Compounds for cancer treatment

Abstract

The present invention relates to modulators of canonical-type of transient receptor potential (TRPC) channels and the polymerization of tubulin, compositions thereof, and methods therewith. The present invention also relates to methods of treating TRPC mediated conditions, such as cancer, using such modulators.

Description

[0001] 1005445281

[0002] Compounds for Cancer T reatment

[0003] Related application

[0004] This application claims priority to Australian provisional application no 2024903018 filed on 20 September 2024, the contents of which are incorporated herein by reference in their entirety.

[0005] Field of the invention

[0006] The present disclosure relates to compounds for cancer treatment, pharmaceutical compositions and methods for using the compounds and compositions of the disclosure.

[0007] Background of the invention

[0008] Cancer is the term given to a collection of related diseases in which abnormal cells divide in an uncontrolled manner, such that they invade nearby tissues. Cancer is recognized as a leading cause of death, with the International Agency for Research on Cancer predicting 23.7 million new cancer cases, and 11.9 million cancer deaths, worldwide in 2030. Despite significant breakthroughs made in the treatment and prevention of a wide variety of cancers, cancer remains a leading cause of death due to its high morbidity and mortality. Moreover, patients who have undergone surgery and subsequent chemotherapy often experience a recurrence.

[0009] Small molecule therapy can be successful for the treatment of some cancers; however, many have a narrow therapeutic index and are not highly selective causing unwanted drug toxicity in a subject. Poor penetrance into sanctuary sites (e.g. CNS) requires small molecules to be administered at high concentrations which can further contribute to toxic side effects. Additionally, many cancers develop drug resistance to some small molecule therapies over time resulting in relapse of the disease. Therefore, a need exists for creating novel small molecules for more successful treatments for cancers and other health conditions.

[0010] In addition, the existence of the blood-brain barrier (BBB) often precludes ready penetration of many anticancer agents into the brain. A consequence of this is that higher concentrations are typically required relative to other cancers when the brain is the target organ. These higher concentrations oftentimes cause serious side effects in other organs. 1005445281

[0011] Therefore, in the treatment of brain cancer and brain metastases, there is a continuous demand for a therapeutic agent that has no difficulty in BBB passage and has an excellent anticancer effect.

[0012] There is a need for improved treatment of brain cancer and brain metastases.

[0013] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.

[0014] Summary of the invention

[0015] The present inventors surprisingly identified that compounds of Formula I or Formula II can provide effective blockers of TRPC3, TRPC6 and / or TRPC7 channels and inhibit the polymerization of tubulin.

[0016] In a first aspect of the disclosure there is provided a compound of Formula I or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

[0017] Formula I wherein:

[0018] R1is a bicyclic system the dashed line ( ) represents an optional double bond (=) or single bond ( — ); wherein when X and CR4are connected by a double bond, then Y and CR4are connected 1005445281 by a single bond, and when X and CR4are connected by a single bond, then Y and CR4are connected by a double bond;

[0019] A1, A2and A3are independently selected from N and CR5; when present R5is independently selected from H, D, halo, Ci-C4alkyl, hydroxy, amino, Ci-C4alkoxy, Ci-C4fluoroalkyl, phenyl, cyano, Ci-C4thioalkyl, and Ci- C4alkylamino;

[0020] X is selected from CH, CD, CH2, CHD, CD2, S and O; and

[0021] Y is selected from CH, CD, CH2, CHD, CD2, S, and N;

[0022] R4is selected from CH3, CD3, CH2D, CHD2, Ci-Cealkyl, hydroxy, amino, Ci- Cealkoxy, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino;

[0023] R2is selected from H, D, halo, cyano, Ci-Cethioalkyl, Ci-Cealkylamino, and C1- Cealkyl;

[0024] R3is: where R6and R7form an optionally substituted ring; the ring formed by R6and R7and the N atom between them comprises 3-9 ring atoms. In a second aspect there is provided a compound according to Formula II or pharmaceutically acceptable salt, solvate or stereoisomer thereof

[0025] Formula II wherein: 1005445281

[0026] A1, A2and A3are independently selected from N and CR12; when present each R12is independently selected from H, D, halo, Ci-C4alkyl, hydroxy, amino, Ci-C4alkoxy, Ci-C4fluoroalkyl, phenyl, cyano, Ci-C4thioalkyl, and Ci- C4alkylamino; X is selected from O and S;

[0027] R8is CH3, CD3, CH2D, CHD2, Ci-Cealkyl, hydroxy, amino, Ci-Cealkoxy, phenyl, cyano, Ci -Cethioalkyl , or Ci-Cealkylamino;

[0028] R9is H, D, halo, cyano, Ci-Cethioalkyl, Ci-Cealkylamino, and Ci-Cealkyl;

[0029] R10and R11form a ring; the ring formed by R10and R11comprises 3-9 ring atoms. In embodiments of the first and second aspect there is provided a compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof, selected from the group consisting of: 1005445281

[0030] In another aspect there is provided a compound, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof having the structure:

[0031] In a further aspect of the disclosure there is provided a pharmaceutical composition comprising a compound of Formula I or II as described herein, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0032] In yet a further aspect of the invention there is provided a method of treating cancer or cancer metastases, comprising administering to a cell, tissue or an individual in need thereof a therapeutically effective amount of a compound of Formula I or II as described herein, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition as described herein.

[0033] In some embodiments the cancer or cancer metastases is responsive to the inhibition of one or more of TRPC3, TRPC6 or TRPC7 ion channel activity, or the cancer or cancer metastases is responsive to tubulin polymerization inhibition, or a combination thereof.

[0034] In a further aspect of the disclosure there is provided a method of inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity, comprising administering to a 1005445281 cell, tissue or an individual in need thereof, a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition as described herein. This thereby treats cancer or cancer metastases that are responsive to inhibition of one or more of TRPC3, TRPC6, TRPC7 ion channel activity. In some embodiments the method inhibits all three of TRPC3, TRPC6 and TRPC7 ion channel activity.

[0035] In another aspect there is provided a method of inhibiting tubulin polymerization, comprising administering to a cell, tissue or an individual in need thereof, a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition as described herein. This thereby treats cancer or cancer metastases responsive to inhibition of tubulin polymerization.

[0036] In another aspect there is provided a method of inhibiting one or more of TRPC3, TRPC6 or TRPC7 ion channel activity, and tubulin polymerization, comprising administering to a cell, tissue or an individual in need thereof, a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition as described herein. This thereby treats cancer or cancer metastases responsive to inhibition of one or more of TRPC3, TRPC6 or TRPC7 ion channel activity and inhibition of tubulin polymerization.

[0037] In another aspect, the invention provides a method of treating a condition or disease responsive to the inhibition of one or more TRPC3, TRPC6, TRPC7 ion channel activity, or tubulin polymerization inhibition, or a combination thereof, in an individual in need thereof, comprising administering a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

[0038] In one embodiment the condition or disease is cancer or cancer metastasis. In preferred embodiments the disease is brain cancer or metastatic brain cancer.

[0039] In one embodiment of this aspect of the invention the condition or disease is cancer or cancer metastasis, preferably brain cancer or metastatic brain cancer, and is responsive to the inhibition of all three of TRPC3, TRPC6 and TRPC7 ion channel activity. 1005445281

[0040] In one embodiment the invention provides a method of preventing metastasis from a cancer, wherein the cancer is responsive to: the inhibition of one or more of TRPC3, TRPC6, TRPC7 ion channel activity; or tubulin polymerisation inhibition; or a combination thereof in an individual in need thereof comprising administering a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

[0041] In another aspect, the present invention also provides a method of inhibiting metastatic progression from a cancer, wherein the cancer is responsive to one or more of TRPC3, TRPC6, TRPC7 ion channel inhibition, tubulin polymerization inhibition, or a combination thereof, comprising administering a therapeutically effective amount of a compound of Formula I or Formula II, or pharmaceutically acceptable salt, stereoisomer, or solvate, thereof, thereby inhibiting metastatic progression in the individual.

[0042] In a preferred embodiment, the disease or condition is brain metastasis wherein the primary cancer is non-small cell lung cancer. Accordingly, in one embodiment there is provided a method of treating glioblastoma, or a non-small cell lung cancer, or a glioblastoma or non-small cell lung cancer that has metastasized, preferably to the brain, or brain metastasis wherein the primary cancer is glioblastoma or non-small cell lung cancer in an individual in need thereof, comprising administering a compound of Formula I or Formula II as described herein or a pharmaceutically acceptable salt, stereoisomer, or solvate, thereof.

[0043] In a further aspect of the disclosure there is provided use of a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in the manufacture of a medicament for: treating cancer or cancer metastases in an individual in need thereof; and / or inhibiting metastatic progression from a cancer; and / or 1005445281 treating a condition or disease responsive to inhibition of one or more TRPC3, TRPC6, TRPC7 ion channel activity, and / or tubulin polymerization inhibition; and / or inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity; and / or inhibiting all three of TRPC3, TRPC6, TRPC7 ion channel activity; and / or inhibiting tubulin polymerization; and / or inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity, and inhibiting tubulin polymerization; and / or treating glioblastoma, or a non-small cell lung cancer, or glioblastoma or a nonsmall cell lung cancer that has metastasized, preferably to the brain, or brain metastasis wherein the primary cancer is glioblastoma or non-small cell lung cancer.

[0044] In a further aspect of the disclosure there is provided a compound of Formula I or II as described herein, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, for use in treating cancer or cancer metastases in an individual in need thereof; and / or inhibiting metastatic progression from a cancer; and / or treating a condition or disease responsive to inhibition of one or more TRPC3, TRPC6, TRPC7 ion channel activity, and / or tubulin polymerization inhibition; and / or inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity; and / or inhibiting all three of TRPC3, TRPC6, TRPC7 ion channel activity; and / or inhibiting tubulin polymerization; and / or inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity, and inhibiting tubulin polymerization; and / or treating glioblastoma, or a non-small cell lung cancer, or glioblastoma or a non- small cell lung cancer that has metastasized, preferably to the brain, or brain metastasis wherein the primary cancer is glioblastoma or non-small cell lung cancer. 1005445281

[0045] In preferred embodiments of these aspects of the invention the cancer is brain cancer, or metastatic brain cancer.

[0046] Preferably in each of the above, the condition or disease is responsive to the inhibition of all three of TRPC3, TRPC6, and TRPC7 ion channel activity. Preferably the condition or disease is also responsive to tubulin polymerization inhibition.

[0047] The diseases or conditions include solid or metastatic cancers including brain, lung, CNS, breast, ovarian, skin, colon, prostate, renal cancers, leukemias, and melanomas as well as all other cancers wherein blocking of or inhibition of TRPC3, TRPC6, TRPC7 ion channel activity or inhibition of tubulin polymerization, or a combination thereof, is beneficial.

[0048] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0049] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

[0050] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0051] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

[0052] Brief description of the drawings

[0053] Figure 1: Compound 6 inhibits tubulin polymerization and assembly in vitro. Porcine tubulin (2 mg / mL) was incubated in 1 x reaction buffer with 0.9 mM GTP and either 3 pM Compound 6, paclitaxel or vinblastine sulfate for 60 min at 37 °C. Tubulin polymerization was tracked using the fluorescence (arbitrary units) of a proprietary fluorophore probe for tubulin assembly as displayed as a function of time. 1005445281

[0054] Figure 2: Compound 6 suppresses breast cancer cell migration and invasion. MDA-MB-231 breast cancer cells were initially seeded and allowed to adhere overnight prior to wounding using a 96-Well Plate Woundmaker tool. The cells were then exposed to 0 - 50 pM Compound 6 for 36 h and their migration or invasion (through 50% Matrigel) was tracked and captured using an Incucyte S3 apparatus. (A) Representative phase contrast images of the migration of cells exposed to 0 - 1 pM for 0 or 36 h post-wounding. (B) The migration and (C) invasion of MDA-MB-231 cells treated with 0 - 50 pM 36 h post-wounding using the % Wound Confluence function of the Incucyte S3 apparatus.

[0055] Figure 3. The pharmacokinetic profile of Compound 6 following single dose delivery. Plasma levels of Compound 6 following single intravenous (IV), oral (PO) or intraperitoneal (IP) dose at 4.2 mg / kg in male mice (n = 3 per dose).

[0056] Figure 4. The pharmacokinetic profile of Compound 6 following twice a day dosing. Plasma and brain levels of Compound 6 following two intraperitoneal (IP) doses at 20 mg / kg in male mice (n = 3 per timepoint).

[0057] Figure 5. Body weight measurements in vehicle and Compound 6 treated animals. Change in body weight following administration of Compound 6 (25 mg / kg twice a day; intraperitoneal) in BALB / c nude mice (n = 10), 20 days post-inoculation with NCI-H1299 tumor cells on the right flank.

[0058] Figure 6. Tumor volume measurements. Mean tumor volumes in BALB / c nude (n = 10) following treatment with vehicle or Compound 6 (25 mg / kg twice a day; intraperitoneal), 20 days post-inoculation with NCI-H1299 tumor cells on the right flank.

[0059] Detailed description of the embodiments

[0060] The inventors have surprisingly discovered that the compounds of the invention can provide effective blockers (i.e. inhibit ion channel activity) of one or more of TRPC3, TRPC6 and / or TRPC7 channels and inhibit the polymerization of tubulin. These dual activities of the compounds make them suitable for use in cancer treatment.

[0061] Importantly these blockers also show brain penetration which is important for the treatment of brain tumors, including metastatic tumors, and other brain diseases and / or disorders where blockade of TRPC3, TRPC6, TRPC7 ion channels and / or inhibition of tubulin polymerization, or any combination thereof, plays a crucial role. It will be 1005445281 understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0062] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0063] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0064] Definitions

[0065] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers, or steps.

[0066] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0067] Thus, for example, a reference to “a salt” may include a plurality of salts and a reference to “at least one heteroatom” may include one or more heteroatoms, and so forth.

[0068] The term “(s)” following a noun contemplates the singular or plural form, or both. For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0069] When any two substituent groups or any two instances of the same substituent group are “independently selected” from a list of alternatives, the groups may be the same 1005445281 or different. For example, if Raand Rbare independently selected from alkyl, fluoro, amino, and hydroxyalkyl, then a molecule with two Ragroups and two Rbgroups could have all groups be an alkyl group (e.g., four different alkyl groups). Alternatively, the first Ra could be alkyl, the second Racould be fluoro, the first Rbcould be hydroxyalkyl, and the second Rbcould be amino (or any other substituents taken from the group). Alternatively, both Raand the first Rbcould be fluoro, while the second Rbcould be alkyl (i.e., some pairs of substituent groups may be the same, while other pairs may be different). In some embodiments, multiple instances of variables that may be selected from a list of alternatives are independently selected.

[0070] A "substituent" as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a "ring substituent" may be a moiety such as a halogen, alkyl group, or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term "substituted," as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, ie, a compound that can be isolated, characterized and tested for biological activity.

[0071] The terms "optionally substituted" or “may be substituted” and the like, as used throughout the specification, denotes that the group may or may not be further substituted or fused (so as to form a polycyclic system), with one or more non-hydrogen substituent groups. Suitable chemically viable substituents for a particular functional group will be apparent to those skilled in the art.

[0072] Examples of substituents include but are not limited to Ci-Cealkyl, Ci-Cehaloalkyl, Ci-Cehaloalkoxy, Ci-Cehydroxyalkyl, Cs-Cyheterocyclyl, Cs-Cycycloalkyl, Ci-Cealkoxy, Ci-Cealkylsulfanyl, Ci-Cealkylsulfenyl, Ci-Cealkylsulfonyl, Ci-Cealkylsulfonylamino, arylsulfonoamino, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino, acyl, carboxy, carbamoyl, aryl, aryloxy, heteroaryl, aminosulfonyl, aroyl, aroylamino, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halo, ureido, Ci-Ceperfluoroalkyl. Preferably the substituents include amino, halo, Ci-Cealkyl, amido, hydroxyl. 1005445281

[0073] As used herein the term "alkyl" refers to a straight or branched chain hydrocarbon radical having from one to twelve carbon atoms, or any range between, i.e. it contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The alkyl group is optionally substituted with substituents.

[0074] Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, and the like.

[0075] As used herein, the terms "Ci-C2alkyl", " Ci-C4alkyl" and " Ci-Cealkyl" refer to an alkyl group, as defined herein, containing at least 1 , and at most 2, 4 or 6 carbon atoms respectively, or any range in between (eg alkyl groups containing 2-5 carbon atoms are also within the range of Ci-Ce).

[0076] The term "cycloalkyl" is intended to include mono-, bi- or tricyclic alkyl groups. In some embodiments, cycloalkyl groups have from 3 to 12, from 3 to 10, from 3 to 8, from 3 to 6, or from 3 to 5 carbon atoms in the ring(s). In some embodiments, cycloalkyl groups have 5 or 6 ring carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0077] In some embodiments, the cycloalkyl group has from 3 to 8, from 3 to 7, from 3 to 6, from 4 to 6, from 3 to 5, or from 4 to 5 ring carbon atoms. As used herein, the terms "Cs-Cecycloalkyl", "C3-C4cycloalkyl" and "Cs-Cycycloalkyl" refer to an alkyl group, as defined herein, containing at least 3, and at most 6, 4 or 7 carbon atoms respectively, or any range in between (eg alkyl groups containing 4-5 carbon atoms are also within the range of Cs-Ce).

[0078] The term “heterocycle” refers to a cycloalkyl group having from 3 to 10 ring atoms (unless otherwise specified), of which 1 , 2, 3 or 4 are ring heteroatoms each heteroatom being independently selected from O, S and N. The term “heterocyclyl” refers to a moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound which moiety. As used herein the term “Cs-Ceheterocyclyl" refers to a heterocycle having 3 to 6 ring members with at least one ring member being a heteroatom.

[0079] The term “carbonyl” refers to the group C=O.

[0080] The term "amino" or "amine" refers to the group -NH2. 1005445281

[0081] The term " Ci-Csalkylamino" refers to an amino group having a hydrogen replaced with a Ci-Csalkyl group, wherein the Ci-Csalkyl group is as herein defined.

[0082] As used herein, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) and the term "halo" refers to the halogen radicals fluoro (-F), chloro (Cl), bromo (-Br), and iodo (-I). Preferably, 'halo' is chloro or bromo.

[0083] As used herein, the term "alkoxy" refers to an alkyl group as defined herein covalently bound via an O linkage. The alkoxy group is optionally substituted with substituents. Examples of "alkoxy" as used herein include, but are not limited to methoxy, ethoxy, propoxy, isoproxy, butoxy, iso-butoxy, tert-butoxy and pentoxy.

[0084] As used herein, the terms " Ci-C2alkoxy", " Ci-C4alkoxy" and " Ci-Csalkoxy" refer to an alkoxy group, as defined herein, containing at least 1 , and at most 2, 4 or 6 carbon atoms respectively, or any range in between (eg alkoxy groups containing 2-5 carbon atoms are also within the range of Ci-Ce).

[0085] As used herein, the term “haloalkyl” refers to an alkyl group as defined herein substituted with at least one halogen.

[0086] As used herein, the terms "Ci-C2haloalkyl", "Ci-C4haloalkyl" and "Ci-Cehaloalkyl" refer to an haloalkyl group, as defined herein, containing at least 1 , and at most 2, 4 or 6 carbon atoms respectively, or any range in between (e.g. haloalkyl groups containing 2- 5 carbon atoms are also within the range of Ci-Ce).

[0087] For example a Ci haloalkyl group could be, but is not limited to, chloromethyl, dichloromethyl, or trichloromethyl.

[0088] As used herein the term "fluoroalkyl" refers to a straight or branched chain hydrocarbon radical having from one to twelve carbon atoms or any range between i.e. it contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms and wherein at least one of the hydrogen atoms is substituted by a fluorine, All of the hydrogen atoms may be substituted by a fluorine. The fluoroalkyl group is optionally substituted with substituents.

[0089] Examples of "fluoroalkyl" as used herein include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, perfluoroethyl and the like. 1005445281

[0090] As used herein the terms " Ci-C2fluoro alkyl", " Ci-C4fluoralkyl" and " Ci- Csfluoroalkyl" refer to a fluoroalkyl group, as defined above, containing at least 1 , and at most 2, 4 or 6 carbon atoms respectively, or any range in between (e.g. fluoroalkyl groups containing 2-5 carbon atoms are also within the range of Ci-Ce).

[0091] The term "Ci-Csthioalkyl" refers to a group in the form of Ci-Csalkyl-SH, wherein the Ci-Csalkyl group is as herein defined.

[0092] The terms “cyano” and “nitrile” refer to the group -CN.

[0093] As used herein “D” refers to deuterium.

[0094] Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space. As used herein, the term "stereoisomer" includes but is not limited to diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures.

[0095] Where the compounds are chiral, the compound may exist as a racemic mixture, predominantly one enantiomer, or only one enantiomer.

[0096] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0097] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1005445281

[0098] 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0099] As used herein, "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material that is included in the composition for a purpose other than pharmaceutical efficacy (this is not intended to exclude materials which may have some biological effect).

[0100] As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of the likelihood of the risk of (or susceptibility to) acquiring a disease or condition, ie preventing at least one of the clinical symptoms of the disease from developing in an individual that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease. Preventing or prevention as used herein may also be in the context of preventing that disease or condition from progressing or preventing a symptom from progressing. Preventing or prevention as used herein in the content of this invention when the subject has cancer may also relate to prevention, or reduction of the likelihood, of a cancer metastasizing. Biological and physiological parameters for identifying such patients are also well-known by physicians.

[0101] The skilled artisan will appreciate that "prevention" is not an absolute term. In particularly preferred embodiments, the methods of the present invention can be to 1005445281 prevent or reduce the severity, or inhibit or minimize progression, of a symptom of a disease or condition as described herein. As such, the methods of the present invention have utility as treatments as well as prophylaxes.

[0102] The terms "treatment" or "treating" of a subject includes delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening the severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the individual; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating. Treatment may not necessarily result in the complete clearance of a disease or disorder but may reduce or minimize complications and side effects of infection and the progression of a disease or disorder. The success or otherwise of treatment may be monitored by, amongst other things, physical examination of the individual, CT scan, MRI, serological or histological markers, blood biomarkers, survival, or quality of life measures.

[0103] As used herein, the term “tumor” refers to any benign or malignant growth including lung, CNS, breast, ovarian, skin, colon, prostate, renal cancers, leukemias, melanomas as well as all other cancers. A malignant tumor is used interchangeably with the term “cancer”.

[0104] The phrase “treatment of cancer” may refer to but is not limited to the slowing of tumor growth, reversal of tumor growth, reduction in tumor size, inhibiting (i.e. slow to some extent and preferably stop) cancer cell infiltration into peripheral organs, prevention of recurrence of cancer, partial or complete remission of cancer, reducing or inhibiting cancer relapse, reduction in metastasis, prevention or inhibition of metastasis, reduction in the risk of metastasis.

[0105] As used herein the term “reducing” (and grammatical variations thereof) refers to a lowering in size, degree, incidence, rate of occurrence, amount or probability. For example, a reduction in risk refers to a lower probability. 1005445281

[0106] As used herein the phrase “reducing the risk of metastasis” includes the reduction of the likelihood of metastasis, inhibition of metastatic progression, reduction in the number of metastasis, delay or slowing the development of metastasis, reduction in the metastasis cell mass, or promotion of metastasis regression compared to the expected metastasis for a non-treated cancer of the same type.

[0107] The term “therapeutical ly-effective amount,” pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with a desired treatment regimen. A therapeutically-effective amount of a compound will vary with the particular compound chosen (e.g. consider the potency, efficacy, and half-life of the compound); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.

[0108] The term “about” is intended to convey that the value in question is not to be taken as being limited to exactly that value, and is intended to include minor variations on the value sufficient for the working of the invention. It is within the skill set of the skilled person to determine the degree of variance from the value that will still achieve the desired result.

[0109] Compounds

[0110] The present invention provides compounds of Formula I and pharmaceutically acceptable salts, solvates and stereoisomers thereof:

[0111] Formula I wherein: 1005445281

[0112] R1is a bicyclic system wherein: the dashed line ( ) represents an optional double bond (=) or single bond ( — );wherein when X and CR4are connected by a double bond, then Y and CR4are connected by a single bond, and when X and CR4are connected by a single bond, then Y and CR4are connected by a double bond;

[0113] A1, A2and A3are independently selected from N and CR5; when present R5is independently selected from H, D, halo, Ci-C4alkyl, hydroxy, amino, Ci-C4alkoxy, Ci-C4fluoroalkyl, phenyl, cyano, Ci-C4thioalkyl, and Ci- C4alkylamino;

[0114] X is selected from CH, CD, CH2, CHD, CD2, S, and O; and

[0115] Y is selected from CH, CD, CH2, CHD, CD2, S, and N;

[0116] R4is selected from CH3, CDs, CH2D, CHD2, Ci-Cealkyl, hydroxy, amino, C1- Cealkoxy, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino;

[0117] R2is selected from H, D, halo, cyano, Ci-Cethioalkyl, Ci-Cealkylamino, and C1- Cealkyl;

[0118] R3is: where R6and R7together with the N atom between them form a ring comprising 3-9 ring atoms.

[0119] In embodiments the ring formed by R6and R7can be mono-cyclic saturated straight-chain alkyl, such as , or mono-cyclic 1005445281 branched-chain alkyl, such bicyclic such or can be variously substituted and branched, such as

[0120] In preferred embodiments R1is: more preferably embodiments R1is: more preferably 1005445281

[0121] In embodiments, R3is 1 -piperidine substituted with 1-4 substituents independently selected from D, halo, Ci-Cealkyl, Ci-Cealkoxy, Ci-Cefluoroalkyl, phenyl, cyano, Ci- Cethioalkyl, and Ci-Cealkylamino. In some particular embodiments, the piperidine ring is substituted with 2-3 substituents selected from these groups.

[0122] In embodiments, R3is 1-piperidine substituted with 1-2 Ci-Cealkyl groups. In some such embodiments, Ci-Cealkyl is methyl. In some particular embodiments, two methyl groups on the same or different ring carbon atoms, and in more particular embodiments 2,2-; 2,3-; 2,5-; 3,3-; 3,5-; or 2,6-dimethyl).

[0123] In embodiments, R3is 3,5-dimethylpiperidine, preferably , more preferably where the relative stereochemistry of the methyl groups is ‘syn’ as in

[0124] In embodiments, R3is 4-morpholine substituted with 1-4 substituents independently selected from D, halo, Ci-Cealkyl, Ci-Cealkoxy, Ci-Cefluoroalkyl, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino. In some particular embodiments, the morpholine ring is substituted with 2-3 substituents selected from these groups.

[0125] In embodiments, R3is 4-morpholine substituted with 1-2 Ci-Cealkyl groups. In some such embodiments, Ci-Cealkyl is methyl. In some particular embodiments, two methyl groups on the same or different ring carbon atoms, and in more particular embodiments 2,2-; 2,3-; 2,5-; 3,3-; 3,5-; or 2,6-dimethyl). 1005445281

[0126] In embodiments, R3is 2,6-dimethylmorpholine, preferably more preferably where the relative stereochemistry of the methyl groups is ‘syn’ as in

[0127] In a further aspect, there is provided a compound of Formula II or a pharmaceutically acceptable salt, solvate or stereoisomer thereof: wherein,

[0128] A1, A2and A3are selected from N and CR12; when present each R12is independently selected from H, D, halo, Ci-C4alkyl, hydroxy, amino, Ci-C4alkoxy, Ci-C4fluoroalkyl, phenyl, cyano, Ci-C4thioalkyl, and Ci- C4alkylamino;

[0129] X is selected from O and S;

[0130] R8is selected from CH3, CD3, CH2D, CHD2, Ci-Cealkyl, hydroxy, amino, C1- Cealkoxy, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino;

[0131] R9is selected from H, D, halo, cyano, Ci-Cethioalkyl, Ci-Cealkylamino, and C1- Cealkyl;

[0132] R10and R11form a ring; the ring formed by R10and R11comprises 3-9 ring atoms. 1005445281

[0133] In one embodiment, compounds of Formula I, or pharmaceutically acceptable salts thereof, may be selected from the group consisting of:

[0134] In a preferred embodiment, the compound of Formula I or Formula II or pharmaceutically acceptable salt, solvate or stereoisomer thereof, is: 1005445281

[0135] The skilled artisan will appreciate that salts of the compounds according to Formula I may be prepared. These salts may be prepared in situ during the isolation and purification of the compound, or by separately treating the purified compound in its free acid or free base form with a suitable base or acid, respectively.

[0136] As used herein, the term "a compound of” a particular formula or "the compound of” a particular formula refers to one or more compounds according to that formula.

[0137] The compound of Formula I or Formula II may exist in solid or liquid form. In the solid state, it may exist in crystalline or noncrystalline form, or as a mixture thereof. The skilled artisan will appreciate that pharmaceutically acceptable solvates may be formed for crystalline compounds wherein solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve non-aqueous solvents such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent incorporated into the crystalline lattice are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The invention includes all such solvates.

[0138] The skilled artisan will further appreciate that certain compounds of the invention that exist in crystalline form, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as "polymorphs." The invention includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, 1005445281 used in making the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions.

[0139] Compositions / formulations

[0140] The compounds of the invention will normally, but not necessarily, be formulated into a pharmaceutical composition prior to administration to a patient. Accordingly, in another aspect the invention is directed to pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable excipient.

[0141] Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

[0142] The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a therapeutically effective amount of a compound of the invention can be extracted and then given to the patient such as with powders, syrups, and solutions for injection.

[0143] Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a therapeutically effective amount of a compound of the invention. When prepared in unit dosage form, the pharmaceutical compositions of the invention typically contain from 1 mg to 1 ,000 mg of a compound of the invention.

[0144] The pharmaceutical compositions of the invention typically contain one compound of the invention. However, in certain embodiments, the pharmaceutical compositions of the invention contain more than one compound of the invention. For example, in certain embodiments the pharmaceutical compositions of the invention contain two compounds of the invention. In addition, the pharmaceutical compositions of the invention may optionally further comprise one or more additional pharmaceutically active compounds (including, e.g., those described herein).

[0145] Combination therapies according to the present invention thus comprise the administration of a compound of Formula I or Formula II or a pharmaceutically acceptable 1005445281 salt thereof, and at least one other therapeutically active agent. The compound(s) of Formula I or Formula II and pharmaceutically acceptable salts thereof, and the other therapeutic agent(s) may be administered together in a single pharmaceutical composition or separately. In this embodiment, the compound(s) of Formula I or Formula II are prepared as a first medicament; and the other therapeutic agent(s) is prepared as a second medicament. Administration may be separate, but may occur simultaneously or sequentially in any order. The amounts of the compound(s) of Formula I or Formula II and pharmaceutically acceptable salts thereof, and the other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. Thus, in a further aspect, there is provided a combination comprising a compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, together with one or more other therapeutic agents.

[0146] The pharmaceutical compositions of the invention typically include more than one pharmaceutically acceptable excipient. However, in certain embodiments, the pharmaceutical compositions of the invention contain one pharmaceutically acceptable excipient.

[0147] As noted earlier, as used herein, "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material that is included in the composition for a purpose other than pharmaceutical efficacy (this is not intended to exclude materials that may have some biological effect). For example, an excipient may be involved in giving form or consistency to the pharmaceutical composition, such as forming a vehicle or carrier for a compound of the invention. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions that would substantially reduce the efficacy of the compound of the invention when administered to a patient and interactions that would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.

[0148] The compound of the invention and the pharmaceutically acceptable excipient or excipients will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration as noted above. For example, dosage forms include those adapted for (1) intranasal administration such as aerosol spray, solution, powder, suspension, emulsion, drops, single or multi-dose, with or without a preservative; 1005445281

[0149] (2) inhalation such as aerosols and solutions; (3) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (4) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; and (5) rectal administration such as suppositories.

[0150] Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms.

[0151] Certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the compound of the invention (or other compounds) once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

[0152] Suitable pharmaceutically-acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

[0153] Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention. In addition, there are many resources available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company, e.g., 18thEd.), Remington: The Science and 1005445281

[0154] Practice of Pharmacy (Lippincott Williams & Wilkins, e.g., 21stEd.), The Handbook of Pharmaceutical Additives (Gower Publishing Limited, e.g., 3rdEd.), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press, e.g., 6thEd.).

[0155] The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company, e.g., 18thEd).

[0156] Tubulin polymerization inhibition

[0157] The protein tubulin is a significant target in disease states characterized by cell proliferation including cancer and inflammation. Tubulin exists as a and p isoforms that polymerize to form structures termed microtubules. Compounds that impair tubulin polymerization disrupt cell division, a process that is dependent on the generation of microtubules to form mitotic spindles. The Vinca alkaloids, including vincristine and vinblastine, are examples of compounds that inhibit tubulin polymerization.

[0158] Compounds that impair microtubule depolymerization also prevent mitosis since they stabilize mitotic spindles, thereby reducing their dynamic nature, a requirement for proper cell division. Disruption of mitosis in this context is associated with apoptosis and cell cycle arrest. Examples of microtubule stabilizers include taxoids such as paclitaxel and cabazitaxel.

[0159] In addition to cancers and inflammation-based diseases, tubulin is also a therapeutic target for treating pathologies dependent on the abnormal formation of blood vessels (neovascularization), such as in cancerous tumors and in ocular myopathy. In these disease states, the microtubules intrinsic to vascular endothelial cells are considered therapeutic targets of the anti-tubulins. Inhibition of tubulin depolymerization within vascular endothelial cells is associated with vascular blockage and cessation of blood flow. In the context of solid tumors, vascular blockage suppresses the delivery of oxygen and nutrients to the tumor, a state that promotes tumor cell necrosis. Neovascular systems are characteristically more sensitive to anti-tubulins given their dependency on microtubule function relative to normal, healthy, vascular endothelial cells. Certain inhibitors of tubulin polymerization target a common binding pocket termed the colchicine- 1005445281 binding pocket of tubulin. Colchicine-binding site inhibitors often demonstrate anti- neovascular activity at a lower in vivo concentration than their anti-proliferative proficiency. Given this dichotomy, colchicine-binding site inhibitors may potentially have dual modalities (i.e. anti-mitotic and anti-vascular activities).

[0160] Drug Resistance

[0161] A common issue in cancer is the development of drug resistance. Certain tumors demonstrate intrinsic resistance to chemotherapy while others develop drug resistance throughout chemotherapy exposure. A significant proportion of tumors exhibit multidrug resistance given their overexpression of drug efflux pumps that are integral membrane proteins. Overexpression of the MDR-1 gene product, P-glycoprotein (P-gp), is associated with decreased intracellular drug levels with an attendant suppression of cancer cell kill. Clinically, multidrug resistance mediated by P-gp drug efflux restricts the utility of various chemotherapies including the anti-microtubule agents, taxol, docetaxol, vinblastine and vincristine. There is a clear need for new drugs that can circumvent multidrug resistance.

[0162] Compounds of the invention may be anti-microtubule agents, as they are inhibitors of microtubulin polymerization. Compounds of the invention may have improved drug resistance properties compared to anti-microtubule agents of the prior art.

[0163] A further significant rationale for the discovery and development of novel microtubule-active agents is that microtubule disruptors often demonstrate activity against p53-defective cancers. Human cancers defective in p53 function constitute approximately half of all cancers in the United States. Compounds of the invention show efficacy against p53-defective cancers.

[0164] TRPC3, 6 and 7 as therapeutic targets in oncology

[0165] The canonical-type of transient receptor potential (TRPC) channels belong to the TRP superfamily and regulate Ca2+homeostasis. They play a critical role in many cellular processes by changing cytosolic free Ca2+concentrations. The TRPC subfamily has seven mammalian members (TRPC1-7) which participate in store-operated Ca2+entry (SOCE) and / or receptor-operated Ca2+entry (ROCE) in cells. In humans, all isoforms are expressed except TRPC2, which is a pseudogene. 1005445281

[0166] TRPC ion channels are coupled to the downstream signalling of Gaq-type G- protein coupled receptors. TRPC channels are assembled as homomeric or heteromeric assembles of subunits; classified into four subsets: TRPC1 , TRPC2, TRPC3 / 6 / 7, and TRPC4 / 5. TRPC3 / 6 / 7 are known to be directly activated by phospholipase C-generated diacylglycerols (DAGs). TRPC3, TRPC6 and TRPC7 subtypes are widely expressed in the brain and implicated in the pathology of various indications involving Ca2+dysregulation including oncology. In particular, Ca2+dysregulation is associated with cancer cell proliferation, migration and invasion. Since the TRPCs promote sustained intracellular Ca2+levels by functioning as channels for the ion, they have emerged as promising therapeutic targets in the context of oncology.

[0167] Expression levels of TRPC3 and 6 are upregulated in multiple tumor types. The upregulation of TRPC3 has been established in gastric cancer specimens, gliomas, breast cancers and ovarian cancer specimens relative to TRPC3 levels in corresponding normal tissues. Likewise, TRPC6 is reportedly overexpressed in gliomas, non-small cell lung cancer, hepatocellular, esophageal and breast cancers when compared to expression levels in normal tissues. In general, TRPC3 and 6 overexpression in these cancers confer an aggressive and malignant phenotype. For example, high TRPC3 expression in glioblastoma cancer correlates with poor survival rates. While TRPC3 and 6 expression promotes tumor cell invasion and migration, key hallmarks of cancer metastases. There are relatively fewer reports concerning the involvement of TRPC7 in the context of oncology, however it was recently highlighted that elevated TRPC7 protein expression levels in lung adenocarcinomas were robustly associated with a clinically lower five-year survival rate.

[0168] A range of molecular and cellular responses to TRPC3, 6 and 7 inhibition have been described following the impairment of channel function using a variety of methods. A widely reported response to TRPC6 impairment, either through siRNA-mediated TRPC6 knockdown or expression of channel-dead dominant-negative TRPC6 mutants, is cancer cell G2 / M arrest with an attendant suppression of cell proliferation. Consistent with this, the blockade of TRPC6 function by pharmacological inhibition also induced arrest at the G2 / M phase of the cell cycle and suppressed cell proliferation. In addition to cell cycle arrest and attenuation of cell proliferation and survival, siRNA-induced TRPC6 knockdown and pharmacological inhibition using benzothiazole amides have been widely associated with anti-invasive and anti-migratory cancer cell activities. Since cell migration 1005445281 and invasion are both critical cellular processes for cancer metastases, there is potential to therapeutically block the metastatic nature of many cancers through TRPC6 inhibition. Lastly, TRPC6 impairment has been associated with an increase associated with the survival of mice intracranially transplanted.

[0169] Like TRPC6, the impairment of TRPC3 and 7 function is also associated with favorable anti-cancer properties, albeit with fewer experimental exemplifications than TRPC6. Pharmacological inhibition of TRPC3 impaired gastric cancer cell growth in vitro and in vivo, likely through attenuation of the CBN2 / GSKp / NFAT2 pathway (Lin, Zheng et al. 2021). It was similarly established that TRPC3 blockade by the pyrazole Pyr3 was associated with the induction of apoptosis and suppression of cell proliferation of triplenegative breast cancer cells, although impairment occurred through a distinct pathway in the form of the RASA4 / MAPK4 signalling cascade. In a separate study, Pyr3 also impaired the proliferation of melanoma cells, a result that further supports the notion of TRPC3 as a therapeutic target in cancer. Blockade of TRPC3 activity through exposure to benzothiazole amides and Pyr3 also attenuated melanoma and gastric cancer cell migration and invasion. While there are few reports of the involvement of TRPC7 as a therapeutic target in cancer, it was recently reported that siRNA-mediated knockdown of TRPC7 inhibited cell cycle progression and cell migration, results that implicate a role for TRPC7 in the cellular processes.

[0170] Advantageously new therapies, such as those described herein, with safe and efficacious oncology agents, wherein the pharmacokinetic / pharmacodynamic properties can provide for potent, selective, long-lasting, and overall controlled delivery of therapeutics, all the while resulting in fewer side-effects to the patient.

[0171] In each of the methods and use aspects of the invention described herein, the compounds of Formula I or II inhibit: all three of TRPC3, TRPC6, and TRPC7 ion channels; one of TRPC3 or TRPC6 or TRPC7 activity;

[0172] TRPC3 and TRPC6 activity;

[0173] TRPC6 and TRPC7 activity;

[0174] TRPC3 and TRPC7 activity. 1005445281

[0175] While inhibition or blocking of one or more of TRPC3, TRPC6, TRPC7 ion channels in each of the abovementioned methods and uses is likely to achieve a therapeutic and / or prophylactic outcome, the compound of Formula I preferably works against all three of them for maximum therapeutic efficacy.

[0176] Methods of use

[0177] The present invention provides a method of treating cancer or cancer metastases, comprising administering to a cell, tissue or an individual in need thereof, the method comprising administering a therapeutically effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, or the pharmaceutical composition as described herein.

[0178] In some embodiments the method or use further comprises an additional anticancer agent, to be sequentially or simultaneously administered to the subject. Additionally or alternatively the subject may also be receiving radiation therapy, chemotherapy, immunotherapy, or a combination thereof. Radiation therapy, chemotherapy, immunotherapy, or a combination thereof may also be utilized prior to administration of the compounds of Formula I or 11 in order to sensitize the tumor or cancer to the treatment.

[0179] In alternative embodiments of the disclosure the methods of uses are to treat or prevent angiogenesis. Both TRPC6 and tubulin are involved in angiogenesis.

[0180] Subjects requiring treatment include those already having a benign, pre- cancerous, non-metastatic, or metastatic tumor as well as those in which the occurrence or recurrence of cancer is to be prevented.

[0181] The objective of treatment may be to reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e. , slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the conditions or symptoms associated with the disorder.

[0182] In one embodiment, the methods of treatment described herein are for the minimization or treatment of a condition or symptom in a subject that is associated with cancer in the subject. 1005445281

[0183] In one embodiment, the method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment. In one embodiment, the cancer is pre-cancerous or pre-neoplastic.

[0184] In one embodiment, the cancer is a secondary cancer or metastases. Secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain. The metastases may be a metastasis from a glioblastoma or non-small cell lung cancer. The metastases may be located in the brain.

[0185] Metastasis is a common and particularly damaging property of cancer and is responsible for approximately 90% of cancer-related deaths in humans. In an unanticipated discovery, certain compounds of Formula I or Formula II demonstrated activity against tubulin polymerization and assembly as well as TRPC3, TRPC6 and TRPC7 antagonism, furnishing the compounds with a unique dual action modality. Compounds of this nature are highly desirable since the activities of tubulin and TRPC3, TRPC6 and TRPC7 are significant in the metastatic process and may have value in blocking this deleterious feature of cancer.

[0186] In these embodiments, the disease or condition is responsive to the inhibition of TRPC3, or TRPC6 or TRPC7 ion-channels, or tubulin polymerization inhibition, or a combination thereof. More preferably, this disease or condition is metastatic cancer. These are malignant cancerous growth including glioblastoma, lung, CNS, breast, ovarian, skin, colon, prostate, renal cancers, leukemias, melanomas as well as all other cancers wherein inhibition or blocking of those channels is beneficial.

[0187] In a further specific embodiment, there is provided a method of treating brain cancers, including but not limited to astrocytoma, glioblastoma, oligodendroglioma, ependymoma and choroid plexus carcinoma, and secondary brain metastases. Chemotherapies for treating brain cancer must demonstrate blood-brain barrier (BBB) permeability and ideally possess features that confer evasion from drug efflux from the brain. Certain compounds of Formula I and Formula II unexpectedly exhibited both BBB permeability and evasion from P-gp-mediated drug efflux, affording them with the potential utility to treat brain cancers and brain cancer metastases. 1005445281

[0188] In another aspect, the method for treating cancer applies to the treatment of cancer cells that are not treated by traditional anti-cancer therapies, wherein the traditional anti-cancer therapies include radiation therapy, chemotherapy, immunooncology therapy, and combinations thereof.

[0189] In another aspect, the method for treating cancer applies to the treatment of cancer cells that have acquired resistance to traditional anti-cancer therapies, wherein the traditional anti-cancer therapies include radiation therapy, chemotherapy, immunooncology therapy, and combinations thereof.

[0190] In certain embodiments, the present invention provides a method of treating cancer or cancer metastases in a subject in need thereof, wherein the cancer or cancer metastases are resistant to radiation therapy, chemotherapy, immunotherapy, or a combination thereof, the method comprising administering a therapeutically effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof to the subject.

[0191] In another embodiment, there is provided a method of sensitizing a tumor in a subject in need thereof to radiation therapy, chemotherapy, immunotherapy, or a combination thereof, the method comprising administering a therapeutically effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof to the subject.

[0192] In one embodiment, the method is useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured.

[0193] In one embodiment, the method is useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, or in the response to treatment. In one embodiment, the cancer is pre-cancerous or pre-neoplastic.

[0194] The compounds of this invention modulate the activity of tubulin polymerization and the activity of TRPC3, TRPC6 and TRPC7 ion channels, hence may provide a beneficial therapeutic impact in methods of therapy. In particular these compounds may be used for treatment of cancers. 1005445281

[0195] The compounds of this invention block the TRPC3, TRPC6 and TRPC7 ion channels and can be used to treat diseases associated with blocking one or a combination of these channels. The compounds of this invention inhibit tubulin polymerization and have utility in diseases where the inhibition of tubulin polymerization is beneficial. The compounds of this invention can be used where dual blockade of the TRPC3, TRPC6, TRPC7 ion channels and tubulin polymerization, or any combination thereof, is beneficial.

[0196] The methods of treatment comprise administering an effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof, stereoisomer or solvate thereof to an individual in need thereof. The compound of Formula I or Formula II that is administered may be any one of the compounds, formulations and compositions thereof described throughout.

[0197] In one aspect, the invention provides a method of inhibiting TRPC3, TRPC6, TRPC7 ion channel or tubulin polymerization, or a combination thereof, comprising administering to a cell, tissue or an individual in need thereof, an effective amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

[0198] In another aspect, the invention provides a method of treating a condition or disease responsive to the inhibition of TRPC3, or TRPC6 or TRPC7 ion channel activity, or tubulin polymerization inhibition, or a combination thereof, in an individual in need thereof, comprising administering a compound of Formula I or Formula II or a pharmaceutically acceptable salt, stereoisomer of solvate thereof.

[0199] Preferably the condition or disease is cancer, for example solid tumors, resistant cancers and cancers that metastasize to the brain, more preferably the cancer is glioblastoma or non-small cell lung cancer with brain metastasis.

[0200] In another aspect, the present invention also provides a method of inhibiting metastatic progression from a cancer responsive to the inhibition of TRPC3, TRPC6, TRPC7 ion channel activity, tubulin polymerization inhibition, or a combination thereof, comprising administering a therapeutically effective amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt stereoisomer or solvate thereof, thereby inhibiting metastatic progression. 1005445281

[0201] The invention also provides a therapeutically effective amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, for use in:

[0202] - treating cancer or cancer metastases in an individual in need thereof; and / or

[0203] - inhibiting metastatic progression from a cancer; and / or

[0204] - inhibiting TRPC3, or TRPC6, or TRPC7 ion channel activity; and / or

[0205] - inhibiting tubulin polymerization activity; and / or

[0206] - inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity, and inhibiting tubulin polymerization; and / or

[0207] - treating a condition or disease responsive to the inhibition of TRPC3, TRPC6, TRPC7 ion channel activity, tubulin polymerization inhibition, or a combination thereof; and / or

[0208] - treating cancers responsive to TRPC3, TRPC6 and TRPC7 ion channel activity, tubulin polymerization activity, or a combination thereof; and / or

[0209] - treating glioblastoma, or a non-small cell lung cancer, or glioblastoma or a nonsmall cell lung cancer that has metastasized, preferably to the brain, or brain metastasis wherein the primary cancer is glioblastoma or non-small cell lung cancer.

[0210] The invention also provides the use of a therapeutically effective amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for:

[0211] - treating cancer or cancer metastases in an individual in need thereof; and / or

[0212] - inhibiting metastatic progression from a cancer; and / or-inhibiting one or more of TRPC3, or TRPC6, or TRPC7 ion channel activity; and / or

[0213] - inhibiting tubulin polymerization and / or

[0214] - inhibiting one or more of TRPC3, TRPC6, TRPC7 ion channel activity, and inhibiting tubulin polymerisation; and / or 1005445281

[0215] - treating a condition or disease responsive to the inhibition of TRPC3, o r TRPC6, or TRPC7 ion channel activity, or tubulin polymerization inhibition or a combination thereof; and / or treating glioblastoma, or a non-small cell lung cancer, or glioblastoma or a nonsmall cell lung cancer that has metastasized, preferably to the brain, or brain metastasis wherein the primary cancer is glioblastoma or non-small cell lung cancer.

[0216] In each of these methods and uses, the disease or condition is responsive to inhibition of TRPC3, TRPC6, TRPC7 ion channel activity, or tubulin polymerization inhibition, or a combination thereof. While inhibition or blocking of one or more of TRPC3, TRPC6, TRPC7 ion channels, or tubulin polymerization activity, in each of the abovementioned methods and uses is likely to achieve a therapeutic and / or outcome, the compound of Formula I or Formula II preferably works against TRPC3, TRPC6, TRPC7 ion channels, or tubulin polymerization activity, or a combination thereof for maximum therapeutic efficacy.

[0217] A “subject” herein is preferably a human subject. It will be understood that the terms “subject” and “individual” and “patient” are interchangeable in relation to an individual requiring treatment according to the present invention.

[0218] Although the invention finds application in humans, the invention is also useful for therapeutic veterinary purposes. The invention is useful for domestic or farm animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals.

[0219] Administration

[0220] The compounds of the invention may be administered by any suitable route of administration, including systemic administration. Systemic administration includes intranasal administration, administration by inhalation, oral administration, parenteral administration, and rectal administration. Parenteral administration refers to routes of administration other than enteral, transdermal, or inhalation, and is typically by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion, optionally including a loading bolus dose. Inhalation refers to administration into the patient's lungs whether inhaled through the mouth or 1005445281 through the nasal passages. In some embodiments, a compound of the invention is administered intravenously, by inhalation, or orally.

[0221] The compounds of the invention may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the skilled artisan.

[0222] The compounds of this invention are delivered to the patient in a therapeutically effective amount for treatment.

[0223] The “therapeutically effective amount” described here relates to an amount of compound which when administered at the desired dosing regimen will produce a therapeutic effect against the particular disease or condition being treated. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body.

[0224] In addition, suitable dosing regimens, including the duration such regimens are administered, for a compound of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated and any comorbidities, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's response to the dosing regimen or over time as individual patient needs change.

[0225] Typical daily dosages may vary depending upon the particular route of administration chosen.

[0226] Additionally, the compounds of the invention may be administered as prodrugs. As used herein, a "prodrug" of a compound of the invention is a functional derivative of the compound which, upon administration to a patient, eventually liberates the compound of 1005445281 the invention in vivo. Administration of a compound of the invention as a prodrug may enable the skilled artisan to do one or more of the following: (a) modify the onset of the compound in vivo, (b) modify the duration of action of the compound in vivo, (c) modify the transportation or distribution of the compound in vivo, (d) modify the solubility of the compound in vivo, and (e) overcome or overcome a side effect or other difficulty encountered with the compound. Typical functional derivatives used to prepare prodrugs include modifications of the compound that are chemically or enzymatically cleaved in vivo. Such modifications, which include the preparation of phosphates, amides, esters, thioesters, carbonates, and carbamates, are well known to those skilled in the art.

[0227] Methods of treatment of the invention may be achieved using the compounds of Formula I or II of the invention as a monotherapy, or in dual or multiple combination therapy with one or more therapeutic agents or therapies. For example, one or more compounds of the invention may be used in combination. One or more compounds of the invention may also be used with one or more other therapeutic agents or therapies. The one or more other therapeutic agents of therapies include but are not limited to: chemotherapies, radiotherapies, immunotherapies, immune-oncology therapies, sedative pain medication, saline, IV nutrition, and combinations thereof.

[0228] The present invention includes the use of a compound of Formula I or Formula II or salt, stereoisomer or solvates thereof as an inhibitor of TRPC3, TRPC6, TRPC7 ion channel over activity, or tubulin polymerization inhibitor, or a combination thereof (e.g. in in vitro or in vivo assays, or in a subject in need thereof). Accordingly, compounds of Formula I or Formula II may be used to identify compounds which inhibit TRPC3, TRPC6, TRPC7 ion channel over activity, or tubulin polymerization activity, or combinations thereof, for example by using a compound of Formula I as a control compound in an assay or model which measures TRPC3, TRPC6, TRPC7 ion channel activity, or tubulin polymerization activity, or combinations thereof, including any biological assays described herein. 1005445281

[0229] Examples

[0230] Where compounds are referred to by number in the example section, the following applies: 1005445281

[0231] Compound names, i.e., IIIPAC names, for compounds described in the instant application were generated using JChem®™ compound naming software.

[0232] Compound Preparation

[0233] The compounds according to Formula I are prepared using conventional organic syntheses. Suitable synthetic routes are depicted below in the following general reaction schemes. All functional groups are as defined in the respective Schemes for corresponding groups unless otherwise defined. For example, "R" is two or more 1005445281 substituents that together form a five-membered ring found as the bicyclic R1of Formula I. Starting materials and reagents depicted below in the general reaction schemes are commercially available or can be made from commercially available starting materials using methods known by those skilled in the art.

[0234] Scheme 1 represents a general reaction scheme for preparing general compounds according to Formula I (depicted as compound 1.6). Treatment of arylthiourea 1.1 with reagent 1.2 in ethanol (or equivalent solvent) provides intermediates 1.3. The intermediate compound 1.3 is treated with base (such as LiOH or equivalent) in a solvent (such as THF or a mixture of THF / MeOH / water) to give intermediate 1.4. Next, reaction of intermediate 1.4 with an amine 1.5, a coupling agent (such as HATLI or EDCI with HOBt) and a base (such as DI PEA or NEts) in a solvent (such as DMF) provides compounds 1.6 according to Formula I.

[0235] Scheme 1

[0236] Scheme 2 represents an alternative general reaction scheme for preparing certain compounds according to Formula I (depicted as compound 2.5). Treatment of halothiazole 2.1 with an amine reagent 2.2 (commercially available or made from commercially available starting materials using methods known to those skilled in the art), a coupling agent (such as EDCI with HOBt) and a base (such as NEts) in a solvent (such as DMF) provides intermediates 2.3. Next, reaction of intermediate 2.3 with an aniline 2.4, a coupling agent (such as HATLI or EDCI with HOBt) and a base (such as DIPEA or NEts) in a solvent (such as DMF) provides compounds 2.5 according to Formula I. 1005445281

[0237] Scheme 2

[0238] Scheme 3 represents a general scheme to incorporate a halogen group onto the thiazole ring. Treatment of amide 3.1 with a halogenating agent (such as / V- chlorosuccinimide) in a solvent (such as THF) yields the desired halogenated compound 3.2.

[0239] Scheme 3

[0240] Experimental

[0241] General information: All evaporations were carried out under reduced pressure with a rotary evaporator. Analytical samples were dried under reduced pressure (1 -5 mmHg) at room temperature. Thin layer chromatography (TLC) was performed on silica gel 60 F254 aluminium-backed plates, spots were visualized by UV light (214 nm and 254 nm). Purification by column and flash chromatography was carried out using silica gel (300-400 mesh). Solvent systems are reported as mixtures by volume. All NMR spectra were recorded on a Bruker 400 (400 MHz) spectrometer.1H chemical shifts are reported in 5 values in ppm with the deuterated solvent as the internal standard. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constant (Hz), integration. LCMS spectra were obtained on an Agilent 1200 series 6110 or 6120 mass spectrometer with electrospray ionization and excepted as otherwise indicated, the general LCMS condition was as follows: Waters 1005445281

[0242] X Bridge C18 column (50 mm x 4.6 mm x 3.5 pm), Flow Rate: 2.0 mL / min, the column temperature: 40 °C.

[0243] The following solvents, reagents or scientific terminology may be referred to by their abbreviations:

[0244] TLC Thin Layer Chromatography mL milliliters mmol millimole h hour or hours min minute or minutes g gram mg milligram eq equivalent rt room temperature, ambient, about 25 °C

[0245] RT Retention Time

[0246] MS mass spectrometry

[0247] ESI Electrospray ionization m / z mass to charge ratio nmr Nuclear Magnetic Resonance

[0248] Hz Hertz

[0249] HPLC high performance liquid chromatography

[0250] LC / MS liquid chromatography / mass spectrometry

[0251] DCM dichloromethane

[0252] EtOAc ethyl acetate

[0253] HATLI hexafluorophosphate azabenzotriazole tetramethyl uronium

[0254] DMF / V, / V-dimethylformamide

[0255] EtOH ethanol

[0256] MeOH methanol 1005445281

[0257] PE petroleum ether

[0258] DOX dioxane

[0259] DI PEA diisopropylethylamine

[0260] NEts triethylamine THF tetrahydrofuran

[0261] EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0262] HOBt hydroxybenzotriazole

[0263] NCS / V-chlorosuccinimide

[0264] NBS / V-bromosuccinimide

[0265] Experimental Section

[0266] Scheme 1a: Experimental procedure for the preparation of Compound 1 (Method 1)

[0267] Experimental procedure for Scheme 1a: Step-1 : A mixture of 2-amino-5-nitrophenol 217-0 (3.0 g, 19.5 mmol) in triethyl orthoacetate (20 mL) was heated to reflux and stirred overnight. After cooling to room temperature, the product 217-A was formed as a precipitate. The precipitate was filtered, washed with PE (50 mL) and dried to provide 217-A as an off-white solid (2.6 g, 75% yield). LC / MS (ESI): m / z=179.2 [M+1]+RT=1.598 min. 1005445281

[0268] Step-2: To a solution of 217-A (2.6 g, 14.6 mmol) in methanol (200 mL) was added a powder of PtC>2 (300 mg). The mixture was stirred under H2 atmosphere for 3 h. After consumption of 217-A, the reaction mixture was filtered through Celite™ to remove PtC>2. The filtrate was concentrated in vacuo to provide 217-B as a brown solid (2.05 g, 95% yield). LC / MS (ESI): m / z=149.3 [M+1]+RT=1.198 min.

[0269] Step-3: To a solution of 217-B (2.3 g, 15.5 mmol) in DCM (100 mL) was added benzoyl isothiocyanate (2.52 g, 15.5 mmol). The mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo to a residue. The residue was purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) to give 217-C (3.1 g, 65% yield) as a brown solid. LC / MS (ESI): m / z=312.3 [M+1]+RT=1.821 min.

[0270] Step-4: To a solution of 217-C (622 mg, 2.0 mmol) in THF (50 mL) was added a solution of NaOH (320 mg, 8.0 mmol) in water (5.0 mL). The mixture was stirred at room temperature overnight. LCMS analysis showed the hydrolysis was finished. The reaction mixture was neutralized with 2N HCI and concentrated in vacuo to dryness. The hangovers was purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) to give 217-D (290 mg, 70% yield) as a yellow solid. LC / MS (ESI): m / z=208.3 [M+1]+RT=1.160 min.

[0271] Step-5: A mixture of 217-D (450 mg, 2.17 mmol) and ethyl 3-bromo-2- oxopropanoate (629 mg, 3.26 mmol) in EtOH (50 mL) was added Na2COs (230 mg, 2.17 mmol). The reaction mixture was allowed to heat at reflux and stirred overnight. After consumption of the starting material, the mixture was evaporated in vacuo to remove the majority of solvent. The final mixture was diluted by water (100 mL) and extracted with EtOAc (3x70 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified with column chromatography on silica gel (eluting with 33% EtOAc in PE) to give 217-E (500 mg, 76% yield) as a grey solid. LC / MS (ESI): m / z=304.2 [M+1 ]+RT=1.802 min.

[0272] Step-6: To a solution of 217-E (500 mg, 1.65 mmol) in H2O (5 mL), MeOH (5 mL) and THF (5 mL) was added LiOH.H2O (600 mg, 15 mmol) and the reaction mixture was stirred at room temperature for 3 hours. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to remove the solvent and acidized with acetic acid. The solid was filtered and washed with water (10 mL) and PE 1005445281

[0273] (10 mL) to give 217-F (330 mg, 73% yield) as a grey solid. LC / MS (ESI): m / z=276.2 [M+1]+RT=1.105 min.

[0274] Step-6: To a solution of 217-F (100 mg, 0.36 mmol) and cis-3,5-dimethylpiperidine (217-G, 50 mg, 0.44 mmol) in DMF (20 mL) was added EDCI (104 mg, 0.54 mmol), HOBT (74 mg, 0.54 mmol) and EtsN (110 mg, 1.09 mmol). The reaction mixture was allowed to stir at room temperature overnight. After consumption of the starting material, the reaction mixture was diluted by water (200 mL) and extracted with EtOAc (3 x 50 mL). The organic extract was washed by water (3 x 100 mL) and brine (100 mL) successively, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to give a residue. The residue was purified with Prep-HPLC to give Compound 1 (54 mg, 41% yield) as an off-white solid.1H NMR (400 MHz, DMSO-d6) 5 10.57 (s, 1 H), 8.24 (d, J=2.0 Hz, 1 H), 7.56 (d, J=8.8 Hz, 1 H), 7.33 (s, 1 H), 7.30 (dd, J=2.0, 8.4 Hz, 1 H), 4.45-4.42 (m, 2H), 2.57- 2.51 (m, 4H), 2.24-2.18 (m, 1 H), 1.87-1.56 (m, 3H), 0.91-0.81 (m, 7H). LC / MS (ESI): m / z=371.0 [M+1]+RT=8.667 min.

[0275] Scheme 1b: Experimental procedure for the preparation of Compound 1 (Method 2)

[0276] Experimental procedure for Scheme 1b:

[0277] Step-1 : To a solution of 2-bromothiazole-4-carboxylic acid 511-A (10 g, 48 mmol) and cis-3,5-dimethylpiperidine 217-G (6.0 g, 53 mmol) in DMF (100 mL) was added EDCI (13.8 g, 72 mmol), HOBt (9.72 g, 72 mmol) and DIEA (12.4 g, 96 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (1000 mL) and then extracted with EtOAc (1000 mL). The organic phase was separated and concentrated in vacuo to give a residue. The residue was purified by column chromatography in silica gel (eluting with 20% EtOAc in PE) to give 217-1 (10 g, 69% yield) as a yellow oil. LC / MS (ESI): m / z=305.0 [M+1]+RT=2.108 min. 1005445281

[0278] Step-2: A mixture of 217-1 (3.02 g, 10 mmol), 217-B (1.50 g, 10.1 mmol), CS2CO3 (6.5 g, 20.0 mmol), Brettphos-Pd-Gs (908 mg, 1.0 mmol) and Xphos (477 mg, 1.0 mmol) in dioxane (100 mL) was allowed to heat to 100 °C and stir overnight under argon atmosphere. The solvent was evaporated in vacuo to a residue. The residue was purified by chromatography in silica gel (eluting with EA) to give a yellow solid that was further purified by Prep-HPLC to give Compound 1 (1.2 g, 32% yield) as an off-white solid.1H NMR (400 MHz, DMSO-d6) 5 10.58 (s, 1 H), 8.25 (d, J=2.0 Hz, 1 H), 7.56 (d, J=8.4 Hz, 1 H), 7.34 (s, 1 H), 7.30 (dd, J=2.0, 8.4 Hz, 1 H), 4.46-4.43 (m, 2H), 2.57-2.51 (m, 4H), 2.24-2.18 (m, 1 H), 1.86-1.59 (m, 3H), 0.910-0.81 (m, 7H). LC / MS (ESI): m / z=371.2 [M+1]+RT=2.005 min.

[0279] Scheme 2: Experimental procedure for the preparation of Compound 2

[0280] Experimental procedure for Scheme 2:

[0281] Step-1 : To a mixture of 217-D (450 mg, 2.17 mmol) and Na2COs (230 mg, 2.174 mmol) in EtOH (50 mL) was added ethyl 3-bromo-2-oxopropanoate (629 mg, 3.26 mmol), and the reaction mixture was allowed to heat to 70 °C and stirred for 6 hours. After consumption of the starting material, the reaction mixture was concentrated and diluted with water (100 mL), extracted with EtOAc (3 x 70 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SC>4 and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 33% EtOAc in PE) to give 217-E (500 mg, 76% yield) as a grey solid. LC / MS (ESI): m / z=304.2 [M+1]+RT=1.803 min.

[0282] Step-2: To a solution of 217-E (500 mg, 1.65 mmol) in H2O (5 mL), MeOH (5 mL) and THF (5 mL) was added UOH.H2O (600 mg, 14.31 mmol), and the reaction mixture 1005445281 was stirred at room temperature for 3 hours. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to remove THF and MeOH, and acidized by acetic acid to give a precipitate. The precipitate was collected by filtration, washed with water (10 mL) and PE (10 mL), and dried to give 217-F (330 mg, 73% yield) as a grey solid. LC / MS (ESI): m / z=276.2 [M+1]+RT=1.105 min.

[0283] Step-3: To a solution of 217-F (100 mg, 0.36 mmol) and 2,6-dimethylmorpholine (51 mg, 0.44 mmol) in DMF (20 mL) was added EDCI.HCI (104 mg, 0.54 mmol), HOBT (74 mg, 0.54 mmol) and EtsN (110 mg, 1 .09 mmol), and the reaction mixture was allowed to stir at room temperature overnight. After consumption of the starting material, the reaction mixture was diluted by water (50 mL) and extracted with EtOAc (3 x 20 mL). The organic extract was washed by water (4 x 50 mL) and brine (50 mL) successively, dried over anhydrous Na2SC>4 and evaporated under reduced pressure to give a residue. The residue was purified by chromatography on silica gel (eluting 50% EtOAc in PE) to give Compound 2 (55 mg, 41 % yield) as a white solid.1H NMR (400 MHz, DMSO-de) 5 10.53 (s, 1 H), 8.20 (d, J=1.6 Hz, 1 H), 7.57 (d, J=8.4 Hz, 1 H), 7.42 (s, 1 H), 7.28 (dd, J1=8.4 Hz, J2=2.0 Hz, 1 H), 4.56-4.36 (m, 2H), 3.68-3.60 (m, 2H), 2.82-2.80 (m, 1 H), 2.58 (s, 3H), 2.45-2.43 (m, 1 H), 1.13 (s, 6H). LC / MS (ESI): m / z=373.2 [M+1]+RT=7.266 min.

[0284] Scheme 3: Experimental procedure for the preparation of Compound 3

[0285] Experimental procedure for Scheme 3:

[0286] Step-1 : To a solution of 2-amino-5-nitrophenol 217-0 (5.00 g, 32.46 mmol) in EtOH (200 mL) was added 1 ,1 ,1 -triethoxyethane (6.32 g, 38.95 mmol) and NH4CI (180 mg, 3.25 1005445281 mmol), and the reaction mixture was heated to reflux and stirred overnight. After consumption of the starting material, the reaction mixture was filtered to give a solid. The solid was washed with EtOH and hexane to give 217-A (5.3 g, 91% yield) as a grey-white solid. LC / MS (ESI): m / z=179.2 [M+1]+RT=1.598 min.

[0287] Step-2: To a solution of 217-A (5.30 g, 29.77 mmol) in MeOH (100 mL) was added PtC>2 (0.50 g), and the reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. After consumption of the starting material, the reaction mixture was filtered through celite to remove the solid and the filtrate was evaporated under reduced pressure to give 217-B (4.40 g, 99% yield) as grey solid. LC / MS (ESI): m / z=149.4 [M+1]+RT=1.201 min.

[0288] Step-3: To a solution of 217-B (2.00 g, 13.42 mmol) in DCM (80 mL) was added benzoyl isothiocyanate (2.19 g, 13.42 mmol), and the reaction mixture was stirred at room temperature overnight. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to give 217-C (4.00 g, 95.7% yield) as a yellow solid. LC / MS (ESI): m / z=312.3 [M+1]+RT=1.821 min.

[0289] Step-4: To a solution of 217-C (1.00 g, 3.20 mmol) in THF (50 mL) was added NaOH aqueous solution (2.0 M, 50 mL), and the reaction mixture was stirred at 60 °C for 5 hours. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to remove THF. The mixture was extracted with EtOAc (3x50 mL), and the combined organic phase was washed with brine (2 x 100 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure to give 217-D (160 mg, 24% yield) as a yellow solid. LC / MS (ESI): m / z=208.3 [M+1]+RT=1.155 min.

[0290] Step-5: To a mixture of 217-D (207 mg, 1.00 mmol) and Na2COs (106 mg, 1.00 mmol) in EtOH (30 mL) was added ethyl 3-bromo-2-oxobutanoate (250 mg, 1.20 mmol) and the reaction mixture was heated to 70 °C and stirred for 4 hours. After consumption of the starting material, the reaction mixture was concentrated and diluted with water (100 mL), extracted with EtOAc (3x50 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) to give 397-A (250 mg, 79% yield) as a yellow oil. LC / MS (ESI): m / z=318.1 [M+1]+RT=1.754 min. 1005445281

[0291] Step-6: To a solution of 397-A (250 mg, 0.78 mmol) in H2O (5 mL), MeOH (5 mL) and THF (5 mL) was added LiOH.H2O (600 mg, 14.31 mmol), and the reaction mixture was stirred at room temperature for 3 hours. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to remove THF and MeOH, and acidized by acetic acid to give a precipitate. The precipitate was collected by filtration, and washed with water (10 mL) and hexane (10 mL) to give 397-B (180 mg, 80% yield) as a grey solid. LC / MS (ESI): m / z=290.1 [M+1]+RT=1.173 min.

[0292] Step-7: To a solution of 397-B (180 mg, 0.62 mmol) and 2,6-dimethylmorpholine (86 mg, 0.75 mmol) in DMF (20 mL) was added EDCI.HCI (178 mg, 0.93 mmol), HOBT (126 mg, 0.93 mmol) and EtsN (189 mg, 1.86 mmol), and the reaction mixture was allowed to stir at room temperature overnight. After consumption of the starting material, the reaction mixture was diluted by water (50 mL) and extracted with EtOAc (3 x 50 mL). The organic extract was washed by water (4 x 100 mL) and brine (100 mL) successively, dried over anhydrous Na2SO4 and evaporated under reduced pressure to give a residue. The residue was purified reversed phase prep-HPLC to give Compound 3 (50 mg, 20% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) 5 10.40 (s, 1 H), 8.18 (d, J=2.0 Hz, 1 H), 7.55 (d, J=8.8 Hz, 1 H), 7.25 (dd, J1=8.8 Hz, J2=2.0 Hz, 1 H), 4.35 (d, J=12.8 Hz, 1 H), 3.97 (d, J=12.8 Hz, 1 H), 3.65-3.56 (m, 2H), 2.80-2.74 (m, 1 H), 2.57 (s, 3H), 2.50-2.45 (m, 1 H), 2.34 (s, 3H), 1.16 (d, J=6.0 Hz, 3H), 1.05 (d, J=6.0 Hz, 3H). LC / MS (ESI): m / z=387.2 [M+1]+RT=7.477 min.

[0293] Scheme 4: Experimental for the preparation of Compound 4

[0294] Experimental procedure Scheme 4:

[0295] Step-1 : To a solution of Compound 1 (140 mg, 0.38 mmol) in dry THF (15 mL) was added NCS (55.6 mg, 0.42 mmol), and the reaction mixture was allowed to stir at 40 °C for 2 days under Ar. atmosphere. After consumption of the starting material, the mixture was evaporated under reduced pressure to remove the THF, diluted by water (100 mL) and extracted with EtOAc (2 x 50 mL). The organic extract was washed by brine (50 mL), 1005445281 dried over anhydrous Na2SO4, evaporated under reduced pressure and purified with reversed phase Prep-HPLC to give Compound 4 (20.0 mg, 13.1% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) 5 10.66 (s, 1 H), 8.10 (d, J=2.0 Hz, 1 H), 7.58 (d, J=8.4 Hz, 1 H), 7.27 (dd, J=8.4, 2.0 Hz, 1 H), 4.43 (d, J=8.8 Hz, 1 H), 3.64 (d, J=10.0 Hz, 1 H), 2.68- 2.64 (m, 1 H), 2.61 (s, 3H), 2.26 (t, J=12.0 Hz, 1 H), 1.82 (d, J=12.8 Hz, 1 H), 1.70-1.56 (m, 2H), 0.93-0.79 (m, 7H). LC / MS (ESI): m / z=405.2 [M+1]+Rt=9.256 min

[0296] Scheme 5: Experimental for the preparation of Compound 5

[0297] Experimental procedure for Scheme 5:

[0298] Step-1 : To a solution of 2-methylbenzo[d]thiazol-6-amine 441-0 (328 mg, 2.0 mmol) in DCM (25 mL) was added benzoyl isothiocyanate (326 mg, 2.0 mmol), and the mixture was stirred at room temperature overnight. The mixture was evaporated in vacuo and purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) to give 441 -A (500 mg, 76% yield) as a brown solid. LC / MS (ESI): m / z=328.2 [M+1]+RT=1.692 min.

[0299] Step-2: To a solution of 441 -A (500 mg, 1.53 mmol) in THF (50 mL) was added a solution of NaOH (320 mg, 8.0 mmol) in water (5.0 mL), and the mixture was stirred at room temperature overnight. Upon completion, the reaction mixture was neutralized with 2N HCI to pH=7.0, concentrated in vacuo to dryness and purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) to give 441 -B (300 mg, 88% yield) as a yellow solid. LC / MS (ESI): m / z=224.2 [M+1 ]+RT=1.060 min.

[0300] Step-3: To a solution of 441-B (300 mg, 1.34 mmol) in EtOH (50 mL) was added ethyl 3-bromo-2-oxopropanoate (290 mg, 1.5 mmol). The mixture was refluxed for two hours and concentrated in vacuo to give a residue. The residue was purified by column 1005445281 chromatography on silica gel (eluting with 50% EtOAc in PE) to give 441 -C (200 mg, 47% yield) as a yellow solid. LC / MS (ESI): m / z=320.2 [M+1]+RT=1.537 min.

[0301] Step-4: To a solution of 441 -C (200 mg, 0.63 mmol) in methanol (25 mL) was added a solution of NaOH (320 mg, 4.0 mmol) in water (5.0 mL), and the mixture was stirred at room temperature overnight. The reaction mixture was neutralized with 2N HCI to pH=7.0 and concentrated in vacuo to dryness to give a residue. The residue (441 -D) was used for the next step without purification. LC / MS (ESI): m / z=292.1 [M+1]+RT=1.16 min.

[0302] Step-5: To a solution of 441 -D (obtained in last step, -0.63 mmol) and 3,5- dimethylpiperidine (75 mg, 0.66 mmol) in DMF (10 mL) was added EDCI.HCI (300 mg, 1.50 mmol), HOBt (200 mg, 1.50 mmol) and triethylamine (303 mg, 3.0 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (100 mL) and then extracted with EtOAc (2 x 50 mL). The organic extract was concentrated in vacuo to give a residue. The residue was purified by reversed phase Prep-HPLC to give Compound 5 (27 mg, 11.1% yield for two steps) as a white solid.1H NMR (400 MHz, CDsOD): 8.55 (d, J=2.0 Hz, 1 H), 7.78 (d, J=8.8 Hz, 1 H), 7.46 (dd, J=8.8 Hz, 2.0 Hz, 1 H), 7.26 (s, 1 H), 4.58-4.51 (m, 2H), 2.79 (s, 3H), 2.62-2.56 (m, 1 H), 2.32- 2.26 (m, 1 H), 1.96-1.68 (m, 3H), 1.00-0.87 (m, 7H). LC / MS (ESI): m / z=387.2 [M+1]+RT=8.969 min.

[0303] Scheme 6: Experimental for the preparation of Compound 6 1005445281

[0304] Experimental procedure for Scheme 6:

[0305] Step-1 : To a solution of 2-bromo-1 ,3-thiazole-4-carboxylic acid (511-A, 500 mg, 2.41 mmol) and 2,6-dimethylmorpholine (397-C, 555 mg, 4.83 mmol) in DMF (10 mL) was added EDCI (925 mg, 4.82 mmol), HOBT (650 mg, 4.82 mmol) and Et3N (730 mg, 7.23 mmol), and the reaction mixture was stirred at room temperature overnight. After consumption of the starting material, the reaction mixture was diluted by water (10 mL) and extracted with EtOAc (2 x 15 mL). The organic extract was washed by water and brine successively, dried over anhydrous Na2SC>4, filtered and evaporated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) to give 511-B (400 mg, 54% yield) as a yellow solid. LC / MS (ESI): m / z=305.2 [M+1]+RT=0.584 min.

[0306] Step-2: A mixture of 511-B (400 mg, 1.31 mmol), 217-B (194 mg, 1.31 mmol), Pd2(dba)3 (241 mg, 0.26 mmol), Xantphos (300 mg, 0.52 mmol), CS2CO3 (1.27 g, 3.90 mmol) in 1 ,4-dioxane (10 mL) was stirred at 100 °C for 1 h under argon atmosphere. After cooling to room temperature, it was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 50% EA in PE) to give Compound 2 (389 mg, 80% yield) as a yellow solid. LC / MS (ESI): m / z=372.3 [M+1]+RT=1.51 min.

[0307] Step-3: A solution of Compound 2 (200 mg, 0.53 mmol) and NCS (57 mg, 0.43 mmol) in THF (8 mL) was stirred at 40 °C overnight. After consumption of the starting material, the reaction mixture was diluted by water (10 mL) and extracted with EtOAc (2 x 15 mL). The organic extract was washed by water (10 mL) and brine (10 mL) successively, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 50% EtOAc in PE) and prep-HPLC to give Compound 6 (86 mg, 40% yield) as a white solid.1H NMR (400 MHz, CD3OD): 5 8.17 (d, J=2.0 Hz, 1 H), 7.50 (d, J=8.8 Hz, 1 H), 7.23 (dd, J=8.8, 2.0 Hz, 1 H), 4.50-4.45 (m, 1 H), 3.81-3.73 (m, 1 H), 3.71- 3.65 (m, 2H), 2.91 (dd, J=13.2, 10.4 Hz, 1 H), 2.62-2.56 (m, 4H), 1.24 (d, J=6.4 Hz, 3H), 1.12 (d, J=6.0 Hz, 3H). LC / MS (ESI): m / z=407.1 [M+1]+RT=7.255 min. 1005445281

[0308] Scheme 7: Experimental for the preparation of Compound 7

[0309] Experimental procedure for Scheme 7:

[0310] Step-1 : A solution of 2-amino-5-nitrophenol (217, 1 g, 6.49 mmol) and 1 ,1 ,1- trimethoxy-2-methylpropane (1.92 g, 13 mmol) was stirred at 100 °C for 4 h. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 20% EtOAc in PE) to give 563-R-1 (1 .2 g, 74.6% yield) as a brown solid. LC / MS (ESI): m / z=207.3 [M+1]+RT=0.871 min.

[0311] Step-2: To a solution of 563-R-1 (800 mg, 10 mmol) in MeOH (25 mL) and H2O (5 mL) was added Fe powder (1 g, 19.4 mmol) and NH4CI (1 g, 19.6 mmol), the reaction mixture was heated to reflux and stirred for 2 h. After consumption of the starting material, the reaction mixture was filtered through a celite pad and evaporated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluting with 30% EtOAc in PE) to give 563-R (700 mg, 100% yield) as a light yellow solid. LC / MS (ESI): m / z=177.3 [M+1 ]+RT=0.638 min.

[0312] Step-3: To a solution of 2-bromo-5-methylthiazole-4-carboxylic acid (563-S, 222 mg, 1.0 mmol) and 397-C (143 mg, 1.20 mmol) in DMF (15 mL) was added EDCI.HCI (287 mg, 1.50 mmol), HOBt (202 mg, 1.50 mmol) and EtsN (303 mg, 3.0 mmol), and the reaction mixture was allowed to stir at room temperature overnight. After all the raw materials were consumed, the reaction mixture was diluted by water (30 mL) and extracted with EtOAc (3 x 20 mL). The organic extract was washed by water (4 x 30 mL) and brine (30 mL) successively, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure and purified by column chromatography on silica gel (eluting with 0%~55% EtOAc in PE) to give 511-1 (212 mg, 66.5% yield) as a colorless oil. LC / MS (ESI): m / z=319.2 [M+H]+RT=0.615 min. 1005445281

[0313] Step-4: To a mixture of 511-1 (200 mg, 0.8 mmol), 563-R (166 mg, 0.94 mmol) and CS2CO3 (780 mg, 2.4 mmol) in 1 ,4-dioxane (10 mL) was added Pd2(dba)3 (146 mg, 0.16 mmol) and Xantphos (184 mg, 0.32 mmol), the reaction mixture was allowed to heat at 100 °C and stir for 3 h under nitrogen atmosphere. After cooling to room temperature, it was diluted by water (50 mL) and extracted with EtOAc (2 x 50 mL). The organic extract was washed by brine (50 mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure to give a residue. The residue was purified by prep-HPLC to give Compound 7 (21 mg, 16.4% yield) as a light yellow solid.1H NMR (400 MHz, CD3OD) 5 8.24 (d, J=2.0 Hz, 1 H), 7.50 (d, J=8.4 Hz, 1 H), 7.22 (d, J=8.4, 2.0 Hz, 1 H), 4.48 (d, J=13.2 Hz, 1 H), 4.08 (d, J=13.2 Hz, 1 H), 3.81-3.76 (m, 1 H), 3.72-3.68 (m, 1 H), 3.28-3.21 (m, 1 H), 2.85 (dd, J=12.8, 10.4 Hz, 1 H), 2.57 (dd, J=12.8, 10.8 Hz, 1 H), 2.39 (s, 3H), 1.44 (d, J=6.8 Hz, 6H), 1.25 (d, J=6.0 Hz, 3H), 1.13 (d, J=6.4 Hz, 3H). LC / MS (ESI): m / z=415.3 [M+1]+RT=9.053 min.

[0314] Scheme 8: Experimental for the preparation of Compound 8

[0315] Experimental procedure for Scheme 8:

[0316] Step-1 : A suspension of 511-B (250 mg, 0.82 mmol), 639-S (146 mg, 0.98 mmol), CS2CO3 (401 mg, 1.23 mmol), Brettphos-Pd-Gs (74 mg, 0.082 mmol) and Xphos (39 mg, 0.082 mmol) in 1 ,4-dioxane (5 mL) was stirred at 100 °C overnight under Ar. atmosphere. After the raw materials were consumed, the reaction mixture was filtered through a celite pad to remove the solid and the filtrate was evaporated under reduced pressure and purified by column chromatography on silica gel (eluting with 9% EtOAc in PE) to give 639-1 (90 mg, 28.3% yield) as a yellow solid. LC / MS (ESI): m / z=389.2 [M+1]+RT=1.598 min.

[0317] Step-2: To a solution of 639-1 (70 mg, 0.18 mmol) in dry THF (10 mL) was added NCS (24 mg, 0.28 mmol) and the reaction mixture was allowed to stir at 40 °C overnight. After consumption of the starting material, the reaction mixture evaporated under reduced 1005445281 pressure and purified by reversed phase Prep-HPLC to give Compound 8 (6 mg, 7.9% yield) as a white solid.1H NMR (400 MHz, DMSO-d6): 5 10.71 (brs, 1 H), 8.35 (d, J=2.4 Hz, 1 H), 7.85 (d, J=8.8 Hz, 1 H), 7.44 (dd, J=8.8, 2.4 Hz, 1 H), 4.34 (d, J = 12.8 Hz, 1 H), 3.72 (d, J=12.8 Hz, 1 H), 3.63-3.57 (m, 2H), 2.86-2.80 (m, 1 H), 2.76 (s, 3H), 2.54-2.52 (m, 1 H), 1.16 (d, J=6.0 Hz, 3H), 1.06 (d, J=6.4 Hz, 3H). LC / MS (ESI): m / z = 423.0 [M+1]+RT =8.705 min.

[0318] Scheme 9: Experimental for the preparation of Compound 9

[0319] Experimental procedure for Scheme 9:

[0320] Step-1 : A mixture of Compound 2 (50 mg, 0.13 mmol) and NBS (26 mg, 0.15 mmol) in THF (10 mL) was stirred at room temperature for 1 h. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure and purified by reversed phase prep-HPLC to give Compound 9 (20 mg, 33.3% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) 5 10.70 (s, 1 H), 8.09 (d, J=2.0 Hz, 1 H), 7.58 (d, J = 8.4 Hz, 1 H), 7.26 (dd, J=8.8, 2.0 Hz, 1 H), 4.35 (d, J=12.8 Hz, 1 H), 3.65-3.54 (m, 3H), 2.82 (dd, J=13.6, 11.2 Hz, 1 H), 2.57-2.54 (m, 4H), 1.16 (d, J=6.0 Hz, 3H), 1.05 (d, J=6.0 Hz, 3H). LC / MS (ESI): m / z=451.0 [M+1]+RT=8.336 min.

[0321] Scheme 10: Experimental for the preparation of Compound 10 1005445281

[0322] Experimental procedure for Scheme 10:

[0323] Step-1 : To a solution of 217 (500 mg, 3.24 mmol) in ethanol (5 mL) was added triethyl orthopropionate (857 mg, 4.87 mmol) and NH4CI (26 mg, 0.49 mmol). The reaction mixture was stirred at 80 °C overnight. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to give crude 647-1 (500 mg, 80% yield) as white solid, which was used for the next step without purification. LC / MS (ESI): m / z=193.3 [M+H]+RT=1.722 min.

[0324] Step-2: To a solution of 647-1 (500 mg, 2.60 mmol) in methanol (10 mL) was added PtC>2 (250 mg). The reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. After all the raw materials are consumed, the reaction mixture was filtered through a celite pad and the filtrate was evaporated under reduced pressure and purified by column chromatography on silica gel (eluting with 0%~50% EtOAc in PE) to give 647-2 (350 mg, 83% yield) as a white solid. LC / MS (ESI): m / z=163.3 [M+H]+RT=1.367 min.

[0325] Step-3: A suspension of 647-2 (200 mg, 1.23 mmol), 511-B (416 mg, 1.36 mmol), CS2CO3 (805 mg, 2.47 mmol), BrettPhos-Pd-Gs (112 mg, 0.12 mmol) and Xphos (59 mg, 0.12 mmol) in 1 ,4-dioxane (10 mL) was heated to 80 °C and stirred overnight under nitrogen atmosphere. After the raw materials are consumed, the reaction mixture was filtered through a celite pad to remove the solid and the filtrate was evaporated under reduced pressure and purified by reversed phase prep-HPLC to give Compound 10 (195 mg, 41% yield) as a white solid.1H NMR (400 MHz, CD3OD) 5 8.26 (d, J=2.0 Hz, 1 H), 7.51 (d, J=8.4 Hz, 1 H), 7.36 (s, 1 H), 7.25 (dd, J=8.4, 2.0 Hz, 1 H), 4.73 (d, J=12.4 Hz, 1 H), 4.46 (d, J=12.4 Hz, 1 H), 3.80-3.69 (m, 2H), 2.96 (q, J=7.6 Hz, 1 H), 2.90-2.84 (m, 1 H), 2.59-2.54 (m, 1 H), 1 .42 (t, J=7.6 Hz, 3H), 1.27-1.19 (m, 6H). LC / MS (ESI): m / z=387.2 [M+1]+R =8.199 min.

[0326] Scheme 11 : Experimental for the preparation of Compound 11 1005445281

[0327] Experimental procedure for Scheme 11 :

[0328] Step-1 : To a solution of Compound 10 (60 mg, 0.15 mmol) in THF (2 mL) was added NCS (20 mg, 0.15 mmol). The reaction mixture was heated to 50 °C and stirred overnight under nitrogen atmosphere. After all the raw materials are consumed, the reaction mixture was evaporated under reduced pressure and purified by reversed phase prep-HPLC to give Compound 11 (25.66 mg, 41% yield) as a white solid.1H NMR (400 MHz, CD3OD) 5 8.18 (d, J=2.0 Hz, 1 H), 7.52 (d, J=8.4 Hz, 1 H), 7.23 (dd, J=8.4, 2.0 Hz, 1 H), 4.50-4.46 (m, 1 H), 3.82-3.70 (m, 3H), 2.99-2.89 (m, 3H), 2.60 (dd, J=13.2, 10.8 Hz, 1 H), 1.42 (t, J=7.6 Hz, 3H), 1.25 (d, J = 6.4 Hz, 3H), 1 .13 (d, J=6.4 Hz, 3H). LC / MS (ESI): m / z=421.0 [M+1]+RT=8.903 min.

[0329] Scheme 12: Experimental for the preparation of Compound 12

[0330] Experimental procedure for Scheme 12:

[0331] Step-1 : To a solution of 650-0 (2.0 g, 10.61 mmol) in ethanol (20 mL) was added triethyl orthoacetate (2.6 g, 15.92 mmol) and NH4CI (86 mg, 1.59 mmol). The reaction mixture was stirred at 100 °C overnight. After consumption of the starting material, the reaction mixture was evaporated under reduced pressure to give crude 647-1 (1.20 g, 53% yield) as a white solid, which was used for the next step without purification. LC / MS (ESI): m / z=213.1 [M+H]+RT=1.743 min.

[0332] Step-2: To a solution of 650-1 (1.20 g, 5.64 mmol) in methanol (15 mL) was added PtC>2 (500 mg). The reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. After the starting materials are consumed, the reaction mixture 1005445281 was filtered through a celite pad and the filtrate was evaporated under reduced pressure to give crude 647-2 (800 mg, 78% yield) as a brown solid, which was used for the next step without purification. LC / MS (ESI): m / z=183.2 [M+H]+RT=1.473 min.

[0333] Step-3: A suspension of 650-2 (400 mg, 2.19 mmol), 511-B (740 mg, 2.42 mmol), CS2CO3 (1.4 g, 4.40 mmol), BrettPhos-Pd-Gs (200 mg, 0.22 mmol) and Xphos (105 mg, 0.22 mmol) in 1 ,4-dioxane (20 mL) was heated to 100 °C and stirred overnight under nitrogen atmosphere overnight. After all the raw materials are consumed, the reaction mixture was filtered through a celite pad to remove the solid and the filtrate was evaporated under reduced pressure and purified by reversed phase prep-HPLC to give 650-3 (60 mg, 6.7% yield) as a white solid. LC / MS (ESI): m / z=407.0 [M+H]+RT=8.301 min.

[0334] Step-4: To a solution of 650-3 (40 mg, 0.10 mmol) in THF (2 mL) was added NCS (13 mg, 0.10 mmol). The reaction mixture was heated to 58 °C and stirred for 2 days under nitrogen atmosphere. After all the raw materials are consumed, the reaction mixture evaporated under reduced pressure and purified by reversed phase prep-HPLC to give Compound 12 (15.14 mg, 35% yield) as a white solid.1H NMR (400 MHz, CD3OD) 5 8.54 (s, 1 H), 7.69 (s, 1 H), 4.48-4.44 (m, 1 H), 3.78-3.63 (m, 3H), 2.89 (dd, J=13.2, 10.4 Hz, 1 H), 2.62 (s, 3H), 2.58 (dd, J=13.2, 10.8 Hz, 1 H), 1.23 (d, J=6.4 Hz, 3H), 1.11 (d, J=6.0 Hz, 3H). LC / MS (ESI): m / z=441.0 [M+1]+RT=8.990 min.

[0335] Biological Activity

[0336] The compounds of the invention inhibit TRPC3, TRPC6, TRPC7 ion channels, tubulin polymerization, or combinations thereof. The compounds may therefore be useful for the prevention or treatment of cancers that are responsive to the inhibition of TRPC3, or TRPC6, or TRPC7 ion channel activity, or tubulin polymerization inhibition, or combinations thereof. The biological activity of compounds of Formula I can be determined using any suitable assay for determining the activity of a candidate compound as a TRPC3, or TRPC6, or TRPC7 inhibitor, or tubulin polymerization inhibitor, or combinations thereof, as well as tissue and in vivo models.

[0337] Biological Example 1: TRPC3, TRPC6, TRPC7 inhibition

[0338] The biological activity of compounds of Formula I were assessed and confirmed to have inhibitory effects by the FLIPR assay. Compounds were screened for activity on 1005445281

[0339] TRPC6 ion channel (Test 1), followed by screening for TRPC3, TRPC6 and TRPC7 (Test 2).

[0340] To assess activity on TRPC6 ion channels, Test 1 was undertaken with carbochol as the agonist in H EK-293 cells stably expressing the human TRPC6 channel (hTRPC6) were seeded at a rate of 15,000 cells / well onto 384 well plate in culture media (Dulbecco's Modified Eagle Medium, 10% Fetal Bovine Serum and 1% G418). Following 16 - 24 hours of seeding, the media was removed, and cells were incubated with a membrane potential sensitive dye (FLIPR® Membrane Potential Assay Kit (blue), Molecular Devices) in assay buffer (Hank's Balanced Salt Solution, 20 mM HEPES, pH 7.4) for 30 mins at 37 °C, followed by addition of serial dilutions of the compounds (0.5% DMSO) for 15 mins at room temperature. The agonist carbachol was added next at ECso concentration and the change in fluorescence dye was captured using the FTIRPTETRAsystem. The ICso values for each compound are presented in Table 1.

[0341] To assess activity on TRPC3, TRPC 7 and reassess activity on TRPC6, in Test 2 compounds were screened for their ability to block TRPC3, TRPC6 and TRPC7 channels in a membrane potential assay. The experiments were performed using H EK-293 stably transfected with either hTRPC3 or hTRPC6 or hTRPC7. 50,000 cells / well were seeded onto 96 well plates in culture media. Following 24 hours of seeding, the media was removed, and cells were incubated with a membrane potential sensitive dye (FLIPR® Membrane Potential Assay Kit (blue), Molecular Devices) in assay buffer (Hank's Balanced Salt Solution, 20 mM HEPES, pH 7.4) for 1 h at 37 °C. Serial dilutions of the compounds (0.5% DMSO) were added to the wells and incubated for 15 minutes at room temperature, followed by the addition of the agonist carbachol at ECso. Change in fluorescence dye was captured using the FLIRP-TETRA system (Molecular Devices). The ICso values for a selected proportion of compounds are presented in Table 1 .

[0342] Both tests confirmed the compounds of the invention have good inhibitory effects on their intended targets.

[0343] Table 1 : Results of the TRPC3, TRPC6 and TRPC7 ion channel assay 1005445281

[0344] Biological Example 2: Cell cytotoxicity assay

[0345] A cell death assay was conducted to assess the cytotoxicity properties of the compounds in a tumor-derived non-small lung cancer NCI-H1299 cell line. The lower the ICso, the more potent the compound is against killing cancer cells. H1299 cells were seeded at a density of 1 ,000 cells / well in a 384-well plate. Twenty-four hours later, cells were treated with test compound for 72 hours in 4-fold serial dilutions with a top test concentration of 4 pM. After the incubation, CellTiter-Glo reagent was added directly to the wells. The plate was shaken for 2 or 5 mins on a plate shaker and then incubated at room temperature for a further 10 mins. Subsequently, 100 pL of the above solution was transferred to a white flat bottom opaque 96 well plate (or opaque-walled clear bottom plates taped with a white bottom seal) and luminescence was recorded on a plate reader.

[0346] Compound 6 was one of the most efficacious compounds described in this invention with an ICso of 48 nM. The ICso values of the compounds of this invention are demonstrated in Table 2. Table 2 : Cytotoxicity of compounds of this invention on lung cancer cell line H1299 1005445281

[0347] Compounds of the invention (Compounds 1-12) showed good cytotoxicity (ICso <1 pM) against H1299 cells.

[0348] Biological Example 3: Tubulin polymerization assay

[0349] Compound 6 was selected to assess the activity on tubulin polymerization in vitro using a fluorescence-based Tubulin Polymerization Assay kit from Cytoskeleton (Catalogue # BK011 P). Compound 6, paclitaxel and vinblastine sulfate were first prepared as 2 mM stock solutions in DMSO prior to dilution in water to 33 pM. Porcine tubulin (2 mg / mL) was mixed with reaction buffer and GTP (80 mM PIPES pH 6.9, 2.0 mM MgCI2, 0.5 mM EGTA, 1.0 mM GTP and 15% glycerol) before immediately placing the reaction mix on ice to preclude premature polymerization. Five pL of each 33 pM preparation of Compound 6, paclitaxel or vinblastine sulfate was added to the wells of an assay plate pre-warmed to 37 °C. The reaction mix (50 pL) was added to each well of the assay plate before transfer of the plate to a temperature-controlled fluorimeter. Fluorescence was measured (Aex and Aem = 360 and 450 nm, respectively) at regular intervals for 60 min at 37 °C. Similar to vinblastine sulfate, an established tubulin polymerization inhibitor, Compound 6 (3 pM) suppressed tubulin polymerization by an average of 82.3% relative to the DMSO vehicle during the 1020 - 3000 s sectional of the 6000 s time course (Figure 1).

[0350] Biological Example 4: Assessing the activity of Compound 6 on the proliferation of multiple tumor-derived cell lines.

[0351] The cytotoxic activity of Compound 6 was assessed on multiple cell lines. Cells were seeded in their optimal medium in 384-multi well cell culture-treated plates. Following overnight incubation at 37 °C, the cells were treated with either Compound 6 1005445281 at concentrations ranging 10 pM - 3 nM with 3-fold dilution intervals or 0.1 % DMSO dispensed using a Nanodrop Tecan D300e machine for 72 h at 37 °C at 5% CO2. Cell plates were then equilibrated to room temperature, and the viability of cells was determined using CellTiter-Glo luminescent reagent based on the amount of ATP present. The IC50S of Compound 6 against 80 cancer cell lines are described in Table 3.

[0352] Table 3 : Cytotoxicity of Compound 6 of this invention on multiple tumor derived cell lines 1005445281 1005445281

[0353] Biological Example 5: Scratch wound assays

[0354] Compound 6 was assessed for its ability to inhibit the invasion and migration of cancer cells.

[0355] Migration assay The wells of an Imagelock 96-well plate (Sartorius, Catalogue # BA-04857) were initially coated with 50 pL collagen I (0.05 mg / mL) and the plate was gently rocked to ensure an even coating of each well. The plate was placed in an incubator at 37 °C, 5% 1005445281

[0356] CO2for 60 min before the contents of each well were aspirated and then washed. MDA- MB-231 cells (100 pL per well at 40,000 cells per well) were seeded and then allowed to adhere overnight. The following day, the Imagelock 96-well plate was carefully removed from the incubator before the 96- Well Plate Woundmaker Tool (Sartorius, Catalogue # 4563) was used to simultaneously generate a wound in each of the wells. After wounding, the media from each well was immediately aspirated and the wells were gently washed with PBS. Finally, culture media with 1% FBS (100 pL) and 0 - 50 pM Compound 6 was added to each well before the plate was transferred to an Incucyte S3. The plate was allowed to warm at 37 °C for 30 min prior to scanning and image capture every 2 h for 36 h using the Incucyte S3. All scans were analyzed using the Incucyte S3’s % Wound Confluence function.

[0357] Invasion assay

[0358] The wells of an Imagelock 96-well plate (Sartorius, Catalogue # BA-04857) were initially coated with 50 pL collagen I (0.05 mg / mL) and the plate was gently rocked to ensure an even coating of each well. The plate was placed in an incubator at 37 °C, 5% CO2 for 60 min before the contents of each well were aspirated and then washed. MDA- MB-231 cells (100 pL per well at 40,000 cells per well) were seeded and then allowed to adhere overnight. The following day, the Imagelock 96-well plate was carefully removed from the incubator before the 96- Well Plate Woundmaker Tool (Sartorius, Catalogue # 4563) was used to simultaneously generate a wound in each of the wells. After wounding, the media from each well was immediately aspirated and the wells were gently washed with PBS. Culture media (100 pL) was then added to each well before cooling the plate to 4 °C for 5 min. The culture media was aspirated and replaced with 50% Matrigel (50 pL per well) and 0 - 50 pM Compound 6 as indicated. The Matrigel was allowed to polymerize by warming the plate at 37 °C for 30 min. Finally, culture media with 1 % FBS (100 pL) and 0 - 50 pM Compound 6 was added to each well before the plate was transferred to an Incucyte S3. The plate was allowed to warm at 37 °C for 30 min prior to scanning and image capture every 2 h for 36 h using the Incucyte S3. All scans were analyzed using the Incucyte S3’s % Wound Confluence function.

[0359] Compound 6 successfully decreased both the migration and invasion of MDA-MB- 231 breast cancer cells (Figure 2). 1005445281

[0360] Biological example 6: MDCK-MDR1 assay for CNS permeability and drug efflux via P-gp protein.

[0361] The MDCK-MDR1 assay was used to evaluate the absorption potential of the compounds. The MDCK-MDR1 cells were cultured in MEM supplemented with 10% FBS and 1% NEAA and seeded 4 days prior to the assay at 0.88x105 / well cell density and maintained in a humidified atmosphere of 5% CO2 at 37 °C. Twenty-four hours prior to the assay the media was replaced.

[0362] For permeability assessment, the culture plate containing the monolayer was washed with HBSS buffer before the transepithelial / transendothelial electrical resistance (TEER) was measured at room temperature. The transport experiment was started by adding 5 pM solution of test compounds in Hank’s Balanced Salt Solution containing luciferase yellow to the apical side (400 pL volume) or the basolateral side (800 pL volume). A sample from the apical chamber (A-B direction) or the basolateral chamber (B-A direction) was taken at t = 0 min. The apical and basolateral plates were incubated at 37 °C for 90 min. After 90 min, the apical plate was separated from the basolateral plate and an aliquot from the basolateral side (A-B direction) or the apical side (B-A direction) was transferred to an opaque plate for the determination of lucifer yellow concentration at t = 90 min. The luciferase yellow concentration of each sample was measured by fluorimetry using excitation and emission wavelengths of 485 nm and 535 nm, respectively. Samples from the donor and receiver chambers were diluted with 0.3% DMSO in Hank’s Balanced Salt Solution and then mixed with ACN and either osalmid or imipramine, which were used as an IS in preparation for analysis by LC-MS / MS. Samples were then assayed for their levels of test compounds using an LC / MS / MS instrument operating in a positive electrospray ionization mode.

[0363] The permeability of Compound 6 was 46.2 x 10-6cm s-1suggestive of high permeability and an efflux ratio of 0.89. An efflux ratio of less than 2 suggests that the compound is not susceptible to efflux via the P-gp protein.

[0364] Biological Example 7: Pharmacokinetics (PK) study

[0365] The pharmacokinetics of Compound 6 was assessed following continuous intravenous (IV), oral (PO) and intraperitoneal (IP) dosing. Mice (C57BL / 6, males, 18 - 20 g) aged 6 - 8 weeks were administered with a single dose of 4.2 mg / kg of Compound 1005445281

[0366] 6 (5 mL / kg) via a tail vein injection, oral gavage or IP injection (formulated in 5% DMAC, 5% Solutol HS and 90% saline). Serial blood samples were drawn via the facial vein into pre-cooled K2EDTA tubes, followed by transcardial perfusion and extraction of fresh brain tissues at 0.083, 0.25, 0.5, 1 , and 2 hours (n = 3 per dose). The blood samples were put on ice and centrifuged to obtain plasma samples (2,000 g, 5 mins, 4 °C) within 15 mins of sample collection.

[0367] The mean plasma and brain concentrations and PK parameters for Compound 6 following single IV, PO and IP dose are described in Table 4 and Figure 3. The half-life of Compound 6 following IV injection was 13 minutes and, the clearance was 3,950 mL / h / kg.

[0368] Table 4: Mean plasma concentrations of Compound 6 in the mouse following a single 4.2 mg / kg intravenous dose.

[0369] To assess brain levels, a separate cohort of mice (C57BL / 6, males, 18 - 20 g) aged 6 - 8 weeks were injected with Compound 6 via tail vein injection (4.2 mg / kg formulated in 5% DMAC + 5% Solutol HS + 90% Saline in water). Blood samples were drawn via the facial vein into pre-cooled K2EDTA tubes at 1 h, and the brain tissue was collected at 1 h following perfusion with saline.

[0370] The plasma and diluted brain samples (1 in 4 dilution in PBS) were mixed with internal standard (diclofenac, 50 ng / mL) in acetonitrile. The mixture was centrifuged (5,800 rpm) and analyzed using the LC-MS / MS technique.

[0371] The plasma to brain ratio was 0.31 at 15 mins post-injection suggesting that Compound 6 readily crossed the blood-brain barrier critical for target engagement in the brain tissue (Table 5). 1005445281

[0372] Table 5: Plasma and brain concentrations of Compound 6 following single IV injection at 4.2 mg / kg in male mice.

[0373] The pharmacokinetics of Compound 6 was assessed following IP dosing. Male mice (C57BL / 6, males, 23 - 25 g) aged 6 - 8 weeks were injected with two doses of Compound 6 at 20 mg / kg, 8 hours apart via IP injection. The plasma and diluted brain samples (1 in 4 dilution in PBS) were mixed with internal standard (diclofenac, 50 ng / mL) in acetonitrile. The mixture was centrifuged (5,800 rpm) and analyzed using the LC- MS / MS technique.

[0374] The plasma and brain levels of Compound 6 are described in Table 6 and Figure 4 with the average brain to plasma ratio being 0.53 at Cmax.

[0375] Table 6: Plasma and brain concentrations of Compound 6 following IP dosing (20 mg / kg, twice a day) in male mice.

[0376] Biological Example 8 : Evaluation of the Efficacy of Compound 6 in NCI- H1299 Human Non-Small Cell Lung Cancer Xenograft Model in BALB / c nude Mice The objective of this study was to evaluate the anti-tumor efficacy of Compound 6 in the NCI-H1299 xenograft model in female BALB / c nude mice. 1005445281

[0377] Animals

[0378] This study utilized female BALB / c nude female mice (Supplier: Beijing Anikeeper Biotech Co., Ltd.), aged 6 to 8 weeks weighing 20 - 25 g with all procedures for animal care and housing were in accordance with the standard, Commission on Life Sciences, National Research Council, Standard operating procedures (SOPs) of Pharmaron, Inc. The mice were kept in laminar flow rooms at constant temperature and humidity with 5 mice in each cage. Animals were housed in polycarbonate cages, sized 300 x 180 x 150 mm3and in an environmentally monitored, well-ventilated room maintained at a temperature of (22 ± 3 °C) and a relative humidity of 40% - 80%. Fluorescent lighting provided illumination for approximately 12 hours per day. The bedding material was soft wood, which was changed once per week and food and water were provided ad libitum. Water samples were periodically analyzed to ensure that no known contaminants were present that could interfere with or affect the outcome of studies.

[0379] Cell Culture

[0380] The NCI-H1299 tumor cell line was maintained in vitro as a monolayer culture in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 in air. The tumor cells were routinely sub-cultured, not to exceed 4-5 passages. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

[0381] Method for Tumor Inoculation and Randomization

[0382] Each mouse was subsequently inoculated subcutaneously on the right flank with NCI-H1299 tumor cells (5 x 106) in 100 pL of medium / Matrigel mixtures (1 :1) for tumor development. Treatment was started when the mean tumor size reached 147 mm3. Mice were dosed with vehicle (5%DMAC + 5%Solutol HS-15 + 90%Saline) or Compound 6 (10 mice / group) twice daily at 25 mg / kg IP spaced 8 h apart until the conclusion of the study. The treatments were administered to the tumor-bearing mice according to the study design shown in Table 7. 1005445281

[0383] Table 7: Study design to assess the efficacy of Compound 6.

[0384] Measurement Parameters

[0385] For routine monitoring, all study animals were monitored not only for tumor growth but also behavior such as mobility, food and water consumption (by cage side checking only), body weight (BW), eye / hair matting and any other abnormal effect. Any mortality and / or abnormal clinical signs were recorded.

[0386] Body Weight

[0387] The body weights of all animals were measured twice a week throughout the study. The measurement dates were specified in the study design. Body weight change, expressed in %, was calculated using the following formula:

[0388] BW change (%) = (BWDay X / BWDay 0) x 100, where BWDay X is BW on a given day, and BWDay 0 is BWon Day zero (initiation of treatment). Table 8 contains the mean body weight measurements from the animals treated with vehicle or Compound 6. No significant differences were seen in body weights in mice treated with vehicle or drug suggesting that Compound 6 doesn’t worsen animal health (Figure 5).

[0389] Table 8: Mean Body Weight (n=10) following administration of vehicle or Compound 6 (25 mg / kg twice per day). 1005445281

[0390] Tumor Measurements

[0391] The measurement of tumor size was conducted twice a week with a caliper and the tumor volume (TV, in mm3) was estimated using the formula: TV = a x b2 / 2 throughout the study, where “a” and “b” are the long and short diameters of a tumor, respectively. The TVs were used for calculation of the tumor growth inhibition (TGI, an indicator of antitumor effectiveness) value using the formula: TGI = (1-T / C) x 100%, where “T” and “C” are the mean relative volumes (% tumor growth) of the tumors in the treated and the control groups, respectively.

[0392] The animals were terminated for sample collection and survival curve when the individual tumor volume exceeded 2,000 mm3.

[0393] Table 9: Difference in mean tumor volumes (n=10), 20 days post-tumor inoculation

[0394] The mean tumor volume was 27% lower in the mice treated with 25 mg / kg Compound 6 twice per day compared to mice treated with vehicle alone (Figure 6).

[0395] Biological Example 9: Comparative examples of cytotoxicity and TRPC3 / 6 / 7 blocking of compounds not of this invention

[0396] Tumor cytotoxicity was tested according to Test 3, where a cell death assay was conducted to assess the cytotoxicity properties of the compounds in a tumor derived nonsmall lung cancer NCI-H1299 cell line. The lower the ICso, the more potent the compound is against killing cancer cells. H1299 cells were seeded at a density of 1 ,000 cells / well in a 384-well plate. Twenty-four hours later, cells were treated with the test compound for 72 hours in 4-fold serial dilutions with a top test concentration of 4 pM. After the incubation, the CellTiter-Glo reagent was added directly to the wells. The plate was shaken for 2 or 5 mins on a plate shaker and then incubated at room temperature for a further 10 mins. Subsequently, 100 pL of the above solution was transferred to a white 1005445281 flat bottom opaque 96 well plate (or opaque-walled clear bottom plates taped with a white bottom seal) and luminescence was recorded on a plate reader.

[0397] TRPC6 channel activity was tested according to Test 1 , where HEK-293 cells stably expressing the human TRPC6 channel (hTRPC6) were seeded at a rate of 15,000 cells / well onto 384 well plates in culture media (Dulbecco's Modified Eagle Medium, 10% Fetal Bovine Serum and 1 % G418). Following 16 - 24 hours of seeding, the media was removed, and cells were incubated with a membrane potential sensitive dye (FLIPR® Membrane Potential Assay Kit (blue), Molecular Devices) in assay buffer (Hank's Balanced Salt Solution, 20 mM HEPES, pH 7.4) for 30 mins at 37 °C, followed by addition of serial dilutions of the compounds (0.5% DMSO) for 15 mins at room temperature. The agonist carbachol was added next at ECso concentration and the change in fluorescence dye was captured using the FTIRPTETRAsystem. The ICso values for each compound are presented in Table 10.

[0398] TRPC3, TRPC6, TRPC7 channel activity was tested using Test 2, where compounds were screened for their ability to block TRPC3, TRPC6 and TRPC7 ionchannels in a membrane potential assay. The experiments were performed using HEK- 293 stably transfected with either hTRPC3 or hTRPC6 or hTRPC7. 50,000 cells / well were seeded onto 96 well plate in culture media. Following 24 hours of seeding, the media was removed, and cells were incubated with a membrane potential sensitive dye (FLIPR® Membrane Potential Assay Kit (blue), Molecular Devices) in assay buffer (Hank's Balanced Salt Solution, 20 mM HEPES, pH 7.4) for 1 h at 37 °C. Serial dilutions of the compounds were added (0.5% DMSO) to the wells and incubated for 15 minutes at room temperature, followed by addition of the agonist carbachol at ECso. Change in fluorescence dye was captured using the FLIRP-TETRA system (Molecular Devices). The ICso values for a selected proportion of compounds are presented in Table 10.

[0399] Table 10: Cytotoxicity on a tumor-derived cell line and TRPC3, TRPC6 and TRPC7 ion channel blocking efficacy of comparative compounds not of the invention. 1005445281

[0400] Examples EX1 , EX2 and EX3 contain 1,3-benzodioxole instead the 2-substituted 1,3-benzoxazole / thiazole of the compounds of the invention. They display moderate to high TRPC3 / 6 / 7 ion-channel blocking activity but lack cancer cell cytotoxicity. The differences show that 2-substituted 1,3-benzoxazole / thiazole is required for suitable cancer cell cytotoxicity properties whilst maintaining TRPC ion-channel blocking activity. 1005445281

[0401] Examples EX4 and EX5 contain 2-halo 1 ,3-benzothiazole or methylhalo 1 ,3- benzoxazole, respectively, instead of the 2-alkyl 1 ,3-benzoxazole / thiazole of the compounds of the invention. They display moderate TRPC6 ion-channel blocking activity but lack cancer cell cytotoxicity. The differences show that 2-alkyl 1 ,3- benzoxazole / thiazole is required for suitable tumor cytotoxicity properties whilst maintaining TRPC ion-channel blocking activity.

[0402] Example EX6 contains 1 ,3- benzoxazole without substitution instead of the 2- substituted 1 ,3-benzoxazole / thiazole of the compounds of the invention. It displays moderate TRPC6 ion-channel blocking activity but lacks cancer cell cytotoxicity. The differences show that 2-substituted 1 ,3-benzoxazole / thiazole is required for suitable cancer cell cytotoxicity properties whilst maintaining TRPC ion-channel blocking activity.

[0403] Examples EX7 and EX8 contain thiadiazole and oxazole, respectively, instead of the thiazole of the compounds of the invention. They display moderate to poor TRPC6 ion-channel blocking activity but lack cancer cell cytotoxicity. The differences show that the thiazole is required for suitable cancer cell cytotoxicity properties whilst maintaining TRPC ion-channel blocking activity.

[0404] Example EX9 contains 5-substitution of the 1 ,3- benzoxazole instead of the 6- substitution of the 1 ,3-benzoxazole / thiazole of the compounds of the invention. It displays moderate TRPC6 ion-channel blocking activity and lacks cancer cell cytotoxicity. The differences show that the 1 ,3-benzoxazole / thiazole is required to be the 6-substitution configuration for suitable activity against the TRPC ion-channels and cancer cell cytotoxicity.

[0405] Biological Example 10 : Evaluation of the Anti-Tumor Efficacy in Human T98G Glioblastoma Cancer Xenograft Model in BALB / c Nude Mice

[0406] The objective of this study is to evaluate the anti-tumor efficacy of compounds of the invention in the T98G xenograft model in female BALB / c nude mice. The example will be conducted with Compound 6.

[0407] Animals

[0408] This study will utilize female BALB / C nude mice (Supplier: Beijing Anikeeper

[0409] Biotech Co., Ltd.), aged 6 to 8 weeks weighing 20 - 25 g. The mice will be kept in laminar 1005445281 flow rooms at constant temperature and humidity with 5 mice in each cage. Animals will be housed in polycarbonate cage, sized 300 x 180 x 150 mm3and in an environmentally monitored, well-ventilated room maintained at a temperature of (22 °C ± 3°C) and a relative humidity of 40% - 80%. Fluorescent lighting provided illumination approximately 12 hours per day with food and water provided ad libitum.

[0410] Cell Culture

[0411] T98G cell lines will be maintained in vitro as monolayer culture in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 in air. The tumor cells will then be routinely sub-cultured for no more than 4 - 5 passages. The cells growing in an exponential growth phase will be harvested and counted for tumor inoculation.

[0412] Method for Tumor Inoculation and Randomization

[0413] Each mouse will subsequently be inoculated subcutaneously on the right flank with T98G tumor cells for tumor development. Treatment will initiate when the mean tumor size reaches 147 mm3. Mice will be administered with vehicle (5% DMAC + 5% Solutol HS-15 + 90% Saline) or 25 mg / kg Compound 6 (10 mice / group) thrice per day via intraperitoneal (IP) injections 8 hours apart.

[0414] Measurement Parameters

[0415] For routine monitoring, all study animals will be monitored for tumor growth, behavior such as mobility, food and water consumption (by cage side checking only), body weight (BW), eye / hair matting and any other abnormal effect. Any mortality and / or abnormal clinical signs will be recorded.

[0416] Body Weight

[0417] The body weights of all animals will be measured twice a week throughout the study. Body weight change, expressed in %, was calculated using the following formula:

[0418] BW change (%) = (BWDay X / BWDay 0) x 100, where BWDay X is BW on a given day, and BWDay 0 is BW on Day zero (initiation of treatment). The mean body weight measurements from the animals treated with vehicle or Compound 6 will be measured. 1005445281

[0419] Tumor Measurements

[0420] The measurement of tumor size will be conducted twice a week with a caliper and the tumor volume (mm3) will be estimated using the formula: TV = a * b2 / 2 throughout the study, where “a” and “b” are the long and short diameters of a tumor, respectively. The TVs were used for calculation of the tumor growth inhibition (TGI, an indicator of antitumor effectiveness) value using the formula: TGI = (1-T / C) * 100%, where “T” and “C” are the mean relative volumes (% tumor growth) of the tumors in the treated and the control groups, respectively.

[0421] The animals will be terminated for sample collection and survival curve when the individual tumor volume exceeds 2,000 mm3.

[0422] The mean tumor volume will be compared between mice treated with vehicle only and Compound 6. It is expected that mice treated with Compound 6 will have smaller tumor volumes compared to mice treated with the vehicle.

[0423] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

1. 1005445281CLAIMS1. A compound of Formula I or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:Formula I wherein:R1is a bicyclic systemthe dashed line ( ) represents an optional double bond (=) or single bond ( — ); wherein when X and CR4are connected by a double bond, Y and CR4are connected by a single bond, and when X and CR4are connected by a single bond, Y and CR4are connected by a double bond;A1, A2and A3are independently selected from N and CR5; when present R5is independently selected from H, D, halo, Ci-C4alkyl, hydroxy, amino, Ci-C4alkoxy, Ci-C4fluoroalkyl, phenyl, cyano, Ci-C4thioalkyl, and Ci-C4alkylamino;X is selected from CH, CD, CH2, CHD, CD2, S, and O;Y is selected from CH, CD, CH2, CHD, CD2, S, and N;R4is selected from CH3, CD3, CH2D, CHD2, Ci-Cealkyl, hydroxy, amino, Ci-Cealkoxy, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino;R2is selected from H, D, halo, cyano, Ci-Cethioalkyl, Ci-Cealkylamino, and Ci-Cealkyl; and1005445281R3is:where R6and R7form an optionally substituted ring; the ring formed by R6and R7and the N atom between them comprises 3-9 ring atoms.

2. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim 1 wherein R3is selected from1005445281, and3. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim 1 or 2 wherein R1is:

4. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of any one of claims 1 or 2 wherein R1is selected from5. The compound, pharmaceutically acceptable salt, solvate or stereoisomer any one of claims 1 to 3 wherein R3is 1 -piperidine, optionally substituted with 1-4 substituents, wherein the substituents are independently selected from D, halo, Ci-Cealkyl, Ci- Cealkoxy, Ci-Cefluoroalkyl, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino.10054452816. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim 5 wherein R3is 1 -piperidine substituted with 1-2 substituents.

7. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of any one of claims 1 to 5 wherein R3is 1 -piperidine substituted with 1-2 Ci-Cealkyl groups.

8. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim 7 wherein R3is substituted with 1-2 methyl groups.

9. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim 8 wherein R3is substituted with 2,2-; 2,3-; 2,5-; 3,3-; 3,5-; or 2,6-dimethyl.

10. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of any one of claims 1 to 7 wherein R3is 3,5-dimethylpiperidine.

11. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of any one of claims 1 to 4 wherein R3is 4-morpholine substituted with 1-4 substituents, preferably 2-3 substituents, wherein the substituents are independently selected from D, halo, Ci-Cealkyl, Ci-Cealkoxy, Ci-Cefluoroalkyl, phenyl, cyano, Ci-Cethioalkyl, and Ci- Cealkylamino.

12. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of any one of claims 1 to 4, or 11 wherein R3is 4-morpholine substituted with 1-2 Ci-Cealkyl groups.

13. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim12 wherein R3is substituted with 1-2 methyl groups.

14. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim13 wherein R3is substituted with 2,2-; 2,3-; 2,5-; 3,3-; 3,5-; or 2,6-dimethyl.

15. The compound, pharmaceutically acceptable salt, solvate or stereoisomer of claim14 wherein R3is 2,6-dimethylmorpholine.

16. A compound according to Formula II, or pharmaceutically acceptable salt, solvate or stereoisomer thereof1005445281Formula II wherein:A1, A2and A3are independently selected from N and CR12; when present each R12is independently selected from H, D, halo, Ci-C4alkyl, hydroxy, amino, Ci-C4alkoxy, Ci-C4fluoroalkyl, phenyl, cyano, Ci-C4thioalkyl, and Ci- C4alkylamino;X is selected from O and S;R8is selected from CH3, CD3, CH2D, CHD2, Ci-Cealkyl, hydroxy, amino, Ci-Cealkoxy, phenyl, cyano, Ci-Cethioalkyl, and Ci-Cealkylamino;R9is selected from H, D, halo, cyano, Ci-Cethioalkyl, Ci-Cealkylamino, and Ci-Cealkyl; andR10and R11together with the N atom between them form a ring; the ring formed by R10and R11comprises 3-9 ring atoms.

17. A compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof according to any one of claims 1 or 16, selected from the group consisting of:100544528118. A compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof having the structure:

19. A pharmaceutical composition comprising a compound according to any one of claims 1-18, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, and a pharmaceutically acceptable excipient.

20. A method of treating cancer or cancer metastases, comprising administering to the individual in need thereof a therapeutically effective amount of a compound according to any one of claims 1-18, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition of claim 19.100544528121. Use of a compound according to any one of claims 1-18, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in the manufacture of a medicament for treating cancer or cancer metastases in an individual in need thereof.

22. A method according to claim 20 or use according to claim 21, wherein the cancer or cancer metastases is responsive to the inhibition of one or more of TRPC3, TRPC6 or TRPC7 ion channel activity, tubulin polymerization inhibition or a combination thereof.

23. A method according to claim 20 or use according to claim 21, wherein the cancer or cancer metastases is responsive to tubulin polymerization inhibition.

24. A method according to claim 20 or use according to claim 21, wherein the cancer or cancer metastases is responsive to the inhibition of TRPC3, TRPC6, and TRPC7 ion channels.

25. A method of inhibiting TRPC3, TRPC6, TRPC7 ion channel activity, or a combination thereof, comprising administering to a cell, tissue or an individual in need thereof, an effective amount of a compound according to any one of claims 1-18, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition of claim 19.

26. A method of inhibiting tubulin polymerization, comprising administering to a cell, tissue or an individual in need thereof, an effective amount of a compound according to any one of claims 1-18, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition of claim 19.

27. A method of inhibiting TRPC3, TRPC6 or TRPC7 ion channel activity, or, tubulin polymerization, or a combination thereof, comprising administering to a cell, tissue or an individual in need thereof, an effective amount of a compound according to any one of claims 1-18, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or the pharmaceutical composition of claim 19.

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