Fused triazolopyrimidine compounds having useful pharmaceutical applications

Abstract

A compound and / or pharmaceutically acceptable salt thereof has the following Formula A. These compounds can be PIKfyve kinase inhibitors.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of US Provisional Application No. 62 / 601,501, filed on March 24, 2017, which is incorporated herein in its entirety. Technical Field

[0003] This disclosure relates to compounds and / or pharmaceutically acceptable salts thereof that can be PIKfyve kinase inhibitors and are useful for the treatment of diseases such as cancer and autoimmune diseases. Background Technology

[0004] PIKfyve generates phosphoinositol kinase 3,5-bisphosphate phosphatidylinositol (PtdIns(3,5)P2) by phosphorylating PtdIns(3)P at the 5-position of the inositol ring.

[0005] PIKfyve kinase is a mammalian orthologue of yeast Fab1 and was first discovered in mammalian cells (Shisheva et al., "Cloning, characterization, and expression of a novel Zn"). 2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells”Mol.Cell.Biol.19(1),pp.623-34,1999). Subsequently, the cDNA and protein of human PYKfyve were cloned and characterized (Cabezas et al., “Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate”). 5-kinase, PIKfyve / Fab1., Gene, 371(1), pp.34-41, 2006). The human PIKfyve gene is located at locus 2q34 on chromosome 2. The protein comprises four major domains: 1) PtdIns(3) P-binding FYVE domain (amino acid residues 150 to 219), 2) membrane-binding DEP domain (residues 365 to 440), 3) chaperone-like domain (residues 559 to 1064), and 4) catalytic phosphatidylinositol kinase homologous domain (residues 1791 to 2085). The intracellular localization of the PIKfyve protein is primarily limited to the membranes of late and early endosomes. Biochemically, PIKfyve exhibits a strong preference for phosphatidylinositol (PtdIns) compared to phosphatidylinositol (PI) substrates and produces PtdIns 5-P and PtdIns 5-P. Two products of 3,5-P2 (Sbrissa et al., “A mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin”, J. Biol. Chem., 274(31), pp. 21589-97, 1999).

[0006] PtdIns 3,5-P2 produced by PIKfyve is essential for maintaining the integrity of the late endocytic membrane (Ikonomov et al., “Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity”, J. Biol. Chem., 277(11), pp. 9206-11, 2002). In addition to PtdIns, PIKfyve has also been reported to possess protein kinase activity and can undergo autophosphorylation (Sbrissa et al., “PIKfyve lipid kinase is a protein kinase: downregulation of 5'-phosphoinositide product formation by autophosphorylation”, Biochemistry, 39(51), pp. 15980-9, 2000).

[0007] The PIKfyve signaling pathway is known to regulate a variety of biological processes primarily through its well-established role in endosomal transport. A key aspect of PIKfyve biology is its involvement in Toll-like receptor signaling, a crucial component of the cellular innate immune system. Therefore, recent studies using the small molecule compound apimod to inhibit IL-12 / IL-23 secretion in response to TLR agonists have attributed this to the compound's ability to inhibit the PtdIns kinase activity of PIKfyve (Cai et al., “PIKfyve, a class III PI kinase, is the target of the small molecular IL-12 / IL-23 inhibitor apilimod and a player in Toll-like receptor signaling,” Chem. Biol., 20(7), pp. 912-921, 2013). Note that apimod is also being investigated as a drug in clinical trials in patients with Crohn's disease or rheumatoid arthritis.

[0008] Dysregulated IL12 / IL23 cytokine production is involved in the pathology of various inflammatory diseases, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and multiple sclerosis. Recent studies have demonstrated that APY0201, another small molecule inhibitor targeting IL12 / IL23 production, is a highly selective inhibitor of PIKfyve (Hayakawa et al., “Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12 / 23 inhibitor, APY0201”, Bioorg. Med. Chem., 22(11), pp. 3021-9, 2014). Furthermore, two novel small molecule inhibitors, AS2677131 and AS2795440, targeting IL-12 production induced by mouse macrophages, have also shown selective inhibition of PIKfyve kinase (Terajima et al., “Inhibition of c-Rel DNA binding is critical for the anti-inflammatory effects of novel PIKfyve inhibitor”, Eur. J. Pharmacol., 780, pp. 93-105, 2016). AS2677131 has also been shown to prevent the development of rheumatoid arthritis in laboratory animals.

[0009] PIKfyve also represents a pharmacological target in cancer. Due to its involvement in cytosolic vacuolation and lysosomal fusion reactions necessary for autophagy and macropeptidoma degradation, inhibition of PIKfyve can lead to the obstruction of lysosomal-dependent nutrient production pathways operating in some cancer types (Kim et al., “Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways”, J. Clin. Invest., 126(11), pp. 4088-4102, 2016). Recent studies have demonstrated that PIKfyve (via PtdIns 5-Ps) can regulate cancer cell migration and invasion by activating the Rho family GTPase Rac1 (Dupuis-Coronas et al., “The nucleophosmin-anaplastic lymphoma kinase oncogene interacts, activates, and uses the kinase PIKfyve to increase invasiveness,” J. Biol. Chem., 286(37), pp. 32105-14, 2011; Oppelt et al., “PIKfyve, MTMR3 and their product PtdIns5P regulate cancer cell migration and invasion through activation of Rac1,” Biochem. J., 461(3), pp. 383-90, 2014). The small molecule inhibitor YM201636 of PIKfyve strongly inhibits cancer cell migration in in vitro models (Oppelt et al.). The antiproliferative effects of apimod observed in several cancer cell lines further support the role of PIKfyve inhibition in anticancer therapy.PIKfyve inhibitors exhibit selective nanomolar-level cytotoxicity in B-cell non-Hodgkin lymphoma but not in normal cells (Gayle et al., “Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma”, Blood, doi: 10.1182 / blood-2016-09-736892, 2017).

[0010] Scientific research collectively supports the selection of PIKfyve as a therapeutic target for pharmacological intervention in several disease conditions, including cancer and autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, and multiple sclerosis. Therefore, those skilled in the art should consider the applicability of the small molecule compounds described in this invention to be applicable to, but not limited to, the aforementioned diseases. Summary of the Invention

[0011] In one aspect, 2,5,7-trisubstituted -[1,2,4]triazolo[1,5-a]pyrimidines, such as compounds of formula I and / or pharmaceutically acceptable salts thereof, are provided:

[0012]

[0013] in

[0014] R1 can be an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted heterocyclic group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, provided that R1 is not cyclohexyl.

[0015] R2 is an alkyl, aryl, heteroaryl, -N=CH-alkyl, -N=CH-aryl, or -N=CH-heteroaryl group, wherein the alkyl, aryl, and heteroaryl groups are optionally substituted.

[0016] R3 and R4 are independently H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, or optionally substituted heterocyclic, provided that R1 is not C when R3 and R4 are such. 1-3 Alkyl groups; or R3 and R4 together with the nitrogen to which they are attached to form optionally substituted heterocyclic groups.

[0017] Compounds of Formula I and / or their pharmaceutically acceptable salts can inhibit PIKfyve kinase.

[0018] On the other hand, a pharmaceutical composition comprising a compound of formula I and / or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier is provided.

[0019] On the other hand, a method is provided for treating an individual suffering from a disease treatable by inhibiting PIKfyve kinase. The method comprises administering to the individual in need a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula I and / or a pharmaceutically acceptable salt thereof, wherein such administration reduces or eliminates symptoms associated with said disease. Attached Figure Description

[0020] Figure 1A-1D The PIKfyve inhibitor was shown to selectively inhibit the growth of cancer cell lines.

[0021] Figure 2A-2B Both APY0201 and compound 1 of this disclosure show that they block the secretion of IL-23.

[0022] Figures 3A-3C APY0201, apimod, and compound 1 of this disclosure induce apoptosis in the ML-2 cancer cell line.

[0023] Figure 4 The dose-response curves of apimod, APY 0201, compound 1 of this disclosure and YM201636 in the PIKfyve kinase inhibition assay are shown.

[0024] Figures 5A-5C The dose-response curves of apimod, APY 0201, compound 1 of this disclosure and YM201636 are shown for different hematologic cancer cell lines and in normal human peripheral blood mononuclear cells (PBMCs). Detailed Implementation

[0025] As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators.

[0026] When a part is a cyclic ring, the term "n-membered" is used to describe the number of ring atoms in the cyclic ring. For example, a 4-membered cycloalkyl group refers to a cycloalkyl group such as cyclobutane that has 4 ring atoms.

[0027] As used alone or in combination herein, the term "alkyl" refers to a straight-chain or branched hydrocarbon containing 1 to 20 carbon atoms, linked only by single bonds and without any cyclic structure. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, and eicosyl. The term "lower alkyl" refers to a straight-chain or branched hydrocarbon containing 1 to 6 carbon atoms, linked only by single bonds and without any cyclic structure, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or pentyl.

[0028] As used alone or in combination herein, the term "aryl" refers to a monocyclic, bicyclic (fused), or tricyclic (fused or spirocyclic) hydrocarbon ring system having a total of 5 to 14 ring atoms. When the aryl group is monocyclic, the monocyclic ring is aromatic and does not contain heteroatoms. When the aryl group is bicyclic or tricyclic, at least one of the bicyclic or tricyclic rings is aromatic and does not contain heteroatoms, and when the other rings are aromatic, the other rings do not contain heteroatoms; however, when the other rings are not aromatic, the other rings may or may not contain heteroatoms. Connection points can be on any ring atom. Examples of aryl groups include, but are not limited to, benzene, naphthalene, indane, 1,2,3,4-tetrahydronaphthalene, benzodihydropyran, isobenzodihydropyran, 1,2,3,4-tetrahydroquinoline, thiobenzodihydropyran 1,1-dioxide, 6,7,8,9-tetrahydro-5H-benzo[7]annulene, and 2,3-dihydrobenzofuran.

[0029] As used alone or in combination herein, the term "cycloalkyl" refers to a monocyclic, bicyclic (fused, bridged, or spirocyclic), or tricyclic (fused or spirocyclic) hydrocarbon ring system having a total of 3 to 14 ring atoms, which is fully saturated or contains one or more unsaturated units, but the individual rings in the monocyclic, bicyclic, or tricyclic hydrocarbon are not aromatic, and the ring atoms are not heteroatoms. The bonding points can be on saturated or unsaturated carbons. A bridged bicyclic cycloalkyl refers to two hydrocarbon rings sharing three or more carbon atoms, separated by a bridge containing at least one atom at the two bridgehead carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, bicyclo[2.2.2]octane, bicyclo[2.2.1]heptane, spiro[2.5]octane, spiro[3.5]nonane, spiro[4.5]decane, and spiro[5.5]undecane.

[0030] As used alone or in combination herein, the term "heterocyclic group" refers to a monocyclic, bicyclic (fused, bridged, or spirocyclic), or tricyclic (fused or spirocyclic) hydrocarbon ring system having 4 to 15 ring atoms, which is fully saturated or contains more than one unsaturated unit, but none of the individual rings in the monocyclic, bicyclic, or tricyclic hydrocarbon is aromatic, and further, at least one of the ring atoms is a heteroatom. A bridged bicyclic heterocyclic group is a bridged bicyclic cycloalkyl group in which at least one carbon atom is replaced by a heteroatom. Examples of heterocyclic groups include, but are not limited to, aziridine, oxadiazine, pyrrolidine, piperidine, morpholine, and tetrahydrofuran. The bonding point can be on a saturated or unsaturated carbon atom or a heteroatom.

[0031] As used alone or in combination herein, the term "heteroaryl" refers to a monocyclic, bicyclic (fused), and tricyclic (fused or spirocyclic) ring system having a total of 5 to 14 ring atoms, wherein at least one ring in the monocyclic, bicyclic, and tricyclic ring systems is aromatic and contains at least one heteroatom selected from S, O, and N. The bonding point can be on any ring atom. Examples of heteroaryl groups include, but are not limited to, furan, thiophene, pyridine, pyrimidine, indole, benzofuran, 4,5,6,7-tetrahydrobenzofuran, 4,5,6,7-tetrahydrobenzo[b]thiophene, and 4,5,6,7-tetrahydro-1H-indole.

[0032] As used alone or in combination herein, the term “optionally substituted alkyl” or a term having the same function refers to an unsubstituted alkyl (or an unsubstituted lower alkyl) or an alkyl group substituted by one, two, or three groups selected from the group consisting of: CN, halogen, -NRR, -NHSO2R, -C(O)NRR, -OR, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), and heteroaryl (e.g., monocyclic heteroaryl), wherein R is independently H, alkyl, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), or heteroaryl (e.g., monocyclic heteroaryl). Similarly, the term "optionally substituted lower alkyl" refers to an unsubstituted lower alkyl or a lower alkyl substituted by one, two or three groups selected from the same group above.

[0033] As used alone or in combination herein, the term “optionally substituted aryl” or a term having the same function refers to an unsubstituted aryl group or an aryl group substituted by one, two, or three groups selected from the group consisting of: alkyl, CN, halogen, -NRR, -NHSO2R, -C(O)NRR, -OR, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), and heteroaryl (e.g., monocyclic heteroaryl), wherein R is independently H, alkyl, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), or heteroaryl (e.g., monocyclic heteroaryl). Similarly, the term "optionally substituted phenyl" refers to an unsubstituted phenyl or a phenyl substituted by one, two, or three groups selected from the same group of groups mentioned above.

[0034] As used alone or in combination herein, the term "optionally substituted heteroaryl" or a term having the same function refers to an unsubstituted heteroaryl or a heteroaryl substituted by one, two, or three groups selected from the group consisting of: alkyl, CN, halogen, -NRR, -NHSO2R, -OR, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), and heteroaryl (e.g., monocyclic heteroaryl), wherein R is independently H, alkyl, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), or heteroaryl (e.g., monocyclic heteroaryl). Similarly, the term "optionally substituted monocyclic heteroaryl" refers to an unsubstituted monocyclic heteroaryl or a monocyclic heteroaryl substituted by one, two, or three groups selected from the same group of groups mentioned above.

[0035] As used alone or in combination herein, the term “optionally substituted cycloalkyl” or a term having the same effect refers to an unsubstituted cycloalkyl group or a cycloalkyl group substituted by one, two, or three groups selected from the group consisting of: alkyl, CN, halogen, -NRR, -NHSO2R, -OR, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), and heteroaryl (e.g., monocyclic heteroaryl), wherein R is independently H, alkyl, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocycloalkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), or heteroaryl (e.g., monocyclic heteroaryl).

[0036] As used alone or in combination herein, the term "optionally substituted heterocyclic group" or a term having the same function refers to an unsubstituted heterocyclic alkyl group or a heterocyclic alkyl group substituted by one, two, or three groups selected from the following: alkyl, CN, halogen, -NRR, -NHSO2R, -OR, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocyclic alkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), and heteroaryl (e.g., monocyclic heteroaryl), wherein R is independently H, alkyl, aryl (e.g., phenyl), cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), heterocyclic alkyl (e.g., aziridine, oxetane, pyrrolidine, piperidine, piperazine, morpholine, and tetrahydrofuran), or heteroaryl (e.g., monocyclic heteroaryl). Similarly, the term "optionally substituted monocyclic heterocyclic group" refers to an unsubstituted monocyclic heterocyclic group or a monocyclic heterocyclic group substituted by one, two or three groups selected from the same group above.

[0037] In this specification, the terms "individual" and "mammal" are used interchangeably. Both refer to humans or animals.

[0038] The compounds disclosed in this specification may exist in the form of pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a non-toxic acidic / anionic or basic / cationic salt form of the compounds disclosed in this specification. Suitable pharmaceutically acceptable salts include acid addition salts, which can be formed, for example, by mixing a solution of the compounds disclosed in this specification with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid.

[0039] Furthermore, when the compounds disclosed in this specification carry an acidic moiety, their suitable pharmaceutically acceptable salts may include: alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and salts formed with suitable organic ligands, such as quaternary ammonium salts. Therefore, representative pharmaceutically acceptable salts include, but are not limited to, acetates, aspirin salts, benzenesulfonates, benzoates, besylates, bicarbonates, bisulfates, tartrates, borates, bromides, calcium, camsylates (or camphorsulphonates), carbonates, chlorides, citrates, clavulanates, dihydrochlorides, ethylenediaminetetraacetate, ethylenedisulfonate, esylate, formate, fumarate, gluconate, glucuronide, glucuronide, glutamate, hexafluorophosphate, hymenate ( Hibenzate, hydrabamine, hydrobromide, hydrobromide, hydrochloride, hydroiodide, iodide, hydroxyethyl sulfonate, isothionate, lactate, malate, maleate, malonate, mandelate, methanesulfonate, methyl sulfate, nitrate, naphthylcarbamate, 2-naphthylsulfonate, nicotinate, nitrate, oleate, orotate, oxalate, dihydroxynaphthyl salt, palmitate, phosphate / bisphosphate / hydrogen phosphate, sucrose, salicylate, stearate, sulfate, succinate, tartrate, toluenesulfonate, and trifluoroacetate. See “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermauth (Wiley-VCH, Weinberg, Germany, 2002).

[0040] The compounds disclosed in this specification may exist in unsolvated or solvated forms. As used herein, the term 'solvate' describes a molecular complex comprising a compound disclosed in this specification and one or more pharmaceutically acceptable solvent molecules such as water, ethanol, DMSO, or other organic solvents. When a compound disclosed in this specification forms a solvate with water, the term 'hydrate' may be used instead of 'solvate'. Pharmaceutically acceptable solvates include hydrates and solvates, wherein the solvent may be substituted with an isotope, such as D2O, d6-acetone, or d6-DMSO.

[0041] In a first aspect, this disclosure relates to compounds of formula I or pharmaceutically acceptable salts thereof:

[0042]

[0043] in

[0044] R1 can be an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted heterocyclic group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, provided that R1 is not cyclohexyl.

[0045] R2 is an alkyl, aryl, heteroaryl, -N=CH-alkyl, -N=CH-aryl, or -N=CH-heteroaryl group, wherein the alkyl, aryl, and heteroaryl groups are optionally substituted.

[0046] R3 and R4 are independently H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, or optionally substituted heterocyclic, provided that R1 is not C when R3 and R4 are such. 1-3 Alkyl groups; or R3 and R4 together with the nitrogen to which they are attached to form optionally substituted heterocyclic groups.

[0047] In some embodiments, R1 is an optionally substituted phenyl group. In some embodiments, R1 is an optionally substituted lower alkyl group. In some embodiments, R1 is an optionally substituted monocyclic heteroaryl group. In some embodiments, R1 is an optionally substituted monocyclic heterocyclic group.

[0048] In some embodiments, R1 is a phenyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments, R1 is a pyridyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments, R1 is a pyrimidinyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments, R1 is a quinolinyl or isoquinolinyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl and lower alkyl groups.

[0049] In some embodiments, R1 is a lower alkyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl groups. In some embodiments, R1 is an azirrobutyl, oxetane, tetrahydrofuran, or pyrrolidinyl group, each optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -OCF3, -O-lower alkyl, and lower alkyl groups.

[0050] In some embodiments, R2 is –N=CH-aryl, –N=CH-heteroaryl, or –N=CH-alkyl, wherein the aryl, heteroaryl, and alkyl groups are each optionally substituted by one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl. In some embodiments, R2 is –N=CH-phenyl, -N=CH-naphthyl, -N=CH-pyridyl, -N=CH-indolyl, or -N=CH-lower alkyl, wherein the phenyl, naphthyl, pyridyl, indolyl, and lower alkyl are each optionally substituted by one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl groups.

[0051] In some embodiments, R3 and R4 are independently lower alkyl groups optionally substituted with one or two groups selected from CF3, OH, CN, NH2, -OCF3, and -O- lower alkyl groups. In some embodiments, R3 and R4, together with the nitrogen to which they are attached, form a monocyclic heterocyclic group, a bicyclic heterocyclic group, or a bicyclic aryl group, each optionally substituted with one or two groups selected from lower alkyl groups. The monocyclic heterocyclic group, bicyclic heterocyclic group, or bicyclic aryl group may contain additional heteroatoms selected from N, O, and S. In some embodiments, the monocyclic heterocyclic group is a 4-membered, 5-membered, 6-membered, or 7-membered heterocyclic group. Examples of monocyclic heterocyclic groups include aziridine, aziridine, pyrrolidine, piperidine, morpholine, piperazine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-dioxide, aziridine, 1,4-oxazacycloheptane, and 1,4-thiaazacycloheptane.

[0052] In some implementations, R1 is in This indicates the connection point with the rest of the molecule.

[0053] In some implementations, R2 is in This indicates the connection point with the rest of the molecule.

[0054] In some embodiments, R3 and R4 are independently methyl, isopropyl, or 2-hydroxyethyl. In some embodiments, R3 and R4, together with the nitrogen to which they are attached, form one of the following rings: in This indicates the connection point with the rest of the molecule.

[0055] In some embodiments, the compound of formula I and / or its pharmaceutically acceptable salt is the compound of formula II and / or its pharmaceutically acceptable salt:

[0056]

[0057] in

[0058] R1 can be an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted heterocyclic group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, provided that R1 is not cyclohexyl.

[0059] R2 is an optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, N=CH-alkyl, N=CH-aryl, or N=CH-heteroaryl, wherein the alkyl, aryl, and heteroaryl groups may be optionally substituted.

[0060] R5, R6, R7, and R8 are independently H or methyl.

[0061] In some embodiments of Formula II, R1 is an optionally substituted phenyl group. In some embodiments of Formula II, R1 is an optionally substituted lower alkyl group. In some embodiments of Formula II, R1 is an optionally substituted monocyclic heteroaryl group. In some embodiments of Formula II, R1 is an optionally substituted monocyclic heterocyclic group.

[0062] In some embodiments of Formula II, R1 is a phenyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments of Formula II, R1 is a pyridyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments of Formula II, R1 is a pyrimidinyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments of Formula II, R1 is a quinolinyl or isoquinolinyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl and lower alkyl groups.

[0063] In some embodiments of Formula II, R1 is a lower alkyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl groups. In some embodiments of Formula II, R1 is an azirrobutyl, oxacyclobutyl, tetrahydrofuran, or pyrrolidinyl group, each optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -OCF3, -O-lower alkyl, and lower alkyl groups.

[0064] In some embodiments of Formula II, R2 is –N=CH-aryl, -N=CH-heteroaryl, or –N=CH-alkyl, wherein the aryl, heteroaryl, and alkyl groups are each optionally substituted by one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl. In some embodiments of Formula II, R2 is –N=CH-phenyl, -N=CH-naphthyl, -N=CH-pyridyl, -N=CH-indolyl, or -N=CH-lower alkyl, wherein the phenyl, naphthyl, pyridyl, indolyl, and lower alkyl are each optionally substituted by one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl.

[0065] In some implementations of Equation II, R1 is... in This indicates the connection point with the rest of the molecule.

[0066] In some implementations of Equation II, R2 is... in This indicates the connection point with the rest of the molecule.

[0067] In some embodiments, the compound of formula I and / or its pharmaceutically acceptable salt is the compound of formula III and / or its pharmaceutically acceptable salt:

[0068]

[0069] in

[0070] R1 can be an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted heterocyclic group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, provided that R1 is not cyclohexyl.

[0071] R9 can be an alkyl group that has been optionally substituted, an aryl group that has been optionally substituted, or a heteroaryl group that has been optionally substituted.

[0072] R3 and R4 are independently H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, or optionally substituted heterocyclic; or R3 and R4 together with the nitrogen to which they are attached form an optionally substituted heterocyclic group.

[0073] In some embodiments of Formula III, R1 is an optionally substituted phenyl group. In some embodiments, R1 is an optionally substituted lower alkyl group. In some embodiments of Formula III, R1 is an optionally substituted monocyclic heteroaryl group. In some embodiments of Formula III, R1 is an optionally substituted monocyclic heterocyclic group.

[0074] In some embodiments of Formula III, R1 is a phenyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments of Formula III, R1 is a pyridyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments of Formula III, R1 is a pyrimidinyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, and lower alkyl groups. In some embodiments of Formula III, R1 is a quinolinyl or isoquinolinyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl and lower alkyl groups.

[0075] In some embodiments of Formula III, R1 is a lower alkyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl groups. In some embodiments of Formula III, R1 is an azirrobutylalkyl, oxacyclobutylalkyl, tetrahydrofuran, or pyrrolidinyl group, each optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -OCF3, -O-lower alkyl, and lower alkyl groups.

[0076] In some embodiments of Formula III, R9 is an aryl, heteroaryl, or alkyl group, each optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl. In some embodiments of Formula III, R9 is a phenyl, naphthyl, pyridyl, indolyl, or lower alkyl group, each optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl.

[0077] In some embodiments of Formula III, R3 and R4 are independently lower alkyl groups optionally substituted with one or two groups selected from CF3, OH, CN, NH2, -OCF3, and -O- lower alkyl groups. In some embodiments of Formula III, R3 and R4 together with the nitrogen to which they are attached form a monocyclic heterocyclic group, a bicyclic heterocyclic group, or a bicyclic aryl group, each optionally substituted with one or two groups selected from lower alkyl groups. The monocyclic heterocyclic group, bicyclic heterocyclic group, or bicyclic aryl group may contain additional heteroatoms selected from N, O, and S. In some embodiments of Formula III, the monocyclic heterocyclic group is a 4-membered, 5-membered, 6-membered, or 7-membered heterocyclic group. Examples of monocyclic heterocyclic groups include aziridine, aziridine, pyrrolidine, piperidine, morpholine, piperazine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-dioxide, aziridine, 1,4-oxazacycloheptane, and 1,4-thiaazacycloheptane.

[0078] In some implementations of Equation III, R1 is... in This indicates the connection point with the rest of the molecule.

[0079] In some implementations of Equation III, R2 is in This indicates the connection point with the rest of the molecule.

[0080] In some embodiments of Formula III, R3 and R4 are independently methyl, isopropyl, or 2-hydroxyethyl. In some embodiments, R3 and R4, together with the nitrogen to which they are attached, form one of the following rings: in This indicates the connection point with the rest of the molecule.

[0081] Within the scope of this disclosure, unless otherwise specified, the embodiments of each of R1-R9 disclosed herein can be combined with each other in any way.

[0082] In some embodiments, the compounds of formula I and / or their pharmaceutically acceptable salts are selected from the following compounds:

[0083]

[0084] In a second aspect, this disclosure relates to pharmaceutical compositions comprising compounds of Formula I and / or pharmaceutically acceptable salts thereof, including various embodiments thereof, as disclosed in this specification. The pharmaceutical compositions may be administered to an individual, alone or in combination with other therapeutically active compounds, agents, drugs, or hormones. In addition to compounds of Formula I and / or pharmaceutically acceptable salts thereof, as disclosed in this specification, the pharmaceutical compositions may contain other known therapeutically effective agents for treating cancer or autoimmune diseases.

[0085] Pharmaceutical compositions can be manufactured using any of a wide variety of methods, including but not limited to conventional mixing, dissolving, granulation, dressing, grinding, emulsification, encapsulation, embedding, and lyophilization. Pharmaceutical compositions can take any of a wide variety of forms, including but not limited to sterile solutions, suspensions, emulsions, lyophilized products, tablets, pills, pellets, capsules, powders, syrups, elixirs, or any other dosage form suitable for administration.

[0086] The pharmaceutical composition may be provided in tablet or capsule form for oral administration, comprising about 1.0 to about 1000 mg of a compound of formula I and / or a pharmaceutically acceptable salt thereof (including various embodiments thereof), such as about 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of a compound of formula I and / or a pharmaceutically acceptable salt thereof (including various embodiments thereof).

[0087] Pharmaceutical compositions may further comprise pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" means any carrier that does not substantially have long-term or permanent harmful effects when administered and encompasses terms such as "pharmaceutically acceptable solvent, stabilizer, diluent, additive, adjuvant, or excipient." Typically, such carriers are mixed with or allowed to dilute or encapsulate the active compound, and said carriers may be solid, semi-solid, or liquid reagents. It should be understood that the active ingredient may be soluble or may be delivered as a suspension in a desired carrier or diluent.

[0088] A wide variety of pharmaceutically acceptable carriers can be used, including but not limited to: aqueous media, such as, for example, water, saline, glycine, and hyaluronic acid; solid carriers, such as, for example, starch, magnesium stearate, mannitol, sodium saccharin, talc, cellulose, glucose, sucrose, lactose, trehalose, and magnesium carbonate; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic agents and absorption delay agents; or any other inactive ingredient. The choice of a pharmaceutically acceptable carrier may depend on the route of administration. Unless any pharmaceutically acceptable carrier is incompatible with the active ingredient, it should be considered for use in a pharmaceutically acceptable composition. Non-limiting examples of the specific uses of such drug carriers can be found in *Pharmaceutical Dosage Forms and Drug Delivery Systems* (edited by Howard C. Ansel et al., Lippincott Williams & Wilkins Publishers, 7th ed., 1999), *Remington: The Science and Practice of Pharmacy* (edited by Alfonso R. Gennaro, Lippincott, Williams & Wilkins, 20th ed., 2000), *Goodman & Gilman's The Pharmacological Basis of Therapeutics* (edited by Joel G. Hardman et al., McGraw-Hill Professional, 10th ed., 2001), and *Handbook of Pharmaceutical Excipients* (Raymond C. Rowe et al., APhA Publications, 4th ed., 2003). These schemes are conventional, and any modifications are entirely within the scope of those skilled in the art and derived from the teachings herein.

[0089] In a third aspect, this disclosure relates to a method for treating an individual suffering from a disease treatable by inhibiting PIKfyve kinase, the method comprising administering to the individual in need a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula I and / or a pharmaceutically acceptable salt thereof (including various embodiments thereof), wherein such administration alleviates or eliminates symptoms associated with said disease.

[0090] The diseases mentioned include various forms of cancer and autoimmune diseases. For example, cancers include multiple myeloma, non-Hodgkin's lymphoma, T-cell lymphoma, and acute myelomonocytic leukemia. Autoimmune diseases include, for example, rheumatoid arthritis, inflammatory bowel disease, psoriasis, and multiple sclerosis.

[0091] The following examples are illustrative in nature and are in no way intended to be limiting.

[0092] Example

[0093] Example 1

[0094] As shown below, compounds of formula I can be prepared by methods known to those skilled in the art.

[0095] The reaction of carboxylic acids with aminoguanidines at high temperature and under acidic conditions forms 3-substituted-1H-1,2,4-triazol-5-amines.

[0096]

[0097] For example, benzoic acid reacts with aminoguanidine (Kurzer, F.; Godfrey, LEA Angewandte Chemie 75, (23) 1157-75 (1963)) to give 3-phenyl-1H-1,2,4-triazol-5-amine:

[0098]

[0099] Such aminotriazoles will react with malonic acid esters or halides under a wide variety of conditions to form 2-substituted-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol (Bioorganic & Medicinal Chemistry Letters 22,(9),3198–3202(2012)):

[0100]

[0101] Thus, 3-phenyl-1H-1,2,4-triazol-5-amine reacts with malonyl chloride to form 2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol:

[0102]

[0103] Using any of a variety of halogenating agents such as phosphoryl chloride, PBr5, thionyl chloride or oxalyl chloride, the two hydroxyl groups can be readily replaced by chlorine or other halogens (Bioorganic & Medicinal Chemistry Letters 22, (9), 3198–3202 (2012)).

[0104]

[0105] Therefore, 2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol readily reacts with phosphoric acid chloride to give 5,7-dichloro-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine:

[0106]

[0107] Under mild conditions, the 7-Cl group is selectively substituted with a secondary amine (US 8957064 B2):

[0108]

[0109] Therefore, 5,7-dichloro-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine reacts with morpholine to give 4-(5-chloro-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine:

[0110]

[0111] Then, under more stringent conditions, the 5-chloro group can be replaced with a strongly nucleophilic amine such as ammonia, methylamine, or hydrazine (JP 04099775 A(1992)):

[0112]

[0113] Thus, 4-(5-chloro-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine reacts with hydrazine hydrate at high temperature to provide 4-(5-hydrazino-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine:

[0114]

[0115] If the amine replacing the 5-chloro group is replaced with hydrazine, it can react with the most common aldehydes to form the corresponding imino compounds:

[0116]

[0117] Therefore, 4-(5-hydrazyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine reacts with aldehydes such as benzaldehyde to give (E)-4-(5-(2-benzylhydrazyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine:

[0118]

[0119] Example 2

[0120] Preparation of (E)-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 1)

[0121] Step 1: Preparation of 3-(pyridin-4-yl)-1H-1,2,4-triazol-5-amine

[0122]

[0123] A homogeneous mixture of isonicotinic acid (13.36 g, 108.5 mmol) and aminoguanidine hydrochloride (5.0 g, 45.2 mmol) in an open vial was heated at 230 °C for 1 h, at which point gas escaping ceased. The cooled residue was purified in water and by chromatography on an Amberlite CG-50–1 resin. Excess isonicotinic acid was removed by elution with water and further eluted with 0.5 M ammonium carbonate solution to give pure 3-(pyridin-4-yl)-1H-1,2,4-triazol-5-amine (5.97 g, 82% yield). [M+H] + =161.8.

[0124] Step 2: Preparation of 5,7-dichloro-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidine

[0125]

[0126] 3-(pyridin-4-yl)-1H-1,2,4-triazol-5-amine (5.0 g, 31.0 mmol) was dissolved in acetonitrile (125 mL), treated with malonyl chloride (4.37 g, 31.0 mmol), and stirred for 2.5 h under an inactive atmosphere, at which point another portion of malonyl chloride (2.18 g, 15 mmol) was added. After another 2 h of stirring, the reaction mixture was partitioned between water and ethyl acetate. The layers were separated, and the aqueous layer was extracted twice. The combined layers were dried and evaporated to dryness to give a crude residue, which was suspended in ice-cold phosphoryl chloride (50 mL). The mixture was heated under reflux for 5 h. The reaction mixture was cooled and most of the solvent was removed under reduced pressure. The residue was partitioned between dichloromethane and water, and the organic layer was dried and evaporated to leave the residue. The crude product was purified by Combi rapid chromatography using ethyl acetate–hexane to give pure 5,7-dichloro-2-(pyridin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidine (0.760 g, yield 9.2%). [M+H] + =265.9.

[0127] Step 3: Preparation of 4-(5-chloro-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine

[0128]

[0129] 5,7-Dichloro-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.520 g, 1.95 mmol) was dissolved in dioxane (10 mL) and treated with morpholine (0.340 g, 3.9 mmol). The reaction mixture was stirred at room temperature for 30 min, during which time the mixture was partitioned between dichloromethane and water. The layers were separated and the aqueous layer was extracted again with dichloromethane. The combined organic layers were dried and evaporated to dryness. The solid residue was ground with a small amount of methanol, filtered, and dried to give pure 4-(5-chloro-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidine-7-yl)morpholine (0.600 g, 97% yield). [M+H] + =316.8.

[0130] Step 4: Preparation of ((E)-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 1)

[0131]

[0132] 4-(5-chloro-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (0.600 g, 1.9 mmol) and hydrazine hydrate (1 mL) were suspended in ethanol (25 mL) in a sealed vial and heated in a microwave reactor at 150 °C for 10 min, followed by a further heating at 120 °C for 10 min. The cooled reaction mixture was partitioned between water and ethyl acetate. The aqueous phase was extracted twice with ethyl acetate, and the combined organic extracts were dried and evaporated to dryness. The crude residue ([M+H)) was... +=313.1) was suspended in methanol (10 mL), and acetic acid (10 μL) and 3-methylbenzaldehyde (0.228 g, 1.9 mmol) were added. The resulting mixture was stirred at room temperature for 30 min, at which point additional methanol (5 mL) and 3-methylbenzaldehyde (0.114 g) were added. After stirring at room temperature for another 60 min, the reaction was filtered, and the resulting solid (.597 g, 76% yield) was dissolved in pyridine and further purified by HPLC to give pure ((E)-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 1) [M+H] + =415.1.

[0133] Example 3

[0134] Preparation of (E)-2,2-dimethyl-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 2)

[0135]

[0136] Using the method described in Example 2, in step 3, 2,2-dimethylmorpholine was used instead of morpholine to prepare (E)-2,2-dimethyl-4-(5-(2-(3-methylbenzylhydrazino)hydrazyl)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine. [M+H] + =443.1.

[0137] Example 4

[0138] Preparation of (E)-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(pyridin-3-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine (compound 3)

[0139]

[0140] Using the method described in Example 2, (E)-4-(5-(2-(3-methylbenzylhydrazino)hydrazyl)-2-(pyridin-3-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine was prepared by replacing isonicotinic acid with nicotinic acid in step 1. [M+H] + =415.1

[0141] Example 5

[0142] Preparation of (E)-4-(5-(2-(3-methoxybenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 4)

[0143]

[0144] Using the method described in Example 2, in step 4, 3-methoxybenzaldehyde was used instead of 3-methylbenzaldehyde to prepare (E)-4-(5-(2-(3-methoxybenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]-triazolo[1,5-a]pyrimidin-7-yl)morpholine. [M+H] + =431.1.

[0145] Example 6

[0146] Preparation of (E)-N-(3-((2-(7-morpholino-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)hydrazine)methyl)phenyl)methanesulfonamide (compound 5)

[0147]

[0148] Using the method described in Example 2, in step 4, 2-phenylethylamine was used instead of hydrazine to prepare (E)-N-(3-((2-(7-morpholino-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)hydrazine)methyl)phenyl)methanesulfonamide. [M+H] + =494.1.

[0149] Example 7

[0150] Preparation of (E)-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(quinolin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine (compound 6)

[0151]

[0152] Using the method described in Example 2, (E)-4-(5-(2-(3-methylbenzyl)hydrazinoyl)-2-(quinolin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine was prepared by replacing isonicotinic acid with quinoline-4-carboxylic acid in step 1. [M+H] + =465.1.

[0153] Example 8

[0154] Preparation of (E)-4-(5-(2-(3-methylbenzyl)hydrazino)-2-(pyrimidin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine (compound 7)

[0155]

[0156] Using the method described in Example 2, (E)-4-(5-(2-(3-methylbenzylhydrazinoyl)hydrazyl)-2-(pyrimidin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine was prepared by replacing isonicotinic acid with pyrimidin-4-carboxylic acid in step 1. [M+H] + =416.1.

[0157] Example 9

[0158] Preparation of (E)-4-(5-(2-((1H-indol-3-yl)methylene)hydrazyl)-2-(pyridin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine (compound 8)

[0159]

[0160] Using the method described in Example 2, in step 4, indole-3-carboxaldehyde was used instead of 3-methylbenzaldehyde to prepare (E)-4-(5-(2-((1H-indol-3-yl)methylene)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine. [M+H] + =440.1.

[0161] Example 10

[0162] Preparation of (E)-4-(5-(2-(pyridin-3-ylmethylene)hydrazyl)-2-(pyridin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine (compound 9)

[0163]

[0164] Using the method described in Example 2, in step 4, pyridine-3-carboxaldehyde was used instead of 3-methylbenzaldehyde to prepare (E)-4-(5-(2-(pyridin-3-ylmethylene)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazol[1,5-a]pyrimidin-7-yl)morpholine. [M+H] + =402.1.

[0165] Example 11

[0166] Preparation of (E)-3-((2-(7-morpholino-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)hydrazine)methyl)benzyl nitrile (compound 10)

[0167]

[0168] Using the method described in Example 2, in step 4, 3-cyanobenzaldehyde is used instead of 3-methylbenzaldehyde to prepare (E)-3-((2-(7-morpholino-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)hydrazine)methyl)benzylnitrile. [M+H] + =426.1.

[0169] Example 12

[0170] Preparation of (E)-4-(5-(2-((6-methoxynaphthyl-2-yl)methylene)hydrazyl)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 11)

[0171]

[0172] Using the method described in Example 2, in step 4, 6-methoxy-2-naphthaldehyde was used instead of 3-methylbenzaldehyde to prepare (E)-4-(5-(2-((6-methoxynaphth-2-yl)methylene)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine. [M+H] + =481.1.

[0173] Example 13

[0174] Preparation of (E)-4-(5-(2-(3-isopropylbenzyl)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine (compound 12)

[0175]

[0176] Using the method described in Example 2, in step 4, 3-isopropylbenzaldehyde was substituted for 3-methylbenzaldehyde to prepare (E)-4-(5-(2-(3-isopropylbenzylene)hydrazino)-2-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)morpholine. [M+H] + =443.1.

[0177] Example 14

[0178] The PIKfyve inhibitor APY0201 selectively inhibits the growth of cancer cell lines.

[0179] The viability of several cancer cell lines was evaluated in the presence of two compounds: the PIKfyve inhibitor APY0201 and Dinaciclib (a CDK inhibitor, also known to those skilled in the art as an effective inhibitor of cancer cell growth). Three cancer cell lines (multiple myeloma KMS12E, non-Hodgkin's lymphoma SU-DHL4, and T-cell lymphoma Hut-78) and normal human peripheral blood mononuclear cells derived from healthy individuals were tested. Cells were plated in 384-well plates in RPMI medium supplemented with 10% fetal bovine serum. Cancer cells were plated at 1000 cells / well and normal cells at 10,000 cells / well, with a total volume of 30 μL / well. Immediately after plating, the test compound was added in duplicate at five concentrations: 10 μM, 1 μM, 0.01 μM, and 0.001 μM. Cells were exposed to the compound for 70 hours in a humidified incubator containing 5% CO2 at 37°C. Cell viability was determined using Presto Blue reagent (Thermo Scientific / Invitrogen). Dinaciclib inhibited the viability of all cell types with similar potency (IC50: 10 nM–15 nM). The PIKfyve inhibitor APY0201 effectively inhibited the viability of three cancer cell lines (IC50: 33 nM–46 nM); however, unlike Dinaciclib, it did not significantly inhibit the viability of normal PBMCs (IC50 > 10 μM), demonstrating a selectivity for cancer cells >100-fold higher than that for normal cells. See also Figure 1A-1D .

[0180] Similarly, the survival rates of cancer cell lines Hut-78 (T-cell lymphoma) and KMS12E (multiple myeloma) as well as normal human peripheral blood mononuclear cells were evaluated in the presence of apimod, APY0201, compound 1 of this disclosure (described in Example 2 of this disclosure), and YM201636. Apimod, APY0201, and compound 1 showed selectivity for cancer cells over normal cells.

[0181]

[0182] See also Figure 5A -C.

[0183] Example 15

[0184] Effects of the PIKfyve inhibitor APY0201 and compound 1 (described in Example 2 of this disclosure) on IL23 secretion in normal human peripheral monocytes stimulated with LPS

[0185] Human PBMCs were seeded in RPMI medium supplemented with 10% FBS at a density of 150,000 cells per well in 96-well plates. Cells were pre-incubated with the compound for 2 h. After pre-incubation, cells were stimulated with 100 ng / mL LPS for 18 h. Secreted IL-23 was measured by ELISA (Human IL-23 Quantikine ELISA Kit, R&D cat#D2300B). Conclusion: APY0201 and compound 1 (designated NSN22769) completely blocked LPS-induced IL-23 secretion from PBMCs. See also Figure 2A and 2B .

[0186] Example 16

[0187] The PIKfyve inhibitor and compound 1 (described in Example 2 of this disclosure) induced apoptosis in the ML-2 cancer cell line.

[0188] Acute myelomonocytic leukemia (AML) cells ML-2 were seeded in 96-well plates at a density of 50,000 cells per well in RPMI medium supplemented with 10% FBS. Cells were exposed to the compound, and the activity of the early apoptosis marker caspase 3 / 7 was measured in the cells at 15 h, 24 h, and 41 h post-exposure. Cysteine ​​aspartate protease activity was measured using the 3 / 7 assay (Promega) according to the manufacturer's protocol. Conclusion: All three compounds, including compound 1 (designated NSN22769), induced cysteine ​​aspartate protease 3 / 7 activation in ML-2 cells, a marker of early apoptosis. See also Figures 3A-3C .

[0189] Example 17

[0190] Biochemical PIKfyve test

[0191] Full-length recombinant human PIKFYVE, expressed as an N-terminal GST fusion protein (265 kDa) in a baculovirus expression system, was obtained from Carna Biosciences (Kobe, Japan). Bodipy-labeled phosphatidylinositol 3-phosphate (PI3P) was obtained from Echelon Biosciences (Salt Lake City, UT USA). 1,2-Dioctyl-sn-glycerol-3-phosphate-L-serine (PS) was purchased from Avanti Polar Lipids (Alabaster, AL US).

[0192] The following steps are used to prepare PI3P / PS substrates. A 10 mM PS stock solution was prepared in chloroform in a glass container. A 1 mM PI3P stock solution was prepared in 50 mM HEPES (pH 7.5). Before the experiment, the PS stock solution was rapidly evaporated under a nitrogen stream, and the dried pellets were resuspended in 50 mM HEPES (pH 7.5) to a final concentration of 20 μM. The resuspended PS and PI3P were mixed at a molar ratio of 10:1 (10 μM PS and 1 μM PIP3). The prepared PI3P / PS mixture was sonicated in an ultrasonic water bath for 15 min (3 times, 5 min each time).

[0193] The kinase reaction was assembled in 384-well Greiner plates with a total volume of 20 μL as follows: The kinase protein was pre-diluted in an analysis buffer containing 25 mM HEPES (pH 7.5), 1 mM DTT, 2.5 mM MgCl2, 2.5 mM MnCl2, and 0.005% Triton X-100, and then dispensed into 384-well plates (10 μL per well). The test compounds were serially pre-diluted in DMSO and added to the protein samples via acoustic dispensing (Labyrinth Echo). The concentration of DMSO was made equal to 1% in all samples. All test compounds were tested in triplicate at 12 concentrations. Control samples (0% inhibition in the absence of inhibitors, DMSO only) and 100% inhibition (in the absence of enzymes) were assembled in quadruplicate and used to calculate the % inhibition in the presence of the compounds. The reaction was initiated by adding 10 μL of ATP-supplemented PI3P / PS substrate. The final enzyme concentration was 2 nM, and the final ATP concentration was 10 μM. The kinase reaction was allowed to proceed at room temperature for 3 hours. After incubation, the reaction was quenched by adding 50 μL of stop buffer (100 mM HEPES, pH 7.5, 0.01% Triton X-100, 20 mM EDTA). The reaction was then measured using a microfluidic electrophoresis instrument (Caliper). The analysis was performed on a terminated plate at Caliper Life Sciences / Perkin Elmer (3000). The change in relative fluorescence intensity of the PI(3)P substrate and PI(3,5)P product peaks was the measured parameter. The activity in each test sample was determined as the product-to-total ratio (PSR): P / (S+P), where P is the peak height of the product and S is the peak height of the substrate.

[0194] The inhibition percentage (P) is determined using the following equation. inh ):

[0195] P inh =(PSR) 0％inh -PSR 化合物 ) / (PSR 0％inh -PSR 100％inh )*100, where: PSR 化合物 PSR is the product / total ratio in the presence of the compound. 0％inh The product / total ratio in the absence of the compound, and PSR 100％inhThe product / total ratio is given in the absence of the enzyme. To determine the IC50 (50% inhibition) of the compound, %-inh cdata (P) was fitted using a 4-parameter sigmoid dose-response model with XLfit software (IDBS). inh (Regarding compound concentration).

[0196] In this experiment, the ability of apimod, APY0201, compound 1 of this disclosure (described in Example 2 of this disclosure), and YM201636 to inhibit PIKfyve kinase was tested. Results were... Figure 4 And as shown in the following table:

[0197]

Claims

1. A compound of formula II and / or a pharmaceutically acceptable salt thereof: in R1 is a pyridyl group optionally substituted with one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl and lower alkyl groups. R2 is -N=CH-indolyl, wherein the indolyl is optionally substituted by one or two groups selected from -F, -Cl, -CN, -OH, -C(O)NH2, -CF3, -NH2, -NHSO2-lower alkyl, -OCF3, -O-lower alkyl, lower alkyl, phenyl, and monocyclic heteroaryl groups. R5, R6, R7, and R8 are independently H or methyl.

2. The compound according to claim 1 and / or its pharmaceutically acceptable salt, wherein R1 is... in This indicates the connection point with the rest of the molecule.

3. The compound according to claim 1 and / or its pharmaceutically acceptable salt, wherein R2 is... in This indicates the connection point with the rest of the molecule.

4. The compound according to claim 1 and / or its pharmaceutically acceptable salt, wherein the compound is selected from the following compounds:

5. A pharmaceutical composition comprising the compound of claim 1 and / or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

6. Use of a pharmaceutical composition comprising the compound of claim 1 and / or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating an individual suffering from a disease treatable by inhibiting PIKfyve kinase, said method comprising administering a therapeutically effective amount of said pharmaceutical composition to the individual in need, wherein such administration reduces or eliminates symptoms associated with said disease. The diseases mentioned are selected from multiple myeloma, non-Hodgkin's lymphoma, T-cell lymphoma, and acute myelomonocytic leukemia.

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