Substituted {1,2,4,}triazolo{1,5-A}pyrimidine compounds and their use for microtubule stabilization

Abstract

The present invention provides nitrile- and alkyne-substituted {1,2,4,}triazolo{1,5-a}pyrimidine compounds, compositions comprising the compounds, and methods of treating neurodegenerative diseases or cancer by administration of the compounds.

Description

[0001] Government Rights

[0002] This invention was made with government support under Grant Nos. AG044332 and AG061173 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Cross - Reference to Related Applications

[0004] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 009,727, filed on April 14, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0005] The present invention relates to compounds and methods for treating cancer or neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal lobar degeneration. Background Art

[0006] Neurodegenerative tauopathies, including Alzheimer's disease (AD), are characterized by the misfolding and aggregation of the microtubule (MT) - associated protein tau. Typically, tau binds to MTs and stabilizes them, thereby maintaining the network of MTs necessary for axonal transport in neurons. In AD, tau sequesters into aggregates, called neurofibrillary tangles (NFTs) and neuropil threads, resulting in reduced MT binding. This loss of tau function and / or the formation of oligomeric or fibrillar tau species is thought to lead to MT destabilization and consequent axonal transport defects, which may result in neuronal dysfunction and death.

[0007] Other neurodegenerative diseases in which MT function may be impaired include frontotemporal lobar degeneration, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, Huntington's disease, multiple sclerosis, and traumatic brain injury (TBI), especially repetitive TBI (rTBI), such as that due to boxer dementia and recurrent soccer concussions, as well as military closed - head injuries, which is also known as chronic traumatic encephalopathy (CTE).

[0008] To treat neurodegenerative diseases that are caused, at least in part, by the misfolding and aggregation of MT - associated protein tau, compounds that can cross the blood - brain barrier and effectively stabilize MTs are needed.

[0009] Since then, [1,2,4]triazolo[1,5-a]pyrimidines and related heterocyclic molecules, initially reported as antifungal agents with microtubule (MT) activity, have attracted extensive attention as potential candidates for a variety of applications, including chemotherapy for cancer and treatment of neurodegenerative diseases. Siweibulin (Compound 1) is an effective anticancer compound. This compound, like vincristine, can interact with tubulin heterodimers and interfere with the exchange rate of guanosine triphosphate (GTP), thereby competing with vincristine for binding to MT, but not competing with paclitaxel or colchicine. See, Zhang, “Synthesis and SAR of [1,2,4]triazolo[1,5-a]pyrimidines, a class of anticancer agents with a unique mechanism of tubulin inhibition”, J. Med. Chem., 2007, 50:319-27.

[0010]

[0011] In contrast to the activity of vincristine / vincaleukoblastine, triazolopyrimidine Compound 2 binds only to MT and not to unpolymerized tubulin heterodimers. See Sáez-Calvo, “Triazolopyrimidines Are Microtubule-Stabilizing Agents that Bind the Vinca Inhibitor Site of Tubulin,” Cell Chemical Biology, 2017, 24, 737-750e6.

[0012]

[0013] Triazolopyrimidine structural modifications can promote MT stabilization or disrupt MT integrity. These differences may have important and unknown effects on the therapeutic applications of triazolopyrimidines, including exhibiting different binding modes.

[0014] There is still a need for appropriately substituted triazolopyrimidine compounds for the treatment of neurodegenerative diseases. SUMMARY OF THE INVENTION

[0015] There are provided substituted {1,2,4,}triazolo{1,5-A}pyrimidine compounds of formula I:

[0016]

[0017] wherein

[0018] R1 is Cl;

[0019] R2 is CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3, CH(tert-butyl)CH3 or CH(methyldiaziridinyl)CH3, where R2 may optionally be mono-deuterated or multi-deuterated;

[0020] R3 is H or alkyl; or R2 and R3 together with the N atom to which they are attached form a C5-C7 heterocycle or 3-methoxy-8-azabicyclo[3.2.1]octan-8-yl;

[0021] R4 is H or F;

[0022] R5 is H or F;

[0023] R6 is iodine, cyano, ethynyl or -C≡C-(CH2) n -R7, where n = 0-3, R7 is H, -OH, -NH2, -NHCH3 or -N(CH3)2, where R6 may optionally be mono-deuterated or multi-deuterated;

[0024] R8 is H or alkyl;

[0025] or a stereochemical isomer thereof;

[0026] or a pharmaceutically acceptable salt thereof.

[0027] Also disclosed is a composition for treating a neurodegenerative disease or cancer, the composition comprising a therapeutically effective amount of a compound according to formula I, and also disclosed is a method for treating a neurodegenerative disease or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to formula I. Detailed Description

[0028] [1,2,4]Triazolo[1,5-a]pyrimidines (TPDs) and related heterocyclic molecules with microtubule (MT) activity have received attention as potential candidates for use in the treatment of neurodegenerative diseases. The present inventors have previously characterized a number of MT-stabilizing TPDs and MT-destabilizing TPDs. For example, U.S. Patent No. 9,649,317 discloses methods of using such compounds. The present disclosure provides new TPDs containing specified C6-phenyl substitutions. Previous studies of the MT-stabilizing properties of TPDs in MT-stabilizing cell-based assays have shown that different substitution patterns can cause the molecule to either promote MT stabilization or, conversely, disrupt MT stabilization. Based on the observation that specific structural features in TPDs are crucial for triggering an increase in MT stability, the inventors conducted a more systematic exploration of the SAR of the TPD scaffold. The SAR work led to the synthesis of new C6-cyano and alkynyl-substituted TPDs, as described herein. Importantly, the currently disclosed molecules have improved MT-stabilizing activity and pharmaceutical properties relative to previously described examples.

[0029] Accordingly, it has now been found that certain classes of microtubule-stabilizing compounds will be useful in the treatment of neurodegenerative diseases, particularly tauopathies, such as, for example, Alzheimer's disease, frontotemporal lobar degeneration, Pick's disease, progressive supranuclear palsy (PSP), and corticobasal ganglionic degeneration. In addition, the compounds of the present invention can also be used in other diseases in which tau pathology is a comorbidity or in which microtubule function is impaired, such as schizophrenia, Parkinson's disease (PD), PD with dementia, dementia with Lewy bodies, and amyotrophic lateral sclerosis.

[0030] The compounds of the present invention can also be used to treat traumatic brain injury (TBI), particularly repetitive TBI (rTBI), such as that due to boxer dementia and recurrent soccer concussions, as well as military closed head injuries (e.g., injuries caused by IEDs), which is also known as chronic traumatic encephalopathy (CTE) and has tauopathy or AD-like pathological features. It is hypothesized that PTSD may also lead to CTE.

[0031] In this disclosure, the singular forms "a", "an", and "the" include plural references, and a reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. Thus, for example, a reference to "a material" refers to at least one of such materials known to those of skill in the art and their equivalents, and so forth.

[0032] When a value is expressed as an approximation using the descriptor "about", it will be understood that the particular value forms another embodiment. In general, the use of the term "about" to indicate an approximation can vary according to the desired characteristics sought by the disclosed subject matter and is to be interpreted based on its function in the specific context in which it is used. A person skilled in the art will be able to interpret this as a routine matter. In some cases, the number of significant digits used for a particular value can be a non-limiting method for determining the range of the term "about". In other cases, the gradient used in a series of values can be used to determine the expected range within which the term "about" is available for each value. If present, all ranges are inclusive and combinable. That is, a reference to a value stated in a range includes each value within that range.

[0033] Unless otherwise specified, when a list is presented, it should be understood that each individual element of the list and every combination of the list should be interpreted as a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0034] As used herein, "alkyl" refers to an optionally substituted saturated straight-chain or branched-chain hydrocarbon group having from about 1 to about 20 carbon atoms (and all combinations and sub-combinations of the ranges and specific numbers of carbon atoms therein). For example, "alkyl" can refer to a substituted or unsubstituted straight-chain or branched-chain hydrocarbon group having from about 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 carbon atoms. In some embodiments, "alkyl" refers to an unsubstituted straight-chain group having from about 1 to 6 carbon atoms. If appropriate, "alkyl" can mean "alkylene"; for example, if X is R1R2 and R1 is referred to as "alkyl", then "alkyl" can be correctly interpreted as "alkylene".

[0035] "Heterocyclic group" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic ring group, which includes two to twelve carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the only heteroatom is the nitrogen atom connected to R2 and R3. Whenever a heteroatom appears herein, a numerical range, such as "3 to 18", refers to each integer within the given range. For example, "3 to 18 ring atoms" means that the heterocyclic group can be composed of 3 ring atoms, 4 ring atoms, etc., up to 18 ring atoms. Unless otherwise specified in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system, which can include a fused or bridged ring system. The heteroatoms in the heterocyclic group can be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group can be connected to the rest of the molecule through any atom of the ring. Examples of heterocyclic groups include, but are not limited to, azepanyl, azocanyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidoneyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. "Heterocyclic group" can also include a bicyclic ring system, where one non-aromatic ring generally has 3 to 7 ring atoms, contains at least 2 carbon atoms in addition to 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, and contains a combination of at least one of the aforementioned heteroatoms; and the other ring, generally having 3 to 7 ring atoms, optionally contains 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, and is not aromatic. The heterocyclic group moiety is optionally substituted with one, two, or three substituents selected from halogen (F, Cl, Br, or I, preferably F), -OH, -OC 1-6 alkyl, -CN, -NH2, -NH(C 1-6 alkyl), -NH(C 1-6 alkyl)2, C 3-8 cycloalkyl, heterocyclic group, aryl, or heteroaryl.

[0036] It will be appreciated that, for clarity, certain features of the invention described herein in the context of different embodiments may also be provided in combination in a single embodiment. That is, each individual embodiment is considered combinable with any other embodiment unless clearly incompatible or excluded, and such combination is considered to be another embodiment. Conversely, for the sake of brevity, the various features of the invention described in the context of a single embodiment may also be provided separately or in any sub-combination. It should also be noted that the claims may be drafted to exclude any optional elements. Accordingly, this statement is intended as a precursor basis for using "negative" limitations or exclusionary terms such as "separately", "only", etc. in combination with the recitation of claim elements. Finally, although an embodiment may be described as part of a series of steps or part of a more general structure, each of the said steps may itself be considered an independent embodiment.

[0037] This compound includes a compound of formula I:

[0038]

[0039] wherein

[0040] R1 is Cl;

[0041] R2 is CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3, CH(tert-butyl)CH3 or CH(methyldiaziridinyl)CH3, wherein R2 may optionally be mono-deuterated or poly-deuterated;

[0042] R3 is H or alkyl; or R2 and R3 together with the N atom to which they are attached form a C5-C7 heterocycle or 3-methoxy-8-azabicyclo[3.2.1]octan-8-yl;

[0043] R4 is H or F;

[0044] R5 is H or F;

[0045] R6 is iodine, cyano, ethynyl or -C≡C-(CH2) n -R7, where n = 0-3, R7 is H, -OH, -NH2, -NHCH3 or -N(CH3)2, wherein R6 may optionally be mono-deuterated or poly-deuterated;

[0046] R8 is H or alkyl;

[0047] or its stereochemical isomers;

[0048] or its pharmaceutically acceptable salts.

[0049] Stereoisomeric forms of the compounds of formula I, such as enantiomers, diastereomers and atropisomers, as well as pharmaceutically acceptable salts of any compound or stereoisomer of formula I are also within the scope of the present invention. As used herein, "stereoisomers" refers to all enantiomerically / diastereomerically pure and enantiomerically / diastereomerically enriched compounds of the present invention. Atropisomers, i.e., stereoisomers resulting from restricted rotation about a single bond, are also within the scope of the term "stereoisomers".

[0050] As used herein, "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention that are generally regarded as safe for pharmaceutical use. Examples include, for example, hydrochloride, sulfate, fumarate, succinate, ascorbate, maleate, mesylate and isethionate.

[0051] R3 is H or alkyl. In certain embodiments, R3 is H. In other embodiments, R3 is alkyl, such as C1-C6 alkyl.

[0052] In certain embodiments of the compounds of the present invention, R6 is iodine. In some of these embodiments, both R4 and R5 are F and R3 is H. In embodiments where both R4 and R5 are F, R2 can be, for example, CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3 or CH(tert-butyl)CH3.

[0053] In certain embodiments of the compounds of the present invention, R6 is cyano. In some of these embodiments, both R4 and R5 are F. In embodiments where both R4 and R5 are F, R2 can be CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3 or CH(tert-butyl)CH3. In other embodiments where both R4 and R5 are F, R2, R3 and the N atom to which they are attached can together form a piperidinyl, azepanyl or azocanyl group.

[0054] In certain other embodiments where R6 is cyano, R4 is H and R5 is F. In some of these embodiments, R2 can be CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3 or CH(tert-butyl)CH3. In some of these embodiments, R2, R3 and the N atom to which they are attached together form a piperidinyl, azepanyl or azocanyl group.

[0055] In other embodiments, R6 is ethynyl. In some of these embodiments, R4 and R5 can both be F. Regarding certain of these embodiments, R2 can be CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3, or CH(tert-butyl)CH3. In certain other of these embodiments, R2, R3, and the N atom to which they are attached together form a piperidinyl, azepanyl, or azocanyl group.

[0056] In certain other embodiments where R6 is ethynyl, R4 is H and R5 is F. In some of these embodiments, R2 is CH(CH3)CF3, CH(isopropyl)CF3, CH(isopropyl)CH3, or CH(tert-butyl)CH3. In certain of these embodiments, R2, R3, and the N atom to which they are attached together form a piperidinyl, azepanyl, or azocanyl group.

[0057] In some embodiments, R6 is -C≡C-(CH2) n -R7. In certain of these embodiments, n is 1 - 3. In some of these embodiments, R4 and R5 are both F.

[0058] Exemplary compounds according to the present disclosure are described in Table 1, as follows:

[0059] Table 1

[0060]

[0061]

[0062] Also provided are compositions for treating neurodegenerative diseases or cancer, which comprise a therapeutically effective amount of a compound according to any of the above embodiments. Also disclosed is a method for treating neurodegenerative diseases or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any of the above embodiments.

[0063] As used herein, the phrase "therapeutically effective amount" refers to the amount of an active compound that elicits a biological or pharmaceutical response sought by a researcher, veterinarian, medical doctor, or other clinician in a tissue, system, animal, individual, or human body, and the biological or pharmaceutical response includes one or more of the following:

[0064] (1) At least partially preventing a disease or disorder or its symptoms; for example, preventing the disease, disorder, or condition in an individual who may be susceptible to the disease, disorder, or condition but has not yet experienced or shown the pathology or symptoms of the disease.

[0065] (2) Suppressing a disease or disorder; for example, suppressing the disease, disorder or impairment in an individual who is experiencing or showing the pathology or symptoms of the disease, disorder or impairment (i.e., including preventing further development of the pathology and / or symptoms); and

[0066] (3) At least partially ameliorating a disease or disorder; for example, ameliorating the disease, disorder or impairment in an individual who is experiencing or showing the pathology or symptoms of the disease, disorder or impairment (i.e., including reversing the pathology and / or symptoms).

[0067] The compounds according to the present disclosure can be provided in the form of a composition formulated for any type of administration. For example, the composition can be formulated for oral administration, topical administration, parenteral administration, enteral administration or administration by inhalation. The active compound can be formulated for neat administration or in combination with conventional pharmaceutical carriers, diluents or excipients which can be liquid or solid. Applicable solid carriers, diluents or excipients can, among other things, act as binders, disintegrants, fillers, lubricants, glidants, compression aids, processing aids, colorants, sweeteners, preservatives, suspension / dispersants, tablet disintegrants, encapsulating materials, film formers or coatings, flavoring agents or printing inks. Any material used for preparing any unit dosage form is preferably pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compound can be incorporated into sustained release formulations and preparations. Administration in this regard includes, in particular, administration by the following routes: intravenous, intramuscular, subcutaneous, intraocular, intra-synovial, trans-epithelial (including transdermal, trans-ocular, sublingual and buccal), topical administration (including trans-ocular, transdermal, trans-rectal and trans-nasal administration by insufflation, aerosol and rectal systems).

[0068] In powders, the carrier, diluent or excipient can be a finely divided solid mixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted into the desired shape and size. For oral therapeutic administration, the active compound can be combined with a carrier, diluent or excipient and used in the form of ingestible tablets, lozenges, troches, capsules, elixirs, suspensions, syrups, cachets, etc. The amount of the active compound in such therapeutically useful compositions is preferably such that a suitable dosage can be obtained.

[0069] Liquid carriers, diluents or excipients can be used to prepare solutions, suspensions, emulsions, syrups, elixirs, etc. The active ingredient of the present invention can be dissolved or suspended in a pharmaceutically acceptable liquid, such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat. The liquid carrier, excipient or diluent can contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, coloring agents, viscosity regulators, stabilizers or osmotic pressure regulators.

[0070] Suitable solid carriers, diluents and excipients can include, for example, calcium phosphate, silica, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, low melting point wax, ion exchange resin, cross-linked carboxymethylcellulose carbon, acacia, pre-gelatinized starch, cross-linked polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar, tragacanth or mixtures thereof.

[0071] For example, for oral, topical or parenteral administration, suitable examples of liquid carriers, diluents and excipients include water (especially containing the additives described above, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, such as diols) and their derivatives, and oils (such as fractionated coconut oil and peanut oil), or mixtures thereof.

[0072] For parenteral administration, the carrier, diluent or excipient can also be an oily ester, such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents or excipients for use in sterile liquid form compositions for parenteral administration. Solutions of the active compound in free base or pharmaceutically acceptable salt form can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary storage and use conditions, these formulations can contain preservatives to prevent the growth of microorganisms.

[0073] Pharmaceutical forms suitable for injection include, for example, sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide ease of injection. It is preferably stable under the conditions of manufacture and storage and preferably protects against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent or excipient can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity is maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it preferably includes isotonic agents, for example, sugars or sodium chloride. The absorption of injectable compositions can be extended by using agents that delay absorption (such as aluminum monostearate and gelatin).

[0074] Sterile injectable solutions can be prepared by incorporating the pharmaceutically appropriate amount of the active compound, as required, with the various other ingredients enumerated above into a suitable solvent and then subjecting the solution to sterile filtration. Generally, dispersions can be prepared by incorporating the sterilized active ingredient into a sterile vehicle (containing the base dispersion medium and the required other ingredients from those enumerated above). In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation can include vacuum drying and freeze-drying techniques, which can yield a powder of the one or more active ingredients plus any additional required ingredients from its previously sterile-filtered solution.

[0075] Thus, the active compound can be present in the compositions and methods in an effective amount by any of the conventional techniques established in the art of medicine. For example, the dosage can be from about 0.1 mg / day to about 500 mg / day. In some embodiments, the dosage can be about 250 mg / kg / day. Thus, the dosage can be about 0.1 mg / day, about 0.5 mg / day, about 1.0 mg / day, about 5 mg / day, about 10 mg / day, about 2 mg / day, about 50 mg / day, about 100 mg / day, about 200 mg / day, about 250 mg / day, about 300 mg / day or about 500 mg / day.

[0076] A method of treating a neurodegenerative disease in a subject, the neurodegenerative disease being characterized by tauopathy or impaired microtubule function in the subject's brain. In certain embodiments, the neurodegenerative disease can be Alzheimer's disease, frontotemporal lobar degeneration, Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, Parkinson's disease (PD), PD with dementia, dementia with Lewy bodies, or amyotrophic lateral sclerosis. In other embodiments, the neurodegenerative disease is traumatic brain injury or post-traumatic stress disorder. The traumatic brain injury can be, for example, repetitive traumatic brain injury or chronic traumatic encephalopathy. In other embodiments, the neurodegenerative disease is schizophrenia.

[0077] A method of treating cancer in a subject according to the present disclosure, the cancer can be carcinoma, sarcoma, melanoma, lymphoma, or leukemia. Common carcinomas include, for example, cancers originating from the skin, lung, breast, pancreas, and other organs and glands. Sarcomas are represented by cancers occurring in bone, muscle, fat, blood vessels, cartilage, or other soft tissues or connective tissues of the body. Exemplary cancers that can be treated according to this method include brain cancer (such as glioma and astrocytoma), bladder cancer, breast cancer, colon or rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer.

[0078] Hereinafter, the present disclosure will be described in more detail by examples intended to illustrate the present disclosure, but the present disclosure is not limited to the examples.

[0079] Example 1 - Synthesis Procedure

[0080] As part of these studies, a total of 73 compounds were synthesized and tested, including seven previously described triazolo[1,5-a]pyrimidines; 25 compounds listed in the patent literature; and 41 structurally novel homologues. In all cases, the triazolo[1,5-a]pyrimidine ring was obtained by a ring condensation reaction between the appropriate diethyl malonate (79 - 95, Scheme 1) and 1H-1,2,4-triazol-5-amine. Then, treatment with phosphorus oxychloride gave the corresponding 5,7-dichlorotriazolo[1,5-a]pyrimidine (96 - 112, Scheme 1). Chemoselective amination at C7 with (S)-1,1,1-trifluoropropan-2-amine gave triazolo[1,5-a]pyrimidines 6 - 22 (Scheme 1).

[0081]

[0082] Protocol 1 Reagents and reaction conditions: a) For 79 - 88, 90 - 94: Ar-X, CuBr, NaH, 1,4-dioxane, 60 - 100 °C, 12 h, 19 - 74%; For 89: Ar-CH2Br, NaH, N,N-dimethylformamide, 0 °C, 1 h, 90%; For 95: Ar-F, K2CO3, N,N-dimethylformamide, 60 °C, 4 h, 86%; b) N-tributylamine, 170 - 180 °C, 2 - 6 h; c) Phosphorus oxychloride, 110 - 130 °C, 6 - 16 h, 46 - 58% over two steps; d) Appropriate amine, N,N-dimethylformamide, room temperature to 90 °C, 1 - 18 h, 12 - 50%; e) Fe, NH4Cl, H2O / MeOH (40 / 50), 80 °C, 2 h, 69%.

[0083] Compounds 61 - 78 were synthesized by reacting an appropriate dichlorotriazolopyrimidine (i.e., 98, 99, 109 or 110) with an amine moiety (Protocol 5), which were modified at two moieties at C6 and C7 relative to control compound 6. In selected cases (i.e., 67 and 68), each atropisomer was obtained by purification by silica gel column chromatography, and its structure was determined by X-ray crystallography (Protocol 5):

[0084]

[0085]

[0086]

[0087] Protocol 5 Reagents and reaction conditions: a) Appropriate amine, N,N-dimethylformamide, room temperature, 1 h, 56 - 86%.

[0088] Example 2 - Materials and Methods

[0089] All solvents were of reagent grade. All reagents were purchased from Aldrich or Acros and used directly. Thin layer chromatography (TLC) was carried out using 0.25 mm pre-coated silica gel plates from E.Merck. Silica gel column chromatography was performed using silica gel 60 (particle size 0.040 - 0.062 mm) supplied by Silicycle and Sorbent Technologies. TLC spots were detected by observing under ultraviolet light. Melting points (mp) were obtained on a Mel-Temp II (model: 1001) and were uncorrected. Infrared (IR) spectra were recorded on a Bruker Alpha spectrometer (part number 1003271 / 03). Proton ( 1H) and carbon ( 13 C) nuclear magnetic resonance (NMR) spectroscopy. Chemical shifts were reported relative to the solvent. High-resolution mass spectrometry was performed using an Agilent 6230 time-of-flight mass spectrometer (TOFMS) with a jet electrospray ionization source (ESI). Single-crystal X-ray structure determination was carried out using a Bruker MicroStar with an APEX II detector, dual bounce microfocus optics, and a Cu rotating anode source. Analytical reverse-phase (Sunfire C18; 4.6 mm × 50 mm, 5 mL) high-performance liquid chromatography (HPLC) was performed using a Gilson HPLC equipped with UV and mass detectors. All samples were analyzed using a linear gradient of 10% to 90% CH3CN in water (over an 8-minute period) and a flow rate of 1 mL / min, with a purity level of >95% unless otherwise stated. Preparative reverse-phase HPLC purification was carried out on a Gilson instrument using a Waters SunFire 18 OBD column (5 μm 19 mm × 50 mm or 19 mm × 100 mm). A linear gradient of 10% to 90% CH3CN in water and a flow rate of 20 mL / min were used for 15 minutes for purification. Unless otherwise stated, all final compounds had a purity greater than 95% as determined by HPLC / MS and NMR.

[0090] General procedure A (Synthesis of diethyl malonate derivatives)

[0091] Diethyl malonate (3.00 equiv) was slowly added to a suspension of NaH (60% in mineral oil) (1.00 equiv) in anhydrous 1,4-dioxane (previously degassed with N2) (at a concentration of 1.75 mol / L of the aryl bromide derivative) at 60 °C under N2 and stirred for 10 minutes. Then, CuBr (1.2 equiv) and the aryl bromide derivative were added, and the reaction was heated at reflux overnight. Then, at room temperature, the reaction was quenched with 12N HCl (1.40 equiv). The mixture was filtered and washed with H2O. The filtrate was extracted with EtOAc (x3). The combined organic extracts were washed with brine, then dried over MgSO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography or preparative reverse-phase high-performance liquid chromatography (HPLC) to give the pure diethyl malonate derivative.

[0092] General procedure B (Synthesis of dichlorotriazolopyrimidine derivatives)

[0093] A mixture of diethyl malonate derivative (1.00 equiv), 3-amino-1,2,4-triazole (1.05 equiv) and tributylamine (1.05 equiv) in a sealed tube was stirred at 170 °C for 2 h. After cooling to 130 °C, toluene was added (1.00 mol / L based on diethyl malonate derivative). Then, the reaction was cooled to 50 °C and an aqueous NaOH solution (50% wt) (160 μL / mmol of diethyl malonate derivative) was added. Thereafter, the mixture was stirred at 0 °C for 10 minutes and filtered. The formed solid was washed with cold toluene and dried. The disodium derivative (1.00 equiv) and POCl3 (17.80 equiv) were mixed in a sealed tube and heated at 130 °C for 6 h. Then, the reaction was quenched with H2O and extracted with EtOAc (×2). The combined organic extracts were washed with water (×5), brine, dried over MgSO4, filtered and concentrated in vacuo. The resulting product was used directly in the next reaction without further purification.

[0094] General procedure C (addition of amine)

[0095] According to the reported procedure (Zhang et al., J. Med. Chem., 2007, 50, 319 - 327), at room temperature, i-Pr2NEt (3.0 equiv) or Et3N (3.0 equiv) (if necessary) and the appropriate amine (1.5 to 3.0 equiv) were added to DMF (0.1 M) containing 5,7-dichloro-6-(2,4,6-trifluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (1.0 equiv). The reaction mixture was stirred for 0.5 - 16 h and diluted with H2O. The organic layer was washed with 1 N hydrochloric acid solution (2x), the aqueous phase was extracted with EtOAc (3x), the combined organic layers were washed with brine (2x), dried (MgSO4), filtered and concentrated. The product was purified by flash chromatography or reverse-phase HPLC.

[0096] General procedure D (Addition of side chains)

[0097] According to the reported procedure (Zhang et al., J. Med. Chem., 2007, 50, 319 - 327; Zhang et al., Bioorg. Med. Chem. 2009, 17, 111 - 8), the amino alcohol (4.0 equiv) was added to a suspension (0.35 M) of NaH (4.0 equiv) in a 2:1 mixture of DMSO and THF. The mixture was heated to 60 °C for 1 h. The resulting solution was treated with a solution of the trifluoroarene (1.0 equiv) in a 2:1 mixture of DMSO and THF (0.5 M). The reaction mixture was stirred at 60 °C for 3 h and monitored by LCMS. If starting material was still present after 3 h, additional amino alcohol (4.0 equiv) and NaH (4.0 equiv) were added successively and the reaction mixture was heated for 16 h. After complete consumption of the starting material, the reaction mixture was cooled to room temperature and diluted with H2O and EtOAc. The organic layer was washed with H2O and brine, and the combined aqueous layers were extracted with EtOAc (x3). The combined organic layers were dried (MgSO4), filtered, and concentrated. The product was purified by reverse - phase HPLC.

[0098]

[0099] (S)-4-(5 - chloro - 7 - ((1,1,1 - trifluoropropan - 2 - yl)amino)-[1,2,4]triazolo[1,5 - a]pyrimidin - 6 - yl)-3,5 - difluorobenzonitrile

[0100] Following general procedure C, using 4-(5,7 - dichloro - [1,2,4]triazolo[1,5 - a]pyrimidin - 6 - yl)-3,5 - difluorobenzonitrile (30 mg, 0.094 mmol) and (S)-1,1,1 - trifluoropropan - 2 - amine (22 mg, 0.190 mmol). Purification by silica gel column chromatography (0 - 40% EtOAc in hexanes) gave the title compound (20 mg, 0.050 mmol, 54%) as a white powder.

[0101] 1 1H NMR (600 MHz, CDCl3) δ 8.41 (s, 1H), 7.44 (t, J = 7.1 Hz, 2H), 6.00 (d, J = 10.7 Hz, 1H), 4.77 (s, 1H), 1.45 (d, J = 6.9 Hz, 3H) ppm.

[0102] 1313C NMR (150 MHz, CDCl3) δ 161.28 (dd, J = 256.4, 5.9 Hz), 160.87 (dd, J = 254.7, 6.0 Hz), 156.84, 155.68, 154.32, 124.46 (q, J = 282.1 Hz), 116.58 (t, J = 11.8 Hz), 116.47 (ddd, J = 67.6, 25.4, 4.1 Hz), 115.79 (d, J = 3.5 Hz), 114.93 (t, J = 20.0 Hz), 90.61, 51.27 (q, J = 32.2 Hz), 29.83, 15.14 ppm.

[0103] IR: 2923, 2239, 1617, 1556, 1141 cm -1 。

[0104] HRMS (ES+): C 15 H9N6ClF5 [M+H] + Calculated, 403.0492; Found, 403.0486.

[0105]

[0106] (S)-4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile

[0107] Following general procedure C, using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (40 mg, 0.13 mmol) and (S)-1,1,1-trifluoropropan-2-amine (31 mg, 0.27 mmol). Purification by reverse-phase HPLC gave the title compound (31 mg, 0.070 mmol, 54%) as a white powder.

[0108] A mixture of atropisomers

[0109] 1 1H NMR (600 MHz, CDCl3) δ 8.41 (s, 1H), 7.68 (ddd, J = 7.8, 3.8, 1.5 Hz, 1H), 7.61 (ddd, J = 8.4, 3.8, 1.5 Hz, 1H), 7.57 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.3 Hz, 1H), 5.94 (d, J = 11.0 Hz, 1H), 5.57 (d, J = 10.8 Hz, 1H), 4.97 (s, 1H), 4.50 (s, 1H), 1.40 (d, J = 6.9 Hz, 3H) ppm.

[0110] 13 13C NMR (150 MHz, CDCl3) δ 161.29 (d, J = 52.0 Hz), 159.61 (d, J = 50.4 Hz), 156.65 (d, J = 24.9 Hz), 155.72 (d, J = 11.0 Hz), 154.20 (d, J = 57.5 Hz), 145.65 (d, J = 62.8 Hz), 135.00 (d, J = 2.0 Hz), 133.84 (d, J = 2.2 Hz), 129.21 (d, J = 4.2 Hz), 124.97 (dd, J = 16.2, 13.1 Hz), 124.67 (qd, J = 282.3, 34.7 Hz), 120.62 (dd, J = 74.3, 25.2 Hz), 116.67 (d, J = 2.8 Hz), 116.33 (dd, J = 9.3, 5.2 Hz), 97.66, 96.61, 51.26 (dq, J = 37.9, 31.8 Hz), 15.13, 14.99 ppm.

[0111] HRMS (ES+): C 15 H 10 ClF4N6 [M + H] + Calcd, 385.0586; Found, 385.0589.

[0112]

[0113] (R)-4-(5-Chloro-7-((1,1,1-trifluoro-3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile

[0114] According to General Procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (55 mg, 0.17 mmol) and (R)-1,1,1-trifluoro-3-methyl-2-butylamine (48 mg, 0.34 mmol) were used. After purification by reverse-phase HPLC, the title compound (26 mg, 0.060 mmol, 36%) was obtained as a white powder.

[0115] 11H NMR (600 MHz, CDCl3, mixture of atropisomers) δ 8.81 (s, 0.5H), 8.69 (s, 0.5H), 7.46 (t, J = 9 Hz, 2H), 7.33 (d, J = 12 Hz, 1H), 6.28 (bs, 1H), 4.00 (bs, 1H), 2.19 (hept, J = 6 Hz, 1H), 1.04 (d, J = 6 Hz, 1H), 0.97 (d, J = 6 Hz, 1H) ppm.

[0116] 13 13C NMR (150 MHz, CDCl3, mixture of atropisomers) δ 162.05, 162.01, 161.79, 161.74, 161.54, 161.49, 160.35, 160.30, 160.10, 160.07, 159.86, 159.814, 158.37, 157.23, 157.16, 155.65, 154.61, 152.15, 146.27, 144.77, 139.79, 133.47, 124.33 (q, J = 283.88 Hz), 117.41, 116.85, 116.82, 116.78, 116.70, 116.68, 116.65, 116.40, 116.17, 116.00, 115.84, 115.30, 115.04, 114.91, 113.00, 109.71, 106.86, 59.28 (q, J = 28.69 Hz), 28.28, 19.80, 16.60 ppm.

[0117] HRMS (ES+): C 18 H 17 ClFN6 [M+H] + Calcd, 429.0659; Found, 429.0655.

[0118]

[0119] (R)-4-(5-Chloro-7-((1,1,1-trifluoro-3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile

[0120] Following general procedure C, using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (51 mg, 0.17 mmol) and (R)-1,1,1-trifluoro-3-methylbutan-2-amine (47 mg, 0.34 mmol). Purification by reverse-phase HPLC gave the title compound (13 mg, 0.060 mmol, 19%) as a white powder.

[0121] 1 1H NMR (600 MHz, CDCl3, mixture of atropisomers) δ 8.42 (s, 0.5H), 7.69 (d, J = 7.9 Hz, 0.5H), 7.63 (d, J = 8.2 Hz, 0.5H), 7.60 (d, J = 8.2 Hz, 0.5H), 7.55 (t, J = 7.5 Hz, 0.5H), 7.48 (t, J = 7.3 Hz, 0.5H), 4.86 (d, J = 10.2 Hz, 0.5H), 4.40 (d, J = 4.1 Hz, 0.5H), 2.14 (hept, J = 6.9 Hz, 1H), 1.02 (d, J = 6.1 Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H).

[0122] 13 13C NMR (150 MHz, CDCl3, mixture of atropisomers) δ 160.44 (dd, J = 253.68, 28.69 Hz), 158.91, 155.66, 155.60, 134.88, 134.50, 129.18, 129.02, 126.68, 124.86, 120.97, 120.81, 120.39, 120.21, 116.74 (t, J = 3.0 Hz), 116.42 (t, J = 13.4 Hz), 116.36 (t, J = 13.4 Hz), 59.08 (q, J = 28.7 Hz), 55.89 (q, J = 28.7 Hz), 28.33, 28.19, 27.75, 19.92, 19.80, 17.11, 16.74, 16.69 ppm.

[0123] HRMS (ES+): C 18 H 17 ClFN6 [M + H] + Calculated value, 411.0754; Found value, 411.0747.

[0124]

[0125] (R)-4-(5-Chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile

[0126] According to General Procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (30 mg, 0.092 mmol) and (R)-3-methylbutan-2-amine (17 mg, 0.190 mmol) were used. After purification by reverse-phase HPLC, the title compound (28 mg, 0.074 mmol, 81%) was obtained as a white powder.

[0127] 1 1H NMR (600 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.11 (t, J = 7.8 Hz, 2H), 7.88 (s, 1H), 1.78 (dq, J = 13.9, 6.8 Hz, 1H), 1.10 (d, J = 6.6 Hz, 3H), 0.74 (dd, J = 6.8, 2.9 Hz, 6H).

[0128] 13 13C NMR (151 MHz, DMSO-d6) δ 160.71 (dd, J = 249.2, 6.8 Hz), 160.38 (dd, J = 249.5, 6.3 Hz), 155.02, 146.81, 117.00 (ddd, J = 26.3, 12.6, 3.9 Hz), 116.48 (d, J = 3.5 Hz), 114.83 (t, J = 12.8 Hz).

[0129] IR: 2966, 2220, 1690, 1562, 1422, 1204 cm -1 .

[0130] HRMS (ES+): C 17 H 16 ClF2N6 [M + H] + Calculated value, 377.1088; Found value, 377.1089.

[0131]

[0132] (R)-4-(5-chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile

[0133] According to general procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (48 mg, 0.15 mmol) and (R)-3-methylbutan-2-amine (37 mg, 0.42 mmol) were used. The mixture was dried under vacuum and purified by silica gel column chromatography to give the atropisomer A (29 mg, 0.081 mmol, 39%) as a white solid and the atropisomer B (23 mg, 0.064 mmol, 30%).

[0134] Atropisomer A

[0135] 1 H NMR (600 MHz, CDCl3) δ 8.35 (s, 1H), 7.63 (dd, J = 12.0, 3.0 Hz, 1H), 7.56 (dd, J = 12.0, 3.0 Hz, 1H), 7.53 (t, J = 9.0 Hz, 1H), 6.29 (d, J = 12.0 Hz, 1H), 3.00 (bs, 1H), 1.64 (oct, J = 6.0 Hz, 1H), 0.99 (d, J = 6 Hz, 3H), 0.80 (d, J = 6 Hz, 3H), 0.77 (d, J = 6 Hz, 3H) ppm.

[0136] 13 C NMR (150 MHz, CDCl3) δ 161.37, 159.70, 156.91, 155.15, 153.15, 145.67, 134.39, 128.71, 128.69, 127.13 (d, J = 16.6 Hz), 120.02 (d, J = 25.7 Hz), 116.98, 115.50 (d, J = 9.1 Hz), 94.53, 55.04, 33.48, 18.16, 17.80, 17.14 ppm.

[0137] Atropisomer B

[0138] 1 H NMR (600 MHz, CDCl3) δ 8.35 (s, 1H), 7.63 (dd, J = 12.0, 3.0 Hz, 1H), 7.56 (dd, J = 12.0, 3.0 Hz, 1H), 7.52 (t, J = 9.0 Hz, 1H), 6.30 (bs, 1H), 3.05 (bs, 1H), 1.61 (oct, J = 6.0 Hz, 1H), 1.05 (d, J = 6 Hz, 3H), 0.79 (d, J = 6 Hz, 3H), 0.75 (d, J = 6 Hz, 3H) ppm.

[0139] 1313C NMR (150 MHz, CDCl3) δ 161.40, 159.73, 156.90, 155.19, 153.57, 145.67, 135.08, 134.40 (d, J = 3.0 Hz), 128.55 (d, J = 4.5 Hz), 127.03 (d, J = 15.1 Hz), 120.22 (d, J = 25.7 Hz), 117.01, 115.45 (d, J = 9.1 Hz), 94.37, 54.85, 33.80, 18.20, 17.90, 17.82 ppm.

[0140] IR: 2963, 2220, 1608, 1570, 1260, 1156 cm -1

[0141] HRMS (ES+): C 18 H 17 ClFN6 [M + H] + Calcd for, 359.1182; found, 359.1184.

[0142]

[0143] (R)-4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile

[0144] According to General Procedure C, using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (20 mg, 0.060 mmol) and (R)-3,3-dimethylbutan-2-amine (13 mg, 0.13 mmol). Purified by reverse-phase HPLC to give the title compound (18 mg, 0.046 mmol, 75%) as a white powder.

[0145] 1 1H NMR (600 MHz, CDCl3) δ 8.36 (s, 1H), 7.45–7.41 (m, 2H), 6.52 (d, J = 10.8 Hz, 1H), 2.94 (s, 1H), 1.02 (d, J = 6.7 Hz, 3H), 0.84 (s, 9H) ppm.

[0146] 1313C NMR (150 MHz, CDCl3) δ 161.33 (dd, J = 253.8, 6.2 Hz), 161.05 (dd, J = 253.8, 6.2 Hz), 157.12, 155.22, 146.03, 116.22 (d, J = 4.0 Hz), 116.13–115.95 (m), 115.95–115.82 (m), 58.57, 34.93, 25.86, 16.56 ppm.

[0147] HRMS (ES+): C 18 H 18 ClF2N6 [M + H] + Calcd for, 391.1244; found, 391.1241.

[0148]

[0149] (R)-4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile

[0150] Following general procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (40 mg, 0.13 mmol) and (R)-3,3-dimethylbutan-2-amine (28 mg, 0.27 mmol) were used. Purification by reverse-phase HPLC gave the title compound (27 mg, 0.072 mmol, 56%) as a white powder.

[0151] Mixture of atropisomers

[0152] 1 1H NMR (600 MHz, CDCl3) δ 8.41–8.34 (m, 1H), 7.67–7.61 (m, 1H), 7.60–7.55 (m, 1H), 7.55–7.49 (m, 1H), 6.37 (s, 1H), 2.94 (m, 1H), 1.01 (d, J = 6.7 Hz, 1H), 0.94 (d, J = 6.7 Hz, 2H), 0.82 (s, 5H), 0.81 (s, 4H) ppm.

[0153] 1313C NMR (150 MHz, CDCl3) δ 161.71–159.28 (m), 156.76, 156.67, 145.96, 135.28 (d, J = 2.4 Hz), 133.96 (d, J = 2.3 Hz), 128.65 (d, J = 4.1 Hz), 128.43 (d, J = 4.1 Hz), 119.96 (dd, J = 37.6, 25.4 Hz), 116.86 (d, J = 2.8 Hz), 115.42 (dd, J = 16.7, 9.3 Hz), 94.27, 94.14, 58.27, 58.07, 35.01, 34.82, 25.77 (d, J = 1.9 Hz), 16.42 (d, J = 2.2 Hz) ppm.

[0154] HRMS (ES+): C 18 H 19 ClFN6 [M + H] + Calcd, 373.1338; found, 373.1338.

[0155]

[0156] 4-(5-Chloro-7-(piperidin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile

[0157] Following general procedure C, using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (48 mg, 0.15 mmol) and piperidine (25 mg, 0.30 mmol). Purification by reverse-phase HPLC gave the title compound (5 mg, 0.010 mmol, 9%), as a white powder.

[0158] 13 13C NMR (150 MHz, CDCl3) δ 160.71 (dd, J = 253.7, 9 Hz), 157.05, 155.60, 155.44, 150.84, 118.10 (t, J = 15.1 Hz), 116.22 (d, J = 6 Hz), 116.04, 115.06 (t, J = 12.1 Hz), 96.75, 51.34, 25.92, 23.57 ppm.

[0159] HRMS (ES+): C 18 H 17 ClFN6 [M + H] + Calcd, 375.0931; found, 375.0931.

[0160]

[0161] 4-(7-(Azepan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (51)

[0162] According to General Procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (20 mg, 0.060 mmol) and azepane (13 mg, 0.13 mmol) were used. After purification by reverse-phase HPLC, the title compound (15 mg, 0.039 mmol, 63%) was obtained as a white powder.

[0163] 1 H NMR (600 MHz, CDCl3) δ 8.38 (s, 1H), 7.40 (d, J = 6.0 Hz, 2H), 3.45–3.39 (m, 4H), 1.78–1.70 (m, 4H), 1.64–1.60 (m, 4H) ppm.

[0164] 13 C NMR (150 MHz, CDCl3) δ 160.74 (dd, J = 253.4, 6.6 Hz), 157.14, 155.26, 152.10, 118.61 (t, J = 19.7 Hz), 116.30–115.97 (m), 115.12 (t, J = 11.9 Hz), 97.32, 53.99, 51.22, 28.27, 28.17, 27.28, 27.07 ppm.

[0165] IR: 2928, 2857, 2238, 1589, 1515, 1422 cm -1 。

[0166] HRMS (ES+): C 18 H 16 ClF2N6 [M+H] + Calculated, 389.1088; Found, 389.1085.

[0167]

[0168] 4-(7-(Azepan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (55)

[0169] According to general procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (40 mg, 0.13 mmol) and azepane (27 mg, 0.27 mmol) were used. Purification by reverse-phase HPLC gave the title compound (27 mg, 0.073 mmol, 56%), as a white powder.

[0170] 1 H NMR (600 MHz, CDCl3) δ 8.34 (s, 1H), 7.61 (dd, J = 7.9, 1.6 Hz, 1H), 7.53 (dd, J = 8.7, 1.6 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 3.38–3.33 (m, 4H), 1.71 (dq, J = 8.0, 4.2 Hz, 4H), 1.60 (p, J = 3.0 Hz, 4H) ppm.

[0171] 13 C NMR (150 MHz, CDCl3) δ 160.85, 159.18, 156.93, 155.27, 155.18, 151.61, 134.44 (d, J = 2.8 Hz), 128.60 (d, J = 3.9 Hz), 128.48, 120.11, 119.94, 117.09 (d, J = 2.8 Hz), 114.68 (d, J = 9.2 Hz), 103.59, 54.05, 28.27, 28.03 ppm.

[0172] IR: 2926, 2235, 1589, 1519, 1446 cm -1 .

[0173] HRMS (ES+): C 18 H 17 ClFN6 [M + H] + Calculated, 371.1182; found, 371.1180.

[0174]

[0175] 4-(7-(azocan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile

[0176] According to general procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (54 mg, 0.17 mmol) and azacyclooctane (19 mg, 0.17 mmol) were used. The mixture was dried under vacuum and purified by silica gel column chromatography to give the title compound (31 mg, 0.077 mmol, 46%) as a white solid.

[0177] 1 H NMR (600 MHz, CDCl3) δ 8.29 (s, 1H), 7.30 (d, J = 6.0 Hz, 2H), 3.36 (t, J = 5.7 Hz, 5H), 1.62 (t, J = 5.8 Hz, 5H), 1.52 (t, J = 5.5 Hz, 6H), 1.44 (d, J = 6.0 Hz, 2H).

[0178] 13 C NMR (151 MHz, CDCl3) δ 160.68 (dd, J = 253.6, 6.5 Hz), 157.23, 155.73, 155.12, 151.06, 118.55 (t, J = 19.8 Hz), 116.38, 116.34, 116.2, 116.19, 115.23 (t, J = 11.8 Hz), 97.29, 52.24, 27.72, 26.80, 24.44 ppm.

[0179] HRMS (ES+): C 18 H 17 ClFN6 [M + H] + Calculated, 403.1244; found, 403.1240.

[0180]

[0181] 4-(7-(Azacyclooctan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile

[0182] According to general procedure C, 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (48 mg, 0.15 mmol) and azacyclooctane (35 mg, 0.30 mmol) were used. The mixture was dried under vacuum and purified by silica gel column chromatography to give the title compound (32 mg, 0.083 mmol, 53%) as a white solid.

[0183] 11H NMR (600 MHz, CDCl3, mixture of atropisomers) δ 8.59 (s, 0.4H), 8.42 (s, 0.3H), 7.80 - 7.77 (m, 0.6H), 7.30 (d, J = 6 Hz, 0.3H), 7.16 (d, J = 12 Hz, 0.4H), 3.41 (t, J = 6 Hz, 1.2H), 3.32 (bs, 1.8H), 3.04 (bs, 1.8H), 1.70 - 1.41 (m, 10H) ppm.

[0184] 13 13C NMR (150 MHz, CDCl3) δ 163.68, 159.94 (dd, J = 250.7, 19.6 Hz), 157.21, 155.29, 154.79, 151.33, 150.39, 148.23, 145.52, 144.37, 142.0, 140.38, 134.19, 133.98, 133.96, 132.10, 130.25, 130.20, 129.25, 128.98, 128.89, 128.86, 128.51, 128.40, 128.33, 128.30, 128.26, 128.21, 126.15, 120.38, 120.30, 120.22, 120.13, 117.09, 116.88, 114.89, 114.83, 114.73, 114.67, 103.26, 99.78, 54.20, 52.27, 51.56, 50.31, 44.80, 27.71, 26.84, 25.57, 25.70, 24.27, 23.95, 23.42, 22.69, 22.55, 22.51, 21.84, 21.52 ppm.

[0185] HRMS (ES+): C 18 H 17 ClFN6 [M + H] + Calcd., 385.1338; Found, 385.1332.

[0186]

[0187] Diethyl 2-(4-cyano-2,6-difluorophenyl)malonate

[0188] A mixture of 3,4,5-trifluorobenzonitrile (1.00 g, 6.37 mmol, 1 equiv), potassium carbonate (1.76 g, 12.7 mmol, 2.00 equiv), and diethyl malonate (1.03 g, 6.43 mmol, 1.01 equiv) in anhydrous DMF was stirred at 65 °C until the starting materials were consumed, as indicated by TLC. The reaction mixture was cooled to room temperature, washed with 1 M HCl (50 mL), and extracted with EtOAc (3x). The combined organic layers were washed with saturated anhydrous NaCl, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (up to 30% EtOAc in hexanes) afforded the title compound (1.40 g, 4.69 mmol, 73%) as a white solid.

[0189] 1 1H NMR (600 MHz, CDCl3) δ 7.30 (d, J = 7.0 Hz, 2H), 5.01 (s, 1H), 4.31–4.24 (m, 4H), 1.29 (t, J = 7.4 Hz, 6H) ppm.

[0190] 13 13C NMR (150 MHz, CDCl3) δ 165.54, 161.02 (dd, J = 254.0, 7.8 Hz), 116.78 (t, J = 18.5 Hz), 116.20 (t, J = 3.4 Hz), 115.94–115.48 (m), 113.88 (t, J = 12.4 Hz), 62.60, 47.24, 13.87 ppm.

[0191] IR: 2856, 2178, 1695 cm -1 .

[0192] LCMS: [M+H] + 298.

[0193]

[0194] Diethyl 2-(4-cyano-2-fluorophenyl)malonate

[0195] 3,4-Difluorobenzonitrile (4.00 g, 28.8 mmol, 1 equiv), potassium carbonate (7.95 g, 57.5 mmol, 2.00 equiv), diethyl malonate (4.66 g, 29.1 mmol, 1.01 equiv) and anhydrous DMF (24 ml) were added to a dry flask and heated at 65 °C until the starting material was consumed according to TLC. The reaction mixture was cooled to room temperature and added to a separatory funnel containing 50 ml of 1N HCl. The mixture was extracted with EtOAc (x3), and the combined organic layers were washed with water and saturated anhydrous NaCl. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. Purification by silica gel column chromatography (up to 30% EtOAc in hexanes) gave the title compound (6.73 g, 24.1 mmol, 84%) as a colorless oil.

[0196] 1 H NMR (600 MHz, CDCl3) δ 7.64 (t, J = 7.6 Hz, 1H), 7.47 (dd, J = 8.0, 1.6 Hz, 1H), 7.38 (dd, J = 9.2, 1.6 Hz, 1H), 4.98 (s, 1H), 4.28–4.18 (m, 4H), 1.26 (t, J = 7.2 Hz, 6H).

[0197] 13 C NMR (150 MHz, CDCl3) δ 166.55, 160.04 (d, J = 252.0 Hz), 131.98 (d, J = 3.5 Hz), 128.26 (d, J = 4.0 Hz), 126.21 (d, J = 14.4 Hz), 119.23 (d, J = 26.0 Hz), 117.28 (d, J = 2.9 Hz), 113.71 (d, J = 9.8 Hz), 62.55, 50.44 (d, J = 2.9 Hz), 14.00.

[0198] IR: 2984, 2236, 1733, 1219 cm -1 .

[0199] LCMS: [M+H] + 280.

[0200]

[0201] 5,7-Dichloro-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine

[0202] According to General Procedure B, diethyl 2-phenylmalonate (780 mg, 3.30 mmol) and 3-amino-1,2,4-triazole (292 mg, 3.47 mmol) were used. Then, using intermediate sodium 6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diolate (740 mg, 2.71 mmol) and POCl3 (7.41 g, 48.4 mmol), the title compound (527 mg, 1.99 mmol, 73%) was obtained as a brown solid.

[0203] 1 1H NMR (500 MHz, CDCl3) δ 8.56 (s, 1H), 7.58–7.51 (m, 3H), 7.38–7.34 (m, 2H).

[0204] 13 13C NMR (126 MHz, CDCl3) δ 156.93, 156.65, 153.35, 139.90, 131.72, 130.07, 129.95, 129.16, 124.01.

[0205] IR (KBr) ν 3437, 1637, 1458, 1383, 1267, 1205, 1180, 867, 802, 764, 740, 698, 652 cm -1 。

[0206] HRMS (ES+): C 11 H7Cl2N4 [M+H] + Calcd, 265.0042; Found, 265.0043.

[0207]

[0208] 4-(5,7-Dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (22)

[0209] According to General Procedure B, diethyl 2-(4-cyano-2,6-difluorophenyl)malonate (500 mg, 1.68 mmol) and 3-amino-1,2,4-triazole (148 mg, 1.77 mmol) were used. Then, using intermediate sodium 6-(4-cyano-2,6-difluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diolate (400 mg, 1.20 mmol) and POCl3 (3.28 g, 21.4 mmol), the title compound (210 mg, 1.20 mmol, 53%) was obtained as a brown solid.

[0210] 11H NMR (600 MHz, CDCl3) δ 8.65 (s, 1H), 7.47 (d, J = 6.2 Hz, 2H) ppm.

[0211] 13 13C NMR (150 MHz, CDCl3) δ 161.21 (d, J = 6.4 Hz), 159.51 (d, J = 6.2 Hz), 157.66, 155.57, 116.85, 116.62–116.30 (m), 115.86 (t, J = 3.5 Hz), 114.64 (t, J = 19.6 Hz) ppm.

[0212] IR: 3087, 2195, 1571, 1338, 1195 cm -1 。

[0213] LCMS: [M+H] + 327.

[0214]

[0215] 4-(5,7-Dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-fluorobenzonitrile (23)

[0216] According to the general procedure B, diethyl 2-(4-cyano-2,6-difluorophenyl)malonate (500 mg, 1.68 mmol) and 3-amino-1,2,4-triazole (148 mg, 1.77 mmol) were used. Then, sodium 6-(4-cyano-2-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diolate (3.00 g, 9.52 mmol) and POCl3 (26.0 g, 169 mmol) were used to obtain the title compound (1.97 g, 9.52 mmol, 67%), which was a brown solid.

[0217] 1 1H NMR (600 MHz, CDCl3) δ 8.61 (s, 1H), 7.68 (dd, J = 7.9, 1.5 Hz, 1H), 7.61 (dd, J = 8.6, 1.5 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H) ppm.

[0218] 1313C NMR (150 MHz, CDCl3) δ 160.47, 158.79, 157.48, 155.52, 153.79, 140.73, 133.32, 128.86 (d, J = 4.6 Hz), 124.68 (d, J = 15.7 Hz), 120.42 (d, J = 24.9 Hz), 116.76 (d, J = 2.9 Hz), 116.69, 116.36 (d, J = 9.3 Hz) ppm.

[0219] Example 3 - Microtubule Stability Assay

[0220] All test compounds were evaluated in the previously described cell-based MT stabilization assay (see Kovalevich, J. et al., (2016) Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer's Disease and Related Tauopathies. J Pharmacol Exp Ther 357, 432 - 450, which is incorporated herein by reference), where compound-dependent changes in tubulin polymerization and total tubulin levels were determined by quantifying acetylated α-tubulin and total tubulin in cell lysates by ELISA after incubation of QBI293 cells with 1 and 10 μM compounds for 4 hours.

[0221] The observation that specific structural features within the triazolo[1,5-a]pyrimidines are crucial for causing an increase in MT stability and quality led to a more systematic exploration of the SAR of the triazolo[1,5-a]pyrimidine scaffold, thereby elucidating the MT-stabilizing SAR of triazolo[1,5-a]pyrimidines modified at C6 and C7. The results of these studies led to the identification of selected homologues with improved in vitro activity and physicochemical properties compared to the existing lead compounds. In particular, these studies have taught us that the desired class I MT-stabilizing effect of triazolo[1,5-a]pyrimidines generally requires the presence of an electron-deficient phenyl ring at C6 (R6 in formula I). Different fluorinated phenyl moieties can be tolerated, resulting in active class I homologues, especially those containing one or two fluorine atoms in the ortho position and unsubstituted or substituted with additional fluorine or other electron-withdrawing groups (such as nitro and nitrile groups) at the para position. In addition, the nature of the moiety attached at C7 plays an important role in determining the potency and cellular phenotype elicited by MT-active triazolo[1,5-a]pyrimidines, and the presence of a substituted nitrogen at this position may be a prerequisite for class I activity. Finally, the effect of the chiral configuration of the branched amine moiety at C7 was also evaluated. In most cases examined, it was found that the chirality of the amine affected the potency of the compound, with one enantiomer generally being more active than the other.

[0222] Examples of the new C6 nitrile- and alkyne-substituted triazolo[1,5-a]pyrimidines have improved in vitro activity relative to the previously described examples, and their general structure is shown in formula I. The activity of the new C6 nitrile-substituted compounds was tested in the MT-stabilizing cell assay described previously (Kovalevich, J., et al. (2016)). As outlined in Table 2 below, 18 examples were synthesized and tested. In all cases, these compounds were confirmed to be active class I triazolo[1,5-a]pyrimidines. In addition, among this series of homologues, selected compounds such as 64, 66, 69, 70, and 75-77 were identified as having improved in vitro potency compared to the lead compound 3. Table 2 provides the fold changes in acetylated α-tubulin and total α-tubulin levels in QBI293 cells after incubation with the test compounds at 1 or 10 μM concentration for 4 hours. The data are presented as fold changes relative to vehicle-treated cells. By one-way ANOVA, *p < 0.05 and **p < 0.01.

[0223] Table 2

[0224]

[0225]

[0226] These results indicate that the C6 nitrile-substituted triazolo[1,5-a]pyrimidine compounds tested according to the present disclosure have improved in vitro activity relative to the previously described examples.

[0227] Example 4 - Plasma and Brain Compound Assays

[0228] CD1 mice (n = 3) were administered both CNDR-51990 and CNDR-51993 (a 1:1 mixture representing two diastereomers 52017(67) and 52016(68)) simultaneously at a dose of 2.5 mg / kg. One hour after administration, the mice were euthanized, blood was collected, and then the mice were perfused with saline. The mouse brains were homogenized in 10 mM ammonium acetate at pH 5.7 (1:2; w / v) using a handheld sonic homogenizer. Mouse plasma was obtained from the blood collected into 1.5 ml tubes containing 0.5 M EDTA solution and centrifuged at 4500 g for 10 minutes at 4 °C. Aliquots (50 μl) of the brain homogenate or plasma were mixed with 0.2 ml of acetonitrile, centrifuged at 15000 x g, and the resulting supernatant was used for subsequent LC-MS / MS analysis. LC-MS-MS analysis was performed by Inotiv, Inc. using a method essentially as described previously (Lou et al., J. Med. Chem. 57:6116-27, 2014). The structure is shown in Table 3 below.

[0229] Table 3

[0230]

[0231] Example 6 - Additional Microtubule-Stabilizing Compounds

[0232] The additional microtubule-stabilizing compounds described in the present disclosure were prepared according to Schemes 1 to 4 below.

[0233]

[0234] Scheme 1 Reagents and reaction conditions: a) DiBAl-H, toluene, -78 °C, 1 h, 51%; b) Bestmann-Ohira reagent, K2CO3, MeOH, 20 °C, 3 h, 20%;

[0235]

[0236] Scheme 2 Reagents and reaction conditions: a) NaNO2, KI, 6M HCl, water, 0 - 20 °C, 3 h, 72%; b) N-tributylamine, 170 °C, 6 h; c) phosphorus oxychloride, 130 °C, 6 h, 60% after two steps; d) for TAL-628, N,N-dimethylformamide, room temperature, 16 h, 40%; for TAL-626 and TAL-627, N,N-dimethylformamide, Et3N, room temperature, 1 h, 91% for TAL-626 and 88% for TAL-627; e) appropriate alkyne, Pd(PPh3)4, CuI, degassed N,N-dimethylformamide, 20 °C, 8 h, 33 - 99%; f) 4M HCl in dioxane, MeOH, 20 °C, 1 h, 99%.

[0237]

[0238] Scheme 3 Reagents and reaction conditions: a) Na2Cr2O7, NaIO4, HNO3, water, 0 - 20 °C, 24 h, 78%; b) LiAlD4, Et2O, 0 °C, 0.5 h, 99%; c) TAL-627, Pd(PPh3)4, CuI, degassed N,N-dimethylformamide, 20 °C, 8 h, 44%.

[0239]

[0240] Scheme 4 Reagents and reaction conditions: a) NaH, CH3I, N,N-dimethylformamide, 0 - 20 °C, 2 h; b) 3-butyn-1-ol, Pd(PPh3)4, CuI, degassed N,N-dimethylformamide, 20 °C, 8 h, 17% after two steps.

[0241] The compounds produced according to the foregoing synthetic schemes were used for microtubule stabilization. The MT stabilizing activities of the triazolopyrimidines are described in Table 4 below. The fold changes in the levels of acetylated α-tubulin (AcTub) and α-tubulin (α-Tub) in QBI293 cells after incubation with the test compounds at 1 or 10 μM concentrations for 4 h. The reported AcTub to α-Tub values represent the fold changes relative to cells treated with the control (DMSO) (by one-way ANOVA, *p < 0.05 and **p < 0.01); the numbers in parentheses represent the AcTub fold changes compared to cells treated with the positive control (i.e., 100 nM siverbrin). A + / - 15% change in the control was considered within the variability range of the daily assay and not significant.

[0242] Table 4

[0243]

[0244]

[0245] Materials and MethodsAll solvents were of reagent grade. All reagents were purchased from Aldrich or Enamine and used directly. Thin-layer chromatography (TLC) was performed on 0.25 mm pre-coated silica gel plates from E. Merck. Silica gel column chromatography was carried out using silica gel 60 (particle size 0.040 - 0.062 mm) supplied by Silicycle and Sorbent Technologies. TLC spots were detected by viewing under ultraviolet light. Proton ( 1 H) and carbon ( 13 C) nuclear magnetic resonance spectra were recorded on a 600 MHz Bruker AVANCE III spectrometer. Chemical shifts were reported relative to the solvent. 1H NMR spectral data are reported as follows: chemical shift [ppm, referenced to protium; s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, dd = doublet of doublets, bs = broad singlet, m = multiplet, coupling constant (Hz) and integration]. High-resolution mass spectrometry was performed using an Agilent 6230 time-of-flight mass spectrometer with a jet electrospray ionization source. HPLC was carried out using a Gilson HPLC equipped with UV and mass detectors. All samples were analyzed using a linear gradient of 10% to 90% ACN in water (over 8 minutes) and a flow rate of 1 mL / min. Preparative reverse-phase HPLC purification was carried out on a Gilson instrument using a Waters SunFire C 18 18 OBD column (5 μm 50 mm or 100 mm). The HPLC purity of all final compounds was found to be greater than 95%.

[0246] General Procedure A CuI (0.15 equiv), triethylamine (3 equiv), and the required alkyne (3 equiv) were sequentially added to a solution (0.2 M) of iodotriazolopyrimidine (1 equiv) in degassed DMF. The mixture was degassed and backfilled with nitrogen, then Tetrakis palladium (0.1 equiv) was added. The mixture was degassed and backfilled with nitrogen three times, stirred at room temperature for 8 h, then quenched with water and extracted twice with EtOAc. The combined organic fractions were washed with brine, dried over MgSO4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / EtOAc) or reverse-phase HPLC (water / ACN + 0.1% FA, (10 to 90%), 20 mL / min, 20 min gradient time) gave the required compound.

[0247] General procedure B: A 4 M solution of HCl in dioxane (18 equiv) was added to a solution of Boc-protected alkynylated TPD (1 equiv) in MeOH (0.1 M). After 3 h at room temperature, the mixture was evaporated under reduced pressure to give the title compound as the HCl salt, a yellow or brown solid.

[0248]

[0249] (R)-5-Chloro-6-(4-ethynyl-2,6-difluorophenyl)-N-(3-methylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine: At 0 °C, DiBAL-H (0.170 mL, 1.1 M, 0.027 g, 0.182 mmol, 1.5 equiv) was added dropwise to a solution of (R)-4-(5-chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorobenzonitrile (0.047 g, 0.125 mmol, 1 equiv) in anhydrous toluene (12 mL). The mixture was stirred at this temperature for 1 h and then quenched with 1 M solution of HCl. The aqueous layer was extracted twice with EtOAc, and then the combined organic fractions were washed with brine, dried over MgSO4 and concentrated under reduced pressure to give the aldehyde intermediate, which was directly dissolved in MeOH (4 mL). K2CO3 (0.025 g, 0.182 mmol, 1.5 equiv) and dimethyl (1-diazo-2-oxopropyl)phosphonate (0.035 g, 0.182 mmol, 1.5 equiv) were added successively, and the mixture was stirred at room temperature for 3 h. Then the reaction was filtered through sintered glass. The filtrate was evaporated under reduced pressure. Purification by silica gel column chromatography (hexane / EtOAc: 90 / 10 to 75 / 25) gave the title compound (0.005 g, 0.012 mmol, 10% over two steps), a pale white solid. 1 1H NMR (600 MHz, CDCl3) δ 8.39 (s, 1H), 7.25 (d, J = 7.5 Hz, 2H), 6.38 (bs, 1H), 3.30 (bs, 1H), 2.71 (d, J = 5.4 Hz, 1H), 1.72 - 1.67 (m, 1H), 1.11 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H) ppm. HRMS (ES+): C 18 1 17 15N5F2Cl [M + H] + Calcd, 376.1135; found, 376.1139.

[0250]

[0251] Diethyl 2-(2,6-difluoro-4-iodophenyl)malonate To a solution of diethyl 2-(4-amino-2,6-difluorophenyl)malonate (2.050 g, 7.136 mmol, 1 equiv) in 6N HCl (12 mL, 71.36 mmol, 10 equiv) cooled to 0 °C was added dropwise a solution of NaNO2 (0.492 g, 7.136 mmol, 1 equiv) in water (2.7 mL). The resulting solution was added dropwise to a solution of KI (4.916 g, 29.62 mmol, 4.15 equiv) in water (5 mL), keeping the temperature at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h, then the reaction was stopped and the mixture was extracted twice with EtOAc. The combined layers were washed successively with 10% Na2S2O3 and brine, then dried over MgSO4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / EtOAc: 100 / 0 to 75 / 25) gave the title compound (2.054 g, 5.159 mmol, 72%) as a yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 7.31 (d, J = 7.0 Hz, 1H), 4.89 (s, 1H), 4.25 (q, J = 7.1 Hz, 4H), 1.27 (t, J = 7.2 Hz, 6H) ppm. 13 13C NMR (151 MHz, CDCl3) δ 166.36, 160.77 (dd, J = 255.2, 7.8 Hz), 121.41 (dd, J = 23.6, 4.8 Hz), 101.61 (dt, J = 20.3, 14.7 Hz), 62.45, 47.22, 14.05 ppm. LCMS: [M+H] + : 339.

[0252]

[0253] 5,7-Dichloro-6-(2,6-difluoro-4-iodophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine In a pressure bottle flushed with nitrogen, TAL-541 (1.920 g, 6.459 mmol, 1 equiv), 1H-1,2,4-triazol-5-amine (0.570 g, 6.782 mmol, 1.05 equiv), and tributylamine (1.620 mL, 6.782 mmol, 1.05 equiv) were charged. The mixture was heated to 170 °C for 3 h. Then 10 mL of toluene was added at 110 °C, and then a 50% NaOH solution (1.023 mL, 19.38 mmol, 3 equiv) was added at 50 °C. After reaching room temperature, the mixture was filtered on sintered glass and rinsed twice with toluene to obtain the bisphenolate (2.068 g, 6.621 mmol, 97%), which was used for the next step without further purification. In a round-bottom flask containing the intermediate bisphenolate (2.000 g, 4.608 mmol, 1 equiv), phosphorus oxychloride (7.670 mL, 82.020 mmol, 17.8 equiv) was added and the mixture was heated to 130 °C for 6 h. The reaction was poured onto ice, the aqueous phase was extracted twice with CH2Cl2, the combined organic fractions were washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / EtOAc: 100 / 0 to 70 / 30) gave the title compound (1.520 g, 3.56 mmol, 77%) as an off-white solid. 1 H NMR (600 MHz, CDCl3) δ 8.61 (s, 1H), 7.52 (d, J = 6.4 Hz, 2H) ppm. 13 C NMR (151 MHz, CDCl3) δ 160.45 (d, J = 6.2 Hz), 158.75 (d, J = 6.2 Hz), 157.28, 156.13, 153.85, 141.28, 122.05 (dd, J = 23.0, 4.2 Hz), 112.35, 109.18 (t, J = 19.8 Hz), 95.68 (t, J = 10.2 Hz) ppm. HRMS (ES+): C 11 H4N4F2ICl2 [M + H] + Calcd, 426.8820; found, 426.8812.

[0254]

[0255] (R)-5-Chloro-6-(2,6-difluoro-4-iodophenyl)-N-(3-methylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (R)-3-Methylbutan-2-amine (0.064 mL, 0.048 g, 0.548 mmol, 1.5 eq) and triethylamine (0.153 mL, 0.111 g, 1.100 mmol, 3 eq) were added to a solution of 5,7-dichloro-6-(2,6-difluoro-4-iodophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.156 g, 0.365 mmol, 1 eq) in DMF (1 mL). The mixture was stirred at room temperature for 1 h. The reaction was quenched with water and the aqueous layer was extracted twice with EtOAc. The combined organic fractions were washed with brine, dried over MgSO4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / EtOAc; 100 / 0 to 70 / 30) gave the title compound (0.161 g, 0.337 mmol, 92%) as an off-white solid. 1 1H NMR (600 MHz, CDCl3) δ 8.32 (s, 1H), 7.46 (d, J = 6.5 Hz, 2H), 6.35 (d, J = 7.8 Hz, 1H), 3.18 (s, 1H), 1.64 (dq, J = 13.2, 6.7 Hz, 1H), 1.06 (d, J = 6.6 Hz, 3H), 0.79 (dd, J = 11.6, 6.8 Hz, 6H) ppm. 13 13C NMR (151 MHz, CDCl3) δ 159.60 (dd, J = 254.9, 8.4 Hz), 159.56 (dd, J = 254.8, 8.4 Hz), 156.51, 153.82, 152.51, 144.76, 120.65 (dt, J = 24.4, 3.7 Hz), 110.04 (t, J = 21.1 Hz), 93.34 (t, J = 10.2 Hz), 87.81, 53.81, 32.50, 16.95, 16.94, 16.77 ppm. HRMS (ES+): C 16 H 16 N5F2ICl [M + H] + Calculated, 478.0101; Found, 478.0098.

[0256]

[0257] (R)-5-Chloro-6-(2,6-difluoro-4-iodophenyl)-N-(3,3-dimethylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (R)-3,3-Dimethylbutan-2-amine hydrochloride (0.075 g, 0.548 mmol, 1.5 equiv) and triethylamine (0.153 mL, 0.111 g, 1.100 mmol, 3 equiv) were added to a solution of 5,7-dichloro-6-(2,6-difluoro-4-iodophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine in DMF (1 mL). The mixture was stirred at room temperature for 1 h. The reaction was quenched by adding water, and the aqueous layer was extracted twice with EtOAc. The combined organic fractions were washed with brine, dried over MgSO4 and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane / EtOAc: 100 / 0 to 70 / 30) gave the title compound (0.158 g, 0.321 mmol, 88%) as an off-white solid. 1 H NMR (600 MHz, CDCl3) δ 8.31 (s, 1H), 7.46 (d, J = 6.4 Hz, 2H), 6.42 (s, 1H), 3.10 (s, 1H), 1.00 (d, J = 6.7 Hz, 4H), 0.82 (s, 9H) ppm. 13 C NMR (151 MHz, CDCl3) δ 160.67 (dd, J = 254.9, 6.2 Hz), 160.40 (dd, J = 254.8, 6.0 Hz), 157.59, 154.89, 153.58, 146.03, 121.82 (dd, J = 21.7, 3.9 Hz), 121.67 (dd, J = 21.7, 3.9 Hz), 111.23 (t, J = 18.2 Hz), 94.41 (t, J = 10.1 Hz), 88.71, 58.10, 34.74, 25.77, 16.56, 16.55 ppm. HRMS (ES+): C 17 H 18 N5F2ICl [M + H] + Calculated, 492.0258; Found, 492.0256.

[0258]

[0259] (S)-5-Chloro-6-(2,6-difluoro-4-iodophenyl)-N-(1,1,1-trifluoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine(S)-1,1,1-Trifluoropropan-2-amine(0.097mL,0.111g,0.984mmol,2.1equiv)was added to a solution of 5,7-dichloro-6-(2,6-difluoro-4-iodophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine(0.200g,0.468mmol,1equiv)in DMF(2mL).The mixture was stirred at 40°C for 24h.The reaction was quenched by the addition of water and the aqueous layer was extracted twice with EtOAc.The combined organic fractions were washed with brine,dried over MgSO4 and concentrated under reduced pressure.Purification by silica gel column chromatography(hexane / EtOAc:100 / 0 to 70 / 30)gave the title compound(0.121g,0.240mmol,51%)as an off-white solid. 1 H NMR(600MHz,CDCl3)δ8.38(s,1H),7.49(d,J=6.6Hz,2H),5.97(d,J=9.6Hz,1H),4.84(bs,1H),1.43(d,J=6.8Hz,3H). 13 C NMR(151MHz,CDCl3)δ160.61(dd,J=257.4,5.9Hz),160.29(dd,J=255.8,6.0Hz),157.41,155.41,154.26,145.87,124.59(q,J=282.1Hz),122.32(dd,J=63.0,3.8Hz),122.16(dd,J=62.9,3.7Hz),109.10(t,J=20.2Hz),95.55(t,J=10.1Hz),91.74,51.08(q,J=32.1Hz),15.16ppm.HRMS(ES+):C 14 H9N5F5ICl[M+H] + Calculated,503.9506;found,503.9502.

[0260]

[0261] (R)-5-Chloro-6-(4-(3-(dimethylamino)prop-1-yn-1-yl)-2,6-difluorophenyl)-N-(3-methylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine was obtained as an off-white solid (0.043 g, 0.099 mmol, 68%) following general procedure A using TAL-626 (0.070 g, 0.147 mmol) and N,N-dimethylprop-2-yn-1-amine (0.047 mL, 0.037 g, 0.440 mmol), purified by reverse-phase HPLC and the pure fractions were extracted with EtOAc. 1 H NMR (600 MHz, MeOD) δ 8.60 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 3.74 (s, 2H), 3.38 (s, 1H), 2.56 (s, 6H), 1.72 (pt, J = 6.4 Hz, 1H), 1.11 (d, J = 6.3 Hz, 3H), 0.79 (d, J = 6.5 Hz, 6H) ppm. HRMS (ES+): C 21 H 24 N6F2Cl [M + H] + Calculated, 433.1714; Found, 433.1715.

[0262]

[0263] (R)-5-Chloro-6-(4-(3-(dimethylamino)prop-1-yn-1-yl)-2,6-difluorophenyl)-N-(3,3-dimethylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine was obtained as an off-white solid (0.043 g, 0.096 mmol, 68%) following general procedure A using TAL-627 (0.070 g, 0.142 mmol) and N,N-dimethylprop-2-yn-1-amine (0.046 mL, 0.036 g, 0.427 mmol), purified by reverse-phase HPLC and the pure fractions were extracted with EtOAc. 1 H NMR (600 MHz, MeOD) δ 8.50 (s, 1H), 7.33 (d, J = 7.3 Hz, 2H), 3.58 (bs, 2H), 3.31 (bs, 1H), 2.42 (s, 6H), 1.08 (d, J = 6.6 Hz, 3H), 0.83 (s, 9H) ppm. HRMS (ES+): C 22 H 26 N6F2Cl [M + H] + Calculated, 447.1870; Found, 447.1872.

[0264]

[0265] (R)-(4-(4-(5-Chloro-7-(3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yn-1-yl)tert-butyl carbamate was prepared according to General Procedure A using TAL-626 (0.040 g, 0.084 mmol) and tert-butyl 3-butyn-1-ylcarbamate (0.043 g, 0.250 mmol). After purification by silica gel column chromatography (hexane / EtOAc: 90 / 10 to 60 / 40), the title compound (0.043 g, 0.083 mmol, 99%) was obtained as a yellow solid. 1 H NMR (600 MHz, CDCl3) δ 8.34 (s, 1H), 7.10 (d, J = 7.9 Hz, 2H), 6.34 (s, 1H), 4.85 (s, 1H), 3.40 (t, J = 5.9 Hz, 2H), 3.19 (s, 1H), 2.66 (t, J = 6.5 Hz, 2H), 1.66–1.62 (m, 1H), 1.47 (s, 9H), 1.06 (d, J = 6.6 Hz, 3H), 0.80 (t, J = 7.4 Hz, 6H) ppm. HRMS (ES+): C 25 H 29 N6F2ClO2 [M+Na] + Calcd, 519.2081; found, 519.2083.

[0266]

[0267] (R)-(4-(4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yn-1-yl)tert-butyl carbamate was prepared according to General Procedure A using TAL-627 (0.040 g, 0.081 mmol) and tert-butyl 3-butyn-1-ylcarbamate (0.041 g, 0.240 mmol). After purification by silica gel column chromatography (hexane / EtOAc: 90 / 10 to 60 / 40), the title compound (0.042 g, 0.079 mmol, 97%) was obtained as a yellow solid. 11H NMR (600 MHz, CDCl3) δ 8.40 (s, 1H), 7.11 (d, J = 7.7 Hz, 2H), 6.43 (bs, 1H), 4.85 (bs, 1H), 3.41 (d, J = 5.1 Hz, 2H), 3.14 (bs, 1H), 2.66 (t, J = 6.4 Hz, 2H), 1.47 (s, 9H), 1.02 (d, J = 6.6 Hz, 3H), 0.84 (s, 9H) ppm. HRMS (ES+): C 26 H 32 N6F2ClO2 [M+H] + Calculated, 533.2238; Found, 533.2238.

[0268]

[0269] (R)-4-(4-(5-Chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yn-1-ol was prepared according to General Procedure A using TAL-626 (0.040 g, 0.084 mmol) and 3-butyn-1-ol (0.019 mL, 0.018 g, 0.250 mmol). After purification by reverse-phase HPLC and lyophilization, the title compound (0.012 g, 0.029 mmol, 34%) was obtained as an off-white solid. 1 1H NMR (600 MHz, MeOD) δ 8.44 (s, 1H), 7.26 (d, J = 8.4 Hz, 2H), 3.77 (t, J = 6.5 Hz, 2H), 3.31 (bs, 1H), 2.68 (t, J = 6.5 Hz, 2H), 1.80–1.66 (m, 1H), 1.11 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 6.7 Hz, 6H) ppm. HRMS (ES+): C 20 H 21 N5F2ClO [M+H] + Calculated, 420.1397; Found, 420.1398.

[0270]

[0271] (R)-5-(4-(5-chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)penta-4-yn-1-ol was obtained as an off-white solid (0.012 g, 0.028 mmol, 49%) following general procedure A using TAL-626 (0.027 g, 0.057 mmol) and 4-pentyn-1-ol (0.016 mL, 0.014 g, 0.170 mmol), and purified by reverse phase HPLC and lyophilized. 1 H NMR (600 MHz, MeOD) δ 8.43 (s, 1H), 7.22 (d, J = 8.4 Hz, 2H), 3.71 (t, J = 6.2 Hz, 2H), 3.31 (bs, 1H), 2.58 (t, J = 7.1 Hz, 2H), 1.84 (p, J = 6.7 Hz, 2H), 1.73 (dq, J = 13.6, 6.8 Hz, 2H), 1.11 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 6.7 Hz, 6H) ppm. HRMS (ES+): C 21 H 23 N5F2ClO [M+H] + Calcd, 434.1554; Found, 434.1553.

[0272]

[0273] (R)-4-(4-(5-chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yn-1-ol was obtained as an off-white solid (0.014 g, 0.032 mmol, 40%) following general procedure A using TAL-627 (0.040 g, 0.081 mmol) and 3-butyn-1-ol (0.018 mL, 0.017 g, 0.240 mmol), and purified by reverse phase HPLC and lyophilized. 1 H NMR (600 MHz, MeOD) δ 8.46 (s, 1H), 7.28 (dd, J = 8.1, 1.5 Hz, 2H), 3.77 (t, J = 6.5 Hz, 2H), 3.31 (bs, 1H), 2.68 (t, J = 6.5 Hz, 2H), 1.07 (d, J = 6.7 Hz, 3H), 0.84 (s, 9H) ppm. HRMS (ES+): C 21 H 23 N5F2ClO [M+H] + Calcd, 434.1554; Found, 434.1556.

[0274]

[0275] (R)-5-(4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)penta-4-yn-1-ol was obtained as an off-white solid (0.036 g, 0.031 mmol, 38%) following general procedure A using TAL-627 (0.040 g, 0.081 mmol) and 4-pentyn-1-ol (0.023 mL, 0.021 g, 0.240 mmol), purified by reverse phase HPLC and lyophilized. 1 H NMR (600 MHz, MeOD) δ 8.46 (s, 1H), 7.25 (dd, J = 8.0, 1.5 Hz, 2H), 3.72 (t, J = 6.2 Hz, 2H), 3.31 (bs, 1H), 2.58 (t, J = 7.1 Hz, 2H), 1.85 (p, J = 6.7 Hz, 2H), 1.08 (d, J = 6.7 Hz, 3H), 0.84 (s, 9H) ppm. HRMS (ES+): C 22 H 25 N5F2ClO [M + H] + Calcd, 448.1710; Found, 448.1711.

[0276]

[0277] (S)-4-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yn-1-ol was obtained as an off-white solid (0.011 g, 0.025 mmol, 39%) following general procedure A using TAL-628 (0.032 g, 0.064 mmol) and 3-butyn-1-ol (0.014 mL, 0.013 g, 0.190 mmol), purified by reverse phase HPLC and lyophilized. 1 H NMR (600 MHz, MeOD) δ 8.50 (s, 1H), 7.24 (d, J = 9.0 Hz, 2H), 5.77 (bs, 1H), 3.76 (t, J = 5.9 Hz, 2H), 3.32 (bs, 1H), 2.68 (t, J = 5.9 Hz, 2H), 1.46 (d, J = 6.5 Hz, 3H) ppm. HRMS (ES+): C 18 H 14 N5F5ClO [M + H] +Calculated value, 446.0802; measured value, 446.0804.

[0278]

[0279] (S)-5-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)penta-4-yn-1-ol was prepared according to General Procedure A using TAL-628 (0.033 g, 0.066 mmol) and 4-pentyn-1-ol (0.020 mL, 0.017 g, 0.200 mmol). After purification by reverse-phase HPLC and lyophilization, the title compound (0.010 g, 0.022 mmol, 33%) was obtained as an off-white solid. 1 H NMR (600 MHz, MeOD) δ 8.50 (s, 1H), 7.21 (d, J = 8.8 Hz, 2H), 5.80 (bs, 1H), 3.71 (t, J = 6.0 Hz, 2H), 3.32 (bs, 1H), 2.57 (t, J = 7.0 Hz, 2H), 1.94–1.71 (m, 2H), 1.46 (d, J = 6.7 Hz, 3H) ppm. HRMS (ES+): C 19 H 16 N5F5ClO [M + H] + Calculated value, 460.0958; measured value, 460.0961.

[0280]

[0281] (R)-(3-(4-(5-Chloro-7-(3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)prop-2-yn-1-yl)(methyl)carbamic acid tert-butyl ester was prepared according to General Procedure A using TAL-626 (0.050 g, 0.105 mmol) and (methyl)(prop-2-yn-1-yl)carbamic acid tert-butyl ester (0.053 g, 0.314 mmol). After purification by silica gel column chromatography (hexane / EtOAc: 90 / 10 to 50 / 50), the title compound (0.020 g, 0.039 mmol, 37%) was obtained as a yellow solid. 11H NMR (600 MHz, CDCl3) δ 8.33 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 6.35 (d, J = 8.1 Hz, 1H), 4.32 (bs, 2H), 3.16 (bs, 1H), 2.99 (s, 3H), 1.65–1.60 (m, 1H), 1.49 (s, 9H), 1.05 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 6.9 Hz, 3H), 0.78 (d, J = 6.9 Hz, 3H) ppm. HRMS (ES+): C 25 H 30 N6F2ClO2 [M+H] + Calcd for, 519.2081; Found, 519.2078.

[0282]

[0283] (R)-(3-(4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)prop-2-yn-1-yl)(methyl)carbamic acid tert-butyl ester was obtained as a yellow solid (0.043 g, 0.081 mmol, 79%) by purification by silica gel column chromatography (hexane / EtOAc: 90 / 10 to 50 / 50) according to the general procedure A using TAL-627 (0.050 g, 0.102 mmol) and (methyl)(prop-2-yn-1-yl)carbamic acid tert-butyl ester (0.052 g, 0.305 mmol). 1 1H NMR (600 MHz, CDCl3) δ 8.32 (s, 1H), 7.12 (d, J = 7.5 Hz, 2H), 6.43 (bs, 1H), 4.31 (bs, 2H), 2.98 (s, 3H), 1.49 (s, 9H), 1.47–1.45 (m, 1H), 1.01 (d, J = 6.7 Hz, 3H), 0.83 (s, 9H) ppm. HRMS (ES+): C 26 H 32 N6F2ClO2 [M+H] + Calcd for, 533.2238; Found, 533.2234.

[0284]

[0285] (R)-4-(4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yne-1,1-d2-1-ol was obtained as an off-white solid (0.031 g, 0.071 mmol, 44%) by reverse-phase HPLC purification and lyophilization according to the general procedure A, using TAL-627 (0.080 g, 0.160 mmol) and 3-butyn-1,1-d2-1-ol (0.035 g, 0.490 mmol). 1 H NMR (600 MHz, MeOD) δ 8.46 (s, 1H), 7.27 (d, J = 7.7 Hz, 2H), 3.32 (bs, 1H), 2.67 (s, 2H), 1.06 (d, J = 6.7 Hz, 3H), 0.82 (s, 9H) ppm. HRMS (ES+): C 21 H 21 N5F2D2ClO [M + H] + Calculated, 436.1679; found, 436.1678.

[0286]

[0287] (R)-6-(4-(4-Aminobut-1-yn-1-yl)-2,6-difluorophenyl)-5-chloro-N-(3-methylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine hydrochloride was obtained as a yellow solid (0.034 g, 0.075 mmol, 97%) by the general procedure B, converting TAL-576 (0.040 g, 0.077 mmol). 1 H NMR (600 MHz, MeOD) δ 8.87 (s, 1H), 7.37 (d, J = 8.3 Hz, 2H), 3.33 (bs, 1H), 3.23 (t, J = 6.7 Hz, 2H), 2.92 (t, J = 6.8 Hz, 2H), 1.77 (dq, J = 13.6, 6.8 Hz, 1H), 1.15 (d, J = 6.6 Hz, 3H), 0.81 (t, J = 7.3 Hz, 6H) ppm. HRMS (ES+): C 20 H 22 N6F2Cl [M + H] + Calculated, 419.1557; found, 419.1560.

[0288]

[0289] (R)-6-(4-(4-Aminobut-1-yn-1-yl)-2,6-difluorophenyl)-5-chloro-N-(3,3-dimethylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine hydrochloride According to General Procedure B, TAL-576 (0.040 g, 0.075 mmol) was converted to the title compound (0.034 g, 0.072 mmol, 97%), a yellow solid. 1 H NMR (600 MHz, MeOD) δ 8.75 (s, 1H), 7.38 (dd, J = 8.8, 3.6 Hz, 2H), 3.33 (bs, 1H), 3.23 (t, J = 6.8 Hz, 2H), 2.92 (t, J = 6.8 Hz, 2H), 1.34–1.30 (m, 1H), 1.10 (d, J = 6.7 Hz, 3H), 0.84 (s, 9H) ppm. HRMS (ES+): C 21 H 24 N6F2Cl [M + H] + Calculated, 433.1714; found, 433.1716.

[0290]

[0291] (R)-5-Chloro-6-(2,6-difluoro-4-(3-(methylamino)prop-1-yn-1-yl)phenyl)-N-(3-methylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine hydrochloride According to General Procedure B, TAL-635 (0.015 g, 0.029 mmol) was converted to the title compound (0.011 g, 0.024 mmol, 84%), a brown solid. 1 H NMR (600 MHz, MeOD) δ 8.59 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 4.23 (s, 2H), 3.31 (bs, 1H), 2.85 (s, 3H), 1.74 (dq, J = 13.4, 6.7 Hz, 1H), 1.11 (d, J = 6.5 Hz, 3H), 0.79 (d, J = 4.4 Hz, 6H) ppm. HRMS (ES+): C 20 H 22 N6F2Cl [M + H] + Calculated, 419.1557; found, 419.1554.

[0292]

[0293] (R)-5-Chloro-6-(2,6-difluoro-4-(3-(methylamino)prop-1-yn-1-yl)phenyl)-N-(3,3-dimethylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine hydrochloride Following general procedure B, TAL-636 (0.035 g, 0.066 mmol) was converted to the title compound (0.028 g, 0.060 mmol, 91%), a brown solid. 1 H NMR (600 MHz, MeOD) δ 8.74 (s, 1H), 7.47 (dd, J = 8.4, 3.1 Hz, 2H), 4.24 (s, 2H), 3.33 (bs, 1H), 2.86 (s, 3H), 1.10 (d, J = 6.7 Hz, 3H), 0.84 (s, 9H) ppm. HRMS (ES+): C 21 H 24 N6F2Cl [M + H] + Calculated, 433.1714; found, 433.1716.

[0294]

[0295] (R)-5-Chloro-6-(4-(4-(dimethylamino)but-1-yn-1-yl)-2,6-difluorophenyl)-N-(3,3-dimethylbutan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine Following general procedure A, using TAL-627 (0.040 g, 0.081 mmol) and N,N-dimethylbut-3-yn-1-amine (0.029 mL, 0.024 g, 0.224 mmol), after purification by reverse-phase HPLC and extraction with EtOAc, the title compound (0.023 g, 0.050 mmol, 61%) was obtained, off-white. 1 H NMR (600 MHz, CDCl3) δ 8.36 (s, 1H), 7.13 (d, J = 6.8 Hz, 2H), 6.44 (bs, 1H), 3.34 (s, 2H), 3.23–3.01 (m, 3H), 2.93 (s, 6H), 1.01 (d, J = 6.3 Hz, 3H), 0.82 (s, 9H) ppm.

[0296]

[0297] (R)-4-(4-(5-chloro-2-methyl-7-(methyl(3-methylbutan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl)but-3-yn-1-ol At 0 °C, NaH (60%, 0.005 g, 0.120 mmol, 1.5 equiv) was added to a solution of TAL-626 (0.037 g, 0.077 mmol, 1 equiv) in DMF (0.8 mL). Then, after 20 min, iodomethane (0.006 mL, 0.013 g, 0.093 mmol, 1.2 equiv) was added. After 2 h at room temperature, the reaction was quenched with water, and the aqueous layer was extracted twice with EtOAc. The combined organic fractions were washed with brine, dried over MgSO4 and concentrated under reduced pressure to give the dimethylated intermediate, which was used without further purification in the next step. Following general procedure A, using the aforementioned intermediate (0.013 g, 0.026 mmol) and 3-butyn-1-ol (0.006 mL, 0.005 g, 0.077 mmol), after purification by reverse-phase HPLC and extraction with EtOAc, the title compound (0.006 g, 0.013 mmol, 17% over two steps) was obtained as off-white. 1 H NMR (600 MHz, CDCl3) δ 7.02 (d, J = 7.1 Hz, 1H), 4.04 (bs, 1H), 3.84 (s, 2H), 3.42 (s, 3H), 2.71 (s, 2H), 2.54 (s, 3H), 1.65–1.60 (m, 1H), 1.05 (d, J = 5.9 Hz, 3H), 0.85 (d, J = 5.5 Hz, 3H), 0.76 (d, J = 5.8 Hz, 3H) ppm.

Claims

1. A compound, which is:

2. The compound according to claim 1, wherein the compound is:

3. The compound according to claim 1, wherein the compound is:

4. The compound according to claim 1, wherein the compound is:

5. Use of a composition comprising a therapeutically effective amount of a compound in the preparation of a medicament for treating a neurodegenerative disease, wherein the compound is:

6. The use according to claim 5, wherein the compound is:

7. The use according to claim 5, wherein the compound is:

8. The use according to claim 5, wherein the compound is:

9. Use of a composition comprising a therapeutically effective amount of a compound in the preparation of a medicament for treating cancer, wherein the compound is:

10. The use according to claim 9, wherein the compound is:

11. The use according to claim 9, wherein the compound is:

12. The use according to claim 9, wherein the compound is:

13. Use of a therapeutically effective amount of a compound in the preparation of a medicament for treating a neurodegenerative disease in a subject in need thereof, wherein the compound is:

14. The use according to claim 13, wherein the compound is:

15. The use according to claim 13, wherein the compound is:

16. The use according to claim 13, wherein the compound is:

17. The use according to any one of claims 13 - 16, wherein the neurodegenerative disease is characterized by Tau proteinopathy or impaired microtubule function in the brain of the subject.

18. The use according to any one of claims 13 - 16, wherein the neurodegenerative disease is Alzheimer's disease, frontotemporal lobar degeneration, Pick's disease, progressive supranuclear palsy (PSP), corticobasal ganglionic degeneration, Parkinson's disease (PD), PD with dementia, dementia with Lewy bodies, or amyotrophic lateral sclerosis.

19. The use according to any one of claims 13 - 16, wherein the neurodegenerative disease is traumatic brain injury or post - traumatic stress disorder.

20. The use according to claim 19, wherein the traumatic brain injury is repetitive traumatic brain injury or chronic traumatic encephalopathy.

21. The use according to any one of claims 13 - 16, wherein the neurodegenerative disease is schizophrenia.

22. Use of a therapeutically effective amount of a compound in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the compound is:

23. The use according to claim 22, wherein the compound is:

24. The use according to claim 22, wherein the compound is:

25. The use according to claim 22, wherein the compound is:

26. The use according to any one of claims 22 - 25, wherein the cancer is brain cancer.

27. The use according to claim 26, wherein the cancer is glioma or astrocytoma.

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