 3-azabicyclo [3.1.0]Hexane hydrochloride (416.7mg, 3.5mmol), K₂CO₃(967.5mg, 7.0mmol) and DMF (4 mL). The tube was sealed and stirred at 130 ℃ for 2 hours. The reaction was cooled to room temperature and poured into cold water (4 mL). The solid formed is collected and dried to give 3- (5-bromopyrimidin-2-yl) -3-azabicyclo [3.1.0]Hexane (480mg, 57.4%) as a white solid. MH⁺240.

Step 2(2- (3-azabicyclo [3.1.0] hex-3-yl) pyrimidin-5-yl) boronic acid

To 3- (5-bromopyrimidin-2-yl) -3-azabicyclo [3.1.0] at-78 deg.C]Hexane (480mg, 2.0mmol) and triisopropylboronic acid ester (0.7mL, 3.0mmol) in THF (6mL) were added dropwise n-BuLi (1.1mL, 2.4M in hexane, 2.6 mmol). The reaction was stirred at-78 ℃ for 2 hours, then quenched with water and warmed to room temperature. The reaction was concentrated and the aqueous residue was extracted with ether (2 × 20 mL). The aqueous layer was separated, adjusted to pH 6 with 1N HCl and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated to give the title product (200mg, 48.5%) as a white solid. MH⁺206.

Step 32 '- (3-azabicyclo [3.1.0] hex-3-yl) - [2,5' -bipyrimidine ] -4-amine

2- (3-azabicyclo [3.1.0] o]Hex-3-yl) pyrimidin-5-yl) boronic acid (150.0mg, 1.2mmol), 2-chloropyrimidin-4-amine (237.9mg, 1.2mmol), Pd (PPh)₃)₂Cl₂(86.0mg, 0.1mmol) and Na₂CO₃A mixture of (245.9mg, 2.3mmol) in 1, 4-dioxane (5mL) and water (1mL) was degassed with nitrogen and stirred at 80 ℃ overnight. The reaction was cooled to room temperature and EA was poured in. Is separated byThe organic phase was washed with water and brine, and Na₂SO₄Dried and concentrated. The residue was dissolved in diethyl ether. The insoluble residue was removed by filtration and the filtrate was concentrated to give 2' - (3-azabicyclo [ 3.1.0)]Hex-3-yl) - [2,5' -bipyrimidine]4-amine (100mg, 33.8%) as a white solid. MH⁺255.

Preparation of 15N- (2'- (3-azabicyclo [3.1.0] hex-3-yl) - [2,5' -bipyrimidine ] -4-yl) -2-chloroacetamide

To 2' - (3-azabicyclo [3.1.0] at 0 deg.C]Hex-3-yl) - [2,5' -bipyrimidine]A solution of (40mg, 0.2mmol) of the (4-amino) in DMF (2mL) was added dropwise to 2-chloroacetyl chloride (0.02mL, 0.3 mmol). The reaction was stirred at room temperature for 2h, then EA was poured in. The organic layer was extracted with water and brine, and Na was added₂SO₄Dried and concentrated to give N- (2' - (3-azabicyclo [3.1.0]]Hex-3-yl) - [2,5' -bipyrimidine]-4-yl) -2-chloroacetamide (50mg, 96.2%) as a yellow solid. MH⁺329.

Preparation of 162 '- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

Step 15-bromo-2- (3- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine

5-bromo-2-chloropyrimidine (441.4mg, 2.3mmol), 3- (trifluoromethyl) pyrrolidine hydrochloride (402.6mg, 2.1mmol), and K were added to a closed tube₂CO₃(635.8mg, 4.6mmol) and DMF (3 mL). The tube was sealed and stirred at 130 ℃ for 2 h; it was then poured into water (4 mL). CollectingSolid and dried to give 5-bromo-2- (3, 3-difluoroazetidin-1-yl) pyrimidine (500mg, 73.7%) as a white solid. MH⁺296.

Step 2(2- (3- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-yl) boronic acid

To a solution of 5-bromo-2- (3- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (500mg, 1.7mmol) and triisopropylborate (0.6mL, 2.5mmol) in THF (6mL) was added n-BuLi (0.9mL, 2.4M in hexane, 2.2mmol) dropwise at-78 ℃. The mixture was stirred at-78 ℃ for 2h, then quenched with water and allowed to warm to room temperature. The reaction was concentrated and the aqueous residue was extracted with ether (2 × 20 mL). The aqueous layer was adjusted to pH 6 with 1N HCl and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated to give the title product (320mg, 72.3%) as a white solid. MH⁺260.

Step 32 '- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

(2- (3- (trifluoromethyl) pyrrolidin-1-yl) pyrimidin-5-yl) boronic acid (320.0mg, 1.2mmol), 2-chloropyrimidin-4-amine (158.1mg, 1.2mmol), Pd (PPh)₃)₂Cl₂(86.0mg, 0.1mmol) and Na₂CO₃A mixture of (260.0mg, 2.5mmol) in 1, 4-dioxane (5mL) and water (1mL) was degassed with nitrogen and stirred at 90 ℃ overnight. The reaction was cooled to room temperature and EA was poured in. The organic phase was washed with water and brine, washed with Na₂SO₄Dried and concentrated. The residue was dissolved in ether. The insoluble residue was removed by filtration and the filtrate was concentrated to give 2'- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-amine (200mg, 52.6%) as whiteA colored solid. MH⁺311.

Preparation of 172-chloro-N- (2'- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidin ] -4-yl) acetamide

To 2'- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine at 0 ℃]A solution of (e) -4-amine (93mg, 0.3mmol) in DMF (2mL) was added dropwise 2-chloroacetyl chloride (0.04mL, 0.45 mmol). The reaction was stirred at room temperature for 2h, then EA was poured in. The organic phase was extracted with water and brine, and Na₂SO₄Drying and concentrating to obtain 2-chloro-N- (2'- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (100mg, 86.4%) as a yellow solid. MH⁺387.

Preparation of 182- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine

Step 12- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-ylboronic acid

To a solution of 5-bromo-2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidine (550mg, 1.95mmol) and triisopropylborate (383mg, 2.74mmol) in THF (5mL) was added n-BuLi (1.1mL, 2.4M in hexanes) dropwise at-78 ℃. The reaction was stirred at-78 ℃ for 2 h. The reaction was quenched with water (5mL) and allowed to warm to room temperature. The reaction was concentrated and the aqueous residue was extracted with ether (2 × 2 mL). The pH of the aqueous layer was adjusted to 5 with 1N HCl and extracted with EA (3X 5 mL). The combined organic phases are washed with Na₂SO₄Dried and concentrated to give 2- (3- (trifluoromethyl) azepineCyclobutan-1-yl) pyrimidin-5-ylboronic acid (450mg, 93.7%) as an off-white solid. MH⁺248.

Step 22- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine

To 2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-ylboronic acid (450mg, 1.82mmol), 2-chloropyrimidin-4-amine (213mg, 1.65mmol) and saturated Na₂CO₃(2.5mL) to a mixture in dioxane (10mL) was added Pd (PPh)₃)₂Cl₂(58mg, 0.08mmol) and degassed three times with nitrogen. The reaction was stirred at 90 ℃ overnight. The reaction was cooled to room temperature and filtered through celite. The filtrate was extracted with EA (2X 4 mL). The combined organic phases are washed with Na₂SO₄Dried and concentrated. The residue was purified by chromatography, using PE: acetone (3:1) was eluted to give 2- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine (350mg, 71.4%) as a white solid. MH⁺297.

Preparation of 192-chloro-N- (2- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-yl) acetamide

To a solution of 2- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-amine (100mg, 0.34mmol) in DMF (2mL) was added 2-chloroacetyl chloride (0.045mL, 0.51mmol) dropwise at 0 ℃. The mixture was stirred at room temperature for 2h, then EA was poured in. The organic phase was extracted with water and brine, and Na₂SO₄Drying and concentrating to obtain 2-chloro-N- (2'- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (70mg, 56%) as a yellow oil. MH⁺373.1.

Preparation of 202 '- (4, 4-difluoropiperidin-1-yl) -2,5' -bipyrimidin-4-amine

The reaction mixture was washed with (2- (4, 4-difluoropiperidin-1-yl) pyrimidin-5-yl) boronic acid (142mg, 0.58mmol), 2-chloropyrimidin-4-amine (68.5mg, 0.53mmol), Pd (PPh3)₂Cl₂(37.3mg，0.05mmol)、K₂CO₃A mixture of (146.8mg, 1.06mmol), 1, 4-dioxane (3mL) and water (0.5mL) was degassed with nitrogen and stirred at 90 ℃ for 2 hours. The resulting mixture was cooled to room temperature and poured into EA. The organic layer was separated, washed with water and brine, and dried over anhydrous Na₂SO₄Dried and concentrated. The residue was purified by column chromatography eluting with DCM/MeOH (50:1) to give 2'- (4, 4-difluoropiperidin-1-yl) - [2,5' -bipyrimidine]-4-amine (15mg, 10%) as a white solid. MH⁺293.

Preparation of 21(S) -2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-amine

Step 1(S) -5-bromo-2- (2-methylpiperidin-1-yl) pyrimidine

To a solution of 5-bromo-2-chloropyrimidine (1.41g, 7.35mmol) in DMF (20mL) was added (S) -2-methylpiperidine (800mg, 8.08mmol) and K₂CO₃(1.52g, 11.03 mmoL). The reaction was stirred at rt overnight. The reaction was poured into ice water and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated. The residue was purified by chromatography, using PE: EA elution (80:1) gave the title product (1.07g, 56.9%) as a white solid。MH⁺240.

Step 2(S) -2- (2-methylpiperidin-1-yl) pyrimidin-5-ylboronic acid

To a solution of (S) -5-bromo-2- (2-methylpiperidin-1-yl) pyrimidine (1.07g, 4.2mmol) and triisopropylboronic acid ester (1.1g, 5.87mmol) in THF (20mL) was added n-BuLi (5.25mL, 1.6M in hexane, 8.4mmol) dropwise at-70 ℃. The reaction was stirred at-70 ℃ for 3h and then quenched with water. The reaction was concentrated and the aqueous residue was extracted with ether (2 × 20 mL). The aqueous phase was adjusted to pH 6 with 1N HCl and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried, and concentrated to give the title product (900mg, 96%) as a white solid, which was used directly without purification. MH⁺222.

Step 3(S) -2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-amine

Mixing (S) -2- (2-methylpiperidin-1-yl) pyrimidin-5-ylboronic acid (752mg, 3.4mmol), 4-amino-2-chloropyrimidine (400mg, 3.09mmol), Pd (PPh)₃)₂Cl₂(216.0mg, 0.3mmol) and Na₂CO₃A mixture of (655mg, 6.18mmol) in 1, 4-dioxane (10mL) and water (2.5mL) was degassed with nitrogen and stirred at 80 ℃ for 2 hours. The reaction was cooled to room temperature and partitioned between EA (20mL) and water (15 mL). The organic phase was washed with water and brine, washed with Na₂SO₄Dried and concentrated. The residue was dissolved in ether. The insoluble residue was removed by filtration and the filtrate was concentrated to give (S) -2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-amine (682mg, 81.8%) as a white solid. MH⁺271.

Preparation of 22(S) -2-chloro-N- (2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-yl) acetamide

To a solution of (S) -2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-amine (400mg, 1.48mmol) in DMF (8mL) at 0 deg.C was added 2-chloroacetyl chloride (0.17mL, 2.24mmol) dropwise. The reaction was stirred at room temperature overnight, then poured into ice-water and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated to give the desired product (510mg, 99%) as a yellow slurry. MH⁺347.

Preparation of 232-chloro-N- (2'- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidinyl ] -4-yl) acetamide (LJ-262-64)

To 2'- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine at 0 DEG C]A solution of-4-amine (400mg, 1.48mmol) in DMF (5mL) was added 2-chloroacetyl chloride (251mg, 2.22mmol) dropwise. After stirring overnight at room temperature, the reaction mixture was distinguished by EA and water. The organic phase was washed with water and brine, washed with Na₂SO₄Drying and concentrating to obtain 2-chloro-N- (2'- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (500mg, 97.4%) as a yellow solid. MH⁺347.

Preparation of 24(S) -2'- (2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine

Step 1(S) -2- (2-Methylpyrrolidin-1-yl) pyrimidin-5-ylboronic acid

To a solution of (S) -5-bromo-2- (2-methylpyrrolidin-1-yl) pyrimidine (7g, 30.8mmol, prepared using the method described in WO 2013/023102) and triisopropylboronic acid ester (8.12g, 43.2mmol) in THF (70mL) was added n-BuLi (28.9mL, 1.6M in hexanes, 46.3mmol) dropwise at-70 ℃. The reaction was stirred at-70 ℃ for 3 h; it was then quenched with water. The reaction was concentrated and the aqueous residue was extracted with ether (2 × 20 mL). The aqueous layer was adjusted to pH 6 with 1N HCl and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated to give the title product (4.8g, 75.3%) as a white solid. MH⁺208.

Step 2(S) -2'- (2-methylpyrrolidin-1-yl) -2,5' -bipyrimidine-4-amine

(S) -2- (2-Methylpyrrolidin-1-yl) pyrimidin-5-ylboronic acid (2.53g, 12.23mmol), 4-amino-2-chloropyrimidine (1.44g, 11.12mmol), Pd (PPh)₃)₂Cl₂(432.0mg, 0.6mmol) and Na₂CO₃A mixture of (2.35g, 22.24mmol) in 1, 4-dioxane (40mL) and water (10mL) was degassed with nitrogen and stirred at 80 ℃ for 2 hours. The reaction was cooled to room temperature and EA was poured in. The organic phase was washed with water and brine, washed with Na₂SO₄Dried and concentrated. The residue was poured into diethyl ether. The insoluble residue was removed by filtration and the filtrate was concentrated to give (S) -2'- (2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine (1.5g, 54%) as a white solid. MH⁺257.

Preparation of 25(S) -2-chloro-N- (2'- (2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidinyl ] -4-yl) acetamide

To (S) -2'- (2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine at 0 DEG C]A solution of-4-amine (256mg, 1mmol) in DMF (5mL) was added 2-chloroacetyl chloride (170mg, 1.5mmol) dropwise. After stirring overnight at room temperature, the reaction mixture was partitioned between EA and water. The organic layer was washed with water and brine, and Na₂SO₄Drying and concentrating to obtain (S) -2-chloro-N- (2'- (2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (280mg, 84.1%) as a yellow solid. MH⁺333.

Preparation of 262-chloro-N- (2'- ((S) -2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide

To a solution of (S) -2'- (2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine (800mg, 3.12mmol) in DMF (15mL) was added 2-chloropropionyl chloride (0.33mL, 3.43mmol) dropwise at 0 ℃. The reaction was stirred at room temperature overnight. The reaction was poured into ice water and extracted with EA (3 × 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated. The crude product was purified by chromatography, eluting with PE: EA (5:1) was eluted to give 2-chloro-N- (2'- ((S) -2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide (600mg, 56%) as a viscous oil. MH⁺347.

Preparation 272- (3-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid

3-methyl-1H-purine-2, 6(3H,7H) -dione (15g, 90mmol), K₂CO₃A mixture of (13.73g, 99mmol), DMF (451mL) and ethyl 2-chloroacetate (9.62mL, 90mmol) was heated at 90 ℃ for 0.5 h. The reaction was cooled to room temperature and water was added(450 mL). To the stirred solution was added LiOH (4.32g, 181mmol) in water (100 mL). The reaction was stirred at room temperature for 1 hour. The reaction was adjusted to pH 4 with 6N HCl. The precipitate formed was collected and dried to give 2- (3-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid (18,670g, 92%) as a white powder.¹H NMR(DMSO-d₆)13.51(s，1H)，8.01(s，1H)，5.03(s，2H)，3.36(s，3H).

Preparation of 282- (1-methyl-3-methyl-d 3-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid

Step 12- (1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid tert-butyl ester

To a solution of 1-methylxanthine (9.049g, 54.4mmol) and potassium carbonate (8.258g, 59.8mmol) in DMF (200mL) was added tert-butyl 2-bromoacetate (8.03mL, 54.4mmol) dropwise, the reaction was stirred at 90 ℃ for 1h and cooled to room temperature, the mixture was poured into water and acidified to pH 4 with HCl (6N, aq.) the mixture was then extracted with EA (200mL × 3), the combined organic layers were washed with water (200mL), washed with Na₂SO₄Dried and concentrated in vacuo. The crude product was purified by chromatography using a MaOH: DCM (2: 100) was eluted to give tert-butyl 2- (1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetate (6.539g, 43%) as a yellow solid. MH⁺281.

Step 22- (1-methyl-3-methyl-d 3-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid tert-butyl ester

To a solution of tert-butyl 2- (1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetate (1.158g, 4.13mmol) in DMF (20.66mL) at room temperature was added K₂CO₃(0.857g, 6.20mmoL) and iodomethane-D3 (0.334mL, 5.37 mmoL). The reaction was heated at 65 ℃ for 2 hours. Additional methyl iodide-D3 (0.2equiv) was added and heating continued for an additional 1 h. The reaction was cooled to room temperature, diluted with water and extracted three times with EA. The combined organic phases were washed with brine, over MgSO₄Dried and concentrated. The residue was purified by chromatography (0-100% EA: hexane) to give tert-butyl 2- (1-methyl-3-methyl-d 3-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetate (1.35g, inpure). MH⁺298. Impure product was used without further purification.

Step 32- (1-methyl-3-methyl-d 3-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid

A solution of tert-butyl 2- (1-methyl-3-methyl-d 3-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetate (1.35g, 4.54mmol) in DCM (27.2mL) was treated with TFA (18.16 mL). The reaction was stirred at room temperature for 2 hours. The reaction was concentrated to an oil, which was used without purification. MH⁺242.

Preparation of 292 '- (6, 6-difluoro-3-azabicyclo [3.1.0] hex-3-yl) - [2,5' -bipyrimidine ] -4-amine

Step 13- (5-Bromopyrimidin-2-yl) -6, 6-difluoro-3-azabicyclo [3.1.0] hexane

5-bromo-2-chloropyrimidine (748.5mg, 3.9mmol), 6-difluoro-3-azabicyclo [3.1.0] was added to a closed tube]Hexane hydrochloride (604.6mg, 3.9mmol), K₂CO₃(1.1g, 7.8mmol) and NMP (3 mL). The mixture was stirred at 130 ℃ for 3 h; it was then cooled to room temperature and poured into water (4 mL). The solid was collected by filtration and dried in vacuo to give 3- (5-bromopyrimidin-2-yl) -6, 6-difluoro-3-azabicyclo [3.1.0]Hexane (1.0g, 93.2% yield) as a white solid. MH⁺276.

Step 2(2- (6, 6-difluoro-3-azabicyclo [3.1.0] hex-3-yl) pyrimidin-5-yl) boronic acid

To 3- (5-bromopyrimidin-2-yl) -6, 6-difluoro-3-azabicyclo [3.1.0] at-78 deg.C]Hexane (1.1g, 4.1mmol) and (i-PrO)₃A solution of B (1.4mL, 6.2mmol) in THF (20mL) was added n-BuLi (3.9mL, 1.6M in hexanes, 6.2mmol) dropwise. The reaction was stirred at-78 ℃ for 2 h; it was then quenched with water and warmed to room temperature. The solvent was removed under reduced pressure and the aqueous layer was washed with ether (2 × 50 mL). The aqueous layer was then adjusted to pH 6 with 1N HCl and extracted with EA (3 × 50 mL). The combined organic layers were washed with brine, washed with Na₂SO₄Drying and concentrating to obtain 2- (6, 6-difluoro-3-azabicyclo [ 3.1.0)]Hex-3-yl) pyrimidin-5-yl) boronic acid (700mg, 72.6% yield) as a white solid.

Step 32 '- (6, 6-difluoro-3-azabicyclo [3.1.0] hex-3-yl) - [2,5' -bipyrimidine ] -4-amine

Reacting (2- (6, 6-difluoro-3-azabicyclo [3.1.0]]Hex-3-yl) pyrimidin-5-yl) boronic acid (241.0mg, 1.0mmol), 2-chloropyrimidin-4-amine (129.0mg, 1.0mmol), Pd (PPh)₃)₄(57.8mg，0.05mmol) and K₂CO₃A mixture of (276.4mg, 2.0mmol) in 1, 4-dioxane (5mL) and water (1mL) was degassed with nitrogen and purged three times. The reaction was heated to 90 ℃ while stirring for 3 hours. The resulting mixture was cooled to room temperature and poured into EA. The organic phase was separated, washed with water and brine, and Na₂SO₄Dried and concentrated. The residue was purified by chromatography, eluting with DCM: MeOH (50:1) to give 2' - (6, 6-difluoro-3-azabicyclo [3.1.0]]Hex-3-yl) - [2,5' -bipyrimidine]4-amine (210mg, 72.3% yield) as a white solid. MH⁺291.

Preparation of 30(2R) -2- (1, 3-dimethyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid

Step 1(S) -methyl 2- (methylsulfonyloxy) propionate

A solution of methyl (S) -2-hydroxypropionate (12.379g, 119mol) and TEA (17.4mL, 125mol) in DCM (100mL) was cooled to 0 deg.C and methanesulfonyl chloride (12.4mL, 125mol) was added dropwise over 1 hour at 0 deg.C. The mixture was stirred at 20 ℃ for 1.5 hours. The resulting mixture was quenched with ice-water (100 mL). The organic layer was separated, washed with water (2 × 50mL) and brine, and Na₂SO₄Dried and concentrated to give the crude product methyl (S) -2- (methylsulfonyloxy) propionate (20.940g, 97%) as a brown oil, which was used without purification.

Step 2(R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid

To 1, 3-dimethyl-3, 4,5, 7-tetrahydro-1H-purine-2, 6-dione (4.588g, 25.5mol) and K at room temperature₂CO₃(7.038g, 51mol, 2eq) to a suspension in DMF (500mL) was added methyl(s) -2- (methylsulfonyloxy) propionate (6.953g, 38.2 mol). The mixture was stirred at room temperature overnight and then saturated NH₄And (4) quenching by Cl. The resulting mixture was extracted with DCM (3 × 300 mL). The combined organic phases were washed with water and brine, washed with Na₂SO₄Dried and concentrated. The brown oily residue (8.633g) was dissolved in dioxane (10 mL). To this solution was added 6N HCl (10mL aqueous). The mixture was refluxed for 2h, cooled to room temperature, and then concentrated to remove dioxane and most of the aqueous phase. The residue was purified by chromatography, eluting with MeOH/DCM (0-10%) to give (R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (6.015g, 93.6% yield) as a white solid.

Synthesis of Compounds of formula (I)

Example 1(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

To 2'- (2, 2-dimethylpyrrolidin-1-yl) - [2,5' -bipyrimidine at room temperature]To a mixture of (4-amine) (4.2g, 15.5mmol) and (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (3.56g, 14.1mmol) in DCM (72mL) was added HOAT (1.92g, 14.1 mmol). The reaction was cooled to 0 ℃ and pyridine (2.23g, 28.2mmol) and DIC (2.67g, 21.2mmol) were added. The reaction was warmed to 25-28 ℃ and stirred overnight. The reaction mixture was quenched with 0.5N HCl. The mixture was added dropwise to N-hexane and the precipitate formed was collected and washed with MeOH to give (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2' - (2, 2-di-N-butyl) ethyl acetateMethylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propionamide (3g, 19%).¹H NMR(CDCl₃)9.78(s，1H)，9.14(s，2H)，8.54(d，J＝5.6Hz，1H)，7.91(s，1H)，7.76(d，J＝5.6Hz，1H)，5.83(q，J＝7.2Hz 1H)，3.77(m，2H)，3.63(s，3H)，3.50(s，3H)，1.96(m，7H)，1.60(s，6H)。

Example 2(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide (Compound 2)

To a mixture of (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (2.44g, 9.67mmol) and (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine (3.3g, 10.6mmol) in DCM (48mL) was added HOAT (1.3g, 9.67mmol) at room temperature. The mixture was cooled to 0 ℃. Pyridine (1.5g, 19.3mmoL) was added dropwise over 30 minutes followed by DIC (1.8g, 14.5 mmoL). The reaction was stirred at 35 ℃ for 16 h; it was then diluted with DCM (100 mL). Saturated NH for the mixture₄Cl (50mL, pre-cooled to 0 ℃) and brine, extracted with Na₂SO₄Dried and concentrated. The residue was purified by chromatography, first with EA: PE (3:2) was eluted, then diluted with DCM: MeOH (30:1) eluted, then recrystallized from EtOH to give the title compound (4.5g, 78%) as a white solid.¹H NMR(DMSO-d₆)11.46(s，1H)，9.22(s，2H)，8.66(d，J＝5.6Hz，1H)，8.31(s，1H)，7.81(d，J＝5.6Hz，1H)，5.82(q，J＝7.2Hz 1H)，5.12(t，1H)，3.70(m，2H)，3.47(s，3H)，3.16(s，3H)，2.10(m，4H)，1.88(d，J＝7.2Hz，3H).MH⁺545. Diastereomeric excess (de): 99%.

Chiral HPLC method conditions: column: CHIRALPAK IB, 150 × 4.6mm, 5 μm; mobile phase: a: hexane (HPLC grade); b: EtOH (HPLC grade); flow rate: 0.8 mL/min; gradient: 30% B for 25 minutes. As a result: the retention time of the desired diastereomer (S) was 14.16 minutes and the other isomer (R) was 9.66 minutes.

Example 3(R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

The mother liquor from example 2 after recrystallization in EtOH was concentrated. The residue was loaded for chiral preparative HPLC purification to give (R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propionamide as a white solid.¹H NMR(DMSO-d₆)11.48(s，1H)，9.25(s，2H)，8.69(d，J＝5.6Hz，1H)，8.33(s，1H)，7.83(d，J＝5.6Hz，1H)，5.81(q，J＝7.2Hz 1H)，5.13(t，1H)，3.71(m，2H)，3.69(s，3H)，3.17(s，3H)，2.14(m，4H)，1.88(d，J＝7.2Hz，3H).MH⁺545.de：99％。*

Chiral HPLC method conditions: column: CHIRALPAK IB, 150 × 4.6mm, 5 μm; mobile phase: a: hexane (HPLC grade); b: EtOH (HPLC grade); flow rate: 0.8 mL/min; gradient: 30% B for 25 minutes.

Example 4(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2- ((R) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

The title compound was prepared using the method of example 2 to give a white solid.¹H NMR(DMSO-d₆)11.46(s，1H)，9.25(s，2H)，8.70(d，J＝5.6Hz，1H)，8.33(s，1H)，7.83(d，J＝5.6Hz，1H)，5.81(q，J＝7.2Hz 1H)，5.14(t，1H)，3.67(m，2H)，3.32(s，3H)，3.14(s，3H)，2.10(m，4H)，1.86(d，J＝7.2Hz，3H).MH⁺545。de 98％*

Chiral HPLC method conditions: column: CHIRALPAK IB, 150 × 4.6mm, 5 μm; mobile phase: a: hexane (HPLC grade); b: EtOH (HPLC grade); flow rate: 0.8 mL/min; gradient: 30% B for 25 minutes.

Example 5(S) -2- (1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2' - ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

To a mixture of (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid (90.4mg, 0.38mmol) and (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine (119mg, 0.38mmol) in DCM (2mL) was added HOAT (104mg, 0.76mmol) at room temperature. The reaction was cooled to 0 ℃. Pyridine (91mg, 1.15mmoL) was added dropwise followed by DIC (97mg, 0.77mmoL) dropwise. The reaction was stirred at 20 ℃ for 16 h; it was then diluted with DCM (10 mL). Saturated NH for the mixture₄Cl and brine, washed with Na₂SO₄Dried and concentrated. The residue was purified by chromatography with DCM: MeOH (30:1) eluted to give the title product (83g, 41%) as a white solid.¹H NMR(DMSO-d₆)11.94(s，1H)，11.44(s，1H)，9.25(s，2H)，8.71(d，J＝5.6Hz，1H)，8.23(s，1H)，7.84(d，J＝5.6Hz，1H)，5.78(q，J＝7.2Hz 1H)，5.14(t，1H)，3.71(m，2H)，3.12(s，3H)，2.20(m，4H)，1.84(d，J＝7.2Hz，3H).MH⁺531.

Example 6(S) -2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purine-7 (6H) - Yl) -N- (2'- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

To a mixture of (S) -2- (3- (difluoromethyl) -1-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (61mg, 0.21mmol) and (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine (310mg, 0.21mmol) in DCM (1mL) was added HOAT (57mg, 0.42mmol) at room temperature. The mixture was cooled to 0 ℃. Pyridine (50mg, 0.63mmoL) was added dropwise followed by DIC (53mg, 0.42 mmoL). The reaction was stirred at 20 ℃ for 16h, then diluted with DCM (10 mL). Saturated NH for the mixture₄Cl and brine, washed with Na₂SO₄Dried and concentrated. The residue was purified by chromatography with DCM: MeOH (30:1) eluted to give the product (45.6mg, 37%) as a white solid.¹H NMR(DMSO-d₆)11.49(s，1H)，9.25(s，2H)，8.70(s，1H)，8.39(s，1H)，7.86(t，J＝5.6Hz，2H)，5.82(q，J＝8Hz 1H)，5.14(t，1H)，3.74(m，2H)，3.16(s，3H)，2.22(m，4H)，1.87(d，J＝8Hz，3H).MH⁺581。

Example 7N- (2'- (3, 3-Difluoroazetidin-1-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-di Methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide

2-chloro-N- (2' - (3, 3-difluoroazacyclo)Butane-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (50.0mg, 0.1mmol), K₂CO₃A mixture of (41.5mg, 0.3mmol), 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (26.5mg,0.1mmol) and TBAI (5.6mg, 0.01mmol) in DMF (3mL) was stirred at 90 ℃ overnight. The reaction was cooled to room temperature and diluted with EA. Water, saturated NH for organic phase₄Cl solution and brine, Na₂SO₄Dried and concentrated. The residue was purified by preparative TLC (DCM/MeOH ═ 30:1) to give N- (2'- (3, 3-difluoroazetidin-1-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide (4.0mg, 5.6%) as a white solid.¹H NMR(DMSO-d₆)11.48(s，1H)，9.26(s，2H)，8.73(d，J＝5.6Hz，1H)，8.08(s，1H)，7.83(s，1H)，5.36(s，2H)，4.60(t，J＝12.2Hz，4H)，3.46(s，3H)，3.19(s，3H).MH⁺485.1.

Example 8N- (2'- (4, 4-Difluoropiperidin-1-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide

N- (2-Chloropyrimidin-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide (87.3mg, 0.3mmol), Pd (PPh)₃)₄(28.9mg, 0.03mmol), (2- (4, 4-difluoropiperidin-1-yl) pyrimidin-5-yl) boronic acid (66.9mg, 0.3mmol) and NaHCO₃A mixture of (21.0mg, 0.3mmol) in 1, 4-dioxane (3mL) and water (0.5mL) was degassed with nitrogen and stirred at 90 ℃ overnight. The reaction was cooled to room temperature and diluted with EA. The organic phase was separated, washed with water and brine, and Na₂SO₄Dried and concentrated. The residue was purified by preparative TLC (DCM/MeOH ═ 30:1) to give N- (2'- (4, 4-difluoropiperidin-1-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purine-7(6H) -yl) acetamide (8.0mg, 6.2%) as a white solid.¹H-NMR(DMSO-d₆)11.44(s，1H)，9.23(s，2H)，8.71(d，J＝5.7Hz，1H)，8.08(s，1H)，7.81(s，1H)，5.36(s，2H)，4.05–3.97(m，4H)，3.46(s，3H)，3.19(s，3H)，2.06(dd，J＝12.5，6.6Hz，4H).MH⁺513.1.

Example 9(S) -N- (2'- (3, 3-difluoroazetidin-1-yl) -2,5' -bipyrimidin-4-yl) -2- (1,3- Dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionamide

To a mixture of (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (57mg, 0.23mmol) and 2'- (3, 3-difluoroazetidin-1-yl) -2,5' -bipyrimidin-4-amine (50mg, 0.19mmol) in DCM (3mL) was added HOAT (31mg, 0.23mmol) at room temperature. The reaction was cooled to 0 ℃ and pyridine (30mg, 0.38mmol) was slowly added dropwise followed by DIC (36g, 0.29 mmol). The reaction was warmed to room temperature, then to 30 ℃ and stirred for 18 hours. The reaction was extracted with water and then saturated NH₄And (4) extracting with Cl. Na for organic phase₂SO₄Dried and concentrated. The residue was purified by preparative HPLC (DCM/MeOH ═ 30:1) to give (S) -N- (2'- (3, 3-difluoroazetidin-1-yl) -2,5' -bipyrimidin-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionamide (10mg, 9%) as a white solid.¹H NMR(DMSO-d₆)11.51(s，1H)，9.26(s，2H)，8.72(d，1H),.8.35(s，1H)，7.86(d，1H)，5.81(q，1H)，4.60(t，4H)，3.47(s，3H)，3.17(s，3H)，1.81(d，3H).MH⁺499.

Example 10N- (2' - (3-azabicyclo [ 3.1.0)]Hex-3-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-) The content of the dimethyl-2,6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide

Reacting N- (2' - (3-azabicyclo [3.1.0]]Hex-3-yl) - [2,5' -bipyrimidine]-4-yl) -2-chloroacetamide (50.0mg, 0.16mmol), K₂CO₃A mixture of (44.3mg, 0.32mmol), 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (28.0mg, 0.16mmol) and TBAI (5.9mg, 0.02mmol) in DMF (3mL) was stirred at 90 ℃ overnight. The reaction was cooled to room temperature and diluted with EA. Water, saturated NH for organic phase₄Cl and brine extraction, Na₂SO₄Dried and concentrated. The residue was purified by preparative TLC (DCM/MeOH ═ 25: 1) to give N- (2' - (3-azabicyclo [ 3.1.0)]Hex-3-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide (2.0mg, 2.9%) as a white solid.¹H NMR(DMSO-d₆)11.41(s，1H)，9.17(s，2H)，8.68(d，J＝5.7Hz，1H)，8.08(s，1H)，7.76(s，1H)，5.36(s，2H)，3.87(d，J＝11.5Hz，2H)，3.59(s，2H)，3.51(s，3H)，3.19(s，3H)，0.82(m，3H)，0.18(s，1H).MH⁺475.

Example 11 2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl)

2-chloro-N- (2'- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (100.0mg, 0.26mmol), K₂CO₃A mixture of (53.7mg, 0.39mmol), 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (46.6mg, 0.26mmol) and TBAI (9.7mg, 0.03mmol) in DMF (3mL) at 90Stir at c overnight. The reaction was cooled to room temperature and diluted with EA. Water, saturated NH for organic phase₄Cl and brine, washed with Na₂SO₄Dried and concentrated. The residue was purified by preparative TLC (DCM/MeOH ═ 30:1) to give 2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (3- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (4.0mg, 2.9%) as a white solid.¹HNMR(DMSO-d₆)11.42(s，1H)，9.22(s，2H)，8.70(d，J＝5.7Hz，1H)，8.08(s，1H)，7.79(s，1H)，5.36(s，2H)，3.91(dd，J＝11.7，8.1Hz，1H)，3.77(s，1H)，3.72–3.61(m，2H)，3.46(s，3H)，3.19(s，3H)，2.35–2.29(m，1H)，2.14(dd，J＝12.9，7.3Hz，1H)，2.00(d，J＝7.9Hz，1H).MH⁺531.

Example 12N- (2'- (3, 3-Difluoroazetidin-1-yl) - [2,5' -bipyrimidine]-4-yl) -2- (1, 3-di Methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide

2-chloro-N- (2- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-yl) acetamide (70mg, 0.19mmol), K₂CO₃A mixture of (51.9mg, 0.38mmol), 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (34.2mg,0.19mmol) and TBAI (11.2mg, 0.019mmol) in DMF (2mL) was stirred at 50 ℃ for 2H. The reaction was cooled to room temperature and diluted with EA. The organic phase was washed with water and brine, washed with Na₂SO₄Dried and concentrated. The crude product was triturated with MeOH, filtered, and dried to give the title product 2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydropyrurin-7-yl) -N- (2- (2- (3- (trifluoromethyl) azetidin-1-yl) pyrimidin-5-yl) pyrimidin-4-yl) acetamide (7.0mg, 5.6%) as a white solid.¹H NMR(DMSO-d₆)9.22(s，2H)，8.71(d，J＝6.0Hz，1H)，8.08(s，1H)，7.81(d，J＝4.8Hz，1H)，7.83(s，1H)，5.36(s，2H)，4.39-4.44(m，2H)，4.12-4.17(m，2H)，3.76–3.78(m，1H)，3.45(s，3H)，3.21(s，3H).MH⁺517.

Example 13(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (3- (trifluoromethyl) azetidin-1-yl) -2,5' -bipyrimidin-4-yl) propanamide

To a mixture of (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (85mg, 0.34mmol) and 2'- (3- (trifluoromethyl) azetidin-1-yl) -2,5' -bipyrimidin-4-amine (100mg, 0.34mmol) in DCM (3mL) was added HOAT (46mg, 0.34mmol) at room temperature. The reaction was cooled to 0 ℃. Pyridine (54mg, 0.68mmol) and DIC (64mg, 0.51mmol) were added dropwise slowly one after the other. The reaction was warmed to 30 ℃ and stirred for 18 hours. The reaction was performed using water (5mL) and saturated NH₄Cl (5 mL). Na for organic phase₂SO₄Dried and concentrated. The residue was purified by chromatography, using PE: EA eluted (1:1) to give (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (3- (trifluoromethyl) azetidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide (60mg, 33.3%) as a white solid.¹H NMR(DMSO-d₆,)11.49(s，1H)，9.22(s，2H)，8.70(d，J＝6.0Hz，1H)，8.34(s，1H)，7.84(d，J＝6.0Hz，1H)，5.81(q，J＝8.0Hz 1H)，4.42(t，J＝8.8Hz，2H)，4.13-4.17(m，2H)，3.75-3.78(m，1H)，3.45(s，3H)，3.18(s，3H)，1.87(d，J＝8.8Hz，3H)，MH⁺531.

Example 14(S) -N- (2'- (4, 4-difluoropiperidin-1-yl) -2,5' -bipyrimidin-4-yl) -2- (1, 3-dimethyl Yl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionamide

To a solution of (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (50mg, 0.2mmol) and 2'- (4, 4-difluoropiperidin-1-yl) -2,5' -bipyrimidin-4-amine (58mg, 0.2mmol) in DCM (3mL) at room temperature was added HOAT (27mg, 0.2 mmol). The reaction was cooled to 0 ℃. Pyridine (32mg, 0.4mmol) and DIC (38mg, 0.3mmol) were added dropwise slowly one after the other. The reaction was warmed to 30 ℃ for 18 hours. The reaction was performed using water (5mL) and saturated NH₄Cl (5 mL). Na for organic phase₂SO₄Dried and concentrated. The residue was purified by preparative TLC run on PE: EtOAc (1:1) was eluted to give (S) -N- (2'- (4, 4-difluoropiperidin-1-yl) -2,5' -bipyrimidin-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionamide (15mg, 14.3%) as a white solid.¹H NMR(DMSO-d₆,)9.22(s，2H)，8.70(d，J＝5.6Hz，1H)，8.34(s，1H)，7.83(d，J＝5.6Hz，1H)，5.81(q，J＝6.8Hz 1H)，4.02(t，J＝5.6Hz，4H)，3.54(s，3H)，3.21(s，3H)，2.01-2.10(m，4H)，1.87(d，J＝6.8Hz，3H)。MH⁺527.2.

Example 15(2S) -N- (2' - (3-azabicyclo [ 3.1.0)]Hex-3-yl) -2,5' -bipyrimidin-4-yl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionamide

The title compound was prepared according to the method of example 14 with 20.7% yield as a white solid.¹H NMR(DMSO-d₆)11.42(s，1H)，9.16(s，2H)，8.66(d，J＝5.6Hz，1H)，8.33(s，1H)，7.79(d，J＝5.6Hz，1H)，5.81(q，J＝7.2Hz 1H)，3.86(d，J＝11.2Hz，2H)，3.57(d，J＝11.6Hz，2H)，3.41(s，3H)，3.17(s，3H)，1.86(d，J＝7.6Hz，3H)，1.70(t，J＝3.6Hz，2H)，0.75-0.80(m，1H)，0.15-0.18(m，1H).MH⁺489.

Example 16(2S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (3- (trifluoromethyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide

The title compound was prepared according to the method of example 14 with 39.1% yield as a white solid.¹H NMR(DMSO-d₆)11.46(s，1H)，9.22(s，2H)，8.69(d，J＝5.6Hz，1H)，8.34(s，1H)，7.81(d，J＝5.6Hz，1H)，5.82(q，J＝7.2Hz，1H)，3.88-3.94(m，1H)，3.69-3.78(m，1H)，3.62-3.67(m，2H)，3.50(s，3H)，3.43-3.49(m，1H)，3.20(s，3H)，2.30-2.35(m，2H)，2.12-2.17(m，1H)，1.87(t，J＝7.2Hz，3H).MH⁺545.2。

Example 17(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-yl) acetamide

To a solution of 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (208mg, 1.16mmol) in DMF (8mL) was added K₂CO₃(320mg, 2.32mmoL) and (S) -2-chloro-N- (2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-yl) acetamide (400mg, 1.16 mmoL). The reaction was stirred at room temperature for 1 h; then ice-water was poured in. The solid precipitate was collected and triturated with MeOH to give (S) -2- (1, 3-dimethylYl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-yl) acetamide (220mg, 45.5%) as a white solid.¹H NMR(DMSO-d₆)11.38(s，1H)，9.17(s，2H)，8.68(d，J＝5.6Hz，1H)，8.07(s，1H)，7.75(s，1H)，5.36(s，2H)，5.12-5.14(m，1H)，4.67-4.71(m，1H)，3.45(s，3H)，3.19(s，3H)，3.00(t，J＝12Hz，1H)，1.57-1.75(m，5H)，1.22-1.40(m，1H)，1.19(d，J＝8Hz，3H).MH⁺491.

Example 182- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2' - (2, 2-dimethylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) acetamide

The title compound was prepared using the method of example 17 with 43.1% yield as a white solid.¹H NMR(CDCl₃)9.59(s，1H)，9.19(s，2H)，8.60(d，J＝5.2Hz，1H)，7.77(s，2H)，5.18(s，2H)，3.77(t，J＝6.4Hz，2H)，3.64(s，3H)，3.47(s，3H)，1.94-1.98(m，4H)，1.604(s，6H)。MH⁺491.

Example 19(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) acetamide

The title compound was prepared using the method of example 17 with 46.7% yield as a white solid.¹H NMR(DMSO-d₆)11.38(s，1H)，9.18(s，2H)，8.68(d，J＝5.2Hz，1H)，8.09(s，1H)，7.78(s，1H)，5.37(s，2H)，4.32(t，J＝5.2Hz，1H)，3.54-3.68(m，2H)，3.47(s，3H)，3.20(s，3H)，1.71-2.11(m，4H)，1.24(d，J＝6.4Hz，3H)。MH⁺477.

Example 20 2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2- ((S) -2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propanamide

To a solution of 1, 3-dimethyl-1H-purine-2, 6(3H,7H) -dione (312mg, 1.73mmol) in DMF (8mL) was added K₂CO₃(477mg, 3.46mmol), 2-chloro-N- (2'- ((S) -2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide (600mg, 1.73 mmol). The reaction was stirred at room temperature for 1 h; then, ice water was poured in. The solid precipitate was collected and washed with MeOH to give 2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide (130mg, 16%) as a white solid.¹H NMR(DMSO-d₆)11.43(s，1H)，9.18(s，2H)，8.67(d，J＝5.6Hz，1H)，8.34(s，1H)，7.78(d，J＝5.6Hz，1H)，5.82(d，J＝7.2Hz，1H)，4.31(t，J＝5.2Hz，1H)，3.53-3.68(m，2H)，3.46(s，3H)，3.19(s，3H)，1.71-2.19(m，7H)，1.23(d，J＝6.4Hz，3H).MH⁺491.

Example 21(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

The mixture of diastereomeric products from example 20 was separated by supercritical fluid chromatography using a chiral column to give (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propionamide as a colorless liquid (retention time 6.0 min). MH⁺491.

Chiral HPLC separation conditions: 2.1X25.0cm ChiralPak IC from Chiral Technologies (West Chester, Pa.) using a mixture of 50% supercritical carbon dioxide and 50% 2:1:1 DCM: hexane: isopropanol at a flow rate of 80 mL/min.

Example 22(R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

The mixture of diastereomeric products from example 20 was separated by supercritical fluid chromatography using a chiral column to give (R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propionamide as a colorless liquid (retention time 4.8 min). MH⁺491.

Chiral HPLC separation conditions: 2.1X25.0cm ChiralPak IC from Chiral Technologies (West Chester, Pa.) using a mixture of 50% supercritical carbon dioxide and 50% 2:1:1 DCM: hexane: isopropanol at a flow rate of 80 mL/min.

Example 232- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2' - ((S) -2-methylpiperidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide

The title product was prepared using the methods of preparation 26 and example 20 with 28.3% yield as a white solid.¹HNMR(CDCl₃)9.73(d，J＝6.4Hz，1H)，9.17(s，2H)，8.56(d，J＝5.6Hz，1H)，7.86(d，J＝7.6Hz，1H)7.79(d，J＝4Hz，1H)，5.76(d，J＝6.4Hz，1H)，4.76(d，J＝12.4Hz，1H)，3.60(s，3H)，3.47(s，3H)，3.02(t，J＝12Hz，1H)，1.90(d，J＝7.2Hz，3H)，1.48-1.78(m，6H)，1.23(d，J＝6.4Hz，3H)。MH⁺505.

Example 24(S) -2- (3-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2' - (2- Methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide

(S) -2'- (2-methylpyrrolidin-1-yl) -2,5' -bipyrimidin-4-amine (249mg, 0.971mmol), 2- (3-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid (436mg, 1.943mmol), and EDC (745mg, 3.89mmol) were added to pyridine (2.43 mL). The reaction was stirred at room temperature for 2 days. The reaction was concentrated and the residue was purified by chromatography to give (S) -2- (3-methyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide (50mg, 11%) as a colourless liquid. MH⁺463。

Example 25N- {2- [2- ((2S) -2-Methylpyrrolidinyl) pyrimidin-5-yl]Pyrimidin-4-yl } -2- (1-methyl-) 3-methyl-d 3-2, 6-dioxo (1,3, 7-trihydropurin-7-yl)) acetamide

2- (1-methyl-3-methyl-d 3-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid (0.200g, 0.829mmol), (S) -2'- (2-methylpyrrolidin-1-yl) - [2,5' -bipyrimidine) was added at room temperature]A mixture of-4-amine (0.213g, 0.829mmol) and EDC (0.318g, 1.658mmol) was dissolved in pyridine (4.15 mL). The reaction was stirred overnight and then diluted with water. The reaction was extracted three times with EA. The organic phase is MgSO₄Dried and concentrated. The residue is purified by chromatography to give N- {2- [2- ((2S) -2-methylpyrrolidinyl) pyrimidin-5-yl]Pyrimidin-4-yl } -2- (1-methyl-3-methyl-d 3-2, 6-dioxo (1,3, 7-trihydropurin-7-yl)) acetamide (66.1mg 17%) as a colorless liquid. MH⁺480.

Example 26((2S) -N- (2- (2- (3-azabicyclo [3.1.0]]Hex-3-yl) pyrimidin-5-yl) thiazol-4-yl) - 2- (3-methyl-2, 6-dioxo-1- (2-oxobutyl) -2, 3-dihydro-1H-purin-7 (6H) -yl) propionamide

The title compound was prepared using the method of example 14 in 8% yield as a white solid.¹H NMR(DMSO_d6)11.47(s，1H)，9.20(s，2H)，8.69(d，J＝5.1Hz，1H)，8.35(s，1H)，7.82(d，J＝5.1Hz，1H)，5.83(s，1H)，4.01(d，J＝12.0Hz，2H)，3.87(d，J＝11.0Hz，2H)，3.46(s，3H)，3.18(s，3H)，2.71(d，J＝11.5Hz，2H)，1.87(d，J＝6.6Hz，3H).MH⁺525.

Example 27(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) acetamide

To 2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetyl chloride (250mg, 0.97mmol) and (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine at 0 deg.C]To a mixture of (4-amine) (332mg, 1.07mmol) in THF (10mL) was added DIPEA (0.509mL, 2.92 mmol). The reaction was stirred at room temperature for 18h, then refluxed for 24 h. The mixture was cooled to room temperature, diluted with water (75mL) and extracted with EA (50mL X3). The combined organic layers were over MgSO₄Dried and concentrated. The residue was purified by preparative TLC (eluting with 100% EA) to give the title compound (17mg, 3.3%) as a white solid.¹H NMR(CDCl₃)9.6(brd s，1H)，9.26(s，2H)，8.61(d，J＝4Hz，1H)，7.96-7.8(m，1H)，7.75(s，1H)，5.22-5.06(m，3H)，5.12(t，1H)，3.89-3.75(m，2H)，3.62(s，3H)，3.46(s，3H)，2.35-2.22(m，2H)，2.18-2.04(m，2H).MH⁺531.

Example 28(R) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((R) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-yl) propanamide

The title compound was prepared using the method of example 2 to give the title compound as a white solid.¹H NMR(DMSO-d₆)11.46(s，1H)，9.22(s，2H)，8.66(d，J＝5.6Hz，1H)，8.31(s，1H)，7.81(d，J＝5.6Hz，1H)，5.82(q，J＝7.2Hz 1H)，5.12(t，1H)，3.70(m，2H)，3.47(s，3H)，3.16(s，3H)，2.10(m，4H)，1.88(d，J＝7.2Hz，3H).MH⁺545

EXAMPLE 29 Large Scale Synthesis of (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purine-7 (6H) -yl) -N- (2'- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]Process for the preparation of (4-yl) propionamide

Step 1(R) -methyl 2- (((trifluoromethyl) sulfonyl) oxy) propionate

A 50L reactor was charged with dichloromethane (30L) under nitrogen atmosphere and stirred. (R) -methyl lactate (1.44kg, 13.83mol) was added followed by 2, 6-lutidine (1.56kg, 14.56 mol). The stirred mixture was cooled to-5 to 5 ℃ using a dry ice/acetone bath. The reactor was carefully filled with trifluoromethanesulfonic anhydride (3.9kg, 13.83mol) using a peristaltic pump, while maintaining an internal temperature of-5 to 5 ℃. This addition requires more than 1 h. After the addition was complete, the reaction was stirred for an additional 1h while maintaining the temperature at 0 to 5 ℃. The reaction was carefully quenched with deionized water (10L) and vigorously stirred for an additional 1 minute. The stirring was stopped and the phases were allowed to separate. The bottom (dichloromethane) layer containing the product was transferred to a holding vessel while the reactor was washed successively with acetone (2x 10L) and dichloromethane (2x 10L). The intermediate triflate was used directly without purification.

Step 2(S) -methyl 2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionate

The dichloromethane product solution of R-methyl 2- (((trifluoromethyl) sulfonyl) -oxy) propionate was back-filled into a clean 50L reactor and the mixture was placed under a nitrogen atmosphere. The mixture was cooled to 0 to 5 ℃ with stirring using a dry ice/acetone bath. Theophylline (2.0kg, 11.1mol) was added to the reactor during cooling. By a peristaltic pump1,1,3, 3-tetramethylguanidine (1.34kg, 11.66mol) was slowly added to the reactor while maintaining the internal temperature below 10 ℃. After the addition was complete, the reaction was stirred for at least 1h while maintaining the reaction temperature at 0 to 10 ℃. An aliquot taken after 30 minutes was tested by HPLC and the reaction was confirmed to be complete. Ice-cooled 0.2N HCl (10L) was added to the reactor to quench the reaction. The mixture was stirred vigorously for 1-2 min. Stirring was stopped and the phases separated. The bottom dichloromethane product layer was transferred to the holding vessel. The upper aqueous layer was removed and discarded. The bottom dichloromethane layer was added back to the reactor and was treated with 5% NaHCO₃Aqueous (10L) solution was extracted while stirring vigorously for 1-2 min. Stirring was stopped and the phases separated. The bottom product layer was transferred to a holding vessel. The upper aqueous layer was removed and discarded. The bottom dichloromethane layer was added back to the reactor. Deionized water (10L) was added to the reactor. The mixture was stirred vigorously for 1 min. Stirring was stopped and the phases separated. The bottom product layer was transferred to a holding vessel. The upper aqueous layer was removed and discarded. The product solution containing dichloromethane was transferred to a rotary evaporator and concentrated in vacuo (bath temperature 30-40 ℃) until most of the dichloromethane distilled to give crude methyl (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionate as a dark viscous syrup. HPLC analysis of the sample confirmed the product and its purity.

Step 3(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid

Methyl (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionate (7.16kg, crude syrup obtained in two batches from steps 1 and 2) was transferred to a rotary evaporation flask. Vacuum was applied and the bottle was rotated at a bath temperature of 30-40 ℃ until no more dichloromethane was distilled. Separately, a 3N aqueous HCl solution (32L, 4.5 equivalents based on 4kg theophylline) was prepared. The residue from the rotary evaporator bottle was transferred to a 50L reactor. The vial was rinsed with a small portion of 3N HCl to remove all crude ester and transferred to the reactor, and the remaining 3N HCl was added to the reactor. The reaction mixture was heated at 70-75 ℃ for at least 16 hours. The reaction state at 16h was checked by HPLC analysis of a small aliquot and was considered complete when the amount of ester was less than 10% compared to the acid product. The mixture was cooled to room temperature while stirring for at least 16 h. The product was collected on a buchner funnel and the solid was washed with ice-cold deionized water (2x 2L). The solid was dried on a vacuum funnel overnight until the mixture became a free flowing solid (2.95 Kg). The crude product was 93.8% pure by HPLC analysis.

A portion of the crude (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (1kg) was added to a 22L reactor. Deionized water (9L, 9volumes) was added to the reactor and stirring was started. The slurry was heated to 95 ℃ and held at that temperature until all solids dissolved. 40g (4% by weight) in 250mL of deionized waterA slurry of activated carbon was added to the hot mixture and stirring was continued for 1h at 90-95 ℃. The hot mixture was carefully transferred from the 22L vessel to a clean 50L reactor through a filter funnel containing a glass microfiber filter. The process was repeated twice with 1kg of crude acid, each filtration into the same 50L reactor. The reactor was cooled to below 30 ℃ while stirring. The solid was filtered and the product was washed with ice cold deionized water (2 × 2L). The product was dried on the filter funnel for at least 12H, then transferred to a vacuum oven and dried to constant weight to give (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (2.25 kg). The purified product was confirmed to be 99.31% pure by HPLC and to have an enantiomeric purity of 100% by chiral HPLC. The total yield of pure (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid was 42.4% based on theophylline starting material.

Step 4(S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine

A reaction vessel equipped with mechanical stirring, reflux condenser, nitrogen inlet, thermocouple and external heating mantle was charged with (S) -2-trifluoromethylpyrrolidine (1598g, 11.49mol), 5-bromo-2-chloropyrimidine (2000g, 10.34mol) and N, N-dimethylacetamide (9L). The stirred mixture was warmed to 50 ℃. When the mixture became a solution, diisopropylethylamine (1633g, 12.64mol) was added and the reaction temperature was increased to 120 ℃. The reaction was stirred for 24-48h until the reaction was complete as confirmed by HPLC. The reaction was cooled to not less than 70 ℃ and the contents were transferred to a second stirred vessel containing water (90L). The mixture was stirred and cooled to 20 ℃, then further cooled to 5 to 10 ℃ and held at that temperature for 2 h. The solid product was collected by filtration and washed with cold water (3 × 5L). The product was dried at 50 ℃ in vacuo to constant weight to give (S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (2898g, 94.6%) as a light brown solid which was confirmed to be-99% pure by HPLC.

Step 5(S) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine

The reaction vessel equipped with mechanical stirring, reflux condenser, nitrogen inlet, thermocouple and external heating mantle was filled with dioxane (8L) and gentle stirring was started. The reaction was charged with (S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (1600g, 5.40mol), 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (2058g, 8.11mol) and potassium acetate (1059g, 10.81 mol). Additional dioxane (17L) was added and nitrogen was bubbled through the mixture. Bis- (triphenylphosphine) palladium chloride catalyst (113.7g, 0.161mol) was added to the reaction. The reaction was heated and nitrogen was continuously bubbled through the mixture. When the reaction temperature reached 50 ℃, nitrogen was not bubbled through the mixture. While maintaining a nitrogen atmosphere, the condenser was vented. The reaction temperature was increased to 95 to 100 ℃ and maintained at this temperature until HPLC analysis indicated the reaction was complete, after about 16-24 h. The reaction was cooled to not less than 60 ℃ and transferred by peristaltic pump to a reactor containing 38 volumes of water. The transfer line was flushed with 0.25 to 1.50 volumes of dioxane. Additional water is added to the reactor as appropriate to promote crystallization of the product. The mixture was cooled to 10 ± 5 ℃ and held for at least 1 hour. The product was collected on a buchner funnel, washed with cold water (3X 2 volumes), and dried under vacuum at 50-60 ℃ until constant weight was reached. This gave (S) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine as a light brown solid, 1964 g. The solid contained 12.7% water and was about 96% pure as determined by HPLC. The yield was estimated to be 1715g, 91.4%. This material was suitable for further reaction without removing residual water.

Step 6(S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine

Wet (S) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine from step 5 (1964g) was tested for water content and stoichiometry adjusted accordingly (1715g, 5.0 mol). Dioxane (16L) was added to a reaction vessel equipped with a heating mantle, thermocouple control, nitrogen inlet, mechanical stirrer and reflux condenser. The compound (S) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (corrected to 1715g, 5.0mol), 4-amino-2-chloropyrimidine (648g, 5mol) and sodium carbonate (962g, 9.08mol) were added to the reaction vessel. Additional dioxane (8L) was added. Nitrogen was bubbled through the solution for about 30-60 minutes, and the condenser was vented. Tetrakis (triphenylphosphine) palladium (230g 0.2mol) was added and the residual catalyst was washed into a reaction vessel containing dioxane (1L). Heating was started and nitrogen bubbling was continued until the mixture reached about 50 ℃. The nitrogen line was then retracted above the surface of the solution, but maintained a nitrogen atmosphere, and the condenser was vented. The temperature was increased to 85-90 ℃ and maintained until the reaction was complete (1-4h), as determined by HPLC. The reaction was cooled to not less than 60n deg.C, and water (18L) was added while maintaining the temperature. The reaction mixture was filtered hot through GF-B glass fiber paper to a filter flask. The filtrate was transferred while warming in the reactor, washing with 1:1 dioxane/water (0.5 to 3L) as required, and the reactor jacket temperature was set at 45 ℃. Water (36L) was then added to the reactor and the temperature was maintained during the addition. The mixture was slowly cooled to 5 ± 5 ℃, and additional water was added to the reactor as needed to maximize crystallization. The temperature was maintained at 5 ± 5 ℃ for at least 2 hours, then the product was collected on a buchner funnel and washed with cold water (3X 3.5L). The product was dried under vacuum at 50-60 ℃ until constant weight was reached to give (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine (1073g, 70%) as an off-white solid.

A portion of this crude product (754g, 2.43mol) was slurried with 3N HCl (15L) and filtered to remove impurities. The acidic solution was extracted with MTBE (4L)) and heptane (4L). These organic extracts are discarded as waste. The acidic solution is alkalinized to pH 9-10 with 50% sodium hydroxide. The mixture was cooled and the precipitate was collected by filtration. The solid was washed with cold water and dried under vacuum to give an off-white solid, 695g, 92% recovery. The residual palladium in this material was 782 ppm. The product was recrystallized from 50% aqueous acetonitrile (8L). The mixture was cooled and the product collected by filtration, washed with cold solvent and dried in vacuo to give 401.7g (53% of the initial 754 g) as an off-white solid, now with 181ppm residual palladium. The off-white solid was dissolved in THF and washed with palladium scavengerMercapto resin (25 g). Removing the solvent to obtain (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]-4-amine as a white solid (390g, 97% recovery), confirmed to be more than 97% pure by HPLC and with less than 10ppm residual palladium.

Step 7(S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide

To a 100L reactor with stirrer, nitrogen inlet and condenser were added dichloromethane (20L), (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) propionic acid (1.0kg, 3.96mol) and N, N-dimethylformamide (14.5mL, 0.2 mol). Additional dichloromethane (10L) was added and the stirred mixture was cooled to 10-15 ℃. Oxalyl chloride (1.51kg, 11.9mol) was added slowly while maintaining the temperature below 25 ℃. The reaction was stirred at 25 ℃ for 30-60 minutes. The solvent was distilled from the reaction under vacuum with a nitrogen bleed and the reactor jacket temperature was increased to 35 ℃ as needed. Additional dichloromethane (20L) was added and the solvent was distilled from the reaction again. The addition and distillation of dichloromethane was repeated. Tetrahydrofuran (10L) was added and it gave a white to beige slurry. To the reaction was added (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine (1.07kg, 3.45mol) and the addition vessel was rinsed into the reaction mixture with tetrahydrofuran (1.5L). The reaction was cooled to 0 ℃ or less and 2, 6-lutidine (0.964L, 8.28mol) was added maintaining the reaction temperature below 5 ℃. The reaction was stirred at about 0 ℃ until deemed complete by HPLC (about 2% of (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidin ] -4-amine remained). After about 14h, the reaction was carefully quenched with 0.2N HCl (40L) and water (20L) while keeping the reaction temperature below 15 ℃. The solid product was collected and washed twice with deionized water. The solid was dried under vacuum at 50-60 ℃ to constant weight to give 1,473g (78%) (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2- (trifluoromethyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide. This product material was combined with 1,435g of product from a similar reaction and recrystallized from 2% aqueous ethanol (86.8L) to give 2,250g of purified (S) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) -N- (2'- ((S) -2- (trifluoro-methyl) pyrrolidin-1-yl) -2,5' -bipyrimidin-4-yl) propionamide as an off-white solid.

Example 30 from (S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine to (S) -2' - (2- (trifluoromethyl) Yl) pyrrolidin-1-yl) - [2,5' -bipyrimidine]Telescopic (telescaled) synthesis of (E) -4-amines

To a reaction vessel filled with dioxane (17.25L) were added (S) -5-bromo-2- (2- (trifluoromethyl) pyrrolidin-1-yl) pyrimidine (1500g, 5.066mol), 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (1937g, 7.628mol) and potassium acetate (998g, 10.17 mol). Additional dioxane (6.25L) was added and nitrogen was bubbled through the mixture while stirring gently for 30 to 60 min. Bis- (triphenylphosphine) palladium chloride catalyst (107g, 0.152mol) was added and washed with dioxane (0.5L) and the reaction was heated to 50 ℃. At this point, nitrogen was no longer bubbled through the mixture, but a nitrogen atmosphere was maintained and the condenser was vented. The reaction temperature was further increased to 95 to 100 ℃ and kept at this temperature until HPLC analysis indicated the reaction was complete (about 24 h).

The reaction was then cooled to 60 ℃ and 4-amino-2-chloropyrimidine (623g, 4.81mol), sodium carbonate (975g, 9.2mol) and water (7.5L) were added. Nitrogen was bubbled through the solution for about 30-60 minutes, and the condenser was vented. Tetrakis (triphenylphosphine) palladium (129g 0.11mol) was added and the residual catalyst was flushed with dioxane (0.5L) to the reaction vessel. Heating was resumed and nitrogen bubbling continued until the mixture reached about 50 ℃. The nitrogen tube was retracted above the surface of the solution at this point, but a nitrogen atmosphere was maintained and the condenser was vented. The temperature was increased to 85-90 ℃ and maintained until the reaction was complete (1-24h), as determined by HPLC. After cooling to not less than 60 ℃, water (18L) was added while maintaining the temperature, and the reaction mixture was hot filtered through GF-B glass fiber paper to a filter flask. The filtrate was transferred while warming in the reactor, washing with 1:1 dioxane/water (0.5 to 3L) as required, and the reactor jacket temperature was set at 45 ℃. Water (42L) was added to the reactor and the mixture was slowly cooled to 5 ± 5 ℃. Additional water was added to the reactor as needed to maximize crystallization and the temperature was held at 5 ± 5 ℃ for at least 2 hours before the product was collected on a buchner funnel and washed with cold water (3X 3.5L). It was then dried under vacuum at 50-60 ℃ until a constant weight was reached to give (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidine ] -4-amine (1266g, 70%) as an off-white solid. This product was purified as described in example 29 (step 7) to prepare (S) -2'- (2- (trifluoromethyl) pyrrolidin-1-yl) - [2,5' -bipyrimidin ] -4-amine, which had a purity similar to that described.

Characterization of the Compounds of the invention

The compounds of the invention are referred to herein by their respective example numbers. For example, a compound prepared by the method described in example 1 may be referred to as "the compound of example 1", "example 1", or "compound 1". All three names may be used interchangeably herein.

EXAMPLE 31 characterization of the solid crystalline form obtained by slurry treatment of the Compound of formula (I)

Certain compounds of the present invention form solvate crystalline forms upon slurry treatment in a solvent or combination of solvents (e.g., water, ethanol, or a combination thereof). For example, compound 2 produces a solvate crystalline form, referred to herein as form a, when slurried at room temperature in ethanol or an aqueous ethanol mixture containing up to 3% water. The solid crystalline product obtained from the slurry was indexed using X-ray powder diffraction (XRPD) to define a unit cell (fig. 1). The observed XPRD peaks are listed in table 1 below.

Table 1X-ray powder diffraction peaks observed for solid crystalline form of compound 2 (form a) obtained from ethanol slurry

Drying the crystals of the compound of formula (I) obtained from slurry treatment can yield other polymorphic forms. For example, crystals obtained from compound 2 slurry treatment in 97% ethanol/3% water were vacuum treated (-80 ℃ for 1 day) to give a stable, anhydrous solid crystalline form, referred to herein as form B. The resulting crystalline form is characterized using a variety of methods, including XRPD, polarized light microscopy, Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA) and Dynamic Vapor Sorption (DVS) with post-DVS XRPD.

Figure 2 shows the XRPD pattern of an anhydrous solid crystalline form sample of compound 2, and the observed peaks are listed in table 2 below. The success index of this sample indicates that it is composed of a single crystalline phase. After the solid crystalline form was stored at ambient conditions for 3 months, the sample was again subjected to XRPD analysis. The resulting XRPD pattern matched the original index map shown in figure 2.

TABLE 2X-ray powder diffraction peaks observed from the anhydrous solid crystalline form of Compound 2 (form B) obtained from an ethanol slurry

Differential Scanning Calorimetry (DSC) and thermogravimetric analysis (TGA) were also performed on the solid crystalline form of anhydrous compound 2 (form B); the data obtained are shown in FIGS. 3 and 4. DSC shows small broad endothermic peaks with the largest peak at 48.5 ℃, which is generally indicated as a volatilization event; however there was no corresponding weight loss event in the TGA. A broad endothermic peak at 48.5 ℃ may indicate the presence of absorbed water in the sample during storage, which is consistent with moisture absorption (DVS) data. The DSC also showed a broad endothermic peak, calculated at 185.4 ℃, which may correspond to a melting event and is consistent with a small TGA weight loss of 0.1% by weight, indicating that the sample may contain small amounts of unidentified volatile components.

Dynamic Vapor Sorption (DVS) analysis of the solid crystalline form of anhydrous compound 2 (form B) was also performed. The resulting isotherm plot is shown in fig. 5 and shows a 0.2% weight loss at equilibrium at 5% Relative Humidity (RH) followed by 2.7% weight reversible adsorption/desorption with negligible hysteresis. Based on this behavior, form B appears to be a variable hydrate, where the water content will depend on the ambient relative humidity. In summary, the XRPD, DSC, TGA and DVS data all agree with form B as a crystalline, variable hydrate species that becomes anhydrous upon drying.

Crystals of compound 2 obtained from ethanol or aqueous ethanol mixtures form growing fine needles. The XRPD pattern of the crystalline material is often complicated by the phenomenon of preferred orientation, since the crystals are predominantly aligned in two directions. The XRPD pattern of the compound of formula (I), e.g., compound 2, obtained from the recrystallized sample therefore appears different from those obtained from the slurry experiments described above. It is known that particle size reduction can reduce the extent of preferred orientation artifacts. FIG. 6 shows recrystallization from ethanol and drying, and micronization to d₉₀Examples of XRPD patterns of solid crystalline form of compound 2 (form B) with values before (light grey curve) and after (dark grey curve) 10 microns.

Example 32 measurement of dynamic solubility of Compounds of formula (I)

The solubility of compounds of formula (I) was tested using the procedure described in Kerns, E.H., J Pharm Sci (2001)90:1838-1858, which is incorporated herein by reference and described below. The data for solubility were obtained by this method for the compound of formula (I) and are included in table 3. The chromatographic data was performed by HPLC using an Xbridge Shield RP18 column with the following column dimensions: 4.6X 30mm, 3.5 μm. The mobile phase consisted of: deionized water (MPA) and 0.1% (v/v) trifluoroacetic acid (MPC) and HPLC-grade acetonitrile (MPB) were added. The mobile phase flow rate was 2.5mL/min, and the column and sample were operated at ambient temperature. UV detection was set at 280 nm. The mobile phase gradients used for all samples used for solubility determination are shown in table 4.

Table 3 exemplary solubilities of selected compounds of formula (I):

an assay sample of the compound of formula (I) was prepared at% v/v-1/19 (i.e. 10 μ L stock solution was added to 190 μ L buffer) by adding the stock solution of the compound of formula (I) to the buffer solution. Three buffer solution systems were prepared: prepared from 50mM sodium acetate in 5% aqueous dextrose (pH 4.0), 75mM sodium phosphate in 1:1 ratio sterile water for injection and 5% aqueous dextrose (pH 7.4), and 50mM sodium bicarbonate in 1:2 ratio sterile water for injection and 5% dextrose in aqueous solution (pH 9.0). The samples were incubated at ambient temperature on a microplate shaker at 300rpm for 24 hours. After incubation, the samples were centrifuged at 13k rpm for 5 minutes at ambient temperature. The resulting supernatant was extracted for HPLC analysis.

TABLE 4 mobile phase gradient for solubility determination

For example, the compounds of the invention may allow acceptable levels of drug to reach therapeutic targets.

Solubility of micronized formulation of compound 2 McIlvain citrate-phosphate buffer formulation (0.2M Na) at pH 2.2 to 8.64 may be further used₂HPO₄And 0.1M citric acid). The samples were stirred for 30 hours and sampled, centrifuged, and analyzed by UPLC. The highest solubility was observed at pH 8.6 and 3.1, while of pHThe effect is narrow, and the range is 0.2 to 1.3 mg/ml. The results are shown in FIG. 10.

Solubility was also determined in a fasted state simulated intestinal fluid (FaSSIF) test. Briefly, compound 2 was added to FaSSIF media (bile acid salt, NaOH (0.420g), NaH2PO4(3.438g), NaCl (6.186g), pH to 6.5, at 25 ℃, and added to 1L volume), stirred for 30 hours and sampled, centrifuged, and analyzed by UPLC. Solubility in the FaSSIF model was determined to be 1.19 mg/ml.

Solubility was further assessed in Simulated Gastrointestinal Fluid (SGF) (HCl 0.1N at 25 ℃). Briefly, the compounds were stirred for 30 hours and sampled, centrifuged, and analyzed by UPLC. The solubility in SGF was determined to be 1.05 mg/ml. The results of these studies indicate that the solubility of compound 2 is not significantly dependent on the pH of the medium, but may have some increased solubility based on the presence of bile acid salts.

The compounds of the invention were also tested for solubility in standard ringer's solution. Briefly, compound solubility was determined by dissolving a standard volume range of 10mM DMSO stock solutions of the compound in standard ringer's solution (145mM NaCl, 4.5mM KCl, 2mM CaCl₂，1mM MgCl₂10mM HEPES, 10mM glucose; pH 7.4, at room temperature). After vortexing and incubation at room temperature for 40 minutes, the solution was filtered, quenched with acetonitrile, and analyzed by liquid chromatography. The solubility limit was determined by comparison with a standard curve. The solubility limit was determined to be greater than 31.3 μ M. Solubility is reported as "greater than" if the increase observed between the last 2 dilutions tested is greater than 2-fold. Table 5 shows the values obtained from the compounds tested.

TABLE 5 solubility in standard ringer's solution of compounds of formula (I)

Compound (I)	DissolutionDegree (nM)
		1	>14300
2	～32400
		4	>31300
5	4370
		6	～23000
13	>77600
		15	16300
19	>18200
		16	>47000
26	>15100

Compound 2 was tested for binding to human, rat, dog and cynomolgus monkey (cynolgus monkey) plasma proteins using an equilibrium dialysis method. Using this method, free compounds are separated from protein-bound compounds by dialysis through a semi-permeable membrane. At a concentration of 1 μ M, compound 2 showed 98.5% binding to human, 95.3% binding to rat, 94.5% binding to dog and 98.0% binding to cynomolgus monkey plasma protein (table 6).

Protein binding was tested at a concentration of 1 μ M. Pooled human, cynomolgus monkey, dog and rat K2EDTA plasma was thawed and centrifuged at 2000x g for 10 min at 4 ℃ to remove particles; any lipids at the top of the supernatant were also removed by aspiration. Plasma was warmed to 37 ℃ for 10 minutes prior to use. Test compounds were added to 2ml plasma in polypropylene plates to a final concentration of 1 μ M. In triplicate, 400 μ Ι aliquots of the spiked plasma were transferred to a Thermo RED dialysis unit and dialyzed against 600 μ Ι PBS buffer. RED apparatus was incubated at 37 ℃ with gentle shaking using Boekel Jittterbug 130000; the plates were also protected from light. After 6 hours dialysis, 50 μ Ι aliquots were removed from RED plates in triplicate, matrix-matched with PBS buffer or blank plasma as needed, and quenched with 4 volumes of ACN containing an internal standard. The extracted sample was then centrifuged at 2000x g for 5 minutes at 4 ℃. The supernatant (50. mu.l) was removed and diluted with 100. mu.l water before LC/MS/MS bioanalysis.

TABLE 6 comparison of binding of Compound 2 and warfarin to plasma proteins

IC in the Presence of Albumin and plasma₅₀Changes in blockade were also studied. The pharmacological effects of drugs are thought to be related to unbound plasma levels, which is known as the "free drug hypothesis". The objective of this study was to evaluate the IC of Compound 2 in blocking human TRPA1(hTRPA1) in the presence of physiologically relevant concentrations of albumin and plasma₅₀See table 7. Due to technical problems, the human form of TRPA1 was used to assess protein binding in all species. The whole-cell patch-clamp technique described by del Camino, D. et al J Neurosci74(2010)30:15165 was used to measure the current through hTRPA1 after activation by Allyl Isothiocyanate (AITC), the active ingredient in mustard oil, in the presence of 1% (w/v) serum albumin or 25% (v/v) plasma from various speciesIncluding human plasma (hPlasma), Human Serum Albumin (HSA), rat plasma (rpasma), Rat Serum Albumin (RSA), dog plasma, and sheep serum albumin (sheep SA). Compound 2 was further diluted from 10mM stock in DMSO to 10 and 100 μ M (in DMSO) and then diluted into ringer's solution at the concentrations mentioned in tables 7 and 8.

Subsequent introduction of compound 2 resulted in dose-dependent and reversible blocking of hTRPA 1. hTRPA1 current was blocked and had an IC in the presence of 25% (v/v) human plasma (hClasma)₅₀95. + -.2 nM, IC in the absence of serum₅₀14 times higher (see table 8). Experiments performed on hTRPA1 in the presence of rat plasma (rpasma) and Rat Serum Albumin (RSA) resulted in blocking potency of compound 2 of 68 ± 8nM and 95 ± 9nM, respectively, indicating a degree of protein binding similar to that observed for human plasma. Blocking IC of Compound 2₅₀Somewhat higher (221 ± 54nM) in the presence of dog plasma. hTRPA1 current was blocked and had IC in the presence of 1% (w/v) sheep serum albumin (sheep SA)₅₀70. + -.10 nM. Both blocking with compound 2 and reversal after clearance were completed within 2-3 minutes. These experiments indicate that compound 2 produces pharmacological effects at lower plasma levels than previously identified compounds, as more free drug is available to interact with the target.

Based on the IC observed in the presence of plasma₅₀Can give compound 2 showed significant binding to plasma proteins in the four species tested, ranging from 90-97%. This represents an improvement over previous compounds of similar potency.

TABLE 7 Albumin and hTRPA1IC of Compound 2 in plasma₅₀Measurement of

TABLE 8 IC of Compound 2 on hTRPA1 from various mammalian sources₅₀Measurement of

Metabolic stability

The metabolic stability of the compounds of formula (I) was determined by standard liver microsome assay. Briefly, metabolic stability is tested by adding the test compound dissolved in DMSO to human, dog or rat liver microsomes. The test was performed using a test compound at an initial concentration of 1. mu.M. The reaction was initiated at 37 ℃ by the addition of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) regenerating component, at which time the aliquot was immediately quenched in ice-cooled acetonitrile/methanol/water solution. The reaction mixture was incubated at 37 ℃ on a shaker and additional aliquots were taken at 7, 15, 30 and 60 minutes. After quenching and centrifugation, the samples were analyzed on HPLC/MS/MS. The results are shown in tables 9, 10 and 11 below.

TABLE 9 half-life and liver clearance of Compounds in human liver microsomes

TABLE 10 half-life and liver clearance of Compounds in dog liver microsomes

TABLE 11 half-life and hepatic clearance of Compounds in rat liver microsomes

Bioavailability of

Bioavailability studies in early rats were performed using solutions of the compounds of the present invention. The compounds are delivered by oral administration as a solution in a suitable vehicle. Example formulations include, but are not limited to: 4% DMSO, 10% SolutolHS-15, and 86% water or 4% DMSO, 5% Tween, 25% cremophor EL. The target concentration was typically 1mg/mL and was administered to non-fasted rats by oral gavage. Absolute bioavailability is the area under the curve (AUC) of the non-intravenous dose-correction divided by the AUC of the intravenous dose correction. The formula for F for the drug administered by the oral route (PO) is given below:

% F ═ AUC PO X dose IV/AUC IV X dose PO

The bioavailability of each rat of the test compound is shown in table 12.

Table 12 bioavailability of compounds in non-fasted rats

Other studies were performed in rats, dogs (beagle dogs) and cynomolgus monkeys using micronized compound 2. Animals received a 10mg/kg oral dose of the compound formulated as a suspension in 0.5% methylcellulose in water for injection, at a target concentration of 1mg/mL, and administered by oral gavage in rats or fasted dogs, and by oral delivery in fasted cynomolgus monkeys, at an administration volume of 10 mL/kg. The% F in rats, dogs, and monkeys in these studies was 85%, 36%, and 19%. Fig. 11 depicts the pharmacokinetic profiles of these species.

A number of in vivo studies were performed to characterize the bioavailability of compound 2. In SD rats, single oral doses of 10mg/kg to 1000mg/kg of Compound 2 were compared in a series of experiments. Compound 2 used in these studies was recrystallized from ethanol and micronized. Based on area under the curve (AUC) and maximum plasma concentration (C)_max) The exposure increased as the dose increased upwards to 1000 mg/kg.

Three studies were performed in dogs (beagle dogs). In one study, three fasted dogs were fed a 10mg/kg suspension of micronized compound 2. Blood samples were collected from these dogs pre-dose and at 0.25, 0.50, 1, 2, 4, 6, 8, 12 and 24 hours post-dose. Blood samples were analyzed to determine plasma levels of compound 2 by LC/MS. In subsequent studies, Pharmacokinetic (PK) parameters were determined after single oral administration of suspensions of micronized compound 2 (recrystallized from ethanol) administered to fasted dogs at dose levels of 10, 100, 300, 600 or 1000 mg/kg. Blood samples were taken pre-dose and at 0.5, 1, 2, 4, 8, 12, 24 and 48 hours post-dose and analyzed to determine plasma levels of compound 2. Based on AUC and C_maxThe exposure of (A) increased as the dose increased upwards to 600 mg/kg.

Three studies were performed in fasted cynomolgus monkeys to determine PK profiles and bioavailability of suspension formulations of compound 2.3 monkeys were given a suspension containing 10mg/kg micronized compound 2 by oral gavage. Monkey blood samples were collected pre-dose and at 0.25, 0.50, 1, 2, 4, 6, 8, 12 and 24 hours post-dose. Compound 2 plasma levels were determined by LC/MS. Additional studies were performed to evaluate PK profiles by administering compound 2 as an oral suspension to fasted monkeys at higher dose levels. Blood samples were collected before and up to 24 hours after dosing. An additional 48 hour blood sample was collected from cynomolgus monkeys dosed with 300mg/kg compound 2. Plasma levels of compound 2 were determined by LC/MS. Based on AUC and C_maxThe exposure increased as the dose increased upwards to 1000 mg/kg.

Another study was conducted to compare PK parameters for capsule and suspension formulations of compound 2. All monkeys were dosed with 250mg of compound 2. Both groups were dosed with capsule formulations: one group was fasted and one group was fed ad libitum. Animals receiving the suspension formulation were dosed using nasogastric tube and fasted. As shown in figure 12 and summarized in table 13 below, the capsule formulation was associated with increased bioavailability compared to the suspension, although the numerical differences were smaller. The bioavailability of compound 2 capsules was numerically greater in fasted monkeys when the PK profile of the capsule formulation was compared in both fasted and fed monkeys.

Table 13 comparative compound 2: PK parameters for capsule and suspension formulations

Compound 2 dose (mg)	Compound 2 formulations	F(％)
			250	Suspension (fasting)	7.8
250	Capsule (fasting)	13
			250	Capsule (feed)	10

Example 33 method for measuring TRPA1 ion channel inhibition

The inhibition of the TRPA1 channel by compounds of formula (I), as shown by measurement of in vitro inhibition of human TRPA1, is provided in the data table shown in table 14 using the procedure described by del Camino et al, J Neurosci (2010)30(45): 15165-. TRPA1 inhibitionAre obtained by this method for the compounds of formula (I) shown, with the relevant data contained in table 14 below. All currents were recorded in a whole-cell configuration using EPC-9 and EPC-10 amplifiers and Patchmaster software (HEKA). The resistance of the patch pipette is 1.5-3M and compensates for up to 75% of the series resistance. Standard pipette solutions were prepared from 140mM CsAP, 10mM EGTA, 10mM HEPES, 2.27mM, 20 mM MgCl₂，1.91mMCaCl₂And up to 0.3mM Na₂GTP formation, where the pH is adjusted to 7.2 with CsOH. In addition, compositions comprising 145mM CsCl, 10mM HEPES, 10mM EGTA and up to 0.3mM Na may be used₂GTP and 1mM MgCl₂The pH was adjusted to 7.2 with CsOH. The standard bath solution contained 150mM NaCl, 10mM HEPES, 10mM glucose, 4.5mM KCl, 1mM EGTA and 3mM MgCl₂Wherein the pH was adjusted to 7.4 with NaOH. In some cases, 2mM CaCl was added₂Instead of EGTA and MgCl₂To a concentration of 1 mM.

Data were collected as follows: the recording was continued at-60 mV or a varying voltage (voltage ramp) of 0mV, starting from the holding potential, was applied every 4 seconds. The continuous recordings were collected at 400Hz and digitally filtered off-line at 10Hz for display. Varying voltages from-100 mV to 100mV were applied for 400ms and data was collected at 10kHz and filtered at 302.9 kHz. The inward and outward currents were analyzed by variation at-80 mV and 80mV, respectively. Hydraulic potential correction is not used.

The solution was switched using a gravity fed continuous focal perfusion system. To achieve rapid temperature changes, two temperature control and perfusion systems are used simultaneously. For temperatures greater than or equal to 22 ℃, a Warner Instruments bipolar temperature controller (TC-344B) and an in-line heater (SHM-8) were used. For temperatures below 22 ℃, a Warner instruments temperature controller (CL-100) and a thermal cooling module (TCM-1) were used. Temperature was confirmed using a thermistor (Wamer instruments, TA-29), where the temperature at the recorded cells was estimated to be +/-2 ℃ of the reported temperature.

Table 14 shows the data obtained from the in vitro tests described above. Antagonism of the compound of formula (I) in the whole cell patch configuration to human TRPA1 ("hTRPA 1") was assessed using the in vitro test protocol described above.

TABLE 14 antagonistic effect of compounds of formula (I) on human TRPA1

Example 34 Effect on Cold ultrasensitivity

Embodiments of the present invention are effective in treating inflammatory pain. Compound 2 was tested by the CFA-induced pain test method. Compound 2 was formulated as a clear solution in 4% DMSO, 10% Solutol, 86% DWI, pH 5.9 for oral administration (PO).

Briefly, the hind paw was sensitized to cold temperature (allodynia) by administering 0.1mL of Complete Freund's Adjuvant (CFA) to the right hind paw. After 3 days, the time it took for the animal to lift its CFA-injected paw was recorded and compared to the time it took for the animal to lift its non-injected normal left hind paw. Animals were placed on the surface of a cold plate (1 ℃) and when the animals showed an abnormality by flinching or lifting their paw from the plate (paw withdrawal latency or PWL), the operator immediately stopped the test. To avoid tissue damage, the maximum cut-off time is 5 minutes. Allodynic animals (mean PWL < 150 seconds for the initial 3 pain behaviors in the case of CFA injected hind paw: difference between normal paw and CFA injected paw ≧ 50%) were included in the study and then randomized into each treatment group. The following day, animals were dosed under blind conditions. After a pretreatment time of 1-2 hours, PWL readings after dosing were again recorded. The efficacy of drug treatment was evaluated by comparing the PWL of the drug treated animals to the PWL of the vehicle-receiving animals.

As shown in fig. 7 and table 15, compound 2 attenuated cold hypersensitivity after oral administration of 0.3 to 10 mg/kg. Positive comparator TRPA1 antagonist compound a also reduced cold hypersensitivity at the higher dose of 150mg/kg delivered by plantar injection. Importantly, the orally delivered vehicle (4% DMSO, 10% Solutol, 86% DWI) had no effect on the withdrawal latency compared to the baseline measurement of predosing.

TABLE 15 reduction of Cold hypersensitivity of Compound 2 at various oral doses

Table 16 summarizes mean plasma levels of compound 2 and compound a. Compound 2 exposure was observed approximately dose-proportional over the entire dose range tested. The reduced plasma binding of compound 2 indicates improved bioavailability of compound 2 compared to compound a in the subject.

TABLE 16 plasma levels of Compound 2 and Compound A

There were no behavioral differences between vehicle and treatment groups (see table 17). Lethargy/slow movement was noted in 5/10 animals treated with positive comparator compound a, however, confirming that compound 2 did not induce significant sedation.

TABLE 17 examination of animal behavior following administration of Compound 2

Compound 4 was also tested using the disclosed method. Compound 4 was formulated as a suspension in 0.5% methylcellulose and administered at the doses indicated in table 18.

TABLE 18 attenuation of Cold hypersensitivity of Compound 4 at various oral doses

In a further study, the efficacy of the compounds of the invention at low doses for the treatment of inflammatory pain was tested. Using the methods disclosed above, Compound 2 is administered orally in the range of 0.1 to 1 mg/kg. Positive comparator TRPA1 antagonist compound a was also tested at a dose of 150mg/kg IP. Compound 2 was formulated as a clear solution in 4% DMSO, 10% Solutol, 86% DWI, pH 5.9 for oral administration (PO) at an administration volume of 10 ml/kg. Oral drug delivery was achieved using a 20-gauge 11/2 "oral feeding needle and a 5cc syringe. Fed rats received a single oral gavage of 0.03, 0.1, 0.3 or 1mg/kg compound 2 or vehicle 2 hours prior to testing.

As shown in table 19 and figure 13, compound 2, when administered orally at 0.1mg/kg, 0.3mg/kg and 1mg/kg, showed a significant reversal of CFA-induced cold hypersensitivity as tested by measuring the paw withdrawal latency. The 0.03mg/kg dose level produced no statistically significant effect. The positive comparator, prototype TRPA1 antagonist compound a, also showed a significant reversal of cold hypersensitivity when administered at 150mg/kg IP.

TABLE 19 CFA-induced reversal of cold-cold hypersensitivity by Compound 2

In summary, these studies indicate that the compounds of the present invention have the potential to effectively treat inflammatory pain after oral administration.

Example 35 formalin model

In Dubuisson et al, Pain (1977) Dec; compound 2 was tested in the formalin-induced pain test reported at 161-74 (2). Dubuisson et al describe methods for assessing pain and analgesia in rats and cats. Briefly, dilute formalin (50 μ L of 3% formalin) was injected into the plantar surface of the rat hind paw. The animals were returned to the observation zone as soon as possible (standard Plexiglass mouse cage) at which time the trained observer recorded the time it took for the animal to exhibit painful behavior (flinching, licking or biting the injected paw/leg) in two different phases. The initial phase of the task (phase I: 0-5min) has a significant component, which relies on direct activation of afferent fibres by formalin and functional TRPA1 (McNamara et al, 2007). The person responsible for counting pain behaviors in a particular study was blinded to the treatment group.

Investigators investigated the effect of orally administered compound 2 at 1, 3, and 10mg/kg on pain behavior in the rat formalin model. Compound 2 was prepared as a solution in 4% DMSO, 10% Solutol HS15, 86% WFI. Animals were dosed orally with vehicle (4% DMSO, 10% Solutol, 86% WFI), or 1, 3, or 10mg/kg of compound 21 hour prior to the plantar injection of formalin. Figure 14 shows the duration of pain behaviour observed in the first 2 minutes (left) or throughout the study period; 5 minutes (right). (each group n 8) (p <0.05, p < 0.01; p < 0.001: one-sided T-test)

Oral administration of compound 2 at 3 and 10mg/kg significantly reduced nociceptive pain responses in phase 1 of the formalin model, as shown in table 20 and figure 14. Animals treated with 1mg/kg compound 2 0-2 minutes after plantar injection of formalin resulted in a reduction in duration of painful behavior of-14% compared to vehicle-treated animals, although the reduction was not statistically significant. Compound 2 resulted in a significant reduction in formalin-induced pain behavior of 0-2 minutes by-72% and-89% when the doses were 3 and 10mg/kg PO, respectively, compared to vehicle treated animals.

A similar reduction in the duration of pain behavior by compound 2 was also observed 0-5 minutes after formalin administration. At 1mg/kg PO, compound 2 reduced pain behavior by-14%, but did not reach statistical significance compared to vehicle-treated animals. Compound 2 significantly reduced the duration of formalin-induced pain behavior by-46% and-60% at 3 and 10mg/kg PO, respectively.

Table 20 dose response of orally administered compound 2 using formalin model

A reduction in the duration of painful behavior was also observed with compound 1 during 0-5 minutes after formalin administration. Compound 1 reduced the duration of formalin-induced pain behavior delivered intravenously to rats at 1mg/kg and 3mg/kg as shown in table 21 and figure 15.

Table 21 dose response of compound 1 administered intravenously using the formalin model

A reduction in the duration of painful behavior was also observed with compound 4 during 0-5 minutes after formalin administration. Compound 4 was formulated in 4% DMSO using the method described for the formalin test above; 5% Tween-80; 20% cremophor EL; and a solution in 71% WFI, and was administered to rats by oral gavage. Compound 4 reduced the duration of formalin-induced pain behavior as shown in table 22.

Table 22 dose response of compound 4 administered orally using formalin model

Compound 4 was formulated as a suspension in 0.5% methylcellulose using the method described in the formalin test above and administered to rats by oral gavage. Compound 4 reduced the duration of formalin-induced pain behavior as shown in table 23.

Table 23 dose response of compound 4 administered orally using the formalin model

The duration of the response was also investigated. Compound 2 was formulated as a solution in 4% DMSO, 10% solutol hs15, and 86% WFI. Rats were treated with compound 2 or vehicle (PO) at an oral dose of 10 mg/kg. Figure 8 shows that pretreatment with an orally administered formulation of compound 2 at 10mg/kg PO significantly reduced the duration of formalin-mediated pain behavior 30 minutes to 6 hours prior to formalin injection.

The 15-6 hour pretreatment with 10mg/kg oral compound 2 resulted in a decrease in the duration of formalin-induced pain behavior of 0-2 minutes post formalin injection of-30-87% compared to vehicle-treated animals, with the maximum decrease in pain behavior observed in the 2 hour pretreatment group, as shown in table 24.

Table 24 duration of pain response following oral administration of compound 2 using formalin model

Compound 2 was also tested in an ovine model of allergic bronchoconstriction and airway hyperresponsiveness, according to the method disclosed in Abraham, W.M Pulm Pharmacol Ther (2008)21: 743-754. Allergic sheep stimulated with ascaris suum showed a significant, biphasic increase in pulmonary Resistance (RL). The first 4 hours is considered Early Asthmatic Response (EAR); the subsequent 4 hours (4-8 hours) were considered to be Late Asthmatic Response (LAR). To assess airway responsiveness (AHR), the cumulative carbachol dose in the respiratory unit that increased pulmonary resistance by 400% relative to the buffered value (PC400) was calculated from the dose-response curve. One breath unit is defined as one breath of a 1% w/v carbachol solution. Pre-stimulated PC400 was obtained 1-3 days prior to the start of dosing.

Compound 2 was formulated as a micronized powder suspended in 0.5% methylcellulose at a concentration of 6mg/ml and orally administered once daily at a dose of 30mg/kg for 4 days at approximately the same time each day. Sheep were orally administered 30mg/kg compound 2 daily for 4 days. 2 hours after the last compound administration, the sheep were subjected to allergen (roundworm) stimulation. Each sheep was restrained in the prone position with its head fixed and then the nasal passages were anesthetized locally. The balloon catheter is threaded through one nostril to the lower esophageal segment. Each sheep was inserted into the endotracheal tube of the cuff via the other nostril. Tracheal and pleural pressures were determined using endotracheal and balloon catheters, respectively. Transpulmonary pressure, i.e., the difference between tracheal and pleural pressures, was measured using a differential pressure sensor catheter system. R_LMeasured by connecting the distal end of the endotracheal tube to a pneumotachometer. Data was collected from 5 to 10 breaths into the computer and used to calculate R_L. Data obtained from the same sheep stimulated with roundworm prior to the start of compound 2 treatment was used to establish baseline values. The control and drug experiments were monitored under the same conditions.

On the day of stimulation and 2 hours after the last compound 2 administration, an aerosol of ascaris suum (82,000 protein nitrogen units/mL) was generated using a nebulizer and delivered to the sheep using a Harvard respirator. R_LWas measured 1 hour prior to stimulation, immediately after roundworm stimulation, and then every hour for 8 hours. Stimulation for 4 hours was considered EAR and stimulation for 4 to 8 hours was considered LAR.

Sheep were also stimulated with aerosolized carbachol, a cholinergic agonist with negative effects on AHR. Carbachol concentrations in respiratory units that increased RL 400% (PC400) measurements were determined 24 hours after roundworm stimulation at 24 hours after no compound 2 administration (historical baseline) or 24 hours after administration of the final dose of compound 2.

Figure 16 shows antigen-induced responses in sheep at baseline (control) levels and after treatment with compound 2(30 mg/kg). Compound 2 did not affect the early airway response peak. However, it significantly attenuated late airway response (85% protection). In the control trial, the mean late airway response was 126 ± 4.7%, whereas in the treatment trial the mean LAR was only 19 ± 2.3% (P ═ 0.002).

Figure 17 further shows the effect of compound 2(30mg/kg) on PC400, which is a measure of airway hyperresponsiveness representing carbachol concentration inducing a 400% increase in pulmonary resistance.

In summary, treatment with compound 2 reduced airway hyperresponsiveness to levels similar to those observed in sheep not stimulated with ascaris suum.

EXAMPLE 36 drug Profile

The compounds of the present invention may not have significant drug/drug interactions and are therefore preferably administered to patients taking multiple drugs.

The ability of compound 2 to inhibit human CYP450 enzymes was evaluated. Compound 1 and compound 2 were tested in a standard P450 Cyp-inhibition luminescence assay. The results are shown in Table 25 below.

TABLE 25 inhibition of CYP450 enzymes by Compounds 1 and 2

Compound 2 achieved maximum blocking of human CYP450 enzyme at 10 μ M for 7 isozymes tested, up to 37%; these values indicate the IC₅₀The calculated value will be>10 μ M (Table 26).

CYP450 response phenotyping of compound 2 was performed by culturing test preparations with human liver microsomes in the presence and absence of selective CYP450 inhibitors. In addition to ketoconazole, the metabolic half-life was not significantly affected by any of the CYP450 inhibitors suggesting that the CYP3a4 isozyme is primarily involved in the in vitro metabolism of compound 2 (fig. 9).

TABLE 26 inhibition of CYP450 by Compound 2 and appropriate reference Compounds at 10. mu.M

1 μ M test concentration

Example 37 hepatotoxicity safety Profile of dogs

Group 3 non-natural male and female beagle dogs were orally fed compound 2 once daily for 5 consecutive days in vehicle (0.5% methylcellulose [400cps ] in deionized water). Each group received 1 dose level. The dose levels in each group were 300, 600 and 1000 mg/kg. The parallel control group received vehicle in the same protocol. The dose volume for all groups was 10 ml/kg. Hepatotoxicity is measured by serum biomarkers for alanine aminotransferase [ ALT ], aspartate aminotransferase [ AST ], alkaline phosphatase [ ALP ], and gamma-glutamyltransferase [ GGT ] representing hepatotoxicity or bile duct injury. Table 27 shows that compound 2 did not raise serum biomarkers in dogs at the indicated dose levels (including the 300mg/kg dose).

TABLE 27 hepatotoxicity safety profile of Compound 2 in beagle dogs

Figure 18 further demonstrates that compound 2 does not significantly elevate serum biomarker levels above the normal range. Figure 19 demonstrates that compound 2 did not significantly elevate serum biomarker levels above baseline measurements as evidenced by% difference from vehicle.

Example 38 hepatotoxicity safety profile in rats

3 groups of SD rats from Charles River Laboratories were orally fed compound 2 dosed once daily in vehicle (0.5% methylcellulose [400cps ]) for 28 consecutive days. Each group received 1 dose level. The dose levels in each group were 30, 100 and 300 mg/kg. The parallel control group received vehicle in the same protocol. The dose volume for all groups was 10 ml/kg. Hepatotoxicity is measured by serum biomarkers for alanine aminotransferase [ ALT ], aspartate aminotransferase [ AST ], alkaline phosphatase [ ALP ], and gamma-glutamyltransferase [ GGT ] representing hepatotoxicity or bile duct injury. Table 28 shows that compound 2 did not raise serum biomarkers in rats at the indicated dose levels (including the 300mg/kg dose).

TABLE 28 hepatotoxicity safety profile of Compound 2 in rats

Figure 20 further confirms that compound 2 did not significantly elevate levels above the normal range. Figure 21 demonstrates that compound 2 did not significantly elevate levels above baseline measurements as evidenced by% difference relative to vehicle.

Example 39 hepatotoxicity safety Profile in monkeys

Compound 2 in vehicle (0.5% methylcellulose, 400cps) was intubated once daily to 4 groups of cynomolgus monkeys for 28 or 29 consecutive days. Each group received 1 dose level. The dose levels in each group were 10, 30, 100 and 300 mg/kg/day. The parallel control group received vehicle in the same protocol. The dose volume for all groups was 10 ml/kg. Hepatotoxicity is measured by serum biomarkers for alanine aminotransferase [ ALT ], aspartate aminotransferase [ AST ], alkaline phosphatase [ ALP ], and gamma-glutamyltransferase [ GGT ] representing hepatotoxicity or bile duct injury. Table 29 shows that compound 2 did not raise serum biomarkers in monkeys at each of the indicated dose levels (including the 300mg/kg dose).

TABLE 29 hepatotoxic safety profile of Compound 2 in cynomolgus monkeys

Figure 22 further confirms that compound 2 did not significantly elevate levels above the normal range. Figure 23 demonstrates that compound 2 did not significantly elevate levels above baseline measurements as evidenced by% difference relative to vehicle.

Equivalents of the same

The contents of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to particular aspects, it is apparent that other aspects and variations may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the following claims be interpreted to embrace all such aspects and their equivalents.

Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. Accordingly, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

While the present invention has been particularly shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (65)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

2. A compound according to the preceding claim, wherein R¹Is C₁-C₆An alkyl group.

3. A compound according to any one of the preceding claims, wherein R¹is-CH₃。

4. A compound according to any one of the preceding claims, wherein R¹Is H.

5. A compound according to any one of the preceding claims, wherein R²Is H.

6. A compound according to any one of the preceding claims, wherein R²Is C₁-C₆An alkyl group.

7. A compound according to any one of the preceding claims, wherein R²is-CH₃，–CD₃or-CHF₂。

8. A compound according to any one of the preceding claims, wherein R¹And R²Each independently is C₁-C₆An alkyl group.

9. A compound according to any one of the preceding claims, wherein R¹And R²Each independently is-CH₃。

10. A compound according to any one of the preceding claims, wherein R¹And R²Each independently is-CH₃And R is³Is H.

11. A compound according to any one of the preceding claims, wherein R³Is H.

12. A compound according to any one of the preceding claims, wherein R³Is C₁-C₆An alkyl group.

13. A compound according to any one of the preceding claims, wherein R³is-CH₃。

14. A compound according to any one of the preceding claims, wherein R¹、R²And R³Each of which is independently C₁-C₆An alkyl group.

15. A compound according to any one of the preceding claims, wherein R¹、R²And R³Each of which is independently-CH₃。

16. A compound of formula (I) according to any one of the preceding claims, wherein the compound is of formula (Ia):

17. a compound of formula (I) according to any one of the preceding claims, wherein the compound is of formula (Ib):

18. a compound according to any one of the preceding claims, wherein R⁴Is a heterocyclic group.

19. A compound according to any one of the preceding claims, wherein the heterocyclyl is a 4 to 8-membered ring.

20. A compound according to any one of the preceding claims, wherein the heterocyclyl is attached via a nitrogen atom.

21. A compound according to any one of the preceding claims, wherein R⁴Is a substituted heterocyclic group.

22. A compound according to any one of the preceding claims, wherein R⁴Selected from the following groups:

23. a compound according to any one of the preceding claims, wherein R⁴Selected from the following groups:

and m is 1.

24. A compound according to any one of the preceding claims, wherein R⁴Selected from the following groups:

25. a compound according to any one of the preceding claims, wherein R⁴Selected from the following groups:

and m is 1.

26. A compound according to any one of the preceding claims, wherein R⁴Selected from the following groups:

27. a compound according to any one of the preceding claims wherein m is 1.

28. A compound according to any one of the preceding claims wherein m is 0.

29. A compound according to any one of the preceding claims, wherein R⁶Is alkyl, haloalkyl or cyano.

30. A compound according to any one of the preceding claims, wherein R⁶Is alkyl or haloalkyl.

31. A compound according to any one of the preceding claims, wherein R⁶is-CF₃。

32. A compound according to any one of the preceding claims, wherein R⁴Selected from the following groups:

33. a compound according to any one of the preceding claims, wherein the compound of formula (I) is of formula (II):

wherein:

n is an integer of 0 to 4; and is

m is an integer from 0 to 4.

34. A compound according to any one of the preceding claims, wherein the compound of formula (I) is of formula (IIa):

wherein:

n is an integer of 0 to 4; and is

m is an integer from 0 to 4.

35. A compound according to any one of the preceding claims wherein the compound of formula (I) is of formula (IIb):

wherein:

n is an integer of 0 to 4; and is

m is an integer from 0 to 4.

36. The compound according to any one of the preceding claims, wherein the compound is selected from the following:

or a pharmaceutically acceptable salt thereof.

37. A compound according to any one of the preceding claims, wherein the compound is:

or a pharmaceutically acceptable salt thereof.

38. The compound of claim 37, wherein the solid crystalline form of the compound has an X-ray powder diffraction pattern comprising characteristic peaks, expressed in terms of 2 Θ, at about 7.67 °, about 12.52 °, about 13.49 °, and about 19.31 °.

39. The compound of claim 37, wherein the solid crystalline form of the compound has an X-ray powder diffraction pattern comprising characteristic peaks, expressed in terms of 2 Θ, at about 9.78 °, about 12.98 °, about 19.20 °, and about 19.67 °.

40. The compound according to any one of the preceding claims, wherein the solid crystalline form of the compound has a melting point greater than or equal to about 150 ℃.

41. The compound according to any one of the preceding claims, wherein the solid crystalline form of the compound has a melting point in the range of about 180 ℃ to about 205 ℃.

42. The compound according to any one of the preceding claims, wherein the solid crystalline form of the compound has a melting point in the range of about 190 ℃ to about 200 ℃.

43. A purified pharmaceutical preparation comprising a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

44. The formulation according to the preceding claim, wherein the compound is:

or a pharmaceutically acceptable salt thereof.

45. The formulation according to any one of claims 43-44, wherein said formulation comprises a diastereomeric excess of greater than or equal to about 99%.

46. The formulation according to any one of claims 43-45, wherein said formulation has a moisture content of less than or equal to about 0.1%.

47. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R is⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

48. A composition for treating a TRPA 1-mediated disease in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkeneA group, an alkynyl group, a hydroxyl group, an amino group, an amide group, a phosphonate group, a carboxyl group, an ether, an alkylthio group, a haloalkyl group, and a cyano group; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

49. A composition for treating pain in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

50. The composition of claim 49, wherein the pain is neuropathic pain.

51. The composition of claim 49, wherein the pain is inflammatory pain.

52. The composition of claim 49, wherein the pain is PDN or CIPN.

53. The composition of claim 49, wherein the pain is visceral pain.

54. The composition of claim 49, wherein the pain is selected from the group consisting of: cancer pain, burn pain, oral pain, crush and injury-induced pain, incision pain, bone pain, sickle cell disease pain, fibromyalgia, and musculoskeletal pain.

55. The composition of claim 49, wherein the pain is derived from hyperalgesia or allodynia.

56. A composition for treating an inflammatory disease in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

57. A composition for treating neuropathy in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl orC₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

58. The composition of claim 57, wherein the neuropathy results from diabetes, chemical injury, chemotherapy, and or trauma.

59. A composition for treating a skin disorder in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

60. The composition of claim 59, wherein the skin disorder is selected from the group consisting of atopic dermatitis, acute pruritus, psoriasis, urticaria, eczema, pompholyx, oral ulcers, and diaper rash.

61. A composition for treating a pulmonary disease in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

62. The composition of claim 61, wherein the pulmonary disease is an obstructive disease.

63. The composition of claim 61, wherein the pulmonary disease is chronic obstructive pulmonary disease or asthma.

64. A composition for treating cough in a subject, the composition comprising an effective amount of a compound of formula (I):

wherein:

R¹is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆Alkynyl optionally substituted by one or more R⁵Substituted by groups;

R³is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl or C₁-C₆An alkynyl group;

R⁴is halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, dialkylamino, cyano, nitro, amide, alkylamide, dialkylamide, sulfinyl, sulfonyl, cyclic, heterocyclic, aryl, or heteroaryl, optionally substituted at one or more positions with 1-4R⁶A group;

R⁵independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, hydroxy, amino, amido, phosphonate, carboxy, ether, alkylthio, haloalkyl, and cyano; and is

R⁶Independently H, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic or heteroaromatic ring, haloalkyl, and cyano.

65. The composition of claim 64, wherein the cough is an allergy-induced cough.