PCSK9 inhibitors and methods of use thereof

Abstract

To provide a method of inhibiting PCSK9.SOLUTION: The present invention provides a method of inhibiting PCSK9, comprising contacting PCSK9 with an inhibitor of PCSK9 that binds to a binding pocket defined by amino acid residues Val589 and Ser636 of human PCSK9.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 794,239, filed on January 18, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] PCSK9, also known as "proprotein convertase subtilisin / kexin type 9", is a member of the secretory proprotein convertase family and plays an important role in cholesterol metabolism. PCSK9 increases the level of circulating LDL cholesterol by enhancing the degradation of LDL receptors independently of its catalytic activity. Secreted PCSK9 binds to the epidermal growth factor domain A (EGFA) of the LDL receptor (LDLR) on the cell surface, and the PCSK9 / LDL receptor complex is internalized into the endosome / lysosome compartment. The enhanced binding affinity of PCSK9 for the LDL receptor at the acidic pH of late endosomes / lysosomes reduces the recycling of the LDL receptor and instead targets the LDL receptor for lysosomal degradation. Studies of genetic interactions have demonstrated that loss-of-function mutations in PCSK9 are associated with low plasma LDL-C levels and a reduced incidence of adverse cardiovascular events.

[0003] Another biological pathway involving the action of PCSK9 on the LDL receptor is the development of septic shock. Septic shock is often a fatal complication of severe microbial infection (sepsis) that induces an uncontrolled systemic inflammatory response and subsequent organ failure. Sepsis is caused by bacterial cell walls containing pathogenic lipid moieties such as lipopolysaccharide (LPS; Gram-negative bacteria). LPS is a potent ligand for mammalian innate immune receptors [Toll-like receptors (TLRs)] and thus occupies a prominent position in septic inflammatory responses (septic shock, or sepsis).

[0004] PCSK9 reduces the uptake of LDL by the LDL receptor in the liver, and as a result, free LDL stimulates the body's immune response to pathogens excessively, causing sepsis. Therefore, inhibition of PCSK9 is beneficial for the retention of hepatic LDL receptors, which leads to systemic pathogen clearance and detoxification of the response to sepsis. However, currently, there is no effective treatment for sepsis or septic shock other than antibiotic therapy.

[0005] Regarding cardiovascular diseases, there are few options for inhibiting PCSK9. Statins actually upregulate PCSK9 in HepG2 cells and primary human hepatocytes through an increase in the expression of SREBP-2, a transcription factor that upregulates both the LDLR and PCSK9 genes. Since an increase in the level of PCSK9 reduces the abundance of LDL receptors on the cell surface, an increase in the dose of statins could not achieve a proportional LDL-cholesterol lowering effect.

[0006] Alirocumab and evolocumab, two monoclonal antibodies (mAbs) that selectively bind to extracellular PCSK9 and prevent its interaction with the LDL receptor, have recently received FDA approval regarding the reduction of LDL-C levels. In clinical trials, alirocumab showed a reduction in LDL levels of approximately 50% compared to placebo. Non-Patent Document 1. Patients taking evolocumab showed a reduction in LDL levels of approximately 60 - 75%. The efficacy of these drugs demonstrates the potential of PCSK9 inhibitors as effective treatments for patients with hypercholesterolemia and other cardiovascular diseases. However, both antibody drugs require intravenous administration and may cause allergic reactions or other harmful immune responses in the body.

[0007] The design and development of effective drugs are often aided by information about how a compound interacts with its biological target, such as PCSK9. Cunningham et al. described the three-dimensional crystal structure of PCSK9 and identified different regions, such as the C-terminal domain, which is more flexible than other sections of the protein. (Non-Patent Document 2). Thus, interest in PCSK9 inhibitors has focused on how various small molecules can interact with surfaces or pockets in PCSK9.

[0008] Cardiovascular diseases are not transient like infectious diseases and often require management over a person's lifetime. Thus, ease of dosing and administration are important factors for patient compliance with maintenance drug therapy. There is a need for highly effective and more easily administered PCSK9 inhibitors, which can be achieved by small molecule PCSK9 inhibitors.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0010] In this specification, a compound of formula (I):

Chemical Formula

[0011] Disclosed herein are methods of treating diseases and conditions that benefit from inhibition of PCSK9. These diseases include, but are not limited to, cardiovascular diseases such as hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, abnormal lipoproteinemia, atherosclerosis, fatty liver, metabolic syndrome, and coronary artery disease.

[0012] Other diseases and conditions that can be treated using the methods described herein include, but are not limited to, sepsis and septic shock.

[0013] This specification provides combination therapies of compounds of formula (I) with monoclonal antibodies, statins and other cardiovascular therapeutics that can enhance cardiovascular therapeutic effects beyond the ability of adjuvant therapy alone. Also provided herein are combination therapies of compounds of formula (I) with antibiotic agents that can reduce the incidence and severity of sepsis and septic shock beyond the ability of adjuvant therapy alone.

[0014] Also disclosed herein is a novel binding site on PCSK9 with which the compounds of formula I interact. Certain features common to these compounds, such as H-bonds of a triple structure between the disclosed compounds and specific residues in the C-terminal domain of PCSK9, contribute to the selectivity and affinity of the compounds for PCSK9. This C-terminal binding site is different from the PCSK9 catalytic domain, which is understood to be the target of many previously identified inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0016] Compound In the present specification, a compound of formula (I):

Chemical formula

[0017] In this specification, the compound of formula (I')

Chemical formula

[0018] It is understood that all of the following and the embodiments of the present specification are embodiments of both formula (I) and formula (I').

[0019] In certain embodiments, A is H, hydroxy, thioalkyl, alkyl, acyloxy, cyano, cycloalkyl, -C(O)OR 6 and -C(O)NR 6 R 7 The compound according to claim 1, selected from

[0020] In certain embodiments, A is H, but in other embodiments, A is alkyl such as thioalkyl. In certain embodiments, A is selected from -SCH3, -SCHF2, and -OCHF2. In some embodiments, A is alkoxy. In other embodiments, A is cycloalkyl. In certain embodiments, B is H.

[0021] In certain embodiments, A and B, together with the carbon atom to which they are attached, form a pyrrolyl or thienyl ring, which is unsubstituted or substituted with one or more alkyls.

[0022] In certain embodiments, X is preferably NR5. In other embodiments, Y is preferably heteroaryl or heterocyclyl.

[0023] In certain embodiments, R 1 and R 1’ are each H. However, when n is 0, R 1 and R 1’ can together with the atom to which they are attached form a 4- to 8-membered cycloalkyl ring. In some embodiments, the cycloalkyl is monocyclic or bicyclic. In other embodiments, R 1 and R 1’ can together with the atom to which they are attached form a 4- to 8-membered cycloalkenyl ring. In some embodiments, the cycloalkyl ring is a cyclopentyl ring such as S,S-cyclopentyl. In some embodiments, the cycloalkyl ring is substituted with hydroxyl or hydroxyalkyl.

[0024] In certain embodiments, R 2 is selected from H, halo, alkyl, alkoxy, amidoalkyl, aminoalkyl, alkylamino, cyano, and hydroxyl. In certain embodiments, R 2 is C 1~3 alkyl. R 2 can be substituted with one or more substituents selected from amino, amide, cyano, hydroxy, and heterocyclyl. In some embodiments, R 2’ is C 1~3 alkyl, but in other embodiments, R 2’ is H.

[0025] In certain embodiments, R 1 and R 2 together with the atom to which they are attached form a 3- to 8-membered cycloalkyl or heterocyclyl ring. In other embodiments, R 1’ and R 2 together with the atom to which they are attached form a 3- to 8-membered cycloalkyl or heterocyclyl ring. In certain embodiments, R 2 and R 2’ together with the carbon atom to which they are attached form a 3- to 8-membered cycloalkyl or heterocyclyl ring.

[0026] In certain embodiments, R 3 is C 1~3 alkyl, but in other embodiments, R 3 is H. In certain embodiments, R 4 is H.

[0027] In certain embodiments, R 2 and R 3 , together with the atom to which they are attached, form a 3- to 8-membered cycloalkyl or heterocyclyl ring. In other embodiments, R 1 and R 5 , together with the atom to which they are attached, form a 6- to 8-membered cycloalkyl or heterocyclyl ring. In other embodiments, R 2 and R 5 , together with the atom to which they are attached, form a 5- to 8-membered cycloalkyl or heterocyclyl ring.

[0028] In certain embodiments, Y is a monocyclic heteroaryl such as, but not limited to, pyridinyl, pyrazinyl, pyrimidinyl, and thiazolyl. In other embodiments, Y is a monocyclic heteroaryl such as, but not limited to, pyridinyl, pyrazinyl, and pyrimidinyl. In some embodiments, Y is selected from triazenyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, and triazolyl. The monocyclic heteroaryl may be unsubstituted or substituted with one or more substituents selected from alkyl, thioalkyl, alkoxy, alkoxycarbonyl, amide, carboxy, cyano, halo, heteroaryl, nitro, sulfonamide, and thioalkyl. In another embodiment, Y is a monocyclic heteroaryl that may be unsubstituted or substituted with one or more substituents selected from alkyl, thioalkyl, alkoxy, alkoxycarbonyl, amide, carboxy, cyano, halo, aryl, heteroaryl, heterocyclyl, nitro, sulfonamide, and thioalkyl.

[0029] In certain embodiments, the monocyclic heteroaryl is substituted with an aryl, heteroaryl or heterocyclyl selected from phenyl, pyridinyl, 2-hydroxypyridinyl, piperidinonyl, 2-hydroxy-1-methylpyridinyl, triazolyl, imidazolidinonyl, pyrimidonyl, 2-hydroxyisoquinolinyl, 3-hydroxypyridazinyl, pyrrolidinonyl, pyrazolyl, and morpholinonyl. In certain preferred embodiments, Y is a 6-membered monocyclic heteroaryl. In some embodiments, the monocyclic heteroaryl is substituted with a heteroaryl or heterocyclyl substituted with one or more substituents selected from halo, CN, alkyl, alkoxy, hydroxy, carboxy, -CO2alkyl, and tetrazolyl. In certain preferred embodiments, the monocyclic heteroaryl is disposed at the para position of A relative to X.

[0030] In other embodiments, Y is a bicyclic heteroaryl such as, but not limited to, benzothiazolyl, benzimidazolyl, benzoxazolyl, triazolopyridinyl, thiazolopyrindinyl, quinolinyl, and quinoxalinyl. The bicyclic heteroaryl may be unsubstituted or substituted with one or more substituents selected from alkyl, haloalkyl, hydroxyalkyl, thioalkyl, alkoxy, alkoxycarbonyl, amide, carboxy, cyano, halo, heteroaryl, nitro, and sulfonamide. In certain embodiments, the bicyclic heteroaryl is unsubstituted or substituted with one or more substituents selected from thioalkyl, alkoxycarbonyl, amide, carboxy, halo, and heteroaryl.

[0031] In certain embodiments, Y is substituted with an amide substituent of the formula -C(O)NR 8 R 9 or -NR 9 C(O)R 10 wherein R 8 and R 9 are each independently selected from H, alkyl, heterocyclyl, and heteroaryl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclic or heteroaryl ring; and R 10 is alkyl.

[0032] In certain embodiments, Y is of the formula -S(O)2NR 8 R 9 or -NR 9 S(O)2R 10 substituted with a sulfonamide substituent; R 8 and R 9 are each independently selected from H, alkyl, and heteroaryl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclic ring; and R 10 is alkyl.

[0033] It is understood that all of the foregoing embodiments of the variable element Y in formula (I) are also embodiments of the variable element Het in formula (I').

[0034] In certain embodiments, R 8 and R 9 are each independently selected from H, methyl, ethyl, triazolyl, and pyrazolyl. In embodiments where one or both of R 8 and R 9 are alkyl, each alkyl is independently unsubstituted or substituted with one or more substituents selected from methyl, methoxy, carboxy, cyano, hydroxy, dimethylamino, ethoxycarbonyl, phenyl, methoxyphenyl, oxadiazolyl, tetrazolyl, 2-methyl-tetrazolyl, triazolyl, 1-methyltriazolyl, 4-methyltriazolyl, and 2,4-dihydro-3H-1,2,4-triazol-3-onyl. In certain embodiments, R 8 and R 9Together with the nitrogen atom to which they are attached, form a heterocyclic ring selected from aziridine, isothiazolidine-1,1-dioxide, azetidine, thiazol-4(5H)-one, morpholine, piperidine, piperazine, pyrrolidine, thiomorpholine-1,1-dioxide, 2-oxa-6-azaspiro[3.3]heptane. In some embodiments, R 8 and R 9 Together with the nitrogen atom to which they are attached, form a heterocyclic ring selected from 2,8-diazaspiro[5.5]undecane, tetrahydroimidazo[1,2-a]pyrazine, octahydropyrazino[2,1-c][1,4]oxazine, tetrahydropyrido[3,4-d]pyrimidine, 2-oxa-8-azaspiro[4.5]decane, tetrahydropyrrolo[3,4-c]pyrazole, thiomorpholine, 2-oxa-7-azaspiro[3.5]nonane, 2,8-diazaspiro[4.5]decane-3-one, tetrahydro-1,7-naphthyridine, 1-oxa-4,9-diazaspiro[5.5]undecane-3-one, tetrahydropyrrolo[3,4-d]imidazole, pyrimidine, 8-oxa-2-azaspiro[4.5]decane, hexahydro-3H-oxazolo[3,4-a]pyrazine-3-one, 1-oxa-7-azaspiro[3.5]nonane, octahydrocyclopenta[c]pyrrole, tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine, 2,7-diazaspiro[4.4]nonane, 2,6-diazaspiro[3.4]octane, 7-oxa-2-azaspiro[3.5]nonane, 1-oxa-8λ 2 -azaspiro[4.5]decane, 2-oxa-6-azaspiro[3.3]heptane, tetrahydrofuran, oxadiazole, triazole, pyridinone, tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-3(2H)-one, piperidinone, 3,6-diazabicyclo[3.1.1]heptane, 5-oxa-2,7-diazaspiro[3.5]nonane, pyrazole, and pyridazin-3(2H)-one.

[0035] In some embodiments, the heterocyclic ring is unsubstituted or substituted with one or more substituents selected from alkyl, alkoxycarbonyl, halo, hydroxy, cyano, carboxy, and heterocyclyl. In certain embodiments, the heterocyclic ring is unsubstituted or substituted with one or more substituents selected from methyl, ethoxycarbonyl, halo, hydroxy, cyano, carboxy, and oxetanyl.

[0036] In certain embodiments, the present invention provides a pharmaceutical suitable for use in a human patient, comprising any of the compounds shown above (e.g., a compound of the present invention such as a compound of formula (I)) and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical may be for use in treating or preventing a condition or disease described herein.

[0037] Any of the disclosed compounds can be used in the manufacture of a medicament for the treatment of any of the diseases or conditions disclosed herein.

[0038] Definitions Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide those of ordinary skill in the art with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below unless otherwise specified.

[0039] In this disclosure, terms such as "comprises," "comprising," "contains," and "has" can have the meanings ascribed to them in United States patent law and can mean "includes," "including," etc.; "consisting essentially of" or "consisting of" similarly have the meanings ascribed to them in United States patent law, and the term is non-limiting and allows for the presence of more than that described so long as the basic or novel features described are not changed by the presence of more than that described, excluding prior art embodiments.

[0040] Unless otherwise specified or apparent from the context, as used herein, the term "or" is understood to be inclusive. Unless otherwise specified or apparent from the context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0041] The term "acyl" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)-, preferably alkyl C(O)-.

[0042] The term "acylamino" is recognized in the art and refers to an amino group substituted with an acyl group and can be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0043] The term "acyloxy" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0044] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, to which oxygen is bonded. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0045] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0046] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl", the latter referring to an alkenyl moiety having a substituent that replaces a hydrogen on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons that are or are not included in one or more double bonds. Further, such substituents include all those envisioned for an alkyl group as described below, provided that stability is not impaired. For example, substitution of an alkenyl group by one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups is envisioned.

[0047] An "alkyl" group or "alkane" is a completely saturated straight-chain or branched-chain non-aromatic hydrocarbon. Typically, a straight-chain or branched-chain alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms, unless otherwise defined. Examples of straight-chain and branched-chain alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. A C1-C6 straight-chain or branched-chain alkyl group is also referred to as a "lower alkyl" group.

[0048] Furthermore, as used throughout this specification, examples, and the claims, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl", the latter referring to an alkyl moiety having substituents that replace one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents, unless otherwise specified, can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic ring moieties. It will be understood by those skilled in the art that moieties substituted on the hydrocarbon chain can themselves be substituted as necessary. For example, substituents of substituted alkyl can include amino, azide, imine, amide, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamide, sulfamoyl, and sulfonate), and silyl groups, as well as substituted and unsubstituted forms of ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate, and ester), -CF3, -CN, etc. Exemplary substituted alkyls are described hereinafter. Cycloalkyl can be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, etc.

[0049] The term "C x~y ", when used with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, means a group containing from x to y carbons in the chain. For example, "C x~yThe term "alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group containing straight-chain and branched-chain alkyl groups containing x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. C0 alkyl represents hydrogen when the group is at the end and a bond when it is internal. "C 2~y alkenyl" and "C 2~y alkynyl" are terms that are similar to the alkyl described above in terms of length and possible substitution, but refer to substituted or unsubstituted unsaturated aliphatic groups each containing at least one double or triple bond.

[0050] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0051] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0052] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl", the latter referring to an alkynyl moiety having a substituent that replaces a hydrogen on one or more carbons of the alkynyl group. Such substituents can be present on one or more carbons that are or are not included in one or more triple bonds. Further, such substituents include all those envisioned for an alkyl group as described above, provided that stability is not impaired. For example, substitution of an alkynyl group with one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups is envisioned.

[0053] As used herein, the term "amide" refers to the group

Chemical formula

[0054] The terms "amine" and "amino" are recognized in the art and include both unsubstituted and substituted amines and their salts, for example,

Chem.

[0055] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0056] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which rings is aromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0057] The term "carbamate" is recognized in the art and the group

Chem.

[0058] As used herein, the terms "carbocyclic" and "carbocyclic ring" refer to saturated or unsaturated rings in which each atom of the ring is carbon. The term carbocyclic ring includes both aromatic carbocyclic rings and non-aromatic carbocyclic rings. Non-aromatic carbocyclic rings include both cycloalkane rings in which all carbon atoms are saturated and cycloalkene rings containing at least one double bond.

[0059] The term "carbocyclic ring" includes monocyclic rings of 5 to 7 members and bicyclic rings of 8 to 12 members. Each ring of a bicyclic carbocyclic ring can be selected from saturated rings, unsaturated rings, and aromatic rings. Carbocyclic rings include bicyclic molecules in which one, two, or more atoms are shared between two rings. The term "fused carbocyclic ring" refers to a bicyclic carbocyclic ring in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocyclic ring can be selected from saturated rings, unsaturated rings, and aromatic rings. In exemplary embodiments, an aromatic ring, such as phenyl, can be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated rings, unsaturated rings, and aromatic bicyclic rings is included in the definition of carbocyclic when the valency permits. Exemplary "carbocyclic rings" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocyclic rings include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocyclic ring" can be substituted at any one or more positions capable of bearing a hydrogen atom.

[0060] A "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms, unless otherwise defined. The second ring of a bicyclic cycloalkyl can be selected from a saturated ring, an unsaturated ring, and an aromatic ring. Cycloalkyl includes bicyclic molecules in which one, two, or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl can be selected from a saturated ring, an unsaturated ring, and an aromatic ring. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0061] As used herein, the term "carbocyclic alkyl" refers to an alkyl group substituted with a carbocyclic group.

[0062] The term "carbonate" is recognized in the art and refers to the group -OCO2-R 10 (wherein R 10 represents a hydrocarbyl group).

[0063] As used herein, the term "carboxy" refers to the group represented by the formula -CO2H.

[0064] As used herein, the term "ester" refers to the group -C(O)OR 10 (wherein R 10 represents a hydrocarbyl group).

[0065] As used herein, the term "ether" refers to a hydrocarbyl group linked to another hydrocarbyl group via oxygen. Thus, an ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. The ether can be either symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocyclic-O-heterocyclic and aryl-O-heterocyclic. The ether includes an "alkoxyalkyl" group, which can be represented by the general formula alkyl-O-alkyl.

[0066] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo, and iodo.

[0067] As used herein, the terms "heteroalkyl" and "heteroarylalkyl" refer to an alkyl group substituted with a heteroaryl group.

[0068] As used herein, the term "heteroalkyl" refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, where no two heteroatoms are adjacent.

[0069] The terms "heteroaryl" and "heteroaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, and these ring structures include at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "heteroaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, and at least one of these rings is a heteroaromatic ring. For example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0070] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0071] The terms "heterocyclyl", "heterocyclic", and "heterocyclic ring" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, and these ring structures contain at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic ring" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, and at least one of these rings is heterocyclic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, etc.

[0072] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclic group.

[0073] As used herein, the term "hydrocarbyl" refers to a group that is bonded through a carbon atom that does not have an =O or =S substituent, typically has at least one carbon-hydrogen bond and mainly a carbon skeleton, but optionally can contain a heteroatom. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for the purposes of this application, but substituents such as acetyl (having an =O substituent on the connecting carbon) and ethoxy (connected through oxygen rather than carbon) are not considered hydrocarbyl. Examples of hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0074] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0075] The term "lower", when used with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, means a group having 10 or fewer non-hydrogen atoms, preferably 6 or fewer, in the substituent. "Lower alkyl" refers to an alkyl group containing, for example, 10 or fewer carbon atoms, preferably 6 or fewer. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are each lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the listing of hydroxyalkyl and aralkyl (in which case, for example, when counting the carbon atoms in an alkyl substituent, the atoms within an aryl group are not counted).

[0076] The terms "polysicyclic", "polycyclic", and "polycyclic ring system" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings". Each of the polycyclic rings may or may not be substituted. In certain embodiments, each ring of the polycyclic ring system contains from 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

[0077] The term "silyl" refers to a silicon moiety to which three hydrocarbyl moieties are attached.

[0078] The term "substituted" refers to a moiety having a substituent that replaces a hydrogen on one or more carbons of a backbone. "Substituted" or "substituted with" is understood to mean that such substitution conforms to the valencies of the substituted atom and the substituent, and includes the implicit condition that the substitution results in a stable compound that does not undergo spontaneous conversion, such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substitution values of an organic compound. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Permissible substituents may be one or more and may be the same or different for a suitable organic compound. For the purposes of the present invention, a heteroatom such as nitrogen may have any permissible substituent of the organic compounds described herein that satisfies the valency of the hydrogen substituent and / or the heteroatom. Substituents include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic ring moiety. It will be understood by those skilled in the art that substituents may themselves be substituted as necessary. Unless specifically described as "unsubstituted", references herein to chemical moieties are understood to include substituted forms. For example, a reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted forms.

[0079] The term "sulfate" is recognized in the art and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0080] The term "sulfonamide" is recognized in the art and has the general formula

Chem.

[0081] The term "sulfoxide" is recognized in the art and refers to the group -S(O)-R 10 (wherein R 10 represents a hydrocarbyl).

[0082] The term "sulfonate" is recognized in the art and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0083] The term "sulfone" is recognized in the art and refers to the group -S(O)2-R 10 (wherein R 10 represents a hydrocarbyl).

[0084] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0085] As used herein, the term "thioester" refers to the group -C(O)SR 10 or -SC(O)R 10 (wherein R 10 represents a hydrocarbyl).

[0086] As used herein, the term "thioether" corresponds to an ether in which oxygen is replaced by sulfur.

[0087] The term "urea" is recognized in the art and has the general formula [Chemistry] may be represented by, wherein R 11 and R 12 each independently represent a hydrocarbyl such as hydrogen or alkyl, or the presence of any of R 11 together with R 12 and intervening atoms completes a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0088] The term "protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, shields, reduces, or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed during a synthetic process as needed. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3 rd Ed., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), and the like. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (esterified) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0089] The present invention includes all pharmaceutically acceptable isotopically labeled compounds as described herein, wherein one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the atomic mass or mass number typically found in nature.

[0090] Examples of isotopes suitable for inclusion in the compounds of the present invention include 2 H and 3 H and other hydrogens, 11 C, 13 C and 14 C and other carbons, 36 Cl and other chlorines, 18 F and other fluorines, 123 I and 125 I and other iodines, 13 N and 15 N and other nitrogens, 15 O, 17 O and 18 O and other oxygens, 32 P and other phosphors, and 35 S and other sulfur isotopes.

[0091] Certain isotopically labeled compounds as disclosed herein, for example those incorporating radioisotopes, are useful in drug and / or substrate tissue distribution studies. The radioisotopes tritium, i.e., 3 H, and carbon-14, i.e., 14 C are useful for this purpose in view of their ease of incorporation and ready means of detection.

[0092] Substitution with heavier isotopes such as deuterium, i.e., 2 H, can provide several therapeutic advantages due to higher metabolic stability, e.g., increased in vivo half-life or decreased required dosage, and may be preferred in certain situations.

[0093] 11 C, 18 F, 15 O, and 13Substitution with a positron-emitting isotope such as N can be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy.

[0094] In certain embodiments, the compounds of the invention can be racemic. In certain embodiments, the compounds of the invention can be enriched in one enantiomer. For example, the compounds of the invention can have greater than about 30% ee, about 40% ee, about 50% ee, about 60% ee, about 70% ee, about 80% ee, about 90% ee, or greater than about 95% ee. In certain embodiments, the compounds of the invention can have two or more stereocenters. In certain such embodiments, the compounds of the invention can be enriched in one or more diastereomers. For example, the compounds of the invention can have greater than about 30% de, about 40% de, about 50% de, about 60% de, about 70% de, about 80% de, about 90% de, or greater than about 95% de.

[0095] In certain embodiments, a therapeutic formulation can be enriched to predominantly provide one enantiomer of a compound (e.g., of formula (I)). A mixture enriched in one enantiomer can contain, for example, at least about 60 mole percent of one enantiomer, or more preferably, at least about 75, about 90, about 95, or about 99 mole percent. In certain embodiments, a compound enriched in one enantiomer is substantially free of the other enantiomer, where substantially free means that the substance constitutes less than about 10%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1% compared to the amount of the other enantiomer in a composition or mixture of compounds. For example, if a composition or mixture of compounds contains about 98 grams of a first enantiomer and 2 grams of a second enantiomer, it would be described as containing about 98 mole percent of the first enantiomer and only about 2% of the second enantiomer.

[0096] In certain embodiments, the therapeutic formulation can be enriched to predominantly provide one diastereomer of the compound (e.g., of formula (I)). A mixture enriched in one diastereomer can contain, for example, at least about 60 mole percent of one diastereomer, or more preferably, at least about 75, about 90, about 95, or about 99 mole percent.

[0097] The term "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, toddlers, juveniles) or adult subjects (e.g., young adults, middle-aged adults or the elderly)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds including commercially relevant birds such as chickens, ducks, geese, turkeys, and / or pigeons. Preferred subjects are humans.

[0098] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample as compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition as compared to an untreated control sample.

[0099] The term "treat" includes prophylactic and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is recognized in the art and includes administration of one or more of the disclosed compositions to a subject. When it is administered prior to the clinical symptoms of an undesirable condition (e.g., a disease or other undesirable state of the subject), the treatment is prophylactic (i.e., it protects the subject against the occurrence of the undesirable condition), while when it is administered after the manifestation of an undesirable condition, the treatment is therapeutic (i.e., it is intended to reduce, reverse, or stabilize an existing undesirable condition or its side effects).

[0100] The term "prodrug" is intended to encompass compounds that are converted under physiological conditions to the therapeutically active agent of the invention (e.g., a compound of formula (I)). A common method for making prodrugs is to include one or more selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by the enzymatic activity of the subject. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the invention. In certain embodiments, some or all of the compounds of formula (I) in the formulations shown above may be replaced with the corresponding suitable prodrugs, e.g., hydroxyls in the parent compound are shown as esters or carbonates or carboxylic acids.

[0101] As used herein, "effective amount" refers to an amount sufficient to achieve the desired biological effect. As used herein, "therapeutically effective amount" refers to an amount sufficient to achieve the desired therapeutic effect. For example, a therapeutically effective amount may refer to an amount sufficient to ameliorate at least one sign or symptom of cancer.

[0102] "Response" to a method of treatment may include, among other things, a decrease or amelioration of negative symptoms, a decrease in the progression of the disease or its symptoms, an increase in beneficial symptoms or clinical outcomes, a reduction of side effects, stabilization of the disease, or a partial or complete recovery from the disease.

[0103] Allosteric inhibitor of PCSK9 The applicant has identified and characterized a previously characterized site of PCSK9 to which the compounds disclosed herein bind. As a result, the applicant has identified a series of chemical features, or pharmacophores, that interact with this site and can inhibit the activity of PCSK9.

[0104] The concept of "pharmacophore" is not recent. It was first introduced in 1909 by Paul Ehrlich as "a molecular framework (pharos) that possesses the essential features involved in the biological activity of a drug (medicament)". Ehrlich, Uber den jetzigen stand der chemotherapie. Chem. Ber. 42:17. This definition was further updated in 1977 by Peter Gund as "a set of structural features of a molecule that are recognized at the receptor site and are involved in the biological activity of that molecule". Gund, Three dimensional pharmacophoric pattern searching. Prog. Mol. Subcell. Biol. 5:117 - 143. More recently, in 1997, the official IUPAC recommendation summarized the concept as follows: "A pharmacophore is a collection of steric and electronic features necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response". Wermuth, C.-G. et al. 1998 Glossary of terms used in medicinal chemistry (IUPAC Recommendations 1998). Pure Appl. Chem. 70:1129 - 1143. Thus, a pharmacophore is not representative of a specific association of particular molecules or functional groups, but rather the understanding that it explains the common molecular interaction ability of a group of compounds towards their target structure. A pharmacophore can be considered as the maximum common element shared by a series of active molecules. A pharmacophore is usually defined by pharmacophore descriptors that include H-bonding, hydrophobic and electrostatic interaction sites, defined by atoms, ring centers, and virtual points.

[0105] Using the structure-activity relationships described below and throughout this specification, Applicants have identified a pharmacophore that binds to PCSK9 and inhibits its activity. PCSK9 has three known domains: a prodomain, a catalytic domain, and a C-terminal domain. Cunningham p414. In the active site of PCSK9, a serine protease catalytic triad of Ser386, His226, and Asp186 has been identified, which interacts with the LDLR and promotes its internalization into the cell and ultimate degradation. Studies of loss-of-function mutations have shown that the C-terminal domain is also required for degradation activity but may not bind to the LDLR. The binding site identified herein is located in the C-terminal domain. Without being bound by any theory, the disclosed inhibitors may act allosterically to modify the conformation and function of the C-terminal domain of PCSK9 at a location other than directly binding to the catalytic domain site of the enzyme.

[0106] The C-terminal domain of PCSK9 has three modules designated M1, M2, and M3. The boundaries of the modules have been described as including slightly different ranges of amino acid residues as studies of PCSK9 progress. The differences in that range mainly serve as positional descriptors and do not imply specific activity of one or more particular residues. In some embodiments, the M1 domain ranges from residues 457-527, the M2 domain ranges from residues 534-601, and the M3 domain ranges from residues 608-679. In other embodiments, the M1 domain ranges from residues 447-530, the M2 domain ranges from residues 531-604, and the M3 domain ranges from residues 608-683.

[0107] Figure 1 provides the crystal structure of the C-terminal domain. Halgren et al., J. Chem. Inf. Model. 2009 49:377-389. In the crystal structure, the C-terminal domain had several disordered segments and a higher level of flexibility. Cunningham, p. 416. Experimental results in the art suggest that the C-terminal domain is involved in the secretion of PCSK9 required for its activity in LDLR degradation. Cunningham, p. 417. The C-terminal domain also plays a role in targeting the PCSK9-LDLR complex to the endosome / lysosome organelles inside the cell for degradation. Saavedra et al., J. Biol. Chem. 2012 287:43492-43501. Mutant studies in which the M2 domain of PCSK9 was removed resulted in the loss of the extracellular activity of the enzyme rather than its intracellular activity. Saavedra, p. 43500. Thus, the binding site described herein located in the C-terminal domain could affect PCSK9 activity in any of several known or unspecified pathways.

[0108] An important feature of the binding site includes a triple structure of hydrogen-bonded atom pairs extending to the M2 and M3 domains as shown in Figure 2. Based on the crystal structure of PCSK9 bound to the disclosed compounds, the compounds of Formula I interact with the binding site as follows.

[0109] [Table 1]

[0110] The numbers of these residue positions are for the most common isoform of PCSK9. An isoform is a peptide or protein that has differences in its amino acid sequence but is translated from the same gene. However, due to the inclusion and deletion of some residues during transcription and translation, the amino acid numbers described herein may vary in different isoforms. Isoforms can also arise due to natural mutations in the gene sequence. The present disclosure includes all isoforms of PCSK9 and their corresponding residue positions in the isoform or other variant sequences. In some embodiments, the isoform of PCSK9 may have about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%, about 99.4%, about 99.6%, about 98.8%, or about 99.9% sequence homology with human PCSK9 having the sequence provided in the Biological Assays section below. Isoform sequences (SEQ ID NOs: 1-6) are provided after the Biological Assays section.

[0111] The disclosed compounds act as both donor B with 2-NH of its 2-aminopyrimidinyl group and acceptor A with pyrimidinyl nitrogen for H-bonding to Val589. Thus, the binding triad structure at the binding site is formed from two PCSK9 residues. The other pyrimidinyl nitrogen binds to Ser636 as acceptor C. The binding distances between each triad structure of the pharmacophore as disclosed herein are about 2.3 Å to about 2.5 Å as shown in Figure 3. For example, acceptor A is 2.3 - 2.4 Å from donor B and 2.3 - 2.5 Å from acceptor C. Donor B is 2.3 - 2.5 Å from acceptor C.

[0112] As shown below, the three nitrogen atoms of the 2-aminopyrimidinyl core of the disclosed compounds interact with the triad structure. Analogs lacking one of these binding characteristics do not show detectable binding to human PCSK9.

Chemical Structure

[0113] Disclosed herein is a method of inhibiting PCSK9, the method comprising contacting PCSK9 with an inhibitor of PCSK9 that binds to the binding pocket defined by the amino acid residues Val589 and Ser636 of human PCSK9. In some embodiments, the inhibitor is an allosteric inhibitor of PCSK9. In some embodiments, the method comprises contacting the surface of a cell that secretes PCSK9 with an inhibitor of PCSK9 that binds to the binding pocket defined by the amino acid residues Val589 and Ser636 of human PCSK9. In certain embodiments, the method comprises binding PCSK9 that is intracellular to an inhibitor of PCSK9 that binds to the binding pocket defined by the amino acid residues Val589 and Ser636 of human PCSK9. In some embodiments, the method comprises contacting a cell that expresses PCSK9 with an inhibitor of PCSK9 that binds to the binding pocket defined by the amino acid residues Val589 and Ser636 of human PCSK9. In other embodiments, the method comprises binding PCSK9 that is in plasma to an inhibitor of PCSK9 that binds to the binding pocket defined by the amino acid residues Val589 and Ser636 of human PCSK9.

[0114] In some embodiments, the inhibitor comprises an H-bond acceptor / donor group having two H-bond acceptor moieties and one H-bond donor moiety arranged to donate an H-bond to and then accept an H-bond from the backbone amide functionality of residue Val589 of human PCSK9 and to accept an H-bond from the hydroxymethyl side chain of Ser636.

[0115] In certain embodiments, the inhibitor comprises an H-bond acceptor / donor group having two H-bond donor moieties and one H-bond acceptor moiety that are arranged to donate an H-bond to and receive an H-bond from the backbone amide functionality of residue Val589 of human PCSK9 and to donate an H-bond to the hydroxymethyl side chain of Ser636.

[0116] In certain embodiments, the inhibitor further a) an H-bond acceptor moiety arranged to bind to amino acid residues Ser642, His643 or Val644, b) an H-bond donor moiety arranged to bind to amino acid residues Ala637 or Thr641, and c) one or more of a cation-π stacking interaction moiety arranged to bind to amino acid residues Arg495 or His591, The H-bond acceptor / donor group is bound to amino acid residues Val589 and Ser636 of human PCSK9. In certain embodiments, the inhibitor further comprises an H-bond acceptor moiety arranged to bind to amino acid residue Glu612. The binding can be a direct H-bond or an indirect H-bond (e.g., an H-bond mediated by water occurs).

[0117] The H-bonds of the triple structure are common to the inhibitory compounds disclosed herein, although some compounds have additional H-bonds to other residues in the pharmacophore. For example, as shown in FIG. 5, compound 43 forms a 2-aminopyrimidine H-bond triple structure common to the pharmacophore, and 2-aminobenzothiazole forms H-bonds with Ala637 and a water molecule. The additional H-bonds are caused by the sulfonamide substituent on the benzothiazole ring. Residue Val644 is an H-bond donor and Ser642 is an H-bond acceptor.

[0118] The crystal structure of compound 60 bound to the pharmacophore shown in Figure 6 shows that in addition to the H-bond triple structure, cation-π stacking interactions occur between Arg495 in the M1 domain and the benzoxazole ring. These cation-π stacking interactions can also occur by His591 (shown as HIE591) in the M2 domain as shown for compound 5 bound to the binding site in Figure 7. Compound 5 has additional H-bonds between His643 and the carbonyl oxygen and between Thr641 and the hydroxyl group hydrogen.

[0119] The versatility of the binding site that binds compounds of various structures is further demonstrated by compound 133. As shown in Figure 8, compound 133 has two 2-aminopyrimidinyl functional groups that interact with the binding site. The pyrimidinyl ring with its thiomethyl substituent binds to a common H-bond triple structure formed by Ser636 and Val589. The pyrimidyl ring substituted with a methyl ester forms five additional H-bonds with Ala637, Val644, and two molecules of water. Cation-π stacking interactions occur between His591 (shown as HIE591) and the pyrimidine ring. In addition, Figure 9 shows the interaction of the N-H of glycine 640 with compound 458B.

[0120] Substitutions around the 2-aminopyrimidinyl core promote an increase in H-bonding or cation-π stacking, thereby increasing the affinity of the compound for the binding site, and thereby the inhibitory effect. These affinity characteristics can be combined and selected. The binding site binds to residues from all three subdomains of the C-terminal domain. The flexibility of the pharmacophore that binds to this binding site provides numerous opportunities for designing compounds such as compounds having a 2-aminopyrimidinyl group or a biological equivalent of the core that has a structure that hydrogen bonds to a triple structure (e.g., having two donors and one acceptor H-bond) and binds one or more of other affinity elements (e.g., cation-π stacking interactions and additional H-bonding sites).

[0121] As used herein, the term "interacts" in the context of an inhibitor associated with one or more residues of PCSK9 refers to a direct or indirect association of the inhibitor with either the backbone of the enzyme or the side chain of any given residue. For example, interactions include covalent bonds, hydrogen bonds, hydrophobic attractions, cation-π interactions, anion-π interactions, and others well known in the art.

[0122] The amino acids that bind to the pharmacophore of the inhibitor, which is a triple structure formed between Val589 and Se636, can also further interact with the amino acid residues in the M1, M2, and M3 domains. In certain embodiments, the inhibitor interacts with at least one residue in the M2 C-terminal domain and at least one residue in the M3 C-terminal domain. In some embodiments, the inhibitor further interacts with at least one residue in the M1 C-terminal domain. In certain embodiments, the inhibitor interacts with at least one residue in the M1 C-terminal domain and at least one residue in the M3 C-terminal domain. In other embodiments, the inhibitor interacts with at least one residue in the M1 C-terminal domain and at least one residue in the M2 C-terminal domain. In certain embodiments, the inhibitor interacts with PCSK9 between amino acid residues 558 - 590 in the M2 C-terminal domain and amino acid residues 631 - 650 in the M3 C-terminal domain.

[0123] In certain embodiments, the inhibitor interacts with at least one residue in beta-strand 3 and beta-strand 5 of the M2 C-terminal domain and at least one residue in beta-strand 3 and beta-strand 4 of the M3 C-terminal domain. In certain embodiments, the inhibitor interacts with PCSK9 generated between amino acid residues 558 - 566 in beta-strand 3 of the M2 C-terminal domain and amino acid residues 587 - 590 in beta-strand 5 of the M2 C-terminal domain. In other embodiments, the inhibitor interacts with a pocket in PCSK9 generated between amino acid residues 631 - 637 in beta-strand 3 of the M3 C-terminal domain and amino acid residues 644 - 650 in beta-strand 4 of the M3 C-terminal domain.

[0124] The structural evaluation of the PCSK9 binding site can be carried out using several techniques well known to those skilled in the art. For example, FIGS. 4-8 show 3D renderings of PCSK9-compound interactions as determined from X-ray crystal structures. Details for the preparation and analysis of the crystal structures are given in the examples below. Analysis of the X-ray data provides 3D renderings of the compounds bound to PCSK9, and the atomic orientations and distances can indicate the types of bonds present, such as covalent bonds, H-bonds, π-stacking, and others.

[0125] Other methods include 3D computer modeling techniques that give computer-generated images of compounds interacting with the substrate. Similar to the crystal structure data, computer modeling programs can provide 3D information in a sphere / rod format showing atoms as a surface shell or in a 3D array. In non-limiting examples, using the computer models of the compounds disclosed herein that bind to PCSK9 to identify how and where the compounds interact with PCSK9 is well within the skill of those skilled in the art.

[0126] Method of Use The PCSK9 gene was identified using genetic mapping techniques on DNA from subjects with autosomal dominant hypercholesterolemia (Abifadel, et al. Nat. Genet. 2003 34:154-6). The encoded protein is a serine protease that is expressed primarily in the liver, intestine, kidney, and nervous system. Without being bound by any particular theory, studies of mutations in the gene have shown that its putative role is in reducing the LDL receptor on the cell surface independent of its catalytic activity (Abifadel, et al. Expert Opin. Ther. Pat. 2010 20:1547-71). Binding of PCSK9 to its receptor leads to their lysosomal degradation. This enhanced degradation results in an increase in the amount of circulating low density lipoprotein LDL (LDL-c). PCSK9 is upregulated by statins, SREBP-1a and SREBP-2, LXR agonists, and insulin, but is downregulated by dietary cholesterol, glucagon, ethinyl estradiol, chenodeoxycholic acid and bile acid-activated farnesoid X receptor (FXR) (Maxwell, et al. J. Lipid Res. 2003 44:2109-19; Persson et al. Endocrinology 2009 150:1140-6; Langhi et al. FEBS Lett. 2008 582:949-55). An increase in the level of PCSK9 reduces the abundance of the LDL receptor on the cell surface, so that an increase in the dose of statin does not achieve a proportionate reduction in LDL-cholesterol. Accordingly, methods are disclosed herein for treating a wide range of cardiovascular diseases and conditions that would benefit from reducing LDL-c by inhibiting PCSK9.

[0127] In certain embodiments, the disclosed methods of inhibiting PCSK9 are performed in a subject in need thereof, thereby treating a PCSK9-mediated disease or disorder. Also disclosed herein are methods of treating or preventing a PCSK9-mediated disease or disorder, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, disclosed herein are methods of treating a PCSK9-mediated disease or disorder, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, disclosed herein are methods of preventing a PCSK9-mediated disease or disorder, comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof. Prevention of cardiovascular events via inhibition of PCSK9 is described, for example, in Robinson et al., Artherosclerosis 2015 243:593-597.

[0128] Exemplary cardiovascular diseases and conditions include, but are not limited to, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, hypoalphalipoproteinemia, metabolic syndrome, diabetic complications, atherosclerosis, stroke, vascular dementia, chronic kidney disease, coronary heart disease, coronary artery disease, retinopathy, inflammation, thrombosis, peripheral vascular disease, or congestive heart failure.

[0129] In certain embodiments, exemplary cardiovascular diseases and conditions include, but are not limited to, hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, abnormal lipoproteinemia, atherosclerosis, fatty liver, metabolic syndrome, and coronary artery disease. In certain embodiments, the disease is hypercholesterolemia such as familial hypercholesterolemia or autosomal dominant hypercholesterolemia. In certain embodiments, the disease is hyperlipidemia. In certain embodiments, the disease is coronary artery disease.

[0130] In certain embodiments, the disclosed methods of treatment can reduce high levels of circulating serum cholesterol such as LDL-cholesterol and VLDL-cholesterol. In addition, the disclosed methods are useful for reducing circulating serum triglycerides, circulating serum lipoprotein A, circulating serum LDL, and atherogenic lipoproteins. In certain embodiments, diseases or conditions treatable with the disclosed compounds and compositions include atherosclerosis and atherosclerotic plaque formation. Subjects having a gain-of-function mutation in the PCSK9 gene also benefit from treatment with the disclosed compounds and compositions that suppress the mutation via inhibition of PCSK9.

[0131] Inhibition of PCSK9 has also shown a therapeutic effect in treating sepsis in a subject. Septic shock is often a life-threatening complication of severe microbial infection (sepsis) that induces an uncontrolled systemic inflammatory response and subsequent organ failure. Sepsis is caused by bacterial cell walls containing pathogenic lipid moieties such as lipopolysaccharide (LPS; Gram-negative bacteria). LPS is a potent ligand for mammalian innate immune receptors [Toll-like receptors (TLRs)] and thus plays a prominent role in septic inflammatory responses (septic shock, or sepsis). PCSK9 reduces the uptake of LPS by the LDL receptor in the liver, such that free LPS overstimulates the body's immune response to pathogens, causing sepsis. Inhibition of PCSK9 is beneficial for the retention of hepatic LDL receptors, which results in systemic pathogen clearance and detoxification of the response to sepsis (see, e.g., Walley et al Sci.Translat.Med.2014 6:1-10).

[0132] Disclosed herein is a method of treating sepsis or septic shock, the method comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the disclosed methods of treatment are useful for increasing LPS uptake. Certain embodiments provide a method of reducing an inflammatory response induced by sepsis or septic shock.

[0133] Pharmaceutical composition The compositions and methods of the present invention can be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human or a non-human mammal. When administered to a subject such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or organic acid esters for injection. In a preferred embodiment, when such a pharmaceutical composition is for administration to a human, particularly for invasive route administration (i.e., a route such as injection or infusion that avoids transport or diffusion through an epithelial barrier), the aqueous solution is free of pyrogens or substantially free of pyrogens. Excipients can be selected, for example, to provide for delayed release of the drug or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized for reconstitution, powders, solutions, syrups, suppositories, injections. The composition can also be present in a transdermal delivery system such as a skin patch. The composition can also be present in a solution suitable for topical administration such as an eye drop.

[0134] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that functions to, for example, stabilize, increase the solubility of, or increase the absorption of a compound such as a compound of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymeric matrix, which can, for example, incorporate the compound of the present invention. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, metabolizable carriers that are relatively easy to prepare and administer.

[0135] As used herein, the phrase "physiologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0136] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0137] The pharmaceutical composition (formulation) can be administered to a subject by any of several routes of administration, for example, orally (e.g., as a water medicine in an aqueous or non-aqueous liquid or suspension, tablet, capsule (including sprinkle capsules and gelatin capsules), bolus, powder, granule, paste for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or intravaginally (e.g., as a pessary, cream or foam); parenterally (e.g., as a sterile liquid or suspension, including intramuscular, intravenous, subcutaneous or intrathecal); nasally; intraperitoneally; subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment or spray applied to the skin, or as an eye drop). The compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, and the patents cited therein.

[0138] The formulation may conveniently be provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of the active ingredient that, when combined with the carrier material, can produce a single dosage form will vary depending on the subject being treated and the particular method of administration. The amount of the active ingredient that, when combined with the carrier material, can produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 1 percent to about 99 percent active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0139] The methods for preparing these formulations or compositions include the step of combining an active compound, such as a compound of the present invention, with a carrier and, optionally, one or more auxiliary components. Generally, the formulations are prepared by uniformly and intimately combining the compound of the present invention with a liquid carrier, or a micronized solid carrier, or both, and then, if necessary, shaping the product.

[0140] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilized products, powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or water-in-oil or oil-in-water liquid emulsions, or elixirs or syrups, or troches (using an inert base such as gelatin and glycerin, or sucrose and acacia) and / or mouthwashes, each containing a predetermined amount of the compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, a lickable preparation or a paste.

[0141] For preparing solid dosage forms for oral administration (including capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is one or more pharmaceutically acceptable monomers such as sodium citrate or dibasic calcium phosphate, and / or mixed with any of the following: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retardants, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain a buffering agent. Solid compositions of the same type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol, etc.

[0142] Tablets can optionally be made by compression or molding using one or more auxiliary components. Compressed tablets can be prepared using binders (such as gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (such as sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfactants or dispersants. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0143] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose in various proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be of a composition that releases the active ingredient only, or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

[0144] Liquid dosage forms useful for oral administration include pharma- ceutically acceptable emulsions, lyophilized for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0145] In addition to the inert diluent, the oral composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfumes and preservatives.

[0146] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0147] Preparations of pharmaceutical compositions for rectal, vaginal or urethral administration may be provided as suppositories, which may be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate, which are solid at room temperature but liquid at body temperature and thus melt in the rectal or vaginal cavity and release the active compound.

[0148] Preparations of pharmaceutical compositions for oral administration may be provided as mouthwashes, or oral sprays, or oral ointments.

[0149] In addition or alternatively, the composition may be formulated for delivery via a catheter, stent, wire, or other intravascular device. Delivery via such devices may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0150] Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing carriers known to be appropriate in the art.

[0151] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as may be required.

[0152] Ointments, pastes, creams and gels may, in addition to the active compound, contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0153] Powders and sprays may, in addition to the active compound, contain excipients such as lactose, talc, silicic acid, aluminium hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0154] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the invention to the body. Such dosage forms may be produced by dissolving or dispersing the active compound in a suitable medium. Penetration enhancers may also be used to increase the flux of the compound across the skin. The rate of such flux may be controlled either by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0155] Ophthalmic formulations, ophthalmic ointments, powders, solutions and the like are also contemplated within the scope of the present invention. Exemplary ophthalmic formulations are described in US Patent Application Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697 and 2005 / 004074 and US Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to or are compatible with tears, aqueous humour or vitreous humour. The preferred route of administration is topical administration (e.g., topical administration such as eye drops or administration via an implant).

[0156] As used herein, the terms "parenteral administration" and "administered parenterally" mean a route of administration other than enteral and topical administration (usually by injection), including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds combined with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous liquids, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents which render the formulation isotonic with the blood of the intended recipient.

[0157] Examples of suitable aqueous and nonaqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0158] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. Furthermore, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0159] In some cases, it is desirable to delay the absorption of a drug from a subcutaneous or intramuscular injection in order to prolong the effect of the drug. This can be achieved by using a liquid suspension of a crystalline or amorphous material having low water solubility. In this case, the absorption rate of the drug depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, the delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.

[0160] Injectable depot forms are prepared by forming a microencapsulated matrix of the compounds of the invention in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0161] For use in the methods of the invention, the active compound can be administered by itself or, for example, as a pharmaceutical composition containing 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0162] The method of introduction can also be provided by a refillable or biodegradable device. For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained-release polymer devices have recently been developed and tested in vivo. Using various biocompatible polymers, including both biodegradable and non-degradable polymers (including hydrogels), implants can be formed for the sustained release of compounds at specific target sites.

[0163] The actual dosage level of the active ingredient in the pharmaceutical composition can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without being toxic to the patient.

[0164] The selected dosage level will depend on a variety of factors including the activity of the specific compound or combination of compounds being used, or esters, salts or amides thereof, the route of administration, the time of administration, the rate of excretion of the specific compound being used, the duration of treatment, other agents, compounds and / or materials used in combination with the specific compound being used, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

[0165] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start with a dosage level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" means a concentration of the compound sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of a compound will vary depending on the weight, sex, age, and medical history of the subject. Other factors that can affect the effective amount include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent administered with the compounds of the invention. A greater total dosage can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those of skill in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, which is incorporated herein by reference).

[0166] In general, a preferred daily dosage of the active compound used in the compositions and methods of the present invention is the amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such effective dosages generally will depend on the factors described above.

[0167] If desired, the effective daily dosage of the active compound can optionally be administered in unit dosage form as separate divided dosages of 1, 2, 3, 4, 5, 6 or more times a day at appropriate intervals throughout the day. In certain embodiments of the invention, the active compound can be administered 2 or 3 times a day. In a preferred embodiment, the active compound is administered once a day.

[0168] In certain embodiments, the compounds of the invention can be used alone or in combination with another type of therapeutic agent. As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic compounds such that a second compound is administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in a subject, which can include a synergistic effect of the two compounds). For example, the different therapeutic compounds can be administered simultaneously or sequentially, either in the same formulation or in separate formulations. In certain embodiments, the different therapeutic compounds can be administered to each other within 1 hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or within 1 week. Thus, a subject receiving such treatment can benefit from the combined effects of the different therapeutic compounds.

[0169] In certain embodiments, co-administration of the compounds of the invention with one or more additional therapeutic agents provides enhanced efficacy compared to the individual administration of each of the compounds of the invention (e.g., a compound of formula I or Ia) or one or more additional therapeutic agents. In certain such embodiments, co-administration provides an additive effect, where the additive effect refers to the sum of the effects of the individual administrations of the compounds of the invention and one or more additional therapeutic agents.

[0170] The present invention includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetraalkylammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.

[0171] Pharmaceutically acceptable acid addition salts can also exist as various solvents such as water, methanol, ethanol, dimethylformamide and the like. Mixtures of such solvates can also be prepared. The source of such solvates can be from the crystallization solvent, inherent to the preparation or crystallization solvent, or associated with such solvents.

[0172] Wetting agents, emulsifying agents and lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants can also be present in the composition.

[0173] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

Example

[0174] Examples of the compounds of formula (I) having useful biological activities, or pharmaceutically acceptable salts thereof, are listed in Tables 1 to 10. 1 The 1H NMR spectra were performed on a Varian MR-400 spectrometer operating at 400 MHz (proton frequency) equipped with a self-shielded Z-gradient coil 5 mm 1H / nX reverse detection broadband probe head, a deuterium digital lock channel unit, and an orthogonal digital detection unit with a transmitter offset frequency shift. Chemical shifts are reported as values in ppm relative to trimethylsilane (TMS) as an internal standard. Coupling constants (J values) are given in Hertz (Hz), and multiplicities are reported using the following abbreviations (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, nd = undetermined).

[0175] A. Analytical Method Method 1 (Acid FA) UPLC Settings Solvent: - A Water containing 0.1% formic acid (high purity via PureLab Option unit) B Acetonitrile containing 0.1% (v / v) formic acid (Far UV grade) Column: - Acquity UPLC HSS C18 1.8um 100×2.1mm. (plus guard cartridge) Flow Rate: - 0.5 mL / min Gradient:

[0176]

Table 2

[0177] Inject 0.5 - 2 μL UV detection via Waters DAD Start range (nm) 210, End range (nm) 400, Resolution (nm) 1.2 MS detection: Waters SQD2, single quadrupole UPLC - MS Scanning range (m / z) for MS data Start (m / z) 100 End (m / z) 700 or 1500 (if necessary) With +ve / -ve switching Ionization is ESI ESI voltage and temperature are Source 150°C, 3.5 kV, Capillary 25 V, Cone

[0178] Method 2 (basic FA) UPLC settings Solvent: - Acetonitrile (Far UV grade) Water containing 10 mM ammonium bicarbonate (high purity via PureLab Option unit) Column: - Acquity UPLC BEH Shield RP18 1.7 μm, 100×2.1 mm. (plus guard cartridge) Flow rate: - 0.5 mL / min Gradient: - A: water / basic, B: MeCN / basic

[0179]

Table 3

[0180] Typical injection 0.5 - 2 μL (concentration about 0.2 - 1 mg / mL). UV detection via Waters DAD Start range (nm) 210, End range (nm) 400, Resolution (nm) 1.2 Other wavelength traces are extracted from the DAD data. MS detection: Waters SQD2, single quadrupole UPLC-MS It is sent to mass spectrometry at approximately 300 μL / min using a flow splitter. Scanning range (m / z) for MS data Start (m / z) 100 End (m / z) 700 or 1500 (as required) With +ve / -ve switching

[0181] Preparative reverse-phase HPLC conditions Preparative HPLC Waters Micromass ZQ / Sample Manager 2767 Photodiode array detector 2996; Column: XTerra preparative MS C18 column (5 μm, 19×150 mm, Waters) Flow rate: 20 mL / min with MS detection UV wavelength: 254 nm. Mobile phase: Solvent A (water: MeCN: HCO2H 95:5:0.05); Solvent B (water: MeCN: HCO2H 5:95:0.05) Gradient:

[0182]

Table 4

[0183] Flash chromatography is carried out using an Isolera MPLC system (manufactured by Biotage) using pre-packed silica gel or reverse-phase cartridges (provided by Biotage or Interchim).

[0184] B. Chemical synthesis The general procedures used in the method for preparing the compounds of the present invention are described below. Scheme 1

Chemical formula

[0185] 2-Chloro-5-(methylthio)pyrimidine (100B) Dimethyldisulfide (13.96 mL, 155.4 mmol, 1.0 eq) was added under nitrogen to a solution of 5-bromo-2-chloropyrimidine (100A) (30 g, 155.4 mmol, 1.0 eq) in anhydrous tetrahydrofuran (700 mL) cooled to -75 °C. To this mixture, a solution of n-butyllithium (2.5 M, 68.4 mL, 170.9 mmol, 1.0 eq) was added dropwise over 1.5 h, maintaining the internal temperature at -70 °C to -75 °C throughout the addition. After the addition was complete, the mixture was stirred at -75 °C for 4.5 h and then quenched by the slow addition of a saturated solution of ammonium chloride (100 mL). The cold bath was removed and the reaction was warmed to room temperature under nitrogen for 18 h. The reaction mixture was diluted with ethyl acetate (500 mL) and washed with water (50 mL) followed by saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting crude pale yellow oil was purified by flash chromatography (eluting with isohexane ~ ethyl acetate, 0 - 50%) to give the desired product 2-chloro-5-(methylthio)pyrimidine as a waxy pale yellow solid (100B). Yield: 7.39 g (29%). 1 H NMR (CDCl3) δ 8.48 (2H, s), 2.54 (3H, s); MS (ESI+) m / z 161 (M + H) + .

[0186] General Method 1 (2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)carbamic acid tert-butyl (100C) (3-Amino-2-methylpropyl) carbamic acid tert-butyl (4.79 g, 25.52 mmol, 1.05 eq) was added to a stirred solution of 2-chloro-5-(methylthio)pyrimidine (100B) (3.89 g, 24.31 mmol, 1.0 eq) and cesium carbonate (11.85 g, 36.46 mmol, 1.4 eq) in anhydrous dimethylformamide (50 mL). The mixture was stirred at room temperature for 18 h and then concentrated under reduced pressure to approximately 20 mL. The solution was diluted with ethyl acetate (100 mL), washed with water (75 mL) and brine (50 mL), and then dried over magnesium sulfate. The solvent was removed under vacuum to give a crude residue, which was purified by flash chromatography (eluting with isohexane~ethyl acetate, 0~50%) to give the desired product, (2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl) carbamic acid tert-butyl (100C) as a pale yellow oil. Yield: 6.59 g (86%) MS (ESI+) m / z 313 (M+H) + .

[0187] (S)-(2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl) carbamic acid tert-butyl (100D) Racemic (2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl) carbamic acid tert-butyl (100C) (5 g) was purified by chiral SFC using the following conditions: YMC amylose-C 30 / 70 MeOH / CO2, 100 mL / min, 120 bar, 40 °C, GLS 40 psi, System 3900 psi, drop 140 bar, Stacker, DAD 245 nm.

[0188] The first eluting isomer, 1.3 min. (R)-(2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl) carbamic acid tert-butyl Yield: 2.05 g. 11H NMR (400 MHz, CDCl3) δ 8.34 (s, 2H), 5.79 (dd, J = 6.7, 6.7 Hz, 1H), 5.18 (dd, J = 5.8, 5.8 Hz, 1H), 3.50 - 3.39 (m, 1H), 3.33 - 3.14 (m, 2H), 3.04 - 2.94 (m, 1H), 2.36 (s, 3H), 1.94 - 1.85 (m, 1H), 1.45 (s, 9H), 0.95 (d, J = 6.9 Hz, 3H). MS (ESI+) m / z 313 (M + H) + .

[0189] The isomer eluting second, 1.7 minutes. (S)-(2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)carbamic acid tert-butyl (100D) Yield: 2.48 g. 1 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 2H), 5.77 - 5.69 (m, 1H), 5.15 (dd, J = 6.5, 6.5 Hz, 1H), 3.50 - 3.40 (m, 1H), 3.33 - 3.16 (m, 2H), 3.03 - 2.94 (m, 1H), 2.36 (s, 3H), 1.95 - 1.85 (m, 1H), 1.47 (s, 9H), 0.95 (d, J = 6.9 Hz, 3H); MS (ESI+) m / z 313 (M + H) + .

[0190] General method 2 (R)-2-Methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (100E) A solution of hydrogen chloride (15 mL, 4 M in 1,4-dioxane) was added to (S)-(2-ethyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)carbamic acid tert-butyl (100D) (600 mg, 2.48 mmol), and the mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum to give the desired product (R)-2-methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (100E) as a pale yellow semi-solid. Yield: 548 mg (100%) HCl salt. 11H NMR (400 MHz, DMSO) δ 8.39 (s, 2H), 8.00 - 7.96 (m, 3H), 7.72 - 7.72 (m, 1H), 3.32 - 3.18 (m, 2H), 2.88 - 2.80 (m, 1H), 2.66 - 2.55 (m, 1H), 2.38 (s, 3H), 2.13 - 2.00 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 213 (M + H) + .

[0191] General method 3 (S)-Ethyl 2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylate (100F) Ethyl 2-chlorobenzo[b]thiazole-6-carboxylate (600 mg, 2.48 mmol, 1.0 equiv) was added under nitrogen to a stirred solution of (R)-2-methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (100E) (548 mg, 2.48 mmol, 1.0 equiv) and triethylamine (1.73 mL, 12.44 mmol, 5.0 equiv) in anhydrous dimethylformamide (20 mL). The mixture was stirred at room temperature for 48 h and then concentrated in vacuo to about 5 mL. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (20 mL) and brine (25 mL) and then dried over magnesium sulfate. The solvent was removed in vacuo to give a crude yellow oil which was purified by flash chromatography (eluting with isohexane ~ ethyl acetate, 0 - 100%) to give the desired (S)-ethyl 2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylate (100F) as an off-white solid. Yield: 647 mg (62%). 11H NMR (400 MHz, CDCl3) δ 8.37 (s, 2H), 8.27 (d, J = 1.5 Hz, 1H), 7.99 (dd, J = 1.8, 8.5 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.02 - 6.98 (m, 1H), 5.83 (dd, J = 6.7, 6.7 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 3.67 - 3.47 (m, 2H), 3.42 - 3.24 (m, 2H), 2.37 (s, 3H), 2.20 - 2.11 (m, 1H), 1.40 (dd, J = 7.2, 7.2 Hz, 3H), 1.07 (d, J = 7.0 Hz, 3H); MS (ESI+) m / z 418 (M + H) + .

[0192] General method 4 (S)-2-((2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylic acid (Example 1) Lithium hydroxide monohydrate (318 mg, 7.75 mmol, 5.0 eq) was added to a stirred solution of ethyl (S)-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylate (100F) (647 mg, 1.55 mmol, 1.0 eq) in ethanol (7 mL) and water (5 mL). The mixture was stirred at ambient temperature for 18 h and then concentrated under reduced pressure. Water (5 mL) was added to the residue and the mixture was acidified to pH ~3 with aqueous hydrochloric acid (2 M). The precipitate formed was collected by filtration, washed with water and then dried under high vacuum to give the desired product (S)-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylic acid (Example 1) as a pale yellow solid.

[0193] General method 5 (S)-2-Methyl-2-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxamido)propanoic acid methyl ester (100G) 1-[(Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 146 mg, 0.385 mmol, 1.5 eq) was added to a solution of (S)-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylic acid (1) (100 mg, 0.257 mmol, 1.0 eq), triethylamine (0.36 mL, 2.57 mmol, 10.0 eq) and methyl 2-amino-2-methylpropanoate hydrochloride (196 mg, 1.28 mmol, 4.9 eq) in dimethylformamide (5 mL), and the reaction mixture was stirred at room temperature for 18 h. The solvent was removed under reduced pressure, and the resulting crude residue was purified by flash chromatography (eluting with isohexane~ethyl acetate, 0~75%) to give the desired (S)-methyl 2-methyl-2-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxamido)propanoate (G) as an off-white solid. Yield: 120 mg (95%). 1 H NMR (400 MHz, MeOD) δ 8.34 (s, 2H), 8.10 (d, J = 1.8 Hz, 1H), 7.77 (dd, J = 1.9, 8.5 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 3.74 (s, 3H), 3.45 (dd, J = 6.3, 22.4 Hz, 4H), 2.34 (s, 3H), 2.29 - 2.16 (m, 1H), 1.59 (s, 6H), 1.08 (d, J = 6.9 Hz, 3H) NH exchangeable protons were not observed; MS (ESI+) m / z 489 (M + H) + .

[0194] (S)-2-Methyl-2-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxamido)propanoic acid (Example 2) A method similar to General Method 4 Lithium hydroxide monohydrate (50 mg, 1.22 mmol, 5.0 eq) was added to a stirred solution of methyl (S)-2-methyl-2-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxamido)propanoate (100G) (120 mg, 0.245 mmol, 1.0 eq) in ethanol (5 mL) and water (5 mL), and the mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum, the residue was diluted with water (3 mL), and acidified to pH ~3 with aqueous hydrochloric acid (2 M). The formed precipitate was collected by filtration, washed with water, and then dried under high vacuum to afford the title compound as an off-white solid.

[0195] The following examples were synthesized using the above procedure.

[0196] [Table 5]

[0197] [Table 6]

[0198] [Table 7]

[0199] [Table 8]

[0200] [Table 9]

[0201] [Table 10]

[0202] [Table 11]

[0203]

Table 12

[0204]

Table 13

[0205]

Table 14

[0206]

Table 15

[0207]

Table 16

[0208] Scheme 2

Chem.

[0209] General method 6 2-Chloro-N-(4-methoxybenzyl)benzo[d]thiazole-6-sulfonamide (101B) (4-Methoxyphenyl)methanamine (134 mg, 0.97 mmol, 1.05 equiv) was added dropwise to a solution of 2-chlorobenzothiazole-6-sulfonyl chloride (101A) (250 mg, 0.932 mmol, 1.0 equiv) and triethylamine (0.39 mL, 2.79 mmol, 3.0 equiv) in tetrahydrofuran (10 mL) at ice-cooled temperature, and the mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to obtain a solid, which was washed with ice-cold water (10 mL) and ice-cold tetrahydrofuran (10 mL), and then dried under vacuum to obtain the desired 2-chloro-N-(4-methoxybenzyl)benzothiazole-6-sulfonamide as a white solid (101B). Yield: 295 mg (85%). 1 H NMR (400 MHz, DMSO) δ 8.56 (d, J = 1.6 Hz, 1H), 8.24 (s, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.90 (dd, J = 1.9, 8.7 Hz, 1H), 7.10 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 3.98 (s, 2H), 3.67 (s, 3H); MS (ESI+) m / z 369 (M + H) + .

[0210] The intermediates in Table 2 were synthesized using conditions similar to those described for intermediate 101B.

[0211]

Table 17

[0212]

Table 18

[0213] (S)-N-(4-Methoxybenzyl)-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzothiazole-6-sulfonamide (101C) The applied methodology was similar to that described in General Method 3. Yield: 298 mg, (69%). 1 1H NMR (400 MHz, DMSO) δ 8.56 (1H, d, J = 1.6 Hz), 8.24 (1H, s), 8.11 (1H, d, J = 8.7 Hz), 7.90 (1H, dd, J = 1.9, 8.7 Hz), 7.10 (2H, d, J = 8.8 Hz), 6.76 (2H, d, J = 8.8 Hz), 3.98 (2H, s), 3.67 (3H, s); MS (ESI+) m / z 545 (M+H) + .

[0214] (S)-2-((2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzothiazole-6-sulfonamide (Example 87) Trifluoroacetic acid (5 mL) was added dropwise to an ice-cooled solution of N-(4-methoxybenzyl)-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzothiazole-6-sulfonamide (101C) (250 mg, 0.459 mmol, 1.0 equiv) in anhydrous dichloromethane (5 mL). The mixture was stirred on ice for 30 minutes and then warmed to ambient temperature over 18 hours. The reaction mixture was diluted with dichloromethane (15 mL) and saturated sodium bicarbonate (15 mL) was added slowly. The organic phase was separated, washed with brine (5 mL), dried over magnesium sulfate, and then concentrated to dryness under reduced pressure. The resulting crude residue was purified by flash chromatography (eluting with DCM~methanol, 0~10%) to give the desired (S)-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzothiazole-6-sulfonamide (Example 87) as a white solid. Yield: 190 mg (97%). 11H NMR (400 MHz, DMSO) δ 8.40 (dd, J = 5.5, 5.5 Hz, 1H), 8.34 (s, 2H), 8.12 (d, J = 1.6 Hz, 1H), 7.66 (dd, J = 2.0, 8.4 Hz, 1H), 7.51 (dd, J = 6.0, 6.0 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.21 (s, 2H), 3.49 - 3.42 (m, 1H), 3.31 - 3.23 (m, 3H), 2.34 (s, 3H), 2.17 - 2.07 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 425 (M+H) + .

[0215] Using the procedure described in Scheme 2, the following examples were prepared according to General Method 3.

[0216] [Table 19]

[0217] [Table 20]

[0218] [Table 21]

[0219] Scheme 3 [Chemical Structure]

[0220] 2-Methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (102A) The applicable methodology was the same as General Method 2. A solution of hydrogen chloride (54 mL, 4 M in 1,4-dioxane) was added to tert-butyl (2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)carbamate (100C) (4.25 g, 13.60 mmol), and the mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum to give the desired 2-methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (102A) as a pale yellow semi-solid. The semi-crude sample was carried on to the next reaction without further purification.

[0221] N 1 -(6-bromobenzo[d]thiazol-2-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (102B) The methodology applied was similar to general method 3. 6-Bromo-2-chlorobenzothiazole (828 mg, 3.33 mmol, 0.95 eq) was added under nitrogen to a stirred suspension of 2-methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (102A) (1.00 g, 3.51 mmol, 1.0 eq) and cesium carbonate (3.43 g, 10.52 mmol, 3.0 eq) in anhydrous dimethylformamide (25 mL). The mixture was stirred at room temperature for 72 h and then concentrated under vacuum to approximately 3 mL. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (20 mL) and brine (25 mL) and then dried over magnesium sulfate. The solvent was removed under vacuum to give a crude yellow oil which was purified by flash chromatography (eluting with isohexane ~ ethyl acetate, 0 - 100%) to give the desired N 1 -(6-bromobenzo[d]thiazol-2-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (102B) as a sticky yellow solid. Yield: 266 mg (18%). 11H NMR (400 MHz, CDCl3) δ 8.37 (s, 2H), 7.66 (d, J = 0.6 Hz, 1H), 7.37 (d, J = 1.9 Hz, 2H), 6.58 (s, 1H), 5.69 (dd, J = 6.3, 6.3 Hz, 1H), 3.67 - 3.23 (m, 4H), 2.37 (s, 3H), 2.17 - 2.09 (m, 1H), 1.06 (d, J = 6.9 Hz, 3H).

[0222] General method 7 tert-Butyl 4-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)-1H-pyrazole-1-carboxylate (102C) N 1 -(6-bromobenzo[d]thiazol-2-yl)-2-methyl-N 3A solution of (5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (102B) (50 mg, 0.12 mmol, 1.0 equiv) was added under nitrogen to a solution of (1-(tert-butoxycarbonyl)-1H-pyrazol-4-yl)boronic acid (27.8 mg, 0.13 mmol, 1.0 equiv), cesium carbonate (58 mg, 0.18 mmol, 1.5 equiv) and tetrakis(triphenylphosphine)palladium(0) (7 mg, 0.01 mmol, 0.05 equiv) in water (0.20 mL) and N,N-dimethylformamide (0.80 mL). The reaction mixture was heated to 90 °C for 16 h. An additional aliquot of (1-(tert-butoxycarbonyl)-1H-pyrazol-4-yl)boronic acid (27.8 mg, 0.13 mmol, 1.0 equiv) and tetrakis(triphenylphosphine)palladium(0) (7 mg, 0.01 mmol, 0.05 equiv) was added to the reaction and the mixture was heated to 90 °C for a further 16 h under nitrogen. The solvent was removed under reduced pressure; water (2 mL) was added and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with water (2 mL) and brine (2 mL), dried through a phase separator and then concentrated in vacuo. The crude residue obtained was purified by flash chromatography (eluting with isohexane~ethyl acetate, 0~75%) to give the desired tert-butyl 4-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)-1H-pyrazole-1-carboxylate (102C) as an off-white solid. The semi-crude product was carried forward to the next reaction without further purification.

[0223] N 1 -(6-(1H-Pyrazol-4-yl)benzo[d]thiazol-2-yl)-2-methyl-N 3 -(5-(Methylthio)pyrimidin-2-yl)propane-1,3-diamine (Example 97) A solution of hydrogen chloride (2 mL, 4 M in 1,4-dioxane) was added to tert-butyl 4-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)-1H-pyrazole-1-carboxylate (102C) (100 mg, 0.12 mmol), and the mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum to give the desired N 1 -(6-(1H-pyrazol-4-yl)benzo[d]thiazol-2-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (Example 97).

[0224] Using the procedure described in Scheme 3, the following examples were prepared according to General Method 7.

[0225]

Table 22

[0226] General Method 8 2-Methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)-N 3 -(6-(oxazol-2-yl)benzo[d]thiazol-2-yl)propane-1,3-diamine (Example 101) N 1 -(6-bromobenzo[d]thiazol-2-yl)-2-methyl-N 3-(5-(Methylthio)pyrimidin-2-yl)propane-1,3-diamine (102B) (90 mg, 0.21 mmol, 1.0 equiv) was added under nitrogen to a solution of 2-(tri-n-butylstannyl)oxazole (0.044 mL, 0.21 mmol, 1.0 equiv) and tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.02 mmol, 0.1 equiv) in N,N-dimethylformamide (0.80 mL). The reaction mixture was heated to 90 °C for 16 h. A further aliquot of 2-(tri-n-butylstannyl)oxazole (0.044 mL, 0.21 mmol, 1.0 equiv) and tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.02 mmol, 0.1 equiv) was added to the reaction mixture and heated to 110 °C for 18 h. The reaction was cooled to room temperature, diluted with water (2 mL), and extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with water (2 mL) and brine (2 mL), filtered through a pad of celite, dried by passing through a phase separator, and concentrated to dryness under vacuum. The resulting crude residue was purified twice by flash chromatography (isohexane~ethyl acetate, eluting first with 0~100% and then with ethyl acetate~methanol, 0~10%) to give a semi-crude residue, which was further purified by reverse-phase preparative HPLC to give the desired 2-methyl-N 1 -(5-(Methylthio)pyrimidin-2-yl)-N 3 -(6-(Oxazol-2-yl)benzo[d]thiazol-2-yl)propane-1,3-diamine (101) was obtained.

[0227] Using the procedure described in Scheme 3, the following examples were prepared according to General Method 8.

[0228]

Table 23

[0229] Scheme 4

Chem.

[0230] Using the procedure described in Scheme 4, the following examples were prepared according to General Method 3.

[0231] [Table 24]

[0232] Scheme 7 [Chemical formula]

[0233] (2,2-Dimethyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)carbamic acid tert-butyl (105A) The applied methodology was similar to that described in General Method 1 except that further heating was required. (3-Amino-2,2-dimethylpropyl)carbamic acid tert-butyl (0.13 g, 0.65 mmol, 1.05 eq) was added to a stirred suspension of 2-chloro-5-(methylthio)pyrimidine (100B) (0.10 g, 0.62 mmol, 1.0 eq) and cesium carbonate (0.24 g, 0.75 mmol, 1.2 eq) in anhydrous N,N-dimethylformamide (1.5 mL), and the mixture was stirred at 80 °C for 4 h. The reaction mixture was concentrated under vacuum, diluted with ethyl acetate (20 mL), washed with water (7.5 mL) and brine (5.0 mL), and then dried by passing through a phase separator. The solvent was removed under vacuum to give the desired (2,2-dimethyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)carbamic acid tert-butyl (105A) as a pale yellow oil. Yield: 0.163 g (81%). 11H NMR (400 MHz, DMSO) δ 8.37 (s, 2H), 7.24 (dd, J = 6.6, 6.6 Hz, 1H), 6.93 (dd, J = 6.3, 6.3 Hz, 1H), 3.20 (d, J = 6.8 Hz, 2H), 2.85 (d, J = 8.7 Hz, 2H), 2.40 (s, 3H), 1.43 (d, J = 3.3 Hz, 9H), 0.83 (s, 6H).

[0234] 2,2 - Dimethyl - N 1 -(5 - (methylthio)pyrimidin - 2 - yl)propane - 1,3 - diamine hydrochloride (105B) The applicable methodology was similar to that described in General Method 2. A solution of hydrogen chloride (5 mL, 4 M in 1,4 - dioxane) was added to tert - butyl (2,2 - dimethyl - 3 - ((5 - (methylthio)pyrimidin - 2 - yl)amino)propyl)carbamate (105A) (0.16 g, 0.50 mmol), and the mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum to give the desired 2,2 - dimethyl - N 1 -(5 - (methylthio)pyrimidin - 2 - yl)propane - 1,3 - diamine hydrochloride (105B) as a pale yellow semi - solid. Yield: 0.13 g (100%) HCl salt. 1 1H NMR (400 MHz, DMSO) δ 8.39 (s, 2H), 7.95 (s, 3H), 7.70 (s, 1H), 3.25 (d, J = 5.5 Hz, 2H), 2.68 - 2.61 (m, 2H), 2.38 (s, 3H), 0.96 (s, 6H).

[0235] N1 - (benzo[d]oxazol - 2 - yl) - 2,2 - dimethyl - N3 - (5 - (methylthio)pyrimidin - 2 - yl)propane - 1,3 - diamine (Example 164) The applicable methodology was similar to that described in General Method 3 except that cesium carbonate was used as the normal base. 2 - Chlorobenzoxazole (0.06 mL, 0.54 mmol, 0.1 equiv) was added to 2,2 - dimethyl - N in anhydrous N,N - dimethylformamide (2.0 mL) 1-(5-(Methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (105B) (0.13 g, 0.49 mmol, 1.0 equiv) and cesium carbonate (0.48 g, 1.48 mmol, 3.0 equiv) were added to a stirred solution under nitrogen. The mixture was stirred at either 80 °C or room temperature for 16 h and then concentrated under vacuum. Water (2.5 mL) was added and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with water (2 mL) and brine (2 mL), passed through a phase separator and dried. The solvent was removed under vacuum to give a crude yellow oil, which was purified by preparative HPLC to give the desired N 1 -(benzodioxazol-2-yl)-2,2-dimethyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (Example 164) was obtained.

[0236] Using the procedure described in Scheme 7, the following examples were prepared according to General Method 3.

[0237]

Table 25

[0238]

Table 26

[0239]

Table 27

[0240] Scheme 8

Chemical Formula

[0241] 3-chloro-N-(2-chlorobenzothiazol-6-yl)propane-1-sulfonamide (106B) Sodium hydride (60% dispersion in mineral oil) (326 mg, 8.15 mmol, 3.0 eq) was added portionwise to an ice-cold solution of 2-chlorobenzothiazol-6-amine (500 mg, 2.71 mmol, 1.0 eq) in N,N-dimethylformamide (25 mL), and the mixture was stirred under ice-cooling for 1 h. A solution of 3-chloropropane-1-sulfonyl chloride (673 mg, 3.80 mmol, 1.4 eq) in N,N-dimethylformamide (3 mL) was added dropwise, and subsequently the reaction mixture was warmed to ambient temperature over 3 h. The reaction mixture was diluted with brine (20 mL) and extracted with ethyl acetate (2×25 mL). The combined organic fractions were combined and concentrated under reduced pressure to give a pale yellow oil, which was purified by flash chromatography (eluting with iso-hexane~ethyl acetate, 0~100%) to afford the desired product 3-chloro-N-(2-chlorobenzo[d]thiazol-6-yl)propane-1-sulfonamide 106B as an off-white gum. Yield: 427 mg (48%). 1 H NMR (400 MHz, MeOD) δ 7.87 - 7.84 (m, 2H), 7.38 (dd, J = 2.3, 8.8 Hz, 1H), 3.67 (dd, J = 6.3, 6.3 Hz, 2H), 2.27 - 2.19 (m, 2H). Note the CH2 protons that were obscured by MeOD.

[0242] 2-(2-Chlorobenzo[d]thiazol-6-yl)isothiazolidine 1,1-dioxide (106C) Sodium hydride (60% dispersion in mineral oil) (98 mg, 2.46 mmol, 2.0 eq) was added to an ice-cooled solution of 3-chloro-N-(2-chlorobenzo[d]thiazol-6-yl)propane-1-sulfonamide (106B) (400 mg, 1.23 mmol, 1.0 eq) in N,N-dimethylformamide (5 mL). The reaction mixture was stirred for 1 h under ice-cooling and then quenched by careful addition of saturated ammonium chloride solution (20 mL). The resulting mixture was extracted with ethyl acetate (3 x 20 mL), and the combined organic phases were washed with water (20 mL) and brine (20 mL) and concentrated in vacuo to give a gum. The crude product was purified by flash chromatography (eluting with isohexane ~ ethyl acetate, 0 - 100%) to give the desired product (106C) as an off-white gum. Yield: 220 mg (62%). 1 H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.38 (dd, J = 2.4, 8.9 Hz, 1H), 3.84 (dd, J = 6.5, 6.5 Hz, 2H), 3.43 (dd, J = 7.5, 7.5 Hz, 2H), 2.63 - 2.55 (m, 2H).

[0243] 2-(2-((2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)isothiazolidine 1,1-dioxide (Example 175) The methodology applied was similar to that described in General Procedure 3. Yield: 25 mg (15%). 11H NMR (400 MHz, DMSO) δ 8.34 (s, 2H), 8.03 (dd, J = 5.6, 5.6 Hz, 1H), 7.57 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 5.9, 5.9 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.14 (dd, J = 2.4, 8.7 Hz, 1H), 3.72 (dd, J = 6.5, 6.5 Hz, 2H), 3.47 (dd, J = 7.5, 7.5 Hz, 2H), 3.45 - 3.38 (m, 1H), 3.30 - 3.22 (m, 3H), 2.44 - 2.36 (m, 2H), 2.35 (s, 3H), 2.13 - 2.06 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 465 (M + H) + .

[0244] Scheme 9

Chemical Structure

[0245] N-(2-Chlorobenzo[d]thiazol-6-yl)methanesulfonamide (107B) Methanesulfonyl chloride (0.055 mL, 0.706 mmol, 1.3 equiv) was added dropwise to an ice-cold solution of 2-chlorobenzothiazol-6-amine (100 mg, 0.54 mmol, 1.0 equiv) and pyridine (0.066 mL, 0.815 mmol, 1.5 equiv) in anhydrous dichloromethane (5 mL). The mixture was stirred at 0 °C for 15 minutes and then warmed to ambient temperature over 1 hour. The reaction mixture was quenched with water (1 mL). The organic phase was removed and concentrated under reduced pressure to afford a pale yellow oil, which was purified by flash chromatography (eluting with iso-hexane ~ ethyl acetate, 0 - 100%) to give the desired N-(2-chlorobenzo[d]thiazol-6-yl)methanesulfonamide (107B) as a pale yellow gum. Yield: 135 mg (94.8%) 11H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H), 3.00 (s, 3H). Aromatic H protons were obscured by CDCl3, and NH-exchangeable protons were not observed.

[0246] N-(2-Chlorobenzo[d]thiazol-6-yl)-N-methylmethanesulfonamide (107C) Sodium hydride (60% dispersion in mineral oil) (31 mg, 0.772 mmol, 1.5 eq) was added portionwise to an ice-cooled solution of N-(2-chlorobenzo[d]thiazol-6-yl)methanesulfonamide (107B) (135 mg, 0.515 mmol, 1.0 eq) in anhydrous tetrahydrofuran (2 mL). The mixture was stirred at room temperature for 2 h. Iodomethane (0.048 mL, 0.772 mmol, 1.5 eq) was added and the mixture was stirred at room temperature for an additional 2 h. Water (1 mL) was added and the solvent was subsequently removed under high vacuum to afford a pale yellow gum, which was purified by flash chromatography (eluting with iso-hexane~ethyl acetate, 0~100%) to give the desired N-(2-chlorobenzo[d]thiazol-6-yl)-N-methylmethanesulfonamide (107C) as a pale yellow gum. Yield: 100 mg (70%) 1 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 7.48 (dd, J = 2.3, 8.8 Hz, 1H), 3.39 (s, 3H), 2.88 (s, 3H).

[0247] N-Methyl-N-(2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)methanesulfonamide (Example 176) The methodology applied was similar to that described in General Method 3. Yield: 89 mg (60%) 11H NMR (400 MHz, CDCl3) δ 8.38 (s, 2H), 7.62 (d, J = 2.0 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.24 (d, J = 3.2 Hz, 1H), 5.74 (dd, J = 6.5, 6.5 Hz, 1H), 3.34 - 3.33 (m, 6H), 2.86 (s, 3H), 2.37 (s, 3H), 2.20 - 2.10 (m, 1H), 1.06 (d, J = 6.9 Hz, 3H). Not all proton exchangeable protons were observed; MS (ESI+) m / z 453 (M + H) + .

[0248] Scheme 10

Chem.

[0249] 4 - (((5 - (methylthio)pyrimidin - 2 - yl)amino)methyl)pyrrolidin - 2 - one (108B) The applied methodology was similar to that described in General Method 1. 5-Aminomethyl-pyrrolidin-2-one (108A) (1.0 g, 8.76 mmol, 1.0 equiv) was added to a stirred solution of 2-chloro-5-(methylthio)pyrimidine (100B) (1.4 g, 8.76 mmol, 1.0 equiv) and cesium carbonate (8.56 g, 26.28 mmol, 3.0 equiv) in anhydrous dimethylformamide (10 mL). The mixture was heated to 50 °C for 18 h and then concentrated under reduced pressure. The resulting solution was diluted with ethyl acetate (50 mL), washed with water (10 mL) and brine (10 mL), and then dried by passing through a phase separator. The solvent was removed under reduced pressure to give a crude residue, which was purified by trituration in methanol to give the desired product 4-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)pyrrolidin-2-one (108B) as a yellow solid. The aqueous phase was concentrated under reduced pressure, combined with the filtrate, and purified by flash chromatography (eluting with dichloromethane~methanol, 0~10%) to give the desired product 4-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)pyrrolidin-2-one (108B) as a yellow solid. The two collections were combined and used in the next step. Yield: 0.74 g (36%). 1 H NMR (400 MHz, CDCl3) δ 8.35 (s, 2H), 5.79 - 5.76 (m, 1H), 5.48 (dd, J = 5.5, 5.5 Hz, 1H), 3.57 - 3.50 (m, 3H), 3.20 (dd, J = 5.3, 9.5 Hz, 1H), 2.89 - 2.84 (m, 1H), 2.53 - 2.46 (m, 1H), 2.37 (s, 3H), 2.16 (dd, J = 6.4, 17.1 Hz, 1H).

[0250] tert-Butyl 4-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)-2-oxopyrrolidine-1-carboxylate (108C) 4-Dimethylaminopyridine (5 mg, 0.04 mmol, 0.1 eq) was added to a stirred suspension of 4-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)pyrrolidin-2-one (108B) (100 mg, 0.4 mmol, 1.0 eq), di-tert-butyl dicarbonate (229 mg, 1.0 mmol, 2.5 eq) and triethylamine (0.146 mL, 1.00 mmol, 2.5 eq) in dichloromethane (4.2 mL). The mixture was stirred at room temperature for 72 h, followed by the addition of water (3.0 mL). The mixture was extracted with ethyl acetate (3 × 5.0 mL), and the combined organic phases were washed with brine (2.5 mL), dried through a phase separator, and the solvent was removed under reduced pressure to give a crude residue, which was purified by reverse-phase chromatography (eluting with 0.1% formic acid solution - acetonitrile, 5 - 100%) to give the desired product tert-butyl 4-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)-2-oxopyrrolidine-1-carboxylate (108C). Yield: 72 mg (53%). 1 H NMR (400 MHz, DMSO) δ 8.35 (s, 2H), 7.62 (t, J = 6.1 Hz, 1H), 3.73 (dd, J = 7.8, 10.4 Hz, 1H), 3.50 - 3.44 (m, 1H), 3.29 (d, J = 6.4 Hz, 2H), 2.60 - 2.55 (m, 2H), 2.36 (s, 3H), 2.28 (dd, J = 9.6, 20.9 Hz, 1H), 1.44 (s, 9H).

[0251] (4-Amino-2-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)-4-oxobutyl)carbamic acid tert-butyl (108D) A solution of ammonium hydroxide (2.2 mL) was added to tert-butyl 4-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)-2-oxopyrrolidine-1-carboxylate (108C) (72 mg, 0.21 mmol), and the mixture was heated at 80 °C for 1.5 h. The mixture was cooled to room temperature and subsequently extracted with dichloromethane (3 × 5 mL). The organic solvent was dried through a phase separator and subsequently removed under reduced pressure to afford the desired product tert-butyl (4-amino-2-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)-4-oxobutyl)carbamate (108D). Yield: 61 mg (86%). 1 H NMR (400 MHz, DMSO) δ 8.33 (s, 2H), 7.32 (s, 1H), 7.25 (dd, J = 6.0, 6.0 Hz, 1H), 6.81 (s, 2H), 3.24 (dd, J = 6.2, 6.2 Hz, 2H), 2.97 (dd, J = 6.0, 6.0 Hz, 2H), 2.36 (s, 3H), 2.18 - 2.08 (m, 1H), 2.04 (d, J = 6.4 Hz, 2H), 1.38 (s, 9H).

[0252] 4-Amino-3-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)butanamide dihydrochloride (108E) The applied methodology was similar to that described in General Procedure 2. A solution of hydrogen chloride (0.7 mL, 4 M in 1,4-dioxane) was added to tert-butyl (4-amino-2-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)-4-oxobutyl)carbamate (108D) (61 mg, 0.17 mmol), and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure to afford the desired product 4-amino-3-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)butanamide dihydrochloride (108E) as a pale yellow solid. Yield: 56 mg (100%) HCl salt.

[0253] 4-((6-(N-(4-Methoxybenzyl)sulfamoyl)benzo[d]thiazol-2-yl)amino)-3-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)butanamide (177) The applicable methodology was similar to that described in General Method 3. 2-Chloro-N-(4-methoxybenzyl)benzo[d]thiazole-6-sulfonamide (101B) (70 mg, 0.19 mmol, 1.10 equiv) was added under nitrogen to a stirred solution of 4-amino-3-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)butanamide dihydrochloride (108E) (56 mg, 0.17 mmol, 1.0 equiv) and triethylamine (0.072 mL, 0.51 mmol, 3.0 equiv) in anhydrous dimethylformamide (2.0 mL). The mixture was stirred at room temperature for 72 h and then concentrated under reduced pressure. Water (2.5 mL) was added and the resulting precipitate was collected by filtration and subsequently washed with methanol. The organic filtrate was concentrated under reduced pressure to give a crude residue, which was purified by flash chromatography (eluting with dichloromethane~methanol, 0~25%) to afford the desired product 4-((6-(N-(4-methoxybenzyl)sulfamoyl)benzo[d]thiazol-2-yl)amino)-3-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)butanamide (Example 177) as an off-white solid. Yield: 25 mg (25%). 1 H NMR (400 MHz, DMSO) δ 8.45 (dd, J = 5.6, 5.6 Hz, 1H), 8.33 (s, 2H), 8.08 (d, J = 1.8 Hz, 1H), 7.88 (dd, J = 6.1, 6.1 Hz, 1H), 7.62 (dd, J = 1.9, 8.5 Hz, 1H), 7.49 - 7.42 (m, 2H), 7.39 - 7.37 (m, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.87 - 6.87 (m, 1H), 6.81 (d, J = 8.7 Hz, 2H), 3.89 (d, J = 6.0 Hz, 2H), 3.70 (s, 3H), 3.48 (d, J = 1.1 Hz, 2H), 3.37 (dd, J = 6.1, 6.1 Hz, 2H), 2.43 - 2.37 (m, 1H), 2.34 (s, 3H), 2.22 - 2.17 (m, 2H).

[0254] 4-((5-(Methylthio)pyrimidin-2-yl)amino)-3-(((6-sulfamoylbenzo[d]thiazol-2-yl)amino)methyl)butanamide (178) Trifluoroacetic acid (0.3 mL) was added dropwise to a solution of 4-((6-(N-(4-methoxybenzyl)sulfamoyl)benzo[d]thiazol-2-yl)amino)-3-(((5-(methylthio)pyrimidin-2-yl)amino)methyl)butanamide (177) (20 mg, 0.03 mmol, 1.0 equiv) cooled to 0 °C in anhydrous dichloromethane (0.3 mL). The mixture was stirred for 30 minutes and then warmed to room temperature over 8 hours. An additional aliquot of trifluoroacetic acid (1 mL) was added and the mixture was stirred for 16 hours. The reaction mixture was concentrated under pressure and then carefully made basic by the addition of saturated aqueous sodium bicarbonate (3 mL). The mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with brine (3 mL), dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The resulting crude residue was purified by flash chromatography (eluting with dichloromethane~methanol, 0~20%) to give the desired product 4-((5-(methylthio)pyrimidin-2-yl)amino)-3-(((6-sulfamoylbenzo[d]thiazol-2-yl)amino)methyl)butanamide (Example 178) as a white solid. Yield: 12 mg (75%). 1 H NMR (400 MHz, DMSO) δ 8.41 (dd, J = 5.5, 5.5 Hz, 1H), 8.33 (s, 2H), 8.13 (d, J = 1.8 Hz, 1H), 7.67 (dd, J = 1.9, 8.5 Hz, 1H), 7.47 - 7.43 (m, 2H), 7.38 (s, 1H), 7.21 (s, 2H), 6.86 (s, 1H), 3.46 - 3.46 (m, 2H), 3.36 (dd, J = 6.2, 6.2 Hz, 2H), 2.39 - 2.38 (m, 1H), 2.34 (s, 3H), 2.21 - 2.17 (m, 2H).

[0255] The following examples were synthesized using the procedure described in Scheme 10.

[0256]

Table 28

[0257] Scheme 11

Chem.

[0258] 4-((2-Chlorobenzo[d]thiazol-6-yl)sulfonyl)morpholine (109B) The applicable methodology was the same as that described in General Method 6, using dichloromethane instead of tetrahydrofuran as the solvent. Yield: 686 mg. 1 H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 1.8 Hz, 1H), 8.10 (d, J = 8.6 Hz, 1H), 7.85 (dd, J = 1.3, 8.6 Hz, 1H), 3.75 (dd, J = 4.7, 4.7 Hz, 4H), 3.04 (dd, J = 4.7, 4.7 Hz, 4H); MS (ESI+) m / z 319 (M + H)+.

[0259] (3-((5-(Difluoromethoxy)pyrimidin-2-yl)amino)-2-methylpropyl)carbamic acid tert-butyl (109D) The applicable methodology was the same as that described in General Method 1. Yield: 433 mg. 1 H NMR (400 MHz, CDCl3) δ 8.16 - 8.15 (m, 2H), 5.05 - 5.05 (m, 1H), 3.47 - 3.38 (m, 1H), 3.31 - 3.17 (m, 2H), 3.04 - 2.95 (m, 1H), 1.96 - 1.86 (m, 1H), 1.60 - 1.58 (m, 1H), 0.96 - 0.94 (m, 3H); MS (ESI+) m / z 333 (M + H)+.

[0260] N1-(5-(Difluoromethoxy)pyrimidin-2-yl)-2-methylpropane-1,3-diamine hydrochloride (109E) A solution of hydrogen chloride (2.7 mL, 4 M in 1,4-dioxane) was added to tert-butyl (3-((5-(difluoromethoxy)pyrimidin-2-yl)amino)-2-methylpropyl)carbamate (109D) (300 mg, 0.903 mmol), and the mixture was stirred at room temperature for 15 minutes. The solvent was removed under vacuum to give the crude title compound N1-(5-(difluoromethoxy)pyrimidin-2-yl)-2-methylpropane-1,3-diamine hydrochloride (109E), which was carried on to the next step without further purification. Yield: 225 mg (quantitative). MS (ESI+) m / z 233 (M+H)+.

[0261] N1-(5-(Difluoromethoxy)pyrimidin-2-yl)-2-methyl-N3-(6-(morpholinosulfonyl)benzo[d]thiazol-2-yl)propane-1,3-diamine (180) The methodology applied was similar to that described in General Method 3. Yield: 225 mg. 1 H NMR (400 MHz, CDCl3) δ 8.21 (s, 2H), 7.97 (d, J = 1.5 Hz, 1H), 7.65 (dd, J = 1.8, 8.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 6.91 (s, 1H), 6.43 (t, J = 71.6 Hz, 1H), 5.72 (dd, J = 6.6, 6.6 Hz, 1H), 3.77 - 3.73 (m, 4H), 3.62 - 3.51 (m, 2H), 3.43 - 3.29 (m, 2H), 3.01 (dd, J = 4.6, 4.6 Hz, 4H), 2.21 - 2.12 (m, 1H), 1.08 (d, J = 6.9 Hz, 3H); (ESI+) m / z 515 (M+H)+.

[0262] The following examples were synthesized according to the procedure described in Scheme 11.

[0263]

Table 29

[0264]

Table 30

[0265] Scheme 12

Chemical formula

[0266] (2-Methyl-3-(pyridin-2-ylamino)propyl)carbamic acid tert-butyl (110B) A solution of tert-butyl 3-amino-2-methylpropylcarbamate (110A) (150 mg, 0.80 mmol, 1.0 equiv) in 1,4-dioxane (2 mL) was added under nitrogen to a solution of 2-bromopyridine (0.076 mL, 0.80 mmol, 1.0 equiv), sodium tert-butoxide (383 mg, 3.98 mmol, 5.0 equiv), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (38 mg, 0.08 mmol, 0.1 equiv), and tris(dibenzylideneacetone)dipalladium(0) (73 mg, 0.08 mmol, 0.1 equiv) in 1,4-dioxane (10 mL). The reaction mixture was heated to 70 °C for 72 h. The solvent was removed under reduced pressure, and the resulting residue was partitioned between water (2 mL) and ethyl acetate (5 mL). The mixture was filtered through celite, and then the aqueous phase was removed and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with water (2 mL) and brine (2 mL), then dried by passing through a phase separator and concentrated to dryness under vacuum. The resulting crude residue was purified by reverse-phase chromatography (10 mM aqueous ammonium bicarbonate solution ~ acetonitrile, eluting with 5 - 95%) to give the desired (2-methyl-3-(pyridin-2-ylamino)propyl)carbamic acid tert-butyl (110B) as an off-white solid in somewhat pure form, which was used in the next step without further purification. Yield: 27 mg (12%). MS (ESI+) m / z 266 (M + H) + .

[0267] 2-Methyl-N 1 -(pyridin-2-yl)propane-1,3-diamine hydrochloride (110C) The methodology applied was similar to that described in General Method 2. A solution of hydrogen chloride (0.4 mL, 4 M in 1,4-dioxane) was added to tert-butyl (2-methyl-3-(pyridin-2-ylamino)propyl)carbamate (108B) (27 mg, 0.10 mmol), and the mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum to afford the desired product 2-methyl-N1-(pyridin-2-yl)propane-1,3-diamine hydrochloride (110C) as a pale yellow semi-solid. The semi-crude sample was transferred to the next reaction without further purification. Yield: 25 mg (assumed to be quantitative %).

[0268] 2-Methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)-N 3 -(pyridin-2-yl)propane-1,3-diamine (Example 187) The methodology applied was similar to that described in General Method 3. 2-Chloro-5-methylsulfanyl-pyrimidine (100B) (17 mg, 0.11 mmol, 1.05 equiv) was added under nitrogen to a stirred solution of 2-methyl-N 1 -(pyridin-2-yl)propane-1,3-diamine hydrochloride (110C) (24 mg, 0.10 mmol, 1.0 equiv) and cesium carbonate (99 mg, 0.30 mmol, 3.0 equiv) in anhydrous N,N-dimethylformamide (0.5 mL). The mixture was heated to 50 °C for 16 h and then concentrated under vacuum. Water (2 mL) was added, and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with water (2 mL) and brine (2 mL) and dried by passing through a phase separator. The solvent was removed under vacuum to afford a crude yellow oil, which was purified by reverse-phase chromatography (10 mM aqueous ammonium bicarbonate ~ acetonitrile, eluting with 5 - 95%) to afford the desired 2-methyl-N1 -(5-(Methylthio)pyrimidin-2-yl)-N 3 -(pyridin-2-yl)propane-1,3-diamine (Example 187) was obtained. Yield: 1.5 mg (5%). 1 H NMR (400 MHz, CDCl3) δ 8.35 (s, 2H), 8.10 (dd, J = 0.6, 3.6 Hz, 1H), 7.41 - 7.35 (m, 1H), 6.54 (dd, J = 5.3, 6.8 Hz, 1H), 6.38 (d, J = 8.4 Hz, 1H), 5.93 - 5.93 (m, 1H), 4.96 - 4.96 (m, 1H), 3.53 - 3.21 (m, 4H), 2.35 (s, 3H), 2.10 - 2.01 (m, 1H), 1.03 (d, J = 6.9 Hz, 3H); MS (ESI+) m / z 290 (M + H) + .

[0269] Scheme 13

Chemical Structure

[0270] Methyl 5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazine-2-carboxylate (188) The applied methodology was similar to that described in General Method 3. Methyl 5-bromopyrazine-2-carboxylate (114 mg, 0.53 mmol, 1.0 equiv) was added to 2-methyl-N in anhydrous N,N-dimethylformamide (2.0 mL) 1A stirred solution of (5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (102A) (150 mg, 0.53 mmol, 1.0 eq) and cesium carbonate (514 mg, 1.58 mmol, 3.0 eq) was added under nitrogen. The mixture was stirred at room temperature for 16 h and then concentrated under vacuum. Water (2.5 mL) was added and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with water (2 mL) and brine (2.5 mL) and then dried through a phase separator. The solvent was removed under vacuum to give a brown oil which was purified by flash chromatography (eluting with iso-hexane ~ ethyl acetate, 0 - 100%). The resulting semi-crude product was further purified by flash chromatography (eluting with dichloromethane ~ methanol, 0 - 10%) to give the desired methyl 5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazine-2-carboxylate (Example 188) as a white solid. Yield: 60 mg (32%). 1 H NMR (400 MHz, CDCl3) δ 8.76 (s, 1H), 8.37 (s, 2H), 7.91 (d, J = 1.4 Hz, 1H), 6.36 - 6.36 (m, 1H), 5.56 (dd, J = 6.0, 6.0 Hz, 1H), 3.95 (s, 3H), 3.60 - 3.51 (m, 2H), 3.40 - 3.26 (m, 2H), 2.38 (s, 3H), 2.10 - 2.03 (m, 1H), 1.04 (d, J = 6.9 Hz, 3H). MS (ESI+) m / z 349 (M + H) + .

[0271] 5-((2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazine-2-carboxylic acid (189) The methodology applied was similar to that described in General Method 4. Lithium hydroxide monohydrate (23 mg, 55 mmol, 5.0 equiv) was added to a stirred solution of methyl 5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazine-2-carboxylate (188) (38 mg, 0.11 mmol, 1.0 equiv) in ethanol (0.4 mL) and water (0.4 mL). The mixture was stirred at ambient temperature for 72 h and then concentrated under reduced pressure. Water (0.5 mL) was added to the residue and the mixture was acidified to pH ~3 with aqueous hydrochloric acid (2 M). The sticky precipitate was collected under filtration and then extracted with ethyl acetate (3 × 3 mL), washed with water (1 mL) and then dried by passing through a phase separator. The solvent was removed under vacuum to afford the desired 5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazine-2-carboxylic acid (Example 189) as a pale yellow solid. Yield: 34 mg (94%); 1 1H NMR (400 MHz, DMSO) δ 8.52 (s, 1H), 8.36 (s, 2H), 7.99 (s, 2H), 7.65 - 7.64 (m, 1H), 3.39 - 3.20 (m, 4H), 2.36 (s, 3H), 2.12 - 2.03 (m, 1H), 0.94 (d, J = 6.8 Hz, 3H), one NH proton was not observed; MS (ESI+) m / z 335 (M + H) + .

[0272] (4-Hydroxypiperidin-1-yl)(5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazin-2-yl)methanone (Example 190) The methodology applied was similar to that described in General Method 5. 1-[(Bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 58 mg, 0.15 mmol, 1.5 equiv) was added to a solution of 5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazine-2-carboxylic acid (189) (34 mg, 0.10 mmol, 1.0 equiv) and 4-hydroxypiperidine (103 mg, 1.02 mmol, 10 equiv) in N,N-dimethylformamide (1 mL), and the reaction mixture was stirred at room temperature for 18 h. The solvent was removed under reduced pressure, and the resulting crude residue was purified by reverse-phase preparative HPLC to give the desired (4-hydroxypiperidin-1-yl)(5-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazin-2-yl)methanone (Example 190) as an off-white solid. Yield: 22 mg (53%). 1 H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.37 (s, 2H), 7.79 (s, 1H), 5.97 - 5.97 (m, 1H), 5.71 - 5.71 (m, 1H), 4.15 - 4.15 (m, 2H), 4.01 - 3.94 (m, 1H), 3.58 - 3.47 (m, 2H), 3.42 - 3.25 (m, 4H), 2.37 (s, 3H), 2.08 (ddd, J = 11.5, 11.5, 5.1 Hz, 1H), 2.00 - 1.94 (m, 2H), 1.62 - 1.60 (m, 3H), 1.04 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 418 (M + H) + .

[0273] Scheme 16

Chemical Structure

[0274] 5-(5-Methyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine (111B) The methodology applied was similar to that described in General Method 7. 2-Bromo-5-methyl-1,3,4-oxadiazole (0.58 g, 3.58 mmol, 1.05 equiv) was added under nitrogen to a solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (111A) (0.75 g, 3.41 mmol, 1.0 equiv), cesium carbonate (3.33 g, 10.22 mmol, 3.0 equiv) and tetrakis(triphenylphosphine)palladium(0) (0.39 g, 0.34 mmol, 0.10 equiv) in water (5.5 mL) and 1,4-dioxane (22.5 mL). The reaction mixture was heated to 100 °C for 16 h. The solvent was removed under vacuum, water (20 mL) was added, and the mixture was extracted with ethyl acetate (6 × 50 mL). The combined organic phases were washed with water (10 mL) and brine (10 mL), dried through a phase separator and then concentrated under vacuum. The resulting crude residue was purified by reverse phase chromatography (10 mM ammonium bicarbonate solution ~ acetonitrile, eluting with 5 - 30%) to give the desired 5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine (111B) as an off-white solid. Yield: 153 mg (25%). 1 H NMR (400 MHz, DMSO) δ 8.49 (d, J = 2.0 Hz, 1H), 7.87 (dd, J = 2.4, 8.8 Hz, 1H), 6.75 (s, 2H), 6.57 (d, J = 8.8 Hz, 1H), 2.53 (s, 3H).

[0275] (2-Methyl-3-((5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)amino)propyl)carbamic acid tert-butyl (111C) tert-Butyl N-(2-methyl-3-oxopropyl)carbamate (111B) (225 mg, 1.21 mmol, 1.2 equiv) was added to a solution of 5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine (114B) (153 mg, 1.00 mmol, 1.0 equiv), acetic acid (0.230 mL, 4.02 mmol, 4.0 equiv) and molecular sieves (4 Å type, 250 mg) in anhydrous dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 5 min, followed by the addition of sodium triacetoxyborohydride (532 mg, 2.51 mmol, 2.5 equiv) all at once. The mixture was stirred at room temperature for 40 h. tert-Butyl N-(2-methyl-3-oxopropyl)carbamate (225 mg, 1.21 mmol, 1.2 equiv) was added and the reaction mixture was stirred at room temperature for a further 72 h. The reaction was quenched by the careful addition of a saturated aqueous solution of sodium hydrogen carbonate (30 mL). The mixture was stirred vigorously for 30 min, followed by isolation of the dichloromethane layer and concentration under reduced pressure to give a gum. The crude product was purified by flash chromatography (eluting with isohexane~ethyl acetate, 0~100%) to give the desired tert-butyl (2-methyl-3-((5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)amino)propyl)carbamate (111C) as an off-white gum. The crude sample was carried on to the next reaction without further purification. Yield: 101 mg (29%).

[0276] 2-Methyl-N 1 -(5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)propane-1,3-diamine hydrochloride (111D) The methodology applied was similar to that described in General Method 2. A solution of hydrogen chloride (1.2 mL, 4 M in 1,4-dioxane) was added to tert-butyl (2-methyl-3-((5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)amino)propyl)carbamate (110C) (101 mg, 0.29 mmol), and the mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum to afford the desired 2-methyl-N 1 -(5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)propane-1,3-diamine hydrochloride (111D) as an off-white solid. The crude sample was carried on to the next reaction without further purification. Yield: 93 mg (assumed to be quantitative %).

[0277] 2-Methyl-N 1 -(5-(5-methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (Example 203) The methodology applied was similar to that described in General Method 3. 2-Chloro-5-methylsulfanyl-pyrimidine (100B, 51 mg, 0.32 mmol, 1.1 equiv) was added to 2-methyl-N in anhydrous N,N-dimethylformamide (2.9 mL) 1-(5-(5-Methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)propane-1,3-diamine hydrochloride (111D) (93 mg, 0.29 mmol, 1.0 equiv) and cesium carbonate (283 mg, 0.87 mmol, 3.0 equiv) were added to a stirred suspension under nitrogen. The mixture was stirred at room temperature for 16 h, heated to 40 °C for a further 16 h and then concentrated in vacuo. Water (10 mL) was added and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water (10 mL) and brine (10 mL) and then dried by passing through a phase separator. The solvent was removed in vacuo to give a yellow oil which was purified by flash chromatography (eluting with isohexane ~ ethyl acetate, 0 - 100%) to give a crude residue which was further purified by reverse phase preparative HPLC to give the desired 2-methyl-N 1 -(5-(5-Methyl-1,3,4-oxadiazol-2-yl)pyridin-2-yl)-N 3 -(5-(Methylthio)pyrimidin-2-yl)propane-1,3-diamine (Example 203) was obtained. Yield: 3.5 mg (3%). 1 H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.8 Hz, 1H), 8.37 (s, 2H), 8.00 (dd, J = 2.3, 8.8 Hz, 1H), 6.47 (d, J = 8.1 Hz, 1H), 5.79 - 5.69 (m, 2H), 3.55 - 3.25 (m, 4H), 2.59 (s, 3H), 2.37 (s, 3H), 2.12 - 2.04 (m, 1H), 1.05 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 372 (M + H) + .

[0278] Scheme 17

Chemical Structure

[0279] Ethyl 2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carboxylate (112B) To the reaction tube, (R)-2-methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (100E) (544 mg, 2.19 mmol), ethyl 2-(6-bromopyridin-3-yl)cyclopropane-1-carboxylate (650 mg, 2.41 mmol), L-proline (101 mg, 0.875 mmol), potassium phosphate (928 mg, 4.37 mmol) and dimethyl sulfoxide (5 mL) were added. The mixture was aerated with nitrogen for 2 minutes, and then copper(I) iodide (83 mg, 0.437 mmol) was added. The tube was sealed under nitrogen and heated at 90 °C overnight. The reaction was cooled, diluted with ethyl acetate (20 mL), passed through a celite pad, and the filtrate was concentrated under vacuum. The resulting residue was diluted with ethyl acetate (30 mL) and water (20 mL), the aqueous phase was separated, and the residue was extracted with ethyl acetate (2 × 30 mL). The combined organics were washed with water (2 × 40 mL) and brine (2 × 50 mL), dried over magnesium sulfate, and then concentrated under vacuum to give the crude title compound ethyl 2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carboxylate (112B) as a light brown oil. (Note Purification via normal phase chromatography failed to resolve some close impurities). Yield: 284 mg. 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 8.21 (dd, J = 2.1, 6.2 Hz, 1H), 7.95 (s, 1H), 7.26 - 7.19 (m, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.33 (d, J = 8.6 Hz, 1H), 4.22 - 4.15 (m, 2H), 3.53 - 3.46 (m, 1H), 3.39 - 3.31 (m, 2H), 3.23 - 3.17 (m, 1H), 2.49 (ddd, J = 11.8, 11.8, 11.8 Hz, 1H), 2.33 (s, 3H), 2.12 - 2.02 (m, 1H), 1.79 - 1.74 (m, 1H), 1.55 - 1.48 (m, 1H), 1.32 - 1.24 (m, 3H), 1.24 - 1.17 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 402 (M + H)+ .

[0280] 2-(6-(((R)-2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carboxylic acid (112C) To a solution of ethyl 2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carboxylate (112B, 274 mg, 0.682 mmol) in ethanol (6 mL) and water (4 mL) was added lithium hydroxide monohydrate (143 mg, 3.41 mmol), and the resulting mixture was stirred at room temperature for 1 h. The mixture was concentrated under vacuum, and the resulting residue was diluted with water (8 mL). The solution was adjusted to pH ca. 2 with 2 M aqueous hydrochloric acid and subsequently extracted with dichloromethane / methanol (20% methanol in dichloromethane, 2 × 10 mL). The combined organic layers were passed through a phase separator cartridge and concentrated under vacuum to give the crude title compound 2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carboxylic acid (112C) as a brown oil, which was used directly without further purification. Yield: 280 mg. MS(ESI+) m / z 374 (M + H) + .

[0281] tert-Butyl 7-(2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropanecarbonyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate (112D) tert-Butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate (65 mg, 0.281 mmol) was added to a solution of 2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carboxylic acid (112C, 70 mg, 0.187 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 86 mg, 0.225 mmol), and triethylamine (0.26 mL, 1.87 mmol) in dimethylformamide (2 mL), and the resulting mixture was stirred at room temperature for 18 h. After completion, the reaction mixture was concentrated under vacuum, and the obtained residue was diluted with ethyl acetate (5 mL) and water (3 mL). The aqueous phase was separated and extracted with ethyl acetate (2 × 5 mL). The combined organics were washed with water (5 mL), brine (2 × 10 mL), dried over magnesium sulfate, and then concentrated under vacuum. The obtained crude residue was purified using column chromatography (eluting with 0–10% methanol in dichloromethane) to give the title compound 7-(2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropanecarbonyl)-2,7-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl (112D) as an off-white solid. Yield: 69 mg. 11H NMR (400 MHz, CDCl3) δ 8.35 (s, 2H), 7.92 (d, J = 1.5 Hz, 1H), 7.15 (dd, J = 2.1, 8.5 Hz, 1H), 6.34 (d, J = 8.6 Hz, 1H), 5.96 (dd, J = 6.2, 6.2 Hz, 1H), 5.00 (s, 1H), 3.73 - 3.63 (m, 5H), 3.49 (s, 4H), 3.38 - 3.29 (m, 2H), 3.26 - 3.18 (m, 2H), 2.37 - 2.30 (m, 4H), 2.10 - 1.98 (m, 1H), 1.89 - 1.82 (m, 1H), 1.61 - 1.53 (m, 2H), 1.44 (s, 9H), 1.29 - 1.23 (m, 2H), 1.21 - 1.14 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z (M+H) + .

[0282] (2-(6-(((R)-2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropyl)(2,7-diazaspiro[3.5]nonan-7-yl)methanone (Example 204) Trifluoroacetic acid (0.2 mL) was added to a stirred solution of tert-butyl 7-(2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropane-1-carbonyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate (112D, 69 mg, 0.119 mmol) in dichloromethane (2 mL), and the mixture was stirred at room temperature for 30 minutes. The solvent was removed under vacuum and subsequently azeotroped with dichloromethane (3 × 5 mL). The resulting crude residue was dissolved in dimethyl sulfoxide (1.5 mL) and purified by preparative HPLC. The resulting solution was dried under vacuum and subsequently lyophilized from an acetonitrile / water mixture to give the title compound (2-(6-(((R)-2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyridin-3-yl)cyclopropyl)(2,7-diazaspiro[3.5]nonan-7-yl)methanone (204) as a fluffy white solid. Yield: 15 mg. 11H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 8.35 (s, 2H), 7.89 - 7.86 (m, 1H), 7.16 (dd, J = 2.0, 8.6 Hz, 1H), 6.36 (d, J = 8.6 Hz, 1H), 5.98 (dd, J = 6.9, 6.9 Hz, 1H), 5.49 - 5.49 (m, 1H), 3.76 (s, 4H), 3.51 - 3.43 (m, 2H), 3.39 - 3.30 (m, 3H), 3.22 (dd, J = 6.6, 13.4 Hz, 2H), 2.38 - 2.31 (m, 4H), 2.11 - 2.01 (m, 1H), 1.89 - 1.79 (m, 5H), 1.59 - 1.52 (m, 1H), 1.27 - 1.14 (m, 1H), 1.03 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 482 (M + H) + .

[0283] According to the procedure described in Scheme 17, the following examples were synthesized.

[0284]

Table 31

[0285] Scheme 18

Chemical formula

[0286] N 1 - (5 - Bromopyrazin - 2 - yl) - 2 - methyl - N3 - (5 - (methylthio)pyrimidin - 2 - yl)propane - 1,3 - diamine (113B) A solution of crude 2-methyl-N-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine hydrochloride (100E) (340 mg, 1.60 mmol, 1.0 eq) in dimethylformamide (8 mL) was added to a suspension of 2,5-dibromopyrazine (457 mg, 1.92 mmol, 1.2 eq) and cesium carbonate (1.56 g, 4.80 mmol, 3.0 eq) in dimethylformamide (2 mL). The reaction mixture was heated to 90 °C for 18 h. The solvent was removed under reduced pressure and the resulting residue was partitioned between water (20 mL) and dichloromethane (50 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (3 × 25 mL). The combined organic phases were washed with brine (25 mL), dried by passing through a phase separator and then concentrated to dryness under vacuum. The crude residue was purified by flash chromatography (eluting with iso-hexane ~ ethyl acetate, 0 - 100%) to give the title compound N 1 -(5-bromopyrazin-2-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (113B) as a pale yellow gum. Yield: 230 mg (38%). 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 8.06 (d, J = 1.3 Hz, 1H), 7.67 (d, J = 1.4 Hz, 1H), 5.67 - 5.51 (m, 2H), 3.55 - 3.31 (m, 3H), 3.25 - 3.12 (m, 1H), 2.37 (s, 3H), 2.04 (s, 1H), 1.03 (d, J = 6.9 Hz, 3H).

[0287] N 1 -(5-(2-methoxypyridin-3-yl)pyrazin-2-yl)-2-methyl-N3-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (209) The methodology applied was similar to that described in General Method 7 (Note: 1,4-dioxane was used instead of dimethylformamide). N 1-A solution of (5-bromopyrazin-2-yl)-2-methyl-N3-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (113B) (50 mg, 0.135 mmol, 1.0 eq) was added under a nitrogen atmosphere to a solution of 2-methoxy-3-phenylboronic acid (23 mg, 0.149 mmol, 1.1 eq), cesium carbonate (132 mg, 0.406 mmol, 3 eq) and tetrakis(triphenylphosphine)palladium(0) (15.6 mg, 0.0135 mmol, 0.1 eq) in water (1 mL) and 1,4-dioxane (2 mL). The reaction mixture was heated to 80 °C for 1 h. The solvent was removed under reduced pressure, water (2 mL) was added, and the mixture was extracted with ethyl acetate (3 × 15 mL). The combined organic phases were washed with brine (10 mL) and concentrated in vacuo. The resulting crude residue was purified by preparative HPLC to give the desired N 1 - (5-(2-Methoxypyridin-3-yl)pyrazin-2-yl)-2-methyl-N3-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (154). Yield: 24 mg (44%). 1 1H NMR (400 MHz, CDCl3) δ 8.77 (d, J = 1.4 Hz, 1H), 8.37 (s, 2H), 8.18 - 8.13 (m, 2H), 7.99 (d, J = 1.5 Hz, 1H), 7.01 (dd, J = 4.9, 7.4 Hz, 1H), 5.69 (dd, J = 6.5, 6.5 Hz, 1H), 5.55 (dd, J = 6.1, 6.1 Hz, 1H), 4.04 (s, 3H), 3.59 - 3.48 (m, 2H), 3.42 - 3.25 (m, 2H), 2.37 (s, 3H), 2.19 - 2.06 (m, 1H), 1.06 (d, J = 6.9 Hz, 3H); MS (ESI+) m / z 398 (M + H) + .

[0288] Scheme 19

Chemical formula

[0289] (2-Methyl-3-oxopropyl)carbamic acid tert-butyl (114B) Attention - Exothermic reaction; Dess-Martin periodinane (2.94 g, 6.94 mmol, 1.3 equiv) was added portionwise over 20 min to a solution of tert-butyl (3-hydroxy-2-methylpropyl)carbamate (1.0 g, 5.34 mmol, 1.0 equiv) in dichloromethane (50 mL), and the mixture was stirred at room temperature for 2 h. The mixture was diluted with dichloromethane (25 mL) and washed with 1 M aqueous sodium dithionite solution (2 x 10 mL) and saturated aqueous sodium hydrogen carbonate solution (2 x 10 mL). The organic phase was passed through a phase separator and dried, and then concentrated under vacuum to give crude tert-butyl (2-methyl-3-oxopropyl)carbamate (114B), which was used immediately in the next step without further purification.

[0290] tert-Butyl (3-((5-bromopyridin-2-yl)amino)-2-methylpropyl)carbamate (114C) The methodology applied was similar to that described in Scheme 13 (for the formation of 109D). The crude product was purified by flash chromatography (eluting with iso-hexane ~ ethyl acetate, 0 - 100%) to give the desired tert-butyl (3-((5-bromopyridin-2-yl)amino)-2-methylpropyl)carbamate (114C) as a pale yellow gum. Yield: 875 mg (47%). (MS(ESI+) m / z 345 (M+H) + .

[0291] tert-Butyl (3-((2’-methoxy-[3,3’-bipyridin]-6-yl)amino)-2-methylpropyl)carbamate (114D) The methodology applied was similar to that described in General Method 7. Crude tert-butyl (3-((2’-methoxy-[3,3’-bipyridin]-6-yl)amino)-2-methylpropyl)carbamate (114D) was used immediately in the next step without further purification. Yield: 110 mg, MS(ESI+) m / z 373 (M+H)+ .

[0292] N1-(2'-Methoxy-[3,3'-bipyridin]-6-yl)-2-methylpropane-1,3-diamine (114E) The applied methodology was similar to that described in General Method 2. Crude N 1 -(2'-Methoxy-[3,3'-bipyridin]-6-yl)-2-methylpropane-1,3-diamine (114E) was immediately transferred to the next step without further purification. Crude yield: 75 mg MS(ESI+) m / z 273 (M+H) + .

[0293] N 1 -(2'-Methoxy-[3,3'-bipyridin]-6-yl)-2-methyl-N3-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (210) The applied methodology was similar to that described in General Method 1. The obtained crude residue was purified by preparative HPLC to give the desired product, N 1 -(2'-Methoxy-[3,3'-bipyridin]-6-yl)-2-methyl-N3-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine as an off-white solid (155). Yield: 8 mg (7%). 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 8.30 (d, J = 2.1 Hz, 1H), 8.11 (dd, J = 1.9, 5.0 Hz, 1H), 7.67 (dd, J = 2.4, 8.7 Hz, 1H), 7.57 (dd, J = 1.9, 7.3 Hz, 1H), 6.95 (dd, J = 5.0, 7.3 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 5.90 (dd, J = 6.3, 6.3 Hz, 1H), 5.11 (dd, J = 6.0, 6.0 Hz, 1H), 3.97 (s, 3H), 3.56 - 3.23 (m, 4H), 2.36 (s, 3H), 2.13 - 2.04 (m, 1H), 1.05 (d, J = 6.9 Hz, 3H); MS(ESI+) m / z 397 (M+H) +.

[0294] Scheme 20

Chemical formula

[0295] (3-([3,3'-Bipyridin]-6-ylamino)-2-methylpropyl)carbamic acid tert-butyl (115A) The applied methodology was the same as that of Scheme 19 (Example 115B) for generating aldehyde (114B), and subsequently the same methodology as General Method 7 was used. Crude N 1 -([3,3'-Bipyridin]-6-yl)-2-methylpropane-1,3-diamine (115A) was immediately transferred to the next step. Yield: 253 mg (63%) 1 1H NMR (400 MHz, DMSO) δ 8.82 (d, J = 1.8 Hz, 1H), 8.48 (dd, J = 1.6, 4.8 Hz, 1H), 8.36 (d, J = 2.1 Hz, 1H), 8.00 - 7.96 (m, 1H), 7.76 (dd, J = 2.6, 8.7 Hz, 1H), 7.44 - 7.40 (m, 1H), 6.90 - 6.76 (m, 2H), 6.62 - 6.59 (m, 1H), 4.10 (q, J = 5.3 Hz, 1H), 3.19 - 3.18 (m, 4H), 3.03 - 2.94 (m, 1H), 2.88 - 2.80 (m, 1H), 1.93 - 1.82 (m, 1H), 1.40 - 1.38 (m, 9H).

[0296] N 1 -([3,3'-Bipyridin]-6-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (211) The applied methodology was the same as the method described in Scheme 13 (regarding the generation of 109D). The obtained crude residue was purified by reverse-phase chromatography (10 mM aqueous ammonium hydrogen carbonate solution ~ acetonitrile, eluting with 5 - 95%) to give the desired product, N 1-([3,3'-Bipyridin]-6-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (211) was obtained. Yield: 32 mg (14%). 1 H NMR (400 MHz, CDCl3) δ 8.77 (1H, d, J = 2.1 Hz), 8.53 (1H, dd, J = 1.6, 4.8 Hz), 8.37 - 8.35 (3H, m), 7.81 - 7.77 (1H, m), 7.63 (1H, dd, J = 2.5, 8.7 Hz), 7.33 (1H, dd, J = 4.6, 7.8 Hz), 6.50 (1H, d, J = 8.7 Hz), 5.93 (1H, dd, J = 6.1, 6.1 Hz), 5.25 (1H, dd, J = 6.1, 6.1 Hz), 3.56 - 3.25 (4H, m), 2.36 (3H, s), 2.18 - 2.00 (1H, m), 1.06 (3H, d, J = 6.9 Hz); MS (ESI+) m / z 367 (M + H) + .

[0297] Scheme 21

Chemical Structure

[0298] 2-Bromo-5-((trimethylsilyl)ethynyl)pyridine (116B) Triethylamine (49 mL, 352.25 mmol, 25 equiv) was added to 2-bromo-5-iodopyridine (4.0 g, 14.09 mmol, 1.0 equiv), followed by the addition of ethynyltrimethylsilane (2.9 mL, 21.13 mmol, 1.5 equiv), copper(I) iodide (270 mg, 1.41 mmol, 0.1 equiv), and bis(triphenylphosphine)palladium(II) dichloride (99 mg, 0.14 mmol, 0.01 equiv). The mixture was stirred at room temperature for 17 h. The mixture was concentrated under vacuum to give a crude gummy material, which was purified by flash chromatography (eluting with iso-hexane ~ ethyl acetate, 0 - 40%) to give the intermediate 2-bromo-5-((trimethylsilyl)ethynyl)pyridine (116B) as a white solid. Yield: 2.5 g (69%). MS (ESI+) m / z 254 / 256 (M+H) + .

[0299] 2-Bromo-5-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-5-yl)pyridine (116C) To a solution of 2-bromo-5-((trimethylsilyl)ethynyl)pyridine (116B) (250 mg, 0.983 mmol, 1.0 equiv) in ethanol (20 mL) was added 1-(azidomethyl)-4-methoxybenzene (177 mg, 1.08 mmol, 1.1 equiv), and the mixture was stirred at room temperature for 1 h. A solution of 1M tetrabutylammonium fluoride in tetrahydrofuran (0.54 mL, 1.08 mmol, 1.1 equiv) was added, and the mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated under vacuum and subsequently purified by flash chromatography (eluting with iso-hexane~ethyl acetate, 0~100%) to give 2-bromo-5-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-5-yl)pyridine (116C) as an off-white solid. Yield: 0.21 g (61%). MS (ESI+) m / z 347 (M+H) + .

[0300] (3-((5-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-5-yl)pyridin-2-yl)amino)-2-methylpropyl)carbamic acid tert-butyl (116D) To a degassed solution of 2-bromo-5-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-5-yl)pyridine (116C) (135 mg, 0.391 mmol, 1.0 equiv) in dimethyl sulfoxide (5 mL) were added tert-butyl (3-amino-2-methylpropyl)carbamate (96 mg, 0.508 mmol, 1.3 equiv), L-proline (18 mg, 0.156 mmol, 0.4 equiv), potassium phosphate (165 mg, 0.782 mmol, 0.2 equiv) and copper(I) iodide (15 mg, 0.078 mmol, 0.2 equiv). The mixture was stirred at 90 °C for 28 h, diluted with water (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic phases were concentrated under vacuum and the crude residue was purified by flash chromatography (eluting with iso-hexane~ethyl acetate, 0~100%) to afford 2-bromo-5-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-5-yl)pyridine (116D) as a light brown solid. Yield: 0.080 g (45%). MS(ESI+) m / z 453 (M+H) + .

[0301] (3-((5-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-5-yl)pyridin-2-yl)amino)-2-methylpropyl)carbamic acid tert-butyl (116E) The applied methodology was similar to that described in General Method 2. Yield: 25 mg (29%). 1 H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 2.0 Hz, 1H), 8.35 (s, 2H), 7.90 (dd, J = 2.4, 8.7 Hz, 1H), 7.52 (s, 1H), 6.94 - 6.90 (m, 2H), 6.44 (d, J = 9.0 Hz, 1H), 5.94 (dd, J = 6.1, 6.1 Hz, 1H), 5.49 (s, 2H), 5.09 (dd, J = 6.3, 6.3 Hz, 1H), 3.81 (s, 4H), 3.53 - 3.22 (m, 4H), 2.35 (s, 3H), 2.10 - 2.01 (m, 1H), 1.03 (d, J = 6.9 Hz, 3H), (NH not observed).

[0302] N 1 -(5-(1H-1,2,3-triazol-5-yl)pyridin-2-yl)-2-methyl-N3-(4-(methylthio)phenyl)propane-1,3-diamine (212) The applied methodology was similar to that described in general method 3. Yield: 18 mg (96%). 1 H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 2.1 Hz, 1H), 8.38 (s, 2H), 7.84 (s, 1H), 7.82 (dd, J = 2.3, 8.7 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 6.17 (dd, J = 6.3, 6.3 Hz, 1H), 5.31 (dd, J = 6.0, 6.0 Hz, 1H), 3.57 - 3.26 (m, 4H), 2.36 (s, 3H), 2.19 - 2.06 (m, 1H), 1.06 (d, J = 6.8 Hz, 3H), (NH not observed); MS (ESI+) m / z 457 (M + H) + .

[0303] Scheme 22

Chemical Structure

[0304] (2-methyl-3-((5-(pyridin-2-yl)pyrazin-2-yl)amino)propyl)carbamic acid tert-butyl (117B) To a solution of 5-(pyridin-2-yl)pyrazine-2-amine (200 mg, 1.16 mmol, 1.0 equiv) in dichloromethane (10 mL) were added tert-butyl (2-methyl-3-oxopropyl)carbamate (117B) (221 mg, 1.17 mmol, 1.0 equiv), 3 Å molecular sieves (750 mg), acetic acid (0.266 mL, 4.65 mmol), and sodium triacetoxyborohydride (616 mg, 2.9 mmol), and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane (25 mL) and filtered. The organic phase was washed with water (25 mL) and brine (25 mL), and then passed through a phase separator cartridge. The solvent was removed under vacuum to afford crude tert-butyl (2-methyl-3-((5-(pyridin-2-yl)pyrazin-2-yl)amino)propyl)carbamate (117B), which was used in the next step without further purification. Yield: 310 mg (78%).

[0305] 2-Methyl-N 1 -(5-(pyridin-2-yl)pyrazin-2-yl)propane-1,3-diamine (117C) The methodology applied was similar to that described in General Method 2. Yield: 200 mg (100%).

[0306] 2-Methyl-N 1 -(5-(methylthio)pyrimidin-2-yl)-N3-(5-(pyridin-2-yl)pyrazin-2-yl)propane-1,3-diamine (213) The methodology applied was similar to that described in General Method 1. Yield: 20 mg (10%). 11H NMR (400 MHz, CDCl3) δ 9.04 (d, J = 1.4 Hz, 1H), 8.62 - 8.61 (m, 1H), 8.37 (s, 2H), 8.10 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 1.4 Hz, 1H), 7.77 - 7.72 (m, 1H), 7.20 (dd, J = 4.8, 6.5 Hz, 1H), 5.75 - 5.63 (m, 2H), 3.58 - 3.50 (m, 2H), 3.42 - 3.28 (m, 2H), 2.37 (s, 3H), 2.19 - 2.06 (m, 1H), 1.06 (d, J = 6.9 Hz, 3H); MS (ESI+) m / z 368 (M + H) + .

[0307] According to the procedure described in Scheme 22, the following examples were synthesized.

[0308]

Table 32

[0309] Scheme 23

Chemical Structure

[0310] 5-(1-Methyl-1H-1,2,3-triazol-4-yl)pyrazin-2-amine (118B) The applicable methodology was similar to that described in General Procedure 7 (note that 1,4-dioxane / water used as the solvent instead of dimethylformamide was used). Yield: 158 mg (73%) m / z 176 (M + H) + .

[0311] (2-Methyl-3-((5-(1-methyl-1H-1,2,3-triazol-4-yl)pyrazin-2-yl)amino)propyl)carbamic acid tert-butyl (118C) The applicable methodology was similar to the method described in Scheme 13 (for the formation of 109D). Crude tert-butyl (2-methyl-3-((5-(1-methyl-1H-1,2,3-triazol-4-yl)pyrazin-2-yl)amino)propyl)carbamate (118C) was immediately transferred to the next step. Yield: 101 mg (34%) MS (ESI+) m / z 347 (M+H) + .

[0312] 2-Methyl-N1-(5-(1-methyl-1H-1,2,3-triazol-4-yl)pyrazin-2-yl)propane-1,3-diamine (118D) The applied methodology was similar to that described in General Method 2. Crude 2-methyl-N 1 -(5-(1-methyl-1H-1,2,3-triazol-4-yl)pyrazin-2-yl)propane-1,3-diamine (106D) was immediately transferred to the next step. Yield: 71 mg (expected) MS (ESI+) m / z 367 (M+H) + .

[0313] 2-Methyl-N1-(5-(1-methyl-1H-1,2,3-triazol-4-yl)pyrazin-2-yl)-N3-(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (218) The applied methodology was similar to that described in General Method 1. Yield: 25 mg (23%). 1 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 3H), 8.30 (s, 1H), 8.05 (dd, J = 2.3, 8.9 Hz, 1H), 7.67 (s, 1H), 6.56 (d, J = 9.2 Hz, 1H), 6.11 - 6.06 (m, 1H), 4.14 (s, 3H), 2.63 (s, 4H), 2.35 (s, 3H), 2.19 - 2.10 (m, 1H), 1.07 (d, J = 6.8 Hz, 3H); MS (ESI+) m / z 371 (M+H) + .

[0314] Scheme 24

Chemical Structure

[0315] N 1 -(5-Bromopyrazin-2-yl)-2-methyl-N 3 -(5-(Methylthio)pyrimidin-2-yl)propane-1,3-diamine (119a) The applied methodology was similar to that described in General Method 3. Yield: 230 mg (38%). 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 8.06 (d, J = 1.3 Hz, 1H), 7.67 (d, J = 1.4 Hz, 1H), 5.67 - 5.51 (m, 2H), 5.30 (s, 2H), 3.55 - 3.31 (m, 3H), 3.25 - 3.12 (m, 1H), 2.37 (s, 3H), 2.04 (s, 1H), 1.03 (d, J = 6.9 Hz, 3H).

[0316] 2-Methyl-N 1 -(5-(Methylthio)pyrimidin-2-yl)-N 3 -(5-(5-(Trifluoromethyl)-1H-pyrazol-4-yl)pyrazin-2-yl)propane-1,3-diamine (219) The applied methodology was similar to that described in General Method 7 (Note: 1,4-dioxane / water was used and no additional aliquots of reagents were added). Yield: 18 mg (31%). 1 H NMR (400 MHz, DMSO) δ 13.70 (s, 1H), 8.35 - 8.34 (m, 2H), 8.26 (d, J = 1.0 Hz, 1H), 8.13 - 8.12 (m, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.55 - 7.44 (m, 1H), 7.23 - 7.18 (m, 1H), 3.35 - 3.18 (m, 4H), 2.35 (s, 3H), 2.11 - 2.02 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H); MS(ESI+) m / z 426 (M + H) + .

[0317] Scheme 25 [Chemical]

[0318] (2-Methyl-3-(pyrazolo[1,5-a]pyrimidin-5-ylamino)propyl)carbamic acid tert-butyl (120A) A microwave vial containing 5-chloropyrazolo[1,5-c]pyrimidine (500 mg, 3.26 mmol, 1 equivalent) and tert-butyl (3-amino-2-methylpropyl)carbamate (100E) (6.13 g, 32.56 mmol, 10 equivalents) was heated to 140 °C for 30 minutes under microwave irradiation. The crude reaction mixture was purified by flash chromatography (eluting with iso-hexane~ethyl acetate, 0~100%) to obtain (2-Methyl-3-(pyrazolo[1,5-a]pyrimidin-5-ylamino)propyl)carbamic acid tert-butyl (120A). Yield: 700 mg (70%). MS(ESI+) m / z 306 (M+H) + .

[0319] 2-Methyl-N 1 -(pyrazolo[1,5-a]pyrimidin-5-yl)propane-1,3-diamine (120B) The applied methodology was similar to that described in General Method 2. MS(ESI+) m / z 229 (M+H) + .

[0320] (2-Methyl-3-(pyrazolo[1,5-a]pyrimidin-5-ylamino)propyl)carbamic acid tert-butyl (220) The applied methodology was similar to that described in General Method 3. Yield: 20 mg (14%). 11H NMR (400 MHz, DMSO) δ 7.34 (s, 2H), 7.27 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 1.8 Hz, 1H), 5.29 (d, J = 7.6 Hz, 1H), 5.08 (d, J = 1.5 Hz, 1H), 2.44 - 2.37 (m, 4H), 1.36 (s, 3H), 1.20 - 1.11 (m, 1H), (2xNH not observed); MS (ESI+) m / z 330 (M + H) + .

[0321] Scheme 26

Chem.

[0322] Methyl 6 - bromonicotinate imidate (121B) Sodium methoxide (0.71 g, 13.22 mmol, 1.1 eq) was added to an ice - cold solution of 6 - bromonicotinonitrile (121A) (2.2 g, 12.02 mmol, 1.0 eq) in dioxane / water (20 mL / 20 mL). The mixture was stirred for 30 minutes under ice - cooling and then warmed to room temperature. After 1 hour, the mixture was diluted with ethyl acetate (200 mL) and water (200 mL). The organic phase was separated and the aqueous phase was further extracted with ethyl acetate (50 mL). The organics were combined and the solvent was removed under vacuum to give crude methyl 6 - bromonicotinate imidate (121B), which was used in the next step without further purification. Yield: 2.5 g (96%).

[0323] 6 - Bromo - N’ - methylnicotinohydrazide (121C) Methylhydrazine (0.73 mL, 13.96 mmol, 1.2 eq) was added to a solution of 6 - bromonicotinimidate (121B) (2.5 g, 11.63 mmol, 1.0 eq) and the mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum to give 6 - bromo - N’ - methylnicotinohydrazide (121C), which was used in the next step without further purification. Yield: 2 g (75%).

[0324] 2-Bromo-5-(1-methyl-1H-1,2,4-triazol-3-yl)pyridine (121D) Formic acid (10 mL, 265 mmol, 30.4 eq) was added to 6-bromo-N'-methylnicotinohydrazide (121C) (2.0 g, 8.73 mmol, 1.0 eq), and the mixture was heated to reflux for 1 h. The reaction mixture was diluted with water (100 mL) and subsequently extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with a saturated aqueous solution of sodium bicarbonate (50 mL), dried over sodium sulfate, and filtered. The solvent was removed under vacuum to give a crude brown residue, which was purified by flash chromatography (eluting with 0 - 10% methanol in dichloromethane) to afford 2-bromo-5-(1-methyl-1H-1,2,4-triazol-3-yl)pyridine (121D) as an off-white solid. Yield: 500 mg (23%). MS (ESI+) m / z 240 (M + H) + .

[0325] (2-Methyl-3-((5-(1-methyl-1H-1,2,4-triazol-3-yl)pyridin-2-yl)amino)propyl)carbamic acid tert-butyl (121E) The applied methodology was similar to that described in General Method 7 (Note 1,4-dioxane / water was used and no additional aliquots of reagents were added). Yield: 125 mg

[0326] 2-Methyl-N 1 -(5-(1-methyl-1H-1,2,4-triazol-3-yl)pyridin-2-yl)-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (221) The applied methodology was similar to that described in General Method 2 for deprotection followed by General Method 1. Yield: 25 mg (18%). 11H NMR (400 MHz, DMSO) δ 8.42 (d, J = 2.0 Hz, 1H), 8.34 (s, 2H), 8.04 (s, 1H), 7.76 (dd, J = 2.4, 8.8 Hz, 1H), 7.48 (dd, J = 6.0, 6.0 Hz, 1H), 6.82 (dd, J = 5.8, 5.8 Hz, 1H), 6.57 (d, J = 8.4 Hz, 1H), 4.16 (s, 3H), 3.32 - 3.17 (m, 4H), 2.35 (s, 3H), 2.09 - 1.99 (m, 1H), 0.93 (d, J = 6.7 Hz, 3H); MS (ESI+) m / z 371 (M + H) + .

[0327] Scheme 27

Chem.

[0328] (3 - ((5 - Bromopyridin - 2 - yl)amino)-2 - methylpropyl)carbamic acid tert - butyl (122B) tert - Butyl N - (2 - methyl - 3 - oxopropyl)carbamate (100E) (1.0 g, 5.34 mmol, 1.0 equiv) was added to a solution of 5 - bromopyridin - 2 - amine (122A) (920 mg, 5.34 mmol, 1.0 equiv), acetic acid (1.2 mL, 21.36 mmol, 4.0 equiv) and molecular sieves (4 Å type, 1.0 g) in anhydrous dichloromethane (50 mL). The reaction mixture was stirred at room temperature for 5 minutes, and then sodium triacetoxyborohydride (2.83 g, 13.35 mmol, 2.5 equiv) was added all at once. The mixture was stirred at room temperature for 5 hours. The reaction was quenched by the careful addition of a saturated aqueous solution of sodium bicarbonate (30 mL). The mixture was stirred vigorously for 30 minutes, and then the dichloromethane layer was separated and washed with an aqueous solution of sodium thiosulfate (1 M, 15 mL). The organic phase was isolated and concentrated under reduced pressure to give semi - crude (3 - ((5 - bromopyridin - 2 - yl)amino)-2 - methylpropyl)carbamic acid tert - butyl (122B) as a light brown gum, which was used in the next reaction without further purification. Yield: 875 mg (47%).

[0329] (3 - ((2’-Methoxy-[3,3’-bipyridin]-6-yl)amino)-2-methylpropyl)carbamic acid tert-butyl (122C) The applicable methodology was similar to that described in General Method 7. Yield: 138 mg.

[0330] N 1 -(2’-Methoxy-[3,3’-bipyridin]-6-yl)-2-methyl-N 3 -(5-(methylthio)pyrimidin-2-yl)propane-1,3-diamine (222) The applicable methodology was similar to that described for General Method 2 for deprotection and the subsequent General Method 1. Yield: 57 mg. 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 8.23 (d, J = 2.0 Hz, 1H), 7.68 (dd, J = 2.3, 8.7 Hz, 1H), 7.32 - 7.27 (m, 2H), 7.03 - 6.95 (m, 2H), 6.48 (d, J = 8.7 Hz, 1H), 5.94 - 5.90 (m, 2H), 3.82 (s, 3H), 3.56 - 3.47 (m, 1H), 3.44 - 3.35 (m, 2H), 3.28 (dd, J = 6.6, 13.4 Hz, 1H), 2.35 (s, 3H), 2.18 - 2.07 (m, 1H), 1.06 (d, J = 6.9 Hz, 3H); MS(ESI+) m / z 396 (M + H) + .

[0331] Scheme 28

Chemical Structure

[0332] 6’-Chloro-2H-[1,3’-bipyridin]-2-one (123B) 2-Amino-5-iodopyridine (123A) (1.12 g, 5.00 mmol, 1.0 equiv) was combined with 2-hydroxypyridine (582 mg, 6.00 mmol, 1.2 equiv), potassium carbonate (760 mg, 5.50 mmol, 1.1 equiv), copper(I) iodide (143 mg, 0.75 mmol, 0.15 equiv) and 8-hydroxyquinoline (110 mg, 0.75 mmol, 0.15 equiv) in anhydrous dimethylsulfonamide (5 mL). The mixture was degassed under a nitrogen stream and heated at 130 °C for 21 h. The reaction mixture was cooled to room temperature and then poured into a mixture of 10% aqueous ammonium hydroxide (100 mL) and ethyl acetate (50 mL). Activated carbon (1 g) was added and the mixture was filtered through a pad of celite and washed with ethyl acetate (2 × 50 mL). The layers were separated and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate, filtered and then concentrated under reduced pressure. The crude pale yellow solid was purified by flash chromatography (eluting with dichloromethane ~ methanol, 0 - 10%) to afford the desired product 6'-chloro-2H-[1,3'-bipyridin]-2-one as an off-white solid (123B). Yield: 245 mg, (26%). 1 H NMR (400 MHz, DMSO) δ 7.88 (1H, d, J = 2.5 Hz), 7.60 (1H, ddd, J = 0.7, 2.1, 6.8 Hz), 7.49 (1H, ddd, J = 2.2, 6.7, 9.1 Hz), 7.41 (1H, dd, J = 2.7, 8.7 Hz), 6.52 (1H, dd, J = 0.4, 8.8 Hz), 6.45 (1H, ddd, J = 0.7, 1.3, 9.2 Hz), 6.28 (1H, ddd, J = 6.7, 6.7, 1.3 Hz), 6.23 (2H, s); MS (ESI+) m / z 188 (M + H) + .

[0333] (2-Methyl-3-((2-oxo-2H-[1,3'-bipyridin]-6'-yl)amino)propyl)carbamic acid tert-butyl (123C) The applied methodology was similar to that described in Scheme 19. 6’-Chloro-2H-[1,3’-bipyridin]-2-one (123B) was used in excess (245 mg, 1.31 mmol, 1.1 equiv). 4 Å molecular sieves were used in the reaction. The crude product was purified by flash chromatography (eluting with dichloromethane ~ methanol, 0 - 7%) to give the desired product tert-butyl (2-methyl-3-((2-oxo-2H-[1,3’-bipyridin]-6’-yl)amino)propyl)carbamate (123C) as a light brown solid. Yield: 249 mg, (58%). MS (ESI+) m / z 359 (M+H) + .

[0334] 6’-((2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)-2H-[1,3’-bipyridin]-2-one (223) The methodology applied was similar to that described in General Method 2 using a (4:1) ratio of 4M HCl in 1,4-dioxane - water, followed by General Method 3 using cesium carbonate (3.0 equiv) combined with triethylamine (2.0 equiv). Yield: 152 mg (57% over 2 steps). 1 H NMR (400 MHz, DMSO) δ 8.35 (2H, s), 7.91 (1H, d, J = 2.6 Hz), 7.61 (1H, ddd, J = 0.6, 2.1, 6.8 Hz), 7.52 - 7.45 (2H, m), 7.40 (1H, dd, J = 2.7, 8.9 Hz), 6.90 (1H, dd, J = 5.8, 5.8 Hz), 6.57 (1H, dd, J = 0.5, 8.9 Hz), 6.45 (1H, ddd, J = 0.7, 1.2, 9.2 Hz), 6.28 (1H, ddd, J = 6.7, 6.7, 1.4 Hz), 3.33 - 3.14 (4H, m), 2.35 (3H, s), 2.10 - 2.00 (1H, m), 0.94 (3H, d, J = 6.8 Hz); MS (ESI+) m / z 383 (M+H) + .

[0335] Using the procedure described in Scheme 28, the following examples were synthesized.

[0336]

Table 33

[0337]

Table 34

[0338]

Table 35

[0339] Scheme 29

Chemical formula

[0340] (2-Methyl-3-((5-(2-oxopyrrolidin-1-yl)pyrazin-2-yl)amino)propyl)carbamic acid tert-butyl (124B) A solution of tert-butyl N-(3-amino-2-methylpropyl)carbamate (100E) (436 mg, 2.20 mmol, 1.1 eq) in anhydrous dimethylsulfonamide (10 mL) was added to 2-bromo-5-(pyrrolidin-1-yl)pyrazine (124A) (510 mg, 2.00 mmol, 1.0 eq), tripotassium phosphate (866 mg, 4.00 mmol, 2 eq), L-proline (94 mg, 0.80 mmol, 0.4 eq) and copper(I) iodide (76 mg, 0.40 mmol, 0.2 eq). The mixture was degassed and maintained under a nitrogen stream and heated at 90 °C for 20 h with stirring. The reaction mixture was cooled to room temperature and then poured into a mixture of water (50 mL) and ethyl acetate (50 mL). The layers were separated and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate, filtered and then concentrated under reduced pressure. The crude product was purified by flash chromatography (eluting with dichloromethane~methanol, 0~6%) to give the desired product, tert-butyl (2-methyl-3-((5-(2-oxopyrrolidin-1-yl)pyrazin-2-yl)amino)propyl)carbamate (124B) as a yellow solid. Yield: 375 mg, (54%). MS (ESI+) m / z 350 (M+H) + .

[0341] 1-(5-((2-Methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)pyrazin-2-yl)pyrrolidin-2-one (233) The methodology applied was similar to that described in General Method 2 using a [4:1] ratio of 4M HCl in 1,4-dioxane-water, followed by General Method 3. Yield: 71 mg (18% over 2 steps). 11H NMR (400 MHz, CDCl3) δ 9.02 (1H, d, J = 1.5 Hz), 8.35 (2H, s), 7.64 (1H, d, J = 1.5 Hz), 5.63 (1H, dd, J = 6.2, 6.2 Hz), 5.24 (1H, dd, J = 5.7, 5.7 Hz), 3.97 (2H, t, J = 7.1 Hz), 3.55 - 3.46 (1H, m), 3.45 - 3.32 (2H, m), 3.28 - 3.19 (1H, m), 2.62 (2H, t, J = 8.1 Hz), 2.36 (3H, s), 2.20 - 2.02 (3H, m), 1.03 (3H, d, J = 6.8 Hz); MS (ESI+) m / z 374 (M + H) + .

[0342] Scheme 30

Chem.

[0343] tert-Butyl 2-(2,2-difluoroethyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (125B) 1,1-Difluoro-2-iodo-ethane (128 mg, 0.67 mmol, 1.2 equiv) was added to a suspension of tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate (125A) (126 mg, 0.56 mmol, 1.0 equiv) and potassium carbonate (231 mg, 1.67 mmol, 3.0 equiv) in dimethylformamide (1.0 mL). The mixture was stirred at 60 °C for 2 h. The reaction mixture was cooled to room temperature, diluted with water and extracted with ethyl acetate. The organic phase was washed with brine, dried by passing through a phase separator and then concentrated under reduced pressure to give the desired tert-butyl 2-(2,2-difluoroethyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (125B) as a pale yellow oil. Yield: 73 mg (45%). 1 1H NMR (400 MHz, CDCl3) δ 5.87 - 5.57 (m, 1H), 3.41 - 3.30 (m, 4H), 3.14 (s, 4H), 2.89 - 2.77 (m, 2H), 1.76 - 1.67 (m, 4H), 1.46 (s, 9H).

[0344] 2-(2,2-Difluoroethyl)-2,7-diazaspiro[3.5]nonane (125C) Trifluoroacetic acid (1.3 mL) was added dropwise to a solution of tert-butyl 2-(2,2-difluoroethyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (125B) (73 mg, 0.25 mmol, 1.0 equiv) in dichloromethane (1.3 mL). The mixture was stirred for 30 minutes and then concentrated to dryness under reduced pressure to afford the desired product 2-(2,2-difluoroethyl)-2,7-diazaspiro[3.5]nonane (125C) as a pale yellow oil, which was used in the next reaction without further purification.

[0345] The intermediates in Table 17 were synthesized using conditions similar to those described for Intermediate 125C.

[0346] [Table 36]

[0347] Scheme 31 [Chemical Structure]

[0348] tert-Butyl 2-(2,2,2-trifluoroethyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (126B). 2,2,2-Trifluoroethyl trifluoromethanesulfonate (153 mL, 1.06 mmol, 1.2 equiv) was added to a suspension of tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate (126A) (200 mg, 0.88 mmol, 1.0 equiv) and cesium carbonate (862 mg, 2.65 mmol, 3.0 equiv) in acetonitrile (2.0 mL), and the mixture was stirred at 80 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The combined organic phases were washed with brine, passed through a phase separator and dried, and then concentrated under reduced pressure to give tert-butyl 2-(2,2,2-trifluoroethyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (126B) as a sticky white solid. The sample was used in the next step without further purification. Yield: 341 mg (quantitative). 1 H NMR (400 MHz, CDCl3) δ 3.38 - 3.30 (m, 4H), 3.19 (s, 4H), 3.01 (q, J = 9.4 Hz, 2H), 1.73 - 1.69 (m, 4H), 1.45 (s, 9H).

[0349] 2-(2,2,2-Trifluoroethyl)-2,7-diazaspiro[3.5]nonane (126C) Trifluoroacetic acid (1.3 mL) was added dropwise to a solution of tert-butyl 2-(2,2,2-trifluoroethyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (126B) (110 mg, 0.357 mmol, 1.0 equiv) in dichloromethane (1.0 mL). The mixture was stirred for 30 min and then concentrated to dryness under reduced pressure to give 2-(2,2,2-trifluoroethyl)-2,7-diazaspiro[3.5]nonane (126C) as a light brown oil, which was used directly in the next step without further purification and was assumed to be in 100% yield.

[0350] Scheme 32

Chemical Structure

[0351] 2-Methyl-5-oxa-2,8-diazaspiro[3.5]nonane (127B) Lithium aluminum hydride (1 M solution in tetrahydrofuran, 1.97 mL, 1.97 mmol, 3.0 eq) was added dropwise under nitrogen to a solution of tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-2-carboxylate (127A) (150 mg, 0.657 mmol, 1.0 eq) in tetrahydrofuran (4.0 mL) cooled to 0 °C. The reaction was stirred at 0 °C for 5 minutes, the cold bath was removed, and the reaction was subsequently heated to 70 °C for 16 hours (boiling was observed at 0 °C and 35 °C). The reaction mixture was cooled to 0 °C and quenched by the slow addition of water (0.075 mL), aqueous sodium hydroxide (15%, 0.075 mL), and then water (0.22 mL). The cold bath was removed, the reaction mixture was stirred for 15 minutes, magnesium sulfate was added, and stirring was continued for 40 minutes. The mixture was passed through a phase separator and dried, washed with tetrahydrofuran and diethyl ether, and then concentrated to dryness under reduced pressure to afford 2-methyl-5-oxa-2,8-diazaspiro[3.5]nonane (127B) as a light brown oil. The sample was transferred to the next reaction without further purification. Yield: Assumed to be 100%. 1 H NMR (400 MHz, CDCl3) δ 3.63 - 3.56 (m, 2H), 3.47 - 3.43 (m, 2H), 3.00 (s, 2H), 2.94 - 2.86 (m, 2H), 2.83 - 2.79 (m, 2H), 2.40 (s, 3H), NH not observed.

[0352] The intermediates in Table 18 were synthesized using conditions similar to those described for Intermediate 127B.

[0353]

Table 37

[0354] Scheme 33

Chem.

[0355] tert-Butyl 6-(dimethylcarbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (128B) The method used was the same as that described in General Method 5 using 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid to obtain the desired product tert-butyl 6-(dimethylcarbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (128B). Yield: 111 mg (quantitative). 1 H NMR (400 MHz, DMSO) δ 3.87 (s, 2H), 3.70 (s, 2H), 3.23 - 3.14 (m, 1H), 2.86 (s, 3H), 2.79 (s, 3H), 2.32 - 2.24 (m, 4H), 1.37 - 1.36 (m, 9H).

[0356] N,N-Dimethyl-2-azaspiro[3.3]heptane-6-carboxamide (128C) Trifluoroacetic acid (2.1 mL) was added dropwise to a solution of tert-butyl 6-(dimethylcarbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (128B) (110 mg, 0.357 mmol, 1.0 eq) in dichloromethane (2.1 mL). The mixture was stirred for 30 minutes and then concentrated to dryness under reduced pressure to give the desired product N,N-dimethyl-2-azaspiro[3.3]heptane-6-carboxamide (128C) as a brown oil. The sample was transferred to the next reaction without further purification and was assumed to be in 100% yield.

[0357] Scheme 34

Chemical Structure

[0358] (S)-Methyl 5-fluoro-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carboxylate (129A) The applicable methodology was the same as that described in General Method 3. Yield: 134 mg (20%). 1 H NMR (400 MHz, CDCl3) δ 8.38 (s, 2H), 8.13 (d, J = 6.9 Hz, 1H), 7.21 (d, J = 12.2 Hz, 1H), 7.12 (s, 1H), 5.62 (t, J = 6.7 Hz, 1H), 3.93 (s, 3H), 3.64 - 3.46 (m, 2H), 3.42 - 3.22 (m, 2H), 2.38 - 2.38 (m, 3H), 2.18 - 2.09 (m, 1H), 1.07 (d, J = 6.9 Hz, 3H); MS (ESI+) m / z 422 (M + H) + .

[0359] (S)-5-Fluoro-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzothiazole-6-carboxylic acid (129B) The applicable methodology was the same as that described in General Method 4. Yield: 98 mg (quantitative). 1 H NMR (400 MHz, DMSO) δ 12.82 (s, 1H), 8.57 - 8.54 (m, 1H), 8.33 - 8.32 (m, 2H), 8.18 (d, J = 7.4 Hz, 1H), 7.50 (t, J = 6.1 Hz, 1H), 7.16 (d, J = 12.5 Hz, 1H), 2.34 (s, 3H), 2.16 - 2.06 (m, 1H), 0.97 - 0.94 (m, 3H). NH exchangeable proton was not observed; MS (ESI+) m / z 408 (M + H) + .

[0360] (S)-6-(5-Fluoro-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzothiazole-6-carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylic acid tert-butyl (129C) The applicable methodology was the same as that described in General Method 5. Yield: 20 mg (54%). 11H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 1.8 Hz, 1H), 8.14 (s, 1H), 7.99 (dd, J = 1.7, 8.5 Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.00 - 6.97 (m, 1H), 5.47 (dd, J = 6.7, 6.7 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 3.63 - 3.49 (m, 2H), 3.40 - 3.23 (m, 2H), 2.47 (s, 3H), 2.17 - 2.09 (m, 1H), 1.40 (dd, J = 7.2, 7.2 Hz, 3H), 1.06 (d, J = 6.9 Hz, 3H); MS (ESI+) m / z 489 (M + H) + .

[0361] (S)-(5-Fluoro-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)(2,6-diazaspiro[3.4]octan-6-yl)methanone formate (Example 240) Trifluoroacetic acid (1 mL) was added dropwise to a solution of tert-butyl (S)-6-(5-fluoro-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazole-6-carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (149) (17 mg, 0.0166 mmol, 1.0 equiv) cooled to 0 °C in anhydrous dichloromethane (1 mL). The mixture was stirred for 30 minutes and then concentrated under reduced pressure. The resulting crude residue was purified by preparative HPLC (formic acid additive) to give the desired product (S)-(5-fluoro-2-((2-methyl-3-((5-(methylthio)pyrimidin-2-yl)amino)propyl)amino)benzo[d]thiazol-6-yl)(2,6-diazaspiro[3.4]octan-6-yl)methanone formate (Example 189) as an off-white solid. Yield: 9 mg (100%). 11H NMR (400 MHz, CDCl3) δ 8.38 - 8.36 (m, 3H), 7.60 - 7.52 (m, 1H), 5.84 - 5.72 (m, 1H), 3.98 (d, J = 9.0 Hz, 1H), 3.84 - 3.57 (m, 6H), 3.50 - 3.24 (m, 4H), 2.86 (s, 12H), 2.38 - 2.37 (m, 3H), 2.29 - 2.13 (m, 3H), 1.09 - 1.04 (m, 3H). No NH-exchangeable protons were observed; MS (ESI+) m / z 502 (M + H) + .

[0362] Using the procedures described in Scheme 34, the following examples were prepared.

[0363]

Table 38

[0364] Scheme 35

Chem.

[0365] 2 - ((3 - ((5 - Mercaptopyrimidin - 2 - yl)amino)-2 - methylpropyl)amino)-N,N - dimethylbenzothiazole - 6 - carboxamide (130A) N,N - Dimethyl - 2 - ((2 - methyl - 3 - ((5 - (methylthio)pyrimidin - 2 - yl)amino)propyl)amino)benzothiazole - 6 - carboxamide (Compound 10, 75 mg, 0.18 mmol), sodium methanethiolate (126 mg, 1.80 mmol, 10.0 equiv) and N - methylpyrrolidine (1.5 mL) were added to a microwave vessel under nitrogen, sealed and then heated to 160 °C for 1 hour. LCMS analysis showed complete conversion to the desired thiol 2 - ((3 - ((5 - ((difluoromethyl)thio)pyrimidin - 2 - yl)amino)-2 - methylpropyl)amino)-N,N - dimethylbenzothiazole - 6 - carboxamide (130A), which was used directly in the next step without purification.

[0366] 2-((3-((5-((Difluoromethyl)thio)pyrimidin-2-yl)amino)-2-methylpropyl)amino)-N,N-dimethylbenzo[d]thiazole-6-carboxamide (244) Potassium hydroxide (201 mg, 3.58 mmol, 20.0 equiv) and water (0.5 mL) were added to a crude thiol solution of 2-((3-((5-((difluoromethyl)thio)pyrimidin-2-yl)amino)-2-methylpropyl)amino)-N,N-dimethylbenzo[d]thiazole-6-carboxamide in N-methylpyrrolidine (1.5 mL), and the mixture was cooled to -70 °C and frozen to a solid. Bromodifluoromethyldiethylphosphonate (40 μL, 0.215 mmol, 1.2 equiv) was added all at once, and the reaction was warmed to ambient temperature over 1 h and then cooled again to -70 °C. A further aliquot of bromodifluoromethyldiethylphosphonate (40 μL, 0.215 mmol, 1.2 equiv) was added, and the mixture was warmed to ambient temperature over 1 h. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with water (20 mL) and brine (25 mL) and then dried over magnesium sulfate. The solvent was removed under reduced pressure to give a crude brown oil, which was purified by flash chromatography (eluting with dichloromethane~methanol, 0~10%) followed by preparative HPLC to give the desired 2-((3-((5-((difluoromethyl)thio)pyrimidin-2-yl)amino)-2-methylpropyl)amino)-N,N-dimethylbenzo[d]thiazole-6-carboxamide (244) as an off-white solid. Yield: 15 mg (19%). 1 H NMR (400 MHz, CDCl3) δ 8.42 - 8.39 (m, 2H), 7.69 (d, J = 1.4 Hz, 1H), 7.55 - 7.52 (m, 1H), 7.36 (dd, J = 1.8, 8.3 Hz, 1H), 6.33 (s, 1H), 6.15 (t, J = 6.7 Hz, 1H), 3.65 - 3.51 (m, 2H), 3.43 - 3.29 (m, 2H), 3.08 (s, 6H), 2.20 - 2.10 (m, 1H), 1.09 - 1.06 (m, 3H); MS (ESI+) m / z 453 (M + H)+ .

[0367] According to the methodology described in Scheme 35, the following examples were prepared.

[0368]

Table 39

[0369] Scheme 36

Chemical Structure

[0370] ((1R,3R)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)carbamic acid rac-tert-butyl (131B) The applicable methodology was the same as that described in General Method 1. Yield: 220 mg (44%). 1 H NMR (400 MHz, CDCl3) δ δ 8.35 - 8.34 (m, 4H), 5.35 (s, 1H), 5.18 - 5.15 (m, 1H), 4.84 - 4.82 (m, 1H), 4.56 - 4.53 (m, 1H), 4.36 (dd, J = 6.9, 13.8 Hz, 1H), 4.26 - 4.18 (m, 1H), 4.02 - 3.94 (m, 2H), 2.55 - 2.47 (m, 1H), 2.36 (d, J = 1.0 Hz, 6H), 2.30 - 2.16 (m, 2H), 2.10 - 1.89 (m, 4H), 1.46 - 1.43 (m, 24H).; MS (ESI+) m / z 325 (M + H) + .

[0371] rac-(1R,3R)-N 1 -(5-(methylthio)pyrimidin-2-yl)cyclopentane-1,3-diamine hydrochloride (131C) The applicable methodology was the same as that described in General Method 2. Yield: 250 mg (quantitative%). MS (ESI+) m / z 225 (M + H) + . It was used in the next step without further purification.

[0372] rac-N,N-Bis(4-methoxybenzyl)-2-(((trans)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)benzo[d]thiazole-6-sulfonamide (131D) The methodology applied was similar to that described in General Method 3. Yield: 52 mg (23%). 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 8.00 (d, J = 1.6 Hz, 1H), 7.74 (dd, J = 1.9, 8.5 Hz, 1H), 7.58 - 7.55 (m, 1H), 7.01 - 6.96 (m, 4H), 6.76 - 6.73 (m, 4H), 5.59 - 5.54 (m, 1H), 5.22 (d, J = 6.9 Hz, 1H), 4.50 - 4.44 (m, 1H), 4.35 - 4.30 (m, 1H), 4.25 (s, 4H), 3.77 - 3.76 (m, 6H), 2.37 - 2.37 (m, 6H), 2.23 - 2.07 (m, 2H); MS (ESI+) m / z 677 (M+H) + .

[0373] rac-2-(((trans)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)benzo[d]thiazole-6-sulfonamide (250) Trifluoroacetic acid (1 mL) was added dropwise to a solution of rac-N,N-bis(4-methoxybenzyl)-2-(((trans)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)benzo[d]thiazole-6-sulfonamide (131D) (45 mg, 66.48 μmol, 1.0 equiv) in anhydrous dichloromethane (1 mL) at ice-cooled temperature. The mixture was warmed to ambient temperature over 18 h and then concentrated to dryness under reduced pressure. The resulting crude residue was purified by preparative HPLC and the obtained clear fractions were lyophilized to afford the desired 2-(((trans)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)benzo[d]thiazole-6-sulfonamide (Example 250) as a white powder. Yield: 23 mg (79%). 1 H NMR (CDCl3) δ 8.36 (s, 2H), 8.17 (d, J = 1.8 Hz, 1H), 7.84 (dd, J = 2.0, 8.5 Hz, 1H), 7.59 - 7.52 (m, 1H), 5.34 (d, J = 6.5 Hz, 1H), 4.79 (s, 2H), 4.50 - 4.43 (m, 1H), 4.34 - 4.27 (m, 1H), 2.37 - 2.37 (m, 6H), 2.22 - 2.07 (m, 2H); MS (ESI+) m / z 437 (M + H) + .

[0374] Scheme 41

Chemical Structure

[0375] Step 1: Example 301C To a solution of Example 301A (1 g, 3.14 mmol) in DMF (30 mL) were added Cs2CO3 (2.05 g, 6.28 mmol) and Example 301B (5.6 g, 62.8 mmol). The mixture was heated to 25 °C for 1 h. TLC detected the consumption of the starting materials. The reaction mixture was filtered and the filtrate (crude Example 301C) was used in the next step without any purification.

[0376] Step 2: Example 301D The solution of Example 301C was treated with Boc2O (1.03 g, 4.72 mmol) and stirred at room temperature for 2 hours. Water (100 mL) was added, followed by extraction with EA (50 mL×2), washing with water and brine, drying over Na2SO4, filtering, concentrating the filtrate under reduced pressure, and then purifying by silica gel chromatography (eluted with petroleum ether / EtOAc = 1 / 5 to 1 / 1) to obtain the desired product (Example 301D, 1.2 g, yield 81%) as a yellow solid. LCMS [M+H] + =573

[0377] Step 3: Example 301E To a solution of Example 301D (1.2 g, 2.54 mmol) in DCM (3 mL), TFA (1 mL) was added at room temperature. After the addition, the reaction mixture was stirred at room temperature for 1 hour. TLC detected that the starting material was consumed, and the mixture was concentrated to obtain the desired product (Example 301E, 945 mg, yield: 100%) as a yellow oil, which was used in the next step without further purification.

[0378] Step 4: Example 301 To a solution of Example 301E (945 mg, 2.54 mmol) in ACN (10 mL), TEA (514 mg, 5.08 mmol) and Example 301F (408 mg, 2.54 mmol) were added at room temperature, and then the mixture was heated to 70 °C for 18 hours. TLC detected that the starting material was consumed. The reaction was concentrated and purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 3 / 1 to 5 / 3) to obtain the desired product Example 301 (780 mg, yield: 61%) as a white solid. LCMS [M+H] + =497. 1 1H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.34 (s, 2H), 8.12 (d, J = 1.6 Hz, 1H), 7.54 (dd, J = 8.5, 1.8 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 3.94 - 3.86 (m, 1H), 3.59 (dd, J = 16.6, 11.9 Hz, 6H), 3.39 (dd, J = 19.4, 5.9 Hz, 4H), 2.85 - 2.81 (m, 4H), 2.33 (s, 3H).

[0379] Scheme 42

Chemical formula

[0380] Step 1: Example 302B A solution of Example 302A (6.77 g, 21 mmol) in THF (200 mL) under a N2 atmosphere was cooled to -65 °C. MeMgBr (21 mL, 63.1 mmol, 3 M in THF) was added dropwise, and the mixture was subsequently stirred at -65 °C for 0.5 h. The reaction mixture was warmed to room temperature over 2 h and quenched by the addition of water (200 mL). After extraction with EtOAc (200 mL × 2), the combined organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (eluting with petroleum ether / EtOAc = 3 / 1) to afford the desired product Example 302B (4 g, 56% yield) as a yellow oil. LCMS [M+H] + = 339

[0381] Step 2: Example 302C A suspension of Pd / C (400 mg) catalyst was introduced into a reactor containing a solution of Example 302B (4 g, 0.012 mmol) in MeOH (40 mL). The vessel was purged with nitrogen followed by hydrogen, and the reaction mixture was stirred at room temperature for 18 h. TLC and LCMS detected the consumption of the starting material. Example 302C (4 g, yield: 100%) was obtained by filtration, concentrated, and used in the next step without any purification. LCMS [M+H] + = 205

[0382] Step 3: Example 302D To a solution of Example 301A (154 mg, 0.48 mmol) in DMF (30 mL) was added Cs2CO3 (313 mg, 0.96 mmol) and Example 302C (982 mg, 4.8 mmol), and the mixture was stirred at 25 °C for 2 h. TLC detected that the starting material had been consumed. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 1 / 1 to 1 / 4) to give the desired product Example 302D (500 mg, yield: 100%) as a white solid. LCMS [M+H] + =487

[0383] Step 4: Example 302E To a solution of Example 302D (486 mg, 2 mmol) in DCM (2 mL) was added TFA (1 mL) at room temperature. After the addition, the reaction mixture was stirred at room temperature for 1 h. TLC detected that the starting material had been consumed, and the mixture was concentrated to give the desired product Example 302E (532 mg, yield: 100%) as a yellow oil, which was used in the next step without further purification.

[0384] Step 5: Example 302 Example 302E (193 mg, 0.5 mmol), TEA (101 mg, 1 mmol) and Example 301F (81 mg, 0.5 mmol) were dissolved in ACN (5 mL), and the mixture was heated to 60 °C for 18 h. LCMS detected the formation of TM. Purification by preparative HPLC gave the desired product Example 302 (15 mg, yield: 6%) as a white solid. LCMS [M+H] + =511 1 H NMR (400 MHz, CDCl3) δ 8.41 (s, 2H), 7.98 (s, 1H), 7.65 (s, 2H), 6.10 (s, 1H), 3.77 - 3.72 (m, 4H), 3.67 - 3.52 (m, 4H), 3.04 - 2.98 (m, 4H), 2.38 (s, 3H), 1.33 (s, 3H).

[0385] Scheme 43

Chemical Structure

[0386] Project 1: Example 303B To a solution of Example 303A (33 g, 500 mmol) and methyl carbonochloridate (49.5 g, 520 mmol) in THF (75 mL) was added KOH (56.1 g, 1000 mmol) in H2O (50 mL) over 30 minutes (maintaining the internal temperature below 40 °C). After the addition, the suspension was stirred at room temperature for 16 hours. The suspension was filtered, the filter cake was washed with EtOH (50 mL × 3), and dried in vacuo to give the desired product Example 303B (67 g, 82.7% yield) as a white solid. LCMS [M+H] + =163

[0387] Project 2: Example 303C To a solution of Example 303B (4 g, 24.52 mmol) in MeOH (300 mL) and HCl (5 mL, 4.0 M in MeOH) was added Pd / C (8 g) at room temperature. The suspension was stirred at room temperature for 44 hours under H2 at 15 psi. LCMS indicated that the starting material was completely consumed and the desired product was detected. The mixture was filtered, and the filter cake was washed with MeOH (30 mL × 3). The filtrate was concentrated under reduced pressure to give the product Example 303C (3.7 g, 54.4% yield) as a yellow solid. LCMS [M+H] + =133.2

[0388] Project 3: Example 303D A solution of Example 303C (2.18 g, 7.84 mmol), Example 301A (0.5 g, 1.57 mmol) and DIEA (13.35 g, 103.5 mmol) in MeCN (100 mL) was heated to 80 °C and stirred for 4 hours. LCMS indicated that the starting material was completely consumed and the desired product was detected. The reaction mixture Example 303D was used for the next step without further purification. LCMS [M+H] + =415.5

[0389] Project 4: Example 303E Boc2O (5 g, 22.9 mmol) was added to the solution of Example 303D at room temperature and stirred for 1 hour. LCMS indicated that the starting material was consumed. The reaction mixture was diluted with DCM (200 mL), washed with water (150 mL × 2) and brine (200 mL × 3), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 2 / 1 to 1 / 9) to give the product Example 303E (680 mg, yield over two steps: 76.7%) as a white solid. LCMS [M+H] + =515.5

[0390] Step 5: Example 303F To a solution of Example 303E (840 mg, 1.63 mmol) in THF (20 mL), CaCl2 (363 mg, 3.27 mmol) and NaBH4 (124 mg, 3.27 mmol) were added at room temperature and the reaction mixture was stirred for 16 hours. The reaction mixture was poured into a saturated aqueous solution of NH4Cl (100 mL), diluted with EtOAc (30 mL), and separated. The aqueous layer was extracted with EtOAc (30 mL × 2), and the combined organic layers were concentrated and purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 3 / 1 to 1 / 9) to give the product Example 303F (383 mg, yield: 48.3%) as a white solid. LCMS [M+H] + =487.6

[0391] Step 6: Example 303G To a solution of Example 303F (553 mg, 1.14 mmol) dissolved in DCM (10 mL), HCl / dioxane (4.0 M, 10 mL) was added at room temperature and the reaction mixture was stirred for 0.5 hour. TLC indicated that the starting material was consumed. The reaction mixture was concentrated under reduced pressure to give the product Example 303G (563 mg, yield: 100%) as a white solid. LCMS [M+H] + =387.5

[0392] Step 7: Example 303 To a solution of Example 303G (563 mg, 1.14 mmol) and K2CO3 (940 mg, 6.82 mmol) in DMF (10 mL) was added Example 301A (183 mg, 1.14 mmol) at room temperature. Next, the reaction mixture was heated to 60 °C and stirred for 16 h. TLC indicated consumption of the starting materials. The mixture was cooled to room temperature, diluted with EtOAc (30 mL), and washed with water (20 mL). The aqueous layer was extracted with EtOAc (20 mL × 4), and the combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 3 / 1 to 1 / 9) to give a crude product, which was further purified by preparative HPLC to afford Product Example 303 as a pale yellow solid (75.2 mg, yield: 12.93%). LCMS [M+H] + =511 1 H NMR (400 MHz, CDCl3) δ 8.37 (s, 2H), 7.96 (s, 1H), 7.66 - 7.58 (m, 2H), 6.82 (s, 1H), 5.87 (s, 1H), 3.83 - 3.73 (m, 5H), 3.59 - 3.44 (m, 5H), 3.01 (s, 4H), 2.38 (s, 3H), 2.14 - 2.08 (m, 1H).

[0393]

Chemical Structure

[0394] Step 1: Example 304B Example 304A (23.6 g, 0.12 mol), NaN3 (15.1 g, 0.23 mol), and NH4Cl (7.5 g, 0.14 mol) were suspended in DMF (250 mL), and the resulting mixture was heated to 80 °C for 1 h. After detecting the completion of the reaction by TLC, EtOAc (1 L) was added, and the organic extract was washed with water (200 mL × 5), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 5 / 1) to afford the desired product Example 304B as a white solid (29 g, yield 100%). LCMS [M+H]+ =247

[0395] Step 2: Example 304C To a solution of Example 304B (29 g, 0.12 mol) in EtOH (400 mL) were added HCl (22 mL) and Pd / C (1 g), and the heterogeneous mixture was stirred at room temperature for 18 h under H2. TLC detected that the starting material was mostly consumed. The mixture was filtered and the filtrate was concentrated to give the desired product Example 304C (21 g, yield: 100%) as a white solid. LCMS [M+H] + =221

[0396] Step 3: Example 304D To a solution of Example 304C (6.6 g, 3 mol) in DMF (50 mL) were added 2-chloro-5-(methylthio)pyrimidine (2.4 g, 15 mmol) and TEA (7.6 g, 75 mmol). The resulting mixture was stirred at 100 °C for 1 h. TLC detected that the starting material was consumed. The reaction mixture was concentrated and purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 1 / 1 - 1 / 4) to give the desired product Example 304D (450 mg, yield: 8%) as a white solid. LCMS [M+H] + =345

[0397] Step 4: Example 304E To a solution of Example 304D (450 mg, 1.3 mmol) in EtOH (5 mL) was added hydrazine hydrate (131 mg, 2.6 mmol) at room temperature. After 3 h, TLC detected that the starting material was consumed, and at that point the reaction mixture was concentrated and purified by silica gel chromatography (eluted with DCM / MeOH = 10 / 1) to give the desired product Example 304E (120 mg, yield: 43%) as a white solid.

[0398] Step 5: Example 304 To a solution of Example 304E (43 mg, 0.20 mmol) and 2-chloro-N,N-dimethylbenzo[d]thiazole-6-sulfonamide (37 mg, 0.13 mmol) in DMF (1 mL) was added DBU (40 mg, 0.26 mmol). The resulting mixture was stirred at 50 °C for 1 h. TLC detected the consumption of the starting material, and the reaction mixture was concentrated and purified by preparative TLC to afford the desired product Example 304 (30 mg, yield: 51%) as a white solid. LCMS [M+H] + =454.9. 1 H NMR (400 MHz, CDCl3) δ 8.35 (s, 2H), 7.99 (d, J = 1.5 Hz, 1H), 7.67 (dd, J = 8.5, 1.7 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 5.97 (s, 1H), 4.11 (dd, J = 9.3, 4.8 Hz, 1H), 3.74 (dd, J = 13.7, 4.3 Hz, 1H), 3.66 (s, 1H), 3.58 (dd, J = 13.6, 6.3 Hz, 2H), 2.70 (s, 6H), 2.37 (s, 3H).

[0399] Scheme 45

Chemical Structure

[0400] Step 1: Example 305 To a solution of Example 304E (30 mg, 0.14 mmol) and 2-chloro-N-methylbenzo[d]thiazole-6-sulfonamide (24.5 mg, 0.09 mmol) in DMF (2 mL) was added DBU (47 mg, 0.18 mmol). After the addition, the mixture was stirred at 50 °C for 1 h. LCMS identified the consumption of the starting material. The mixture was cooled to room temperature, diluted with EtOAc (5 mL), and washed with brine (5 mL). The organic layer was concentrated and purified by preparative TLC to afford the desired product Example 305 (23.8 mg, yield: 60%) as a white solid. LCMS [M+H] + =440.9 11H NMR (400 MHz, MeOD-d4) δ 8.35 (s, 2H), 8.07 (s, 1H), 7.69 (dd, J = 8.0, 1H), 7.50 (d, J = 8.0 Hz, 1H), 4.06 (m, 0.46H), 3.57 (m, 4H), 2.51 (s, 3H), 2.32 (s, 3H).

[0401] Scheme 46

Chem.

[0402] Example 306 (30 mg, yield: 50%) was prepared as a white solid in a similar manner starting from Example 306A. LCMS [M+H] + = 454.9. 1 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 2H), 7.98 (d, J = 1.5 Hz, 1H), 7.66 (dd, J = 8.5, 1.7 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 6.04 (t, J = 5.6 Hz, 1H), 4.11 (dd, J = 9.3, 5.1 Hz, 1H), 3.74 - 3.64 (m, 2H), 3.61 - 3.54 (m, 2H), 2.70 (s, 6H), 2.36 (s, 3H).

[0403] Scheme 47

Chem.

[0404] Step 1: Example 307A To a solution of Example 306E (100 mg, 0.47 mmol) in DMF (0.5 mL) was added DBU (89 mg, 0.58 mmol) and methyl 2-chlorobenzo[d]thiazole-6-carboxylate (89 mg, 0.39 mmol). The mixture was heated to 50 °C for 1 h. After identifying by TLC that the starting material was consumed, the mixture was concentrated and purified by silica gel chromatography (eluted with petroleum ether / EtOAc = 1 / 1 - 1 / 4) to give the desired product Example 307A (50 mg, yield: 26%) as a white solid. LCMS [M+H]+ =406

[0405] Step 2: Example 307B To a solution of Example 307A (50 mg, 0.12 mmol) in THF (1 mL) was added LiOH (0.4 mL, 1 M in water), and the resulting mixture was stirred at room temperature for 18 h. After identifying by TLC that the starting material had been consumed, the mixture was concentrated and purified by silica gel chromatography (eluted with DCM / MeOH = 10 / 1) to give the desired product Example 307B (5 mg, yield: 11%) as a white solid. LCMS [M+H] + =392

[0406] Step 3: Example 307 A solution of Example 307B (10 mg, 0.025 mmol) was charged with dimethylamine hydrochloride (2.5 mg, 0.031 mmol), TEA (8 mg, 0.075 mmol) and HBTU (14 mg, 0.038 mmol). The resulting mixture was stirred at room temperature for 1 h. After detecting by TLC that the starting material had been consumed, the mixture was concentrated and purified by preparative TLC to give the desired product Example 307 (2 mg, yield: 19%) as a white solid. LCMS [M+H] + =419. 1 H NMR (400 MHz, CDCl3) δ 8.36 (s, 2H), 7.67 (s, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.35 (dd, J = 8.3, 1.5 Hz, 1H), 5.91 (s, 1H), 4.14 - 4.00 (m, 1H), 3.72 - 3.64 (m, 2H), 3.53 (dd, J = 13.8, 6.3 Hz, 2H), 3.07 (s, 6H), 2.37 (s, 3H).

[0407] Scheme 48

Chemical Structure

[0408] Step 2: Example 308A A Schlenk tube equipped with a magnetic stir bar was charged with TBAI (5 g, 13.6 mmol), cyclopentene (9.25 g, 136 mmol), and O-phthalimide (10 g, 68 mmol) in 250 mL of benzene. After adding a solution of 65% TBHP (18.8 g, 136 mmol), the vial was sealed and the reaction mixture was stirred at 80 °C for 12 h. After cooling to room temperature, the reaction mixture was diluted in ethyl acetate and washed with brine. The aqueous phase was extracted with ethyl acetate. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with PE / ethyl acetate = 5 / 1) to afford the desired compound Example 308A (6.5 g) as a white solid. LCMS [M+H] + =214. 1 H NMR (400 MHz, CDCl3) δ 2.08 - 2.17 (m, 1H), 2.32 - 2.47 (m, 1H), 2.41 - 2.53 (m, 1H), 2.80 - 2.88 (m, 1H), 5.33 - 5.46 (m, 1H), 5.61 - 5.70 (m, 1H), 6.07 - 6.16 (m, 1H), 7.67 - 7.75 (m, 2H), 7.80 - 7.87 (m, 2H).

[0409] Step 2: Example 308B To a solution of Example 308A (5 g, 23.5 mmol) in THF (25 mL) was added 50% hydrazine hydrate in H2O (3.52 g, 35.2 mmol). The mixture was stirred at 70 °C for 2 h. Next, the mixture was filtered and concentrated under reduced pressure. Di-tert-butyl dicarbonate (10.2 g, 47 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with PE / ethyl acetate = 5 / 1) to afford the desired compound Example 308B (550 mg) as a white solid. LCMS [M+H] + =184.

[0410] Step 3: Example 308C To a solution of Example 308B (700 mg, 3.825 mmol) in DCM (5 mL) was added m-CPBA (790 mg, 4.59 mmol) portionwise at 0 °C. After the addition, the mixture was stirred at room temperature overnight. The resulting mixture was cooled to 0 °C, and m-chlorobenzoic acid was filtered off and washed with additional cold DCM. The combined filtrate and washings were stirred with 20% NaHSO3 for 30 minutes. The DCM layer was separated, extracted with 3N NaOH (3×30 mL), saturated NaCl (30 mL), and dried over Na2SO4. Evaporation left a white solid, which was purified by silica gel column chromatography (eluting with PE / ethyl acetate = 5 / 1) to give the desired compound Example 308C (455 mg) as a white solid. LCMS [M+H] + =144. 1 H NMR (400 MHz, CDCl3) δ 1.07-1.17 (m, 1H), 1.48 (s, 9H), 1.66-1.76 (m, 1H), 1.89-1.96 (m, 1H), 2.05-2.16 (m, 1H), 3.41-3.50 (m, 1H), 3.53 (br s, 1H), 4.07-4.26 (m, 1H), 4.63-4.77 (m, 1H).

[0411] Step 4: Example 308D A mixture of Example 308C (445 mg, 2.28 mmol), NaN3 (297 mg, 4.57 mmol), NH4Cl (61 mg, 1.14 mmol), 2-methoxyethanol (5 mL) and H2O (1 mL) was stirred in a bath maintained at 80 °C for 16 hours. The resulting solution was evaporated to dryness, and the residue was dissolved in H2O (5 mL). This solution was saturated with NaCl and subsequently extracted with DCM (4×5 mL). The DCM solution was evaporated, and the residue was purified by silica gel column chromatography (eluting with MeOH / DCM = 3% - 5%) to give the desired compound Example 308D (420 mg) as a colorless oil. LCMS [M+H] + =188.

[0412] Step 5: Example 308E A suspension of Example 308D (420 mg, 1.74 mmol) and Pd / C (catalytic amount) in EtOH (5 mL) was stirred at room temperature for 16 h under a H2 atmosphere. The mixture was filtered and concentrated in vacuo. The residue was dried and used directly in the next step without further purification. Example 308E (320 mg). LCMS [M+H] + =217.

[0413] Step 6: Example 308F A mixture of Example 308E (150 mg, 0.694 mmol), 2-chloro-5-(methylthio)pyrimidine (111 mg, 0.694 mmol), and DIPEA (180 mg, 1.4 mmol) in DMSO (5 mL) was stirred at 130 °C for 3 h. The resulting solution was cooled to room temperature, poured into water, and extracted with EtOAc (3 × 10 mL). The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with PE / EA = 2 / 1) to afford the desired compound, Example 308F (100 mg), as a colorless oil. LCMS [M+H] + =341. 1 1H NMR (400 MHz, CDCl3) δ 1.45 (s, 9H), 1.75 - 1.85 (m, 1H), 2.13 - 2.21 (m, 1H), 2.23 - 2.30 (m, 1H), 2.38 (s, 3H), 3.83 - 4.05 (m, 3H), 5.33 (br s, 1H), 5.57 (br s, 1H), 8.35 (s, 2H).

[0414] Step 7: Example 308G To a solution of Example 308F (100 mg, 0.294 mmol) in DCM (3 mL) was added 4M HCl in dioxane (3 mL). The mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure, dried, and used directly in the next step without further purification. Example 308G (70.6 mg). LCMS [M+H] + =241.

[0415] Step 8: Example 308H A mixture of Example 308G (70.6 mg, 0.256 mmol), tert-butyl 6-((2-chlorobenzo[d]thiazol-6-yl)sulfonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (130 mg, 0.294 mmol), and DIPEA (99 mg, 0.768 mmol) in DMF (4 mL) was stirred at 40 °C for 2 days. The resulting solution was cooled to room temperature, poured into water, and extracted with EtOAc (3 × 10 mL). The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with MeOH / DCM = 5%) to afford the desired compound, Example 308H (70 mg) as a white solid. LCMS [M+H] + =648.

[0416] Step 9: Example 308 To a solution of Example 308H (70 mg, 0.108 mmol) in DCM (3 mL) was added 4M HCl in dioxane (3 mL). The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and purified by preparative HPLC to afford the title compound, Example 308 (20 mg) as a white solid. LCMS [M+H] + =548. 1 H NMR (400 MHz, DMSO-d6) δ 1.47-1.56 (m, 1H), 1.63-1.73 (m, 1H), 1.97 (t, J = 6.98 Hz, 2H), 2.06-2.23 (m, 2H), 2.31-2.41 (m, 3H), 3.19 (t, J = 6.98 Hz, 2H), 3.35 (s, 2H), 3.68 (t, J = 6.04 Hz, 4H), 3.95-4.02 (m, 1H), 4.15 (br.s., 1H), 4.38 (br.s., 1H), 7.48 (d, J = 8.33 Hz, 1H), 7.59 (dd, J = 8.33, 1.88 Hz, 2H), 8.16 (d, J = 1.88 Hz, 1H), 8.37 (s, 2H), 8.49 (d, J = 7.79 Hz, 2H), 8.66 (br.s., 1H).

[0417] Scheme 49

Chemical Structure

[0418] Project 1: Example 309A Sodium cyclopentadienide (2 M in THF, 50 mL, 100 mmol, 1 equiv) was added dropwise to a solution of benzyl chloromethyl ether (90%, 23 g, 130 mmol, 1.3 equiv) in DMF (200 mL) at -40 °C. After vigorous stirring for 20 min at -40 °C, the reaction mixture was poured into a 2:1 mixture of pentane / ice-cold water (900 mL). After shaking and phase separation, the organic layer was washed twice with 150 mL of cold water, dried over Na2SO4 with stirring, and the temperature was maintained below 0 °C to avoid isomerization of the double bond. After removal of the drying agent by filtration, pentane was removed in vacuo at 0 °C to give (benzyloxymethyl)cyclopenta-2,4-diene 1 as a pale orange oil. The crude material obtained was maintained at 0 °C under argon, diluted with THF (160 mL), cooled to -78 °C, and added dropwise via cannula at -78 °C to a suspension of (-)-Ipc2BH (1 M solution in THF, 100 mL, 100 mmol, 1 equiv) in THF (400 mL). The mixture was slowly warmed to -10 °C and stirred at that temperature for 3 days. Next, the reaction was quenched by the addition of MeOH (40 mL), followed by the addition of 3 M aqueous NaOH (40 mL) and 30% H2O2 (40 mL). After vigorous stirring for 24 h at room temperature, THF was removed under reduced pressure, and the remaining aqueous suspension was partitioned between EtOAc (400 mL) and brine (200 mL). After extraction, the organic layer was dried over Na2SO4 and concentrated in vacuo. The crude orange oil was purified by column chromatography (eluent: 9:1 - 8:2 heptane:EtOAc) to give Example 309A (4.8 g, 23.53 mmol, 23%) as a pale yellow oil. Rf = 0.29 (eluent: 7:3 heptane:EtOAc); 1H NMR (400 MHz, CDCl3) δ 2.23 - 2.32 (m, 1H), 2.35 (s, 1H), 2.63 - 2.73 (m, 1H), 2.81 - 2.88 (m, 1H), 3.28 (t, J = 8.9 Hz, 1H), 3.53 (dd, J = 5.4, 9.1 Hz, 1H), 4.29 (td, J = 4.1, 7.0 Hz, 1H), 4.52 (s, 2H), 5.53 - 5.58 (m, 1H), 5.70 - 5.74 (m, 1H), 7.24 - 7.37 (m, 5H).

[0419] Step 2: Example 309B To a solution of Example 309A (4.8 g, 23.53 mmol, 1 equiv) in anhydrous THF (100 mL) was added NaH (50% in mineral oil, 1.13 g, 28.2 mmol, 1.2 equiv) at 0 °C, and the mixture was stirred at room temperature for 20 min. Next, benzyl bromide (BnBr, 3.6 mL, 30.5 mmol, 1.3 equiv) and tetrabutylammonium iodide (TBAI, 100 mg, 0.3 mmol, 0.01 equiv) were added at 0 °C, and the reaction mixture was stirred at room temperature. After 15 h, crushed ice was carefully added, and the mixture was stirred for 30 min. After extraction with EtOAc (150 mL), the organic layer was washed with H2O (150 mL), brine (150 mL), dried over Na2SO4, and concentrated under vacuum. Purification by column chromatography (eluent: 98:2 - 95:5 heptane:EtOAc) gave Example 309B (6.3 g, 21.4 mmol, 80%) as a colorless syrup. Rf = 0.41 (eluent: 9:1 heptane:EtOAc). LCMS [M+H] + =295.1 H NMR (500 MHz, CDCl3) δ 2.42 (d, J = 17.4 Hz, 1H), 2.65 - 2.70 (m, 1H), 3.07 (brs, 1H), 3.33 and 3.44 (ABX, JAB = 9.2 Hz, JAX = 5.7 Hz, JBX = 7.3 Hz, 2H), 4.08 (ddd, J = 3.0, 3.3, 7.0 Hz, 1H), 4.51 (d, J = 3.4 Hz, 2H), 4.54 (s, 2H), 5.64 - 5.66 (m, 1H), 5.74 - 5.75 (m, 1H), 7.22 - 7.34 (m, 10H).

[0420] Step 3: Example 309C A 0.5 M solution of 9-BBN (88 mL, 44 mmol) in THF was added dropwise to a solution of Example 309B (6.50 g, 22.0 mmol) in anhydrous THF (10 mL) at 0 °C under nitrogen. The reaction mixture was slowly warmed to room temperature overnight. The reaction mixture was cooled to 0 °C and treated successively with EtOH (7 mL), 3 N NaOH solution (20 mL), and H2O2 (33%, 20 mL). The resulting mixture was stirred at room temperature overnight. The resulting residue was filtered and washed with EtOAc (200 mL). Water (150 mL) was added to this suspension, and after phase separation, the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were dried (Na2SO4) and concentrated to dryness. The crude product was purified on silica gel (eluent: 1:1 heptane:EtOAc) to give Example 309C (6.0 g, 19.3 mmol, 87%) as a yellow oil. LCMS [M+H] + =313.1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.25 (m, 10H, CH-arom.), 4.52 (s, 2H, CH2-benzyl), 4.49 (d, 1H, J = 11.8 Hz, CHH-benzyl), 4.44 (d, 1H, J = 11.8 Hz, CHH-benzyl), 4.33 - 4.28 (m, 1H, H-1), 4.07 (ddd, 1H, J = 6.6 Hz, 6.6 Hz, 4.1 Hz, H-3), 3.53 (dd, 1H, J = 9.0 Hz, 4.2 Hz, OCHH), 3.49 (d, 1H, J = 9.0 Hz, 4.3 Hz, OCHH), 2.35 - 2.25 (m, 2H, H-4, H-5a), 2.05 (dddd, 1H, J = 13.5 Hz, 6.7 Hz, 3.5 Hz, 1.7 Hz, H-2a), 1.89 - 1.82 (m, 1H, H-2b), 1.52 - 1.46 (m, 1H, H-5b).

[0421] Step 4: Example 309D Compound Example 309C (6.0 g, 19.3 mmol) was dissolved in dry pyridine (30 mL) and cooled to 0 °C. TosCl (5.5 g, 28.9 mmol) was added portionwise over 30 minutes. After the addition, the reaction mixture was stirred at room temperature for 18 hours. The suspension was diluted with ethyl acetate (300 mL) and H2O (200 mL). The organic phase was separated, washed with saturated NH4Cl solution (3 × 200 mL), brine (100 mL), and dried over MgSO4. The solvent was evaporated, and the residue was purified by flash chromatography on silica gel (hexane / ethyl acetate 5:1) to give (1R,3S,4R)-3-(benzyloxy)-4-((benzyloxy)methyl)cyclopentyl 4-methylbenzenesulfonate (1.4 g, 3.0 mmol, 15%) as a colorless oil. Rf = 0.26 (20% ethyl acetate in hexane). LCMS [M+H] + = 467.1 H NMR (400 MHz, CDCl3) δ 7.79 (m, 2H), 7.38 - 7.27 (m, 12H), 5.05 - 4.99 (m, 1H), 4.50 (s, 2H), 4.45 (s, 2H), 3.96 - 3.92 (m, 1H), 3.48 - 3.40 (td, 2H, J = 6.3 Hz), 2.45 (s, 3H), 2.33 - 2.20 (m, 2H), 2.14 - 2.01 (m, 2H), 1.69 - 1.62 (m, 1H).

[0422] (1R,3S,4R)-3-(Benzyloxy)-4-((benzyloxy)methyl)cyclopentyl 4-methylbenzenesulfonate (1.4 g, 3.0 mmol) was dissolved in dry DMF (20 mL), and NaN3 (2.1 g, 15.4 mmol) was added. The mixture was stirred at 60 °C for 14 hours. After the addition of ethyl acetate (300 mL), the organic layer was washed with saturated NaHCO3 solution (2 × 100 mL) and brine (100 mL), and dried over MgSO4. The solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel (hexane / ethyl acetate 10:1) to give Example 309D (1.0 g, 2.95 mmol, 95%) as a colorless oil. Rf = 0.54 (20% ethyl acetate in hexane). LCMS [M+H] + = 338. 11H NMR (400 MHz, CDCl3) δ 7.39 - 7.25 (m, 10H), 4.52 (dd, J = 26.0, 18.6 Hz, 2H), 4.49 (s, 2H), 3.97 - 3.89 (m, 1H), 3.86 (td, J = 7.0, 5.1 Hz, 1H), 3.44 (d, J = 5.5 Hz, 2H), 2.54 - 2.43 (m, 1H), 2.24 (td, J = 13.8, 6.8 Hz, 1H), 1.99 (dddd, J = 13.4, 8.7, 4.7, 1.3 Hz, 1H), 1.84 (dddd, J = 28.2, 20.8, 9.5, 4.2 Hz, 2H).

[0423] Project 5: Example 309E Compound Example 309D (1.0 g, 2.95 mmol) was dissolved in dry CH2Cl2 (20 mL) and cooled to -78 °C. A solution of 1 M BCl3 in CH2Cl2 (40 mL) was added dropwise via a dropping funnel over 45 minutes, and the mixture was stirred at -78 °C for 3 hours and then warmed to room temperature. The reaction was quenched with dry MeOH (20 mL) at -78 °C and warmed to room temperature overnight. The solvent was evaporated, and the residue was purified on silica gel (hexane / ethyl acetate 1:1) to give (1S,2R,4S)-4-azido-2-(hydroxymethyl)cyclopentanol (420 mg, 2.67 mmol, 90%) as a yellow oil. LCMS [M+H] + = 158. 1 1H NMR (400 MHz, CDCl3) δ 4.07 (dd, J = 13.3, 6.0 Hz, 1H), 4.02 - 3.95 (m, 1H), 3.79 (ddd, J = 10.4, 5.2, 3.0 Hz, 1H), 3.56 (dd, J = 10.4, 8.0 Hz, 1H), 2.36 - 2.20 (m, 4H), 1.99 - 1.92 (m, 1H), 1.77 (dddd, J = 14.0, 6.0, 4.5, 1.6 Hz, 1H), 1.58 (ddd, J = 13.9, 9.8, 6.8 Hz, 1H).

[0424] (1S,2R,4S)-4-Azido-2-(hydroxymethyl)cyclopentanol (0.42 g, 2.67 mmol) was dissolved in dry DMF (10 mL), and imidazole (198 mg, 2.94 mmol) was added at room temperature. After the addition of TBDPSCl (808 mg, 2.94 mmol) portionwise at 0 °C, the mixture was stirred at room temperature for 16 h. The reaction was diluted with CH2Cl2 (200 mL), washed once with saturated NH4Cl (70 mL) and brine (50 mL), and dried over MgSO4. The solvent was removed under vacuum, and the crude product was purified by silica gel (hexane / ethyl acetate 10:1) to give Example 309E (630 mg, 1.6 mmol, 60%) as a yellow oil. LCMS [M+H] + = 360. 1 H NMR (400 MHz, CDCl3) δ 7.69 - 7.66 (m, 4H), 7.49 - 7.40 (m, 6H), 4.15 (dd, J = 7.0, 7.0 Hz, 1H), 4.02 - 3.99 (m, 1H), 3.81 (dd, J = 4.8, 4.8 Hz, 1H), 3.59 (dd, J = 7.2, 7.2 Hz, 1H), 2.35 - 2.28 (m, 2H), 1.91 - 1.88 (m, 1H), 1.83 - 1.78 (m, 1H), 1.70 - 1.62 (m, 1H), 1.08 (s, 9H).

[0425] Step 6: Example 309F 10% Pd / C (63 mg) was added to a suspension of Compound Example 309E (630 g, 1.6 mmol) in dry EtOH (100 mL). The reaction was evaporated twice to exchange the inert gas atmosphere and then connected to two balloons filled with H2. The suspension was stirred vigorously at room temperature for 15 h. The palladium catalyst was removed by using a PTFE-filter (Whatman Puradisc), and the solvent was removed under reduced pressure. The amine (588 g, 100%) was obtained as a colorless liquid and used without further purification. LCMS [M+H] + = 370.

[0426] To a solution of the amine (588 mg, 1.6 mmol) in DCM (50 mL) at 0 °C was added Boc2O (700 mg, 3.2 mmol) in DCM (10 mL). The mixture was stirred at 0 °C and then at room temperature for 2 h. The reaction mixture was concentrated and the crude product was purified by silica gel (hexane / ethyl acetate 5:1) to give Example 309F (650 mg, 1.38 mmol, 87%) as a colorless oil. LCMS [M+H] + =470. 1 H NMR (400 MHz, CDCl3) δ 7.68 - 7.65 (m, 4H), 7.48 - 7.40 (m, 6H), 4.97 (br s, 1H), 4.20 (m, 1H), 4.02 (br s, 1H), 3.74 (dd, J = 4.2, 4.2 Hz, 1H), 3.51 (dd, J = 8.4, 8.4 Hz, 1H), 2.29 - 2.22 (m, 2H), 1.78 - 1.57 (m, 3H), 1.45 (s, 9H), 1.07 (s, 9H).

[0427] Step 7: Example 309G To a solution of Compound Example 309F (630 mg, 1.34 mmol), DIEA (300 mg, 2.28 mmol) and DMAP (278 mg, 2.28 mmol) in DCM (50 mL) was added TosCl (384 mg, 2.0 mmol) at 0 °C. The mixture was stirred at room temperature for 14 h and then concentrated to dryness. The crude product was purified by silica gel (hexane / ethyl acetate 10:1) to give (1S,2R,4S)-4-((tert-butoxycarbonyl)amino)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopentyl 4-methylbenzenesulfonate (590 mg, 0.94 mmol, 70%) as a colorless oil. LCMS [M+H] + =625.

[0428] (1S,2R,4S)-4-((tert-Butoxycarbonyl)amino)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopentyl 4-methylbenzenesulfonate (590 g, 0.94 mmol) was dissolved in dry DMF (10 mL), and NaN3 (92 mg, 1.4 mmol) was added. The mixture was stirred at 60 °C for 14 h. After the addition of ethyl acetate (100 mL), the organic layer was washed with saturated NaHCO3 solution (2 × 50 mL) and brine (50 mL), and dried over MgSO4. The solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel (hexane / ethyl acetate 5:1) to give tert-butyl ((1S,3R,4S)-3-azido-4-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopentyl)carbamate (460 mg, 0.93 mmol, 99%) as a colorless oil. Rf = 0.54 (20% ethyl acetate in hexane). LCMS [M+H] + = 496. 1 H NMR (400 MHz, CDCl3) δ 7.72 - 7.67 (m, 4H), 7.46 - 7.40 (m, 6H), 4.45 (br s, 1H), 4.20 (m, 1H), 4.08 (br s, 1H), 3.79 - 3.72 (m, 1H), 3.68 - 3.62 (m, 1H), 2.41 - 2.35 (m, 2H), 1.80 - 1.47 (m, 5H), 1.46 (s, 9H), 1.08 (s, 9H).

[0429] 10% Pd / C (46 mg) was added to a suspension of tert-butyl ((1S,3R,4S)-3-azido-4-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopentyl)carbamate (460 g, 0.93 mmol) in dry EtOH (50 mL). The reaction was evaporated twice to exchange the inert gas atmosphere, and then connected to two balloons filled with H2. The suspension was stirred vigorously at room temperature for 15 h. The palladium catalyst was removed by using a PTFE-filter (Whatman Puradisc), and the solvent was removed under reduced pressure. Amine Example 309G (460 mg, 0.93 mmol, quantitative) was obtained as a colorless liquid and used without further purification. LCMS [M+H]+ =470

[0430] Step 8: Example 309H To a solution of Compound Example 309G (70 mg, 0.17 mmol) in THF (5 mL) was added 1 M TBAF (1.7 mL, 0.17 mmol) at 0 °C under N2, and the mixture was subsequently stirred at room temperature for 2 hours. The reaction mixture was concentrated to give crude product 13, which was the next step. LCMS [M+H] + =231.

[0431] Step 9: Example 309I A mixture of Compound Example 309H (crude, 0.17 mmol), 2-chloro-N,N-dimethylbenzo[d]thiazole-6-sulfonamide (46 mg, 0.17 mmol) and DIEA (65 mg, 0.51 mmol) in DMSO was stirred at 60 °C overnight. The mixture was purified by preparative HPLC to give Compound Example 309I (38 mg, 47%) as a white solid. LCMS [M+H] + =472.

[0432] Step 10: Example 309J A solution of 4 M HCl / dioxane (5 L) was added to Compound Example 309I (38 mg, 0.08 mmol) in DCM (1 mL), and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated to give Compound Example 309J (30 mg, 100%) as a white solid. LCMS [M+H] + =372.

[0433] Step 16: Example 309 A mixture of Compound Example 309J (30 mg, 0.08 mmol), 2-chloro-5-(methylthio)pyrimidine (15 mg, 0.09 mmol) and DIEA (34 mg, 0.25 mmol) in DMSO (2.5 mL) was stirred at 120 °C for 4 hours. The mixture was purified by preparative HPLC to give Compound Example 309 (5 mg, 12%) as a white solid. LCMS [M+H] + =496. 11H NMR (400 MHz, CD3OD) δ 8.36 (s, 2H), 8.09 (d, J = 1.6 Hz, 1H), 7.68 (dd, J = 2.0, 1.6 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 4.62 (br s, 5H), 4.54 (br m, 1H), 3.63 - 3.58 (m, 2H), 2.70 (s, 3H), 2.69 - 2.65 (m, 1H), 2.37 (s, 3H), 2.36 - 2.28 (m, 1H), 2.16 - 2.10 (m, 1H), 2.05 - 1.99 (m, 1H), 1.86 - 1.82 (m, 1H).

[0434] Scheme 50

Chem.

[0435] Step 1: Example 310A To a mixture of tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate (1.5 g, 7.5 mmol) and 2-chloro-5-(methylthio)pyrimidine (1.3 g, 8.3 mmol) in dimethyl sulfoxide (28 mL) was added N,N-diisopropylethylamine (3.7 mL, 22.5 mmol) at room temperature. The resulting mixture was stirred at 130 °C for 6.5 h under a N2 atmosphere. Next, water (90 mL) and ethyl acetate (160 mL) were added to the mixture. The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (PE:EA = 20:1 to PE:EA = 6:1) to give Example 310A (1.8 g, 76% yield) as a yellow oil. LCMS [M+H] + = 325.

[0436] Step 2: Example 310B To a mixture of Example 310A (1.8 g, 5.7 mmol) in methanol (2 mL), HCl / dioxane (8.0 mL, 4 mol / L) was added at room temperature. The resulting mixture was stirred at room temperature for 2.5 h under a N2 atmosphere. Next, the mixture was evaporated to obtain Example 310B (1.5 g, yield 100%) as a brown solid. LCMS [M+H] + =225.

[0437] Step 3: Example 310C To a mixture of Example 310B (1.5 g, 5.7 mmol) and ethyl 2-chlorobenzo[d]thiazole-6-carboxylate (1.4 g, 5.7 mmol) in DMSO (28 mL), DIEA (2.9 mL, 17.3 mmol) was added at room temperature. The resulting mixture was stirred at 80 °C for 18 h under a N2 atmosphere. Next, water (80 mL) and ethyl acetate (150 mL) were added to the mixture. The combined organic layers were washed with brine (90 mL), dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (PE:EA = 30:1~5:1) to obtain Example 310C (1.9 g, yield 77%) as a yellow solid. LCMS [M+H] + =430.

[0438] Step 4: Example 310D To a mixture of Example 310C (1.9 g, 4.4 mmol) in ethanol (20 mL) and water (10 mL), lithium hydroxide monohydrate (372 mg, 8.9 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 18 h under a nitrogen atmosphere. Next, the mixture was concentrated. Water (60 mL) and HCl (3 mol / L) were added to the residue until pH = 5. The precipitate was filtered and the filter cake was dried to obtain Example 310D (1.5 g, yield 84%) as a brown solid. LCMS [M+H] + =402. 11H NMR (400 MHz, DMSO-d6) δ 8.49 - 8.47 (d, J = 6.8 Hz, 1H), 8.34 (s, 2H), 8.26 - 8.25 (d, J = 1.6 Hz, 1H), 7.81 - 7.79 (m, 1H), 7.55 - 7.53 (d, J = 7.2 Hz, 1H), 7.40 - 7.38 (d, J = 8.4 Hz, 1H), 4.39 - 4.33 (m, 2H), 2.35 (s, 3H), 2.21 - 2.19 (m, 2H), 2.12 - 2.10 (m, 2H), 1.98 - 1.94 (t, J = 13.6 Hz, 1H).

[0439] Project E: Example 310 A mixture of Example 310D (30 mg, 0.075 mmol), dimethylamine (6.75 mg, 0.15 mmol), and HATU (28.5 mg, 0.075 mmol) in DCM was stirred at room temperature for 2 hours. The resulting solution was concentrated under reduced pressure. The residue was purified by preparative HPLC to give the title compound, Example 310 (20 mg), as a yellow solid. LCMS [M+H] + = 429. 1 1H NMR (600 MHz, DMSO-d6) δ 1.54 - 1.64 (m, 2H), 1.98 (t, J = 6.75 Hz, 2H), 2.09 - 2.16 (m, 1H), 2.18 - 2.27 (m, 1H), 2.36 (s, 3H), 2.97 (s, 6H), 4.35 (br s, 2H), 7.30 (dd, J = 8.35, 1.49 Hz, 1H), 7.40 (d, J = 8.24 Hz, 1H), 7.57 (br s, 1H), 7.74 - 7.81 (m, 1H), 8.35 (s, 2H), 8.58 (br s, 1H).

[0440] Using the above procedure, the following examples were synthesized.

[0441]

Table 40

[0442]

Table 41

[0443]

Table 42

[0444]

Table 43

[0445]

Table 44

[0446]

Table 45

[0447]

Table 46

[0448]

Table 47

[0449]

Table 48

[0450]

Table 49

[0451]

Table 50

[0452]

Table 51

[0453]

Table 52

[0454]

Table 53

[0455]

Table 54

[0456]

Table 55

[0457]

Table 56

[0458]

Table 57

[0459]

Table 58

[0460]

Table 59

[0461]

Table 60

[0462] Scheme 51

Chemical formula

[0463] Step 1: Example 371A A mixture of 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.0 g, 4.5 mmol), 3-bromo-2-methoxypyridine (930 mg, 4.9 mmol), Pd(dppf)Cl2 (320 mg, 0.45 mmol) and Na2CO3 (950 mg, 9.0 mmol) in dioxane:H2O = 4:1 (50 mL) was stirred at 110 °C overnight under a N2 atmosphere. The mixture was cooled to room temperature and water (50 mL) was added. The mixture was extracted with EtOAc (20 mL × 3), the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated; the residue was purified by chromatography on silica gel eluting with petroleum ether:EtOAc = 5:1 to give Example 371A (660 mg, 72%) as a white solid.

[0464] Step 2: Example 371 A mixture of (1S,3S)-N1-(5-(methylthio)pyrimidin-2-yl)cyclopentane-1,3-diamine hydrochloride (100 mg, 0.38 mmol), Example 371A (86 mg, 0.42 mmol) and Cs2CO3 (376 mg, 1.15 mmol) in DMSO (5 mL) was stirred at 130 °C for 2 days. The mixture was cooled to room temperature and water (10 mL) was added. The mixture was extracted with EtOAc (10 mL × 5), the combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by preparative HPLC to give Example 371 (10 mg, 6.4%). LCMS [M+H] + = 409. 1 1H NMR (400 MHz, CD3OD) δ 8.41 (s, 2H), 8.20 - 8.17 (m, 2H), 8.09 (s, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.13 - 7.07 (m, 2H), 4.51 - 4.49 (m, 1H), 4.29 - 4.27 (m, 1H), 3.99 (s, 1H), 2.41 - 2.33 (m, 2H), 2.38 (s, 3H), 2.19 - 2.15 (m, 2H), 1.78 - 1.75 (m, 1H).

[0465] Scheme 52 [Chemistry]

[0466] Process 1: Example 372A A mixture of 2-fluoro-4-iodopyridine (200 mg, 0.9 mmol), pyridin-2(1H)-one (102 mg, 1.1 mmol), CuI (17 mg, 0.09 mmol), N,N'-dimethyl-1,2-cyclohexanediamine (19 mg, 0.17 mmol), and K3PO4 (381 mg, 1.8 mmol) in DMSO (5 mL) was stirred at 100 °C for 3 hours under a N2 atmosphere. The mixture was cooled, and water (20 mL) was added. The mixture was extracted with EtOAc (20 mL × 3), and the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, concentrated to obtain a crude product, and purified by chromatography on silica gel eluting with petroleum ether:EtOAc = 5:1 to 1:1 to give Example 372A (97 mg, 57%) as an off-white solid.

[0467] Process 2: Example 372 A mixture of Example 372A (50 mg, 0.19 mmol), (1S,3S)-N1-(5-(methylthio)pyrimidin-2-yl)cyclopentane-1,3-diamine hydrochloride (40 mg, 0.21 mmol), and Cs2CO3 (185 mg, 0.57 mmol) in DMSO (3 mL) was stirred at 130 °C for 2 days. The mixture was cooled, and water (10 mL) was added. The mixture was extracted with EtOAc (10 mL × 5), and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC to give Example 372 (10 mg, 13%). LCMS [M+H] + = 395. 11H NMR (400 MHz, CD3OD) δ 8.39 (s, 2H), 7.99 (d, J = 7.2 Hz, 1H), 7.70 - 7.64 (m, 2H), 7.20 (s, 1H), 7.05 - 7.03 (m, 1H), 7.67 (d, J = 9.2 Hz, 1H), 6.58 - 6.54 (m, 1H), 4.51 - 4.47 (m, 1H), 4.29 - 4.27 (m, 1H), 2.42 - 2.33 (m, 2H), 2.38 (s, 3H), 2.20 - 2.14 (m, 2H), 1.78 - 1.74 (m, 1H).

[0468] The following examples were synthesized using the above procedure.

[0469]

Table 61

[0470] Scheme 54

Chemical Structure

[0471] General Method 1 6’-Chloro-5-methoxy-2H-[1,3’-bipyridin]-2-one (Example 400A) 2-Chloro-5-iodopyridine (191 mg, 0.8 mmol, 1.0 equiv), 5-methoxypyridin-2(1H)-one (100 mg, 0.8 mmol, 1.0 equiv), CuI (15 mg, 0.08 mmol, 0.1 equiv), N,N’-dimethyl-1,2-cyclohexanediamine (23 mg, 0.16 mmol, 0.2 equiv) and K3PO4 (339 mg, 1.6 mmol, 0.2 equiv) in DMSO (5 mL) were stirred at 120 °C overnight under N2 atmosphere. The mixture was cooled to room temperature and water (20 mL) was added. The mixture was extracted with EtOAc (20 mL × 2). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, concentrated, and purified by chromatography on silica gel eluting with PE:EA = 1:1 to give 400A (60 mg, 0.23 mmol, 32%) as an off-white solid.

[0472] General procedure 2 5-Methoxy-6'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridin]-2-one (Example 400) A mixture of 400A (53 mg, 0.22 mmol, 1.0 eq), 131C (50 mg, 0.22 mmol, 1.0 eq), tBuXphos Pd G3 (18 mg, 0.022 mmol, 0.1 eq) and tBuOK (49 mg, 0.44 mmol, 0.2 eq) in dioxane (5 mL) was stirred at 110 °C overnight under a N2 atmosphere. The mixture was cooled to room temperature and filtered. The filtrate was concentrated and purified by preparative TLC, followed by preparative HPLC to afford compound 400 (50 mg, 53%, TFA salt) as an off-white solid. LCMS [M+H] + = 425. 1 H NMR (400 MHz, CD3OD) δ 8.36 (s, 2H), 8.09 (s, 1H), 7.95 (d, J = 9.6 Hz, 1H), 7.50 (d, J = 9.6 Hz, 1H), 7.23 (s, 1H), 7.10 (d, J = 9.6 Hz, 1H), 6.61 (d, J = 9.6 Hz, 1H), 4.48 - 4.46 (m, 1H), 4.25 - 4.23 (m, 1H), 3.73 (s, 3H), 2.40 - 2.35 (m, 5H), 2.16 - 2.13 (m, 2H), 1.76 - 1.74 (m, 2H).

[0473] The following examples were synthesized using the above procedure.

[0474]

Table 62

[0475] Scheme 55

Chem.

[0476] 5-Hydroxy-6'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridin]-2-one (Example 402) To a solution of 400 (50 mg, 0.12 mmol, 1.0 equiv) in DCM (10 mL) was added BBr3 (1 mL) at 0 °C. The mixture was stirred overnight at room temperature, quenched with MeOH, and then concentrated. The residue was purified by preparative TLC, followed by preparative HPLC to give the compound Example 402 (50 mg, 53%, TFA salt) as an off-white solid. 1 H NMR (400 MHz, CD3OD) δ 8.37 (s, 2H), 8.06 (s, 1H), 7.94 (d, J = 9.6 Hz, 1H), 7.45 (d, J = 9.6 Hz, 1H), 7.12 - 7.09 (m, 2H), 6.58 (d, J = 9.6 Hz, 1H), 4.49 - 4.46 (m, 1H), 4.31 - 4.28 (m, 1H), 2.42 - 2.30 (m, 5H), 2.17 - 2.13 (m, 2H), 1.79 - 1.73 (m, 2H). [M+H] + = 411.

[0477] Scheme 56

Chemical Structure

[0478] 4-Hydroxy-5'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2H-[1,2'-bipyridin]-2-one (Example 403) The title compound was obtained from the starting material 4-(benzyloxy)pyridin-2(1H)-one by the same methods as described in General Method 1, General Method 2, and General Method 3. 11H NMR (400 MHz, CD3OD) δ 8.32 (s, 2H), 7.89 (d, J = 2.2 Hz, 1H), 7.45 (d, J = 7.5 Hz, 2H), 7.39 (dd, J = 9.0, 2.7 Hz, 1H), 6.60 (d, J = 8.6 Hz, 1H), 6.13 (dd, J = 7.5, 2.6 Hz, 1H), 5.88 (d, J = 8.6, 2.5 Hz, 1H), 4.48 - 4.25 (m, 2H), 2.35 (s, 3H), 2.32 - 2.19 (m, 2H), 2.06 - 1.92 (m, 2H), 1.70 - 1.52 (m, 2H). [M+H] + = 411.49.

[0479] Scheme 57

Chem.

[0480] General Procedure 4 6'-Chloro-4-methoxy-3,3'-bipyridine (404) A mixture of (6-chloropyridin-3-yl)boronic acid (1.0 g, 6.4 mmol, 1 equiv), 3-bromo-4-methoxypyridine (1.2 g, 6.4 mmol, 1 equiv), Pd(dppf)Cl2 (468 mg, 0.64 mmol, 0.1 equiv) and Na2CO3 (1.3 g, 12.8 mmol, 2 equiv) in dioxane (8 mL) and water (2 mL) was stirred at 105 °C overnight under a N2 atmosphere. The mixture was cooled to room temperature and quenched with water (10 mL). The mixture was extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel eluting with petroleum ether:EtOAc = 5:1 to afford compound 404 (200 mg, 14%) as a brown solid.

[0481] Using the above procedure, the following examples were synthesized.

[0482]

Table 63

[0483] Scheme 58 [Chemical formula]

[0484] 2-Chloro-5-(2,2,2-trifluoroethoxy)pyrimidine (408) To a solution of 2-chloropyrimidin-5-ol (0.5 g, 3.8 mmol, 1.0 equiv) and Cs2CO3 (1.49 g, 4.6 mmol, 1.2 equiv) in DMF (20 mL) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (0.97 g, 4.2 mmol, 1.1 equiv). The resulting suspension was stirred at room temperature for 16 h and then partitioned between EtOAc (30 mL) and water (80 mL). The separated aqueous layer was extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give 408 (0.74 g), which was used directly in the next step. LCMS [M+H] + = 213.

[0485] Scheme 59 [Chemical formula]

[0486] 2-Chloro-5-(difluoromethoxy)pyridine (409) A solution of 2-chloropyrimidin-5-ol (101.6 g, 0.76 moL, 1.0 equiv) in DMF (2000 mL) was charged with Cs2CO3 (300 g, 0.92 moL, 1.2 equiv) and then stirred at room temperature for 1.5 h. Sodium 2-chloro-2,2-difluoroacetate (340 g, 2.3 moL, 3.0 equiv) was added and the reaction mixture was stirred at 100 °C for 3.5 h. The reaction mixture was poured into water (5 L) and extracted with EA (3 × 1 L). The combined organic phases were dried over anhydrous Na2SO4 and concentrated. The residue was purified by flash chromatography on silica gel (PE:EA = 10:1) to give 409 (70 g).

[0487] Scheme 60 [Chemical formula]

[0488] 2-Chloro-5-cyclopropylpyrimidine (410A) To a solution of 5-bromo-2-chloropyrimidine (100 g, 518 mmol, 1.0 equiv) in dioxane (1.5 L), cyclopropylboronic acid (53 g, 616 mmol, 1.2 equiv) and Pd(dppf)Cl2 (10 g, 13.7 mmol, 0.03 equiv) were added Cs2CO3 (250 g, 769 mmol, 1.5 equiv), and the mixture was stirred at 110 °C for 12 h under N2. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluting with hexane:ethyl acetate = 15:1) to give 410A (55 g) as a yellow solid. LCMS [M+H] + = 155.

[0489] General procedure 5 ((1S,3S)-3-((5-Cyclopropylpyrimidin-2-yl)amino)cyclopentyl)carbamic acid tert-butyl (410B) A mixture of 410A (40 g, 260 mmol, 1 equiv), ((1S,3S)-3-aminocyclopentyl)carbamic acid tert-butyl (55 g, 275 mmol, 1.05 equiv) and DIPEA (105 g, 814 mmol, 3.13 equiv) in DMSO (400 mL) was stirred at 110 °C for 12 h under N2. Then, the mixture was cooled to room temperature and diluted with water (1000 mL). The resulting mixture was extracted with EtOAc (100 mL × 3), and the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel (eluting with hexane:ethyl acetate = 5:1 to 4:1) to give compound 410B (55 g) as a pale solid. LCMS [M+H] + = 319.

[0490] General procedure 6 (1S,3S)-N 1 -(5-Cyclopropylpyrimidin-2-yl)cyclopentane-1,3-diamine (410C) To a solution of 410B (44 g, 13.8 mmol, 1.0 eq) in MeOH (250 mL) was added dropwise HCl (4 M in dioxane, 250 mL), and the resulting solution was stirred at room temperature for 2.5 h. After completion of the reaction, the mixture was concentrated to dryness under vacuum. The residue was redissolved in MeOH (500 mL), and an ion exchange resin (Ambersep® 900 OH - type) was added to adjust the pH to about 8. The mixture was filtered off, and the filtrate was concentrated to give 410C (44.5) as a yellow oil. LCMS [M+H] + = 219.

[0491] The following examples were synthesized using the above procedure.

[0492]

Table 64

[0493] Scheme 61

Chemical Structure

[0494] 2-Bromo-5-(methylthio)pyridine (416) A solution of 2,5-dibromopyridine (1 g, 4.22 mmol, 1 eq) in THF (20 mL) was cooled to -78 °C under N2. Next, n-BuLi (2.5 M, 1.77 mL, 4.43 mmol, 1.05 eq) was added dropwise at -78 °C. After stirring the reaction mixture for 20 min, 1,2-dimethyldisulfane (0.411 mL, 4.64 mmol, 1.1 eq) was added slowly. After stirring the reaction mixture for an additional 1 h, it was quenched with saturated NH4Cl. The reaction mixture was extracted with EA (100 mL) and water (100 mL), and subsequently washed with brine. The organic phase was dried over anhydrous Na sIt was dried with SO4. The mixture was filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 10:1) to obtain 416 (86.8 mg). ESI [M+H] + =204.1 1 1H NMR (400 MHz, CDCl3) δ 8.25 (t, J = 6.2 Hz, 1H), 7.41 (ddd, J = 8.9, 8.3, 1.6 Hz, 2H), 2.58 - 2.40 (m, 3H).

[0495] Scheme 62

Chemical Structure

[0496] (1S,3S)-N1-(Thieno[3,2-b]pyridin-5-yl)cyclopentane-1,3-diamine (417) The title compound could be synthesized from the starting material 5-chlorothieno[3,2-b]pyridine by the same methods as described in General Method 2 and General Method 6.

[0497] Using the above procedure, the following examples were synthesized.

[0498]

Table 65

[0499] Scheme 63

Chemical Structure

[0500] General Procedure 7 3-(6-Chloropyridin-3-yl)pyrimidin-4(3H)-one (419) A solution of pyrimidin-4(3H)-one (1.5 g, 15.61 mmol, 1.0 equiv), (6-chloropyridin-3-yl)boronic acid (2.9 g, 18.73 mmol, 1.2 equiv), Cu(OAc)2 (7.9 g, 43.71 mmol, 2.8 equiv), pyridine (2.5 mL, 31.22 mmol, 2.0 equiv) and 4 Å molecular sieves (8 g) in DCM (60 mL) was stirred at room temperature for 48 h. The mixture was filtered through Celite, the filtrate was concentrated and purified by flash column (petroleum ether:EtOAc:1:1) to give Example 419 as a yellow solid (170 mg, yield 5.3%). 1 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 2.6 Hz, 1H), 8.15 (s, 1H), 7.97 (d, J = 6.8 Hz, 1H), 7.80 (dd, J = 8.4, 2.8 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 6.59 (dd, J = 6.8, 0.8 Hz, 1H); MS (ESI+) m / z 208.0 (M + H) +

[0501] The following examples were prepared using the procedure described in Scheme 63.

[0502]

Table 66

[0503] Scheme 64

Chemical formula

[0504] (E)-Ethyl 3-(2-fluoropyridin-4-yl)acrylate (423A) A solution of ethyl 2-(ethoxy(propoxy)phosphoryl)acetate (8.6 g, 38.4 mmol, 1.2 equiv) in THF was added dropwise to a suspension of 60% NaH (1.54 g, 38.4 mmol, 1.2 equiv) in THF (15 mL) at 0 °C under a N2 atmosphere. The mixture was stirred at 0 °C for 25 minutes, and then a solution of 2-fluoroisonicotinaldehyde (4.0 g, 32.0 mmol, 1.0 equiv) in DMF (15 mL) was added. The resulting mixture was stirred at room temperature for 16 hours and then quenched with a saturated aqueous solution of NH4Cl at 0 °C. The aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography (PE~PE:EA = 1:15) to give 423A as a white solid (3.0 g, yield 50%). LCMS: m / z 196 [M+H] + , rt 2.890 min. 1 1H-NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.2 Hz, 1H), 7.59 (d, J = 16.1 Hz, 1H), 7.29 - 7.26 (m, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.60 (d, J = 16.0 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H).

[0505] Ethyl 3-(2-fluoropyridin-4-yl)propanoate (423B) To a solution of 423A (3.0 g, 20.5 mmol) in EtOH (20 mL) was added 10% Pd / C (400 mg). The reaction system was purged with H2, and the mixture was stirred under a H2 atmosphere overnight. The Pd / C was filtered off, and the filtrate was concentrated under vacuum to give 423B (2.3 g, yield 76.7%), which was used directly. LCMS: m / z 198.1 [M+H] + , rt 2.801 min. 1 1H-NMR (400 MHz, CDCl3) δ 8.12 (d, J = 5.5 Hz, 1H), 7.04 (d, J = 4.8 Hz, 1H), 6.78 (d, J = 4.0 Hz, 1H), 4.13 (p, J = 6.8 Hz, 2H), 3.03 - 2.97 (m, 2H), 2.66 (t, J = 7.6 Hz, 2H), 1.24 (t, J = 6.7 Hz, 3H).

[0506] 3-(2-Oxo-1,2-dihydropyridin-4-yl)propanoic acid (423C) Concentrated HCl (5 mL) was added to a 100 mL flask containing 423B (1.53 g, 7.77 mmol). The mixture was heated to 100 °C for 16 h. Next, the mixture was cooled to room temperature and 2 mL of water was added. Solid NaHCO3 was added portionwise to adjust the pH to 6. The mixture was extracted with 30% i-PrOH in CHCl3, and the combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by column chromatography (MeOH / DCM) to give 423C (1.6 g, 91% yield), which was used directly in the next step. LCMS: m / z 167.8 [M+H] + ; 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 5.1 Hz, 1H), 7.05 (dt, J = 5.3, 1.7 Hz, 1H), 6.82 - 6.76 (m, 1H), 3.01 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H).

[0507] Methyl 3-(2-oxo-1,2-dihydropyridin-4-yl)propanoate (423D) 98% H2SO4 (0.15 mL) was added to a 100 mL flask containing a solution of crude 423C (1.6 g) in MeOH (28 mL). The reaction mixture was heated to 90 °C for 16 h and then cooled to room temperature. Saturated aqueous NaCl was added to the above mixture, and it was extracted with 30% i-PrOH in CHCl3. The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by column chromatography (MeOH:DCM = 1:15) to give the product 423D (702 mg, 55% yield). LCMS: m / z 182.1 [M+H] + .

[0508] General Procedure 8 Methyl 3-(6'-fluoro-2-oxo-2H-[1,3'-bipyridin]-4-yl)propanoate (423) To a sealed tube were added 423D (100.0 mg, 0.55 mmol, 1.0 equiv), 2-fluoro-5-iodopyridine (147 mg, 0.66 mmol, 1.2 equiv), CuI (21.0 mg, 0.10 mmol, 0.2 equiv), N,N'-dimethyl-1,2-cyclohexanediamine (15.5 mg, 0.10 mmol, 0.2 equiv), and K2CO3 (151.0 mg, 1.10 mmol, 2.0 equiv). Dioxane (3 mL) was added, and the resulting mixture was purged with N2 and stirred at 110 °C for 16 h. Next, the mixture was diluted with dichloromethane and filtered. The filtrate was washed with water and separated. The aqueous phase was extracted three times with dichloromethane. The combined organic layers were dried over Na2SO4, concentrated, and purified by silica gel chromatography to afford 423 (42 mg, 27.6% yield). LCMS: m / z 278.08 [M+H] +

[0509] Scheme 65

Chemical Structure

[0510] Methyl 6'-fluoro-2-oxo-2H-[1,3'-bipyridine]-5-carboxylate (424A) A mixture of 6-fluoropyridin-3-amine (1.1 g, 10 mmol, 1.0 equiv) and methyl 2-oxo-2H-pyran-5-carboxylate (1.5 g, 10 mmol, 1.0 equiv) in EtOH (10 mL) was stirred at reflux overnight and then cooled to room temperature. The resulting precipitate was collected by filtration and purified by chromatography on silica gel eluting with petroleum ether:EtOAc = 2:1 to afford compound 424A (440 mg, 18%) as an off-white solid.

[0511] Methyl 6'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2-oxo-2H-[1,3'-bipyridine]-5-carboxylate (424B) 424B was obtained from the starting materials 131C and 424A in the same manner as described above.

[0512] General procedure 9 6’-(((1S,3S)-3-((5-(Methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2-oxo-2H-[1,3’-bipyridin]-5-carboxylic acid (Example 424) To a solution of Compound 424B (100 mg, 0.22 mmol, 1.0 equiv) in MeOH:H2O = 5:1 (10 mL) was added NaOH (35 mg, 0.88 mmol, 4.0 equiv). The mixture was stirred at 50 °C for 3 h, then cooled to room temperature and acidified to pH = 5 - 6. The resulting mixture was concentrated and redissolved in THF (50 mL). The solid was filtered off, the filtrate was concentrated, and triturated with EA:MeOH = 5:1 to afford Example 424 (90 mg, 93%) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ: 8.35 (s, 2H), 8.25 (s, 1H), 8.10 (1H), 7.87 (d, J = 9.2 Hz, 1H), 7.56 (s, 1H), 6.87 (s, 1H), 6.51 (d, J = 9.2 Hz, 1H), 4.36 - 4.33 (m, 2H), 2.35 (s, 3H), 2.22 - 2.10 (m, 2H), 2.01 - 1.93 (m, 2H), 1.57 - 1.55 (m, 2H).

[0513] The following examples were synthesized using the above procedure.

[0514] [Table 67]

[0515] [Table 68]

[0516] [Table 69]

[0517] Scheme 66 [Chemistry]

[0518] 2-Chloro-5-((trimethylsilyl)ethynyl)pyridine (431A) To a degassed solution of 2-chloro-5-iodopyridine (5 g, 20.88 mmol, 1.0 equiv) in triethylamine (35 mL), ethynyltrimethylsilane (3.2 mL, 22.97 mmol, 1.1 equiv), CuI (397.7 mg, 2.09 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (1.5 g, 2.09 mmol, 0.1 equiv) were added. The reaction mixture was stirred at room temperature for 16 h under nitrogen. Water (150 mL) was added and the system was extracted with Et2O (100 mL × 2). The combined organic layers were dried over Na2SO4, filtered, and then concentrated under reduced pressure. The resulting crude 431A (6.2 g, black solid) was used for the next step without further purification. MS (ESI+) m / z 209.9 (M+H) +

[0519] 2-Chloro-5-ethynylpyridine (431B) A solution of 431A (crude, 20.88 mmol, 1.0 equiv) and K2CO3 (2.9 g, 20.88 mmol, 1.0 equiv) in methanol (50 mL) was stirred at room temperature for 2 h. After removal of the solvent under reduced pressure, DCM was added (150 mL) and the mixture was filtered. The filtrate was concentrated and purified by flash column (petroleum ether:EtOAc = 10:1) to give compound 432B as a yellow solid (1.0 g, 34.8% yield over 2 steps). MS (ESI+) m / z 138.1 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ: 8.55 (d, J = 2.1 Hz, 1H), 7.98 (dd, J = 8.4, 2.4 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 4.56 (s, 1H).

[0520] 2-Chloro-5-(1-((trimethylsilyl)methyl)-1H-1,2,3-triazol-4-yl)pyridine (431C) A solution of 431B (345 mg, 2.54 mmol, 1.0 equiv), (azidomethyl)trimethylsilane (327 mg, 2.54 mmol, 1.0 equiv), CuI (48 mg, 0.25 mmol, 0.1 equiv), and NEt3 (513 mg, 5.08 mmol, 2.0 equiv) in THF (10 mL) was stirred at room temperature for 16 h. Next, the reaction mixture was concentrated to give 431C (673 mg), which was used without further purification.

[0521] 2-Chloro-5-(1-methyl-1H-1,2,3-triazol-4-yl)pyridine (431) To a solution of 431C in THF (10 mL) was added TBAF (0.80 g, 3.0 mmol, 1.2 equiv), and the resulting solution was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (PE:EA = 1:1) to give 431 (150 mg).

[0522] Scheme 67

Chemical formula

[0523] 2-Chloro-5-(iodoethynyl)pyridine (432A) LDA (4.4 mL, 8.73 mmol, 1.2 equiv) was added dropwise to a solution of 431B (1.0 g, 7.27 mmol, 1.0 equiv) in THF (15 mL) at -78 °C under nitrogen. The mixture was stirred at -78 °C for 0.5 h, and then a solution of iodine (2.0 g, 8.00 mmol, 1.1 equiv) in THF (10 mL) was added dropwise. The resulting solution was slowly warmed to room temperature, stirred for an additional 5 h, and then quenched by the addition of saturated ammonium chloride solution (25 mL). The organic layer was separated, and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were washed with Na2S2O3 (25 mL × 2 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The resulting residue was purified by flash column (petroleum ether:EtOAc:20:1) to give 432A as a yellow solid (1.56 g, yield 82.1%). MS (ESI+) m / z 263.9 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ: 8.51 (d, J = 2.0 Hz, 1H), 7.94 (dd, J = 8.3, 2.4 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H).

[0524] 2-Chloro-5-(5-iodo-1-((trimethylsilyl)methyl)-1H-1,2,3-triazol-4-yl)pyridine (432B) CuI (108.4 mg, 5.69 mmol, 1.0 equiv) and Et3N (1.6 mL, 11.39 mmol, 2.0 equiv) were stirred in THF (60 mL) at room temperature for 1 h under nitrogen. A solution of 432A (1.5 g, 5.69 mmol, 1.0 equiv) and (azidomethyl)trimethylsilane (735.8 mg, 5.69 mmol, 1.0 equiv) in THF (20 mL) was added to the above catalyst solution all at once. Next, the mixture was stirred at room temperature for 16 h. The reaction was quenched by the addition of 10% ammonium chloride solution (15 mL) and concentrated. The residue was washed with water (30 mL) and EtOAc (8 mL) to give 432B as a yellow solid (1.6 g, yield 72.7%), which was used directly in the next step. 11H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 2.3 Hz, 1H), 8.14 (dd, J = 8.3, 2.5 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 3.80 (s, 2H), 0.00 (s, 9H). MS (ESI+) m / z 393.0 (M+H) +

[0525] 2-Chloro-5-(5-iodo-1-methyl-1H-1,2,3-triazol-4-yl)pyridine (432C) To a solution of 432B (1.6 g, 4.07 mmol, 1.0 equiv) in THF (70 mL) was added water (0.15 mL, 8.15 mmol, 2.0 equiv), and then TBAF (4.9 mL, 4.89 mmol, 1.2 equiv) was added dropwise at 0 °C. The resulting reaction mixture was stirred at 0 °C for 15 minutes, poured into water (100 mL), and extracted with DCM (300 mL). The separated organic layer was washed with brine (80 mL), dried over Na2SO4, and concentrated. The residue was purified by flash column (petroleum ether:EtOAc:DCM: 2:1:1) to give 432B as a yellow solid (810 mg, yield 62.3%). MS (ESI+) m / z 320.9 (M+H) + ; 1 1H NMR (400 MHz, DMSO-d6) δ: 8.91 (d, J = 2.1 Hz, 1H), 8.32 (dd, J = 8.4, 2.5 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 4.13 (s, 3H).

[0526] 2-Chloro-5-(5-fluoro-1-methyl-1H-1,2,3-triazol-4-yl)pyridine (432) A suspension of 406B (800 mg, 2.50 mmol, 1.0 equiv) and KF (1.5 g, 25.00 mmol, 10.0 equiv) in acetonitrile / water (14 mL, 1:1) was reacted in a microwave reactor at 160 °C for 20 minutes. After evaporation under reduced pressure, the residue was dissolved in DCM (300 mL) and filtered. The filtrate was concentrated and purified by flash column (petroleum ether:EtOAc:DCM: 2:1:1) to give Example 432 as a yellow solid (220 mg, yield 41.4%). MS (ESI+) m / z 213.0 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 2.4 Hz, 1H), 8.17 (dd, J = 8.3, 2.4 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 4.01 (s, 3H).

[0527] Scheme 68

Chemical Structure

[0528] (6-Bromopyridin-3-yl)carbamic acid 4-nitrophenyl (433A) To a solution of 6-bromopyridin-3-amine (600 mg, 3.47 mmol, 1.0 equiv) in acetonitrile (20 mL) was added dropwise carbonyldichloridic acid 4-nitrophenyl (768.9 mg, 3.81 mmol, 1.1 equiv in 4 mL acetonitrile), maintaining the temperature of the system below 40 °C. After the addition, the mixture was stirred at room temperature for an additional 30 minutes and a yellow precipitate was observed. The precipitate was filtered and washed with acetonitrile (2 mL) to give 433A as a yellow solid (1.1 g, purity ~50%), which was used in the next step without further purification.

[0529] General Procedure 10 3-(6-Bromopyridin-3-yl)-1-methylimidazolidine-2,4-dione (Example 433) A solution of methyl 2-(methylamino)acetate hydrochloride (454.1 mg, 3.25 mmol, 1.0 equiv) and DIPEA (1.7 mL, 9.76 mmol, 3.0 equiv) in acetonitrile (15 mL) was stirred at room temperature for 15 minutes. 433A (1.1 g, 3.25 mmol, 1.0 equiv) was added and the resulting system was stirred at room temperature for an additional 10 minutes. The mixture was concentrated and the residue was purified by flash column (petroleum ether:EtOAc:1:1) to afford Compound Example 433 as a yellow oil (510 mg, 54.6% yield over 2 steps). MS(ESI+) m / z 270.1(M+H) + ; 1 H NMR(400MHz,DMSO-d6)δ 8.48-8.52(m,1H),7.82-7.89(m,2H),4.19(s,2H),3.00(s,3H).

[0530] Scheme 69

Chemical formula

[0531] (2-Methoxyethyl)glycine methyl ester (434A) To a solution of 2-methoxyethan-1-amine (2.9 mL, 33.36 mmol, 1.0 equiv) in THF (40 mL), Et3N (9.3 mL, 66.90 mmol, 2.0 equiv) was added dropwise, followed by the addition of methyl 2-bromoacetate (2.8 mL, 29.58 mmol, 0.9 equiv). The reaction mixture was stirred at room temperature for 19 hours. The reaction mixture was diluted with EA and subsequently washed with water and brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM:MeOH = 20:1) to afford 434A (830 mg) as a colorless liquid. 1 H NMR(400MHz,DMSO-d6)δ 3.62(s,3H),3.38-3.34(m,4H),3.23(s,3H),2.65(t,J=5.6Hz,2H).

[0532] 3-(6-Bromopyridin-3-yl)-1-(2-methoxyethyl)imidazolidine-2,4-dione (Example 434) Example 434 was obtained as a yellow solid from starting materials 434A and 433A in the same manner as described above. 1 H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 2.6 Hz, 1H), 7.74 (dd, J = 8.5, 2.7 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 4.22 (s, 2H), 3.70 - 3.64 (m, 2H), 3.64 - 3.60 (m, 2H), 3.39 (s, 3H). ESI(M+H) + = 314.3

[0533] Scheme 70

Chemical Structure

[0534] 1-(4-Iodophenyl)pyridin-2(1H)-one (435) A solution of 1,4-diiodobenzene (1.0 g, 3.0 mmol, 1.0 equiv), pyridin-2(1H)-one (288 mg, 3 mmol, 1.0 equiv), CuI (58 mg, 0.3 mmol, 0.1 equiv) and K2CO3 (828 mg, 6 mmol, 2.0 equiv) in DMSO (10 mL) was stirred at 130 °C for 2 h under N2. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (30 mL). The organic mixture was washed with water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 10:1~EA) to give 200 mg of Example 435 as a white solid. ESI(M+H) + = 298.09

[0535] Scheme 71

Chemical Structure

[0536] Methyl 3-(2-chloropyridin-4-yl)-2,2-dimethylpropanoate (436A) A solution of methyl isobutyrate (3.3 g, 32.0 mmol, 2.08 equiv) in THF (25 mL) was added dropwise to a solution of LDA (17 mL, 34.0 mmol, 2.2 equiv) in THF (50 mL) at -78 °C over 15 min under a N2 atmosphere. The resulting mixture was stirred at -78 °C for 45 min and then treated with a solution of 2-chloro-4-(chloromethyl)pyridine (2.5 g, 15.4 mmol, 1.0 equiv) in THF (6 mL) over 5 min. The cold bath was removed and the reaction mixture was stirred at room temperature for 18 h. 1.0 N aqueous hydrochloric acid was added dropwise to the above solution (50 mL) to quench the reaction. The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water, dried over sodium sulfate, and concentrated under vacuum. The crude product was purified by column chromatography to afford compound 436A (3.2 g) as a yellow oil. LCMS: m / z 228.0. 1 H NMR (400 MHz, CDCl3) δ 8.33 - 8.21 (m, 1H), 7.16 - 7.07 (m, 1H), 6.98 (dd, J = 5.1, 1.5 Hz, 1H), 3.68 (s, 3H), 2.84 (s, 2H), 1.21 (s, 6H).

[0537] Methyl 2,2-dimethyl-3-(2-oxo-1,2-dihydropyridin-4-yl)propanoate (436B) A solution of 436A (1.2 g, 5.3 mmol, 1.0 equiv) and sodium acetate (868 mg, 10.6 mmol, 2.0 equiv) in acetic acid (5.3 mL) was heated at 160 °C for 1 h in a microwave reactor. The mixture was concentrated under vacuum and the residue was poured into water. The aqueous phase was extracted twice with 15% isopropanol in DCM. The combined organic layers were washed with saturated aqueous NaHCO3, dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by flash chromatography on silica gel (DCM:MeOH = 20:1) to afford 436B (330 mg) as a white solid. ESI[M+H] + = 210.24.

[0538] General procedure 11 Methyl 3-(6’-chloro-2-oxo-2H-[1,3’-bipyridin]-4-yl)-2,2-dimethylpropanoate (436C) A suspension of 436B (330 mg, 1.58 mmol, 1.0 equiv), 2-chloro-5-iodopyridine (567 mg, 2.37 mmol, 1.5 equiv), N1,N2-dimethylcyclohexane-1,2-diamine (44.8 mg, 0.316 mmol, 0.2 equiv), CuI (60 mg, 0.316 mmol, 0.2 equiv) and K3CO3 (436 mg, 3.16 mmol, 2.0 equiv) in dioxane (8 mL) was stirred at 110 °C overnight under N2. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EtOAc = 3:1 to PE:EtOAc = 1:1) to afford 436C (350 mg) as a yellow oil. ESI[M+H] + = 321.77.

[0539] Scheme 72

Chemical formula

[0540] (E)-4-((Dimethylamino)methylene)isochroman-1,3-dione (437A) Phosphoryl chloride (10 mL, 107 mmol, 2.1 equiv) was added dropwise to a solution of 2-(carboxymethyl)benzoic acid (10 g, 50 mmol, 1.0 equiv) in DMF (100 mL) with stirring at 0 °C. The resulting mixture was stirred for an additional 1 h and then poured into ice water. The precipitate formed was collected by filtration, washed with water to afford 437A as a yellow solid (10 g).

[0541] Methyl 1-oxo-1H-isochromene-4-carboxylate (437B) Dry hydrogen chloride gas was passed through a stirred solution of 437A (6.2 g, 0.03 mmol, 1.0 eq) in methanol (180 mL) at room temperature for 2 h. The solution was heated under reflux for 2 h and then concentrated under reduced pressure. The residue was diluted with water and then extracted with DCM (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The crude residue was purified by column chromatography on silica gel (PE:EA = 3:1) to give 437B (1.9 g).

[0542] Methyl 2-(6-chloropyridin-3-yl)-1-oxo-1,2-dihydroisoquinoline-4-carboxylate (Example 437) A solution of 437B (0.83 g, 0.41 mmol, 1.0 eq) and 6-chloropyridin-3-amine (0.53 g, 0.41 mmol, 1.0 eq) in AcOH (15 mL) was heated to 120 °C and stirred for 2 h. The system was cooled to room temperature and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (PE:EA = 10:1) to give Example 437 (400 mg).

[0543] Scheme 73 [Chemical formula]

[0544] General Procedure 12 2-(6-Chloropyridin-3-yl)pyridazin-3(2H)-one (438) A mixture of 2-chloro-5-iodopyridine (5.95 g, 25 mmol, 1.0 equiv), pyridazin-3(2H)-one (2.52 g, 26.3 mmol, 1.05 equiv), CuI (475 mg, 2.5 mmol, 0.1 equiv), trans-N,N'-dimethyl-1,2-cyclohexanediamine (534 mg, 3.76 mmol, 0.15 equiv) and K2CO3 (6.9 g, 50 mmol, 2.0 equiv) in DMSO (25 mL) was stirred at 120 °C overnight under a N2 atmosphere. The mixture was cooled to room temperature and filtered. The filtrate was diluted with water and extracted with EA (200 mL × 2). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by chromatography on silica gel eluting with PE:EA = 5:1 to 1:1 to give 438 (3.6 g, 69%) as a white solid.

[0545] Scheme 74

Chemical formula

[0546] Methyl 2-(6-chloropyridin-3-yl)-3-cyanopropanoate (439A) To a cold (-78 °C) solution of methyl 2-(6-chloropyridin-3-yl)acetate (3.0 g, 16.2 mmol, 1.0 equiv) in THF (30 mL) was added dropwise LiHMDS (24.24 mL, 24.24 mmol, 1.5 equiv). The reaction mixture was stirred at -78 °C for 2 h. 2-Bromoacetonitrile (1.7 mL, 24.24 mmol, 1.5 equiv) was added dropwise at -78 °C. The reaction mixture was stirred at -78 °C for an additional 2 h and then quenched with water. The reaction mixture was extracted 3 times with EA. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 2:1) to give 439A (1.46 g) as a yellow oil. ESI(M+H) + =225.3

[0547] 3-(6-Chloropyridin-3-yl)pyrrolidin-2-one (439B) To a cold (0 °C) solution of 439A (700 mg, 3.1 mmol, 1.0 equiv) and CoCl2 (370 mg, 1.56 mmol, 0.5 equiv) in THF / water (6 mL / 3 mL) was added NaBH4 (590 mg, 15.6 mmol, 5.0 equiv) at 0 °C under N2. The reaction mixture was stirred for 2 h while warming the temperature to room temperature. Next, the reaction was quenched with saturated NH4Cl and filtered through celite. The filtrate was extracted three times with DCM. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 1:1) to afford 439B (360 mg) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 2.5 Hz, 1H), 7.64 (dd, J = 8.3, 2.5 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 3.65 (t, J = 9.4 Hz, 1H), 3.54 - 3.50 (m, 2H), 2.66 (ddd, J = 13.5, 9.4, 4.9 Hz, 1H), 2.32 - 2.15 (m, 1H). ESI(M+H) + = 197.2.

[0548] 3-(6-Chloropyridin-3-yl)-1-methylpyrrolidin-2-one (Example 439) To a cold (0 °C) solution of 439B (210 mg, 1.1 mmol, 1.0 equiv) in THF (10 mL) was added 60% NaH (64 mg, 1.6 mmol, 1.5 equiv). The reaction mixture was stirred at 0 °C for 15 min, then iodomethane (0.053 mL, 0.8 mmol, 0.8 equiv) in THF (0.5 mL) was added dropwise. The resulting solution was stirred at 0 °C for an additional 2 h and quenched with saturated NH4Cl. The system was partitioned and separated, and subsequently the aqueous phase was extracted three times with EA. The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 1:4) to afford Example 439 (80 mg) as a brown oil. ESI(M+H) + = 211.2

[0549] The following examples were synthesized using the above procedure.

[0550]

Table 70

[0551]

Table 71

[0552]

Table 72

[0553]

Table 73

[0554]

Table 74

[0555]

Table 75

[0556]

Table 76

[0557]

Table 77

[0558] Scheme 75

Chemical formula

[0559] 1-(2-(((1S,3S)-3-((5-(Methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)pyrimidin-5-yl)pyridin-2(1H)-one (Example 459) The title compound was obtained from the starting material 2-chloro-5-iodopyrimidine by a method similar to those described in General Method 5 and General Method 1. 1 H NMR (400 MHz, CD3OD) δ 8.41 - 8.40 (d, J = 2.4 Hz, 2H), 8.34 (s, 2H), 7.63 - 7.59 (m, 2H), 6.63 - 6.61 (d, J = 8.8 Hz, 1H), 6.49 - 6.46 (m, 1H), 4.49 - 4.41 (m, 2H), 2.40 - 2.39 (d, J = 1.2 Hz, 3H), 2.31 - 2.25 (m, 2H), 2.08 - 2.05 (t, J = 13.6 Hz, 2H), 1.69 - 1.64 (m, 2H).

[0560] Using the above procedure, the following examples were synthesized.

[0561]

Table 78

[0562] Scheme 76

Chem.

[0563] (E)-3-(2-(Methoxycarbonyl)phenyl)acrylic acid (461A) A mixture of methyl 2-formylbenzoate (2.5 g, 15.23 mmol, 1.0 equiv), malonic acid (1.8 g, 17.67 mmol, and 1.16 equiv), morpholine (0.15 mL), and pyridine (4 mL) was stirred at 100 °C for 4 h. After cooling to room temperature, the resulting solution was poured into a mixture of crushed ice (50 g) and 35% aq. HCl (25 mL). The precipitate was filtered and washed with water (25 mL × 2). Next, the white solid was recrystallized from methanol to give 461A (2.0 g, yield 63.7%). 11H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 15.9 Hz, 1H), 7.99 (dd, J = 7.8, 1.1 Hz, 1H), 7.61 - 7.68 (m, 1H), 7.53 - 7.60 (m, 1H), 7.41 - 7.51 (m, 1H), 6.33 (d, J = 15.9 Hz, 1H), 3.95 (s, 3H).

[0564] (E)-Methyl 2-(3-azido-3-oxoprop-1-en-1-yl)benzoate (461B) To a solution of 461A (1.2 g, 5.82 mmol, 1.0 equiv) and Et3N (1.6 mL, 11.64 mmol, 2.0 equiv) in toluene (15 mL) was added dropwise DPPA (1.2 mL, 5.53 mmol, 0.95 equiv). The mixture was stirred continuously at room temperature for 16 h. The solution was concentrated and purified by flash column (petroleum ether:EtOAc = 10:1) to give 461B as a white solid (1.0 g, yield 74.6%). 1 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 15.8 Hz, 1H), 8.00 (dd, J = 7.8, 0.9 Hz, 1H), 7.53 - 7.67 (m, 2H), 7.42 - 7.53 (m, 1H), 6.31 (d, J = 15.8 Hz, 1H), 3.95 (s, 3H).

[0565] Methyl 1-oxo-1,2-dihydroisoquinoline-5-carboxylate (461) A solution of 461B (500 mg, 2.16 mmol, 1.0 equiv) in diphenylmethane (3 mL) was stirred at 80 °C for 1 h under nitrogen. Next, the mixture was stirred continuously at 240 °C for 3 h. After cooling to room temperature, the mixture was purified by flash column (petroleum ether:EtOAc: 2:1) to give a crude product, which was further purified by preparative HPLC to give Example 461 as a white solid (30 mg, yield 6.8%). MS (ESI+) m / z 204.0 (M+H) + ; 11H NMR (400 MHz, DMSO-d6) δ 11.51 (br.s., 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.27 (d, J = 6.9 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.24 - 7.46 (m, 2H), 3.90 (s, 3H).

[0566] Scheme 77 [Chemical formula]

[0567] 2-Methoxy-5-(1-methyl-1H-pyrazol-4-yl)pyridine (462A) A suspension of (6-methoxypyridin-3-yl)boronic acid (820 mg, 5.4 mmol, 1.0 equiv), 4-bromo-1-methyl-1H-pyrazole (1.04 g, 6.4 mmol, 1.2 equiv), Pd(dppf)Cl2 (392.3 mg, 0.54 mmol, 0.1 equiv) and Cs2CO3 (3.5 g, 10.8 mmol, 2.0 equiv) in dioxane / water (40 mL / 10 mL) was stirred at 110 °C for 16 h under N2. The reaction mixture was partitioned between ethyl acetate and water. The organic phase was separated, washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM:MeOH = 20:1) to afford 462A (662.4 mg) as a yellow solid. ESI(M+H) + = 190.1.

[0568] 5-(1-Methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (Example 462) To a solution of 462A (200 mg, 1.1 mmol) in EtOH (0.5 mL) was added HBr solution (40% in H2O, 2.5 mL). The reaction mixture was stirred at 80 °C for 20 h. The reaction mixture was cooled to room temperature and basified by dropwise addition of aqueous NH3. The solvent was evaporated under reduced pressure and the crude residue was purified by flash chromatography (DCM:MeOH:NH4OH = 10:1:0.1) to afford Example 462 (120 mg) as a grey solid.1 1H NMR (400 MHz, CD3OD) δ 7.94 (dd, J = 9.4, 2.6 Hz, 1H), 7.77 (d, J = 2.3 Hz, 1H), 7.65 (s, 1H), 7.38 (d, J = 1.1 Hz, 1H), 6.57 (dd, J = 21.7, 9.3 Hz, 1H), 3.76 (s, 3H). ESI (M+H) + = 176.1.

[0569] Scheme 78

Chem.

[0570] General method 13 (1S,3S)-N 1 -(5-Iodopyridin-2-yl)-N 3 -(5-(Methylthio)pyrimidin-2-yl)cyclopentane-1,3-diamine (463A) A suspension of 131C (150 mg, 0.669 mmol, 1.0 equiv), 2-Fluoro-5-iodopyridine (178.9 mg, 0.802 mmol, 1.2 equiv) and K2CO3 (277.2 mg, 2.006 mmol, 3.0 equiv) in DMSO (5 mL) was stirred at 140 °C for 16.5 h under N2. The reaction mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate, washed with water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 1:1) to give 463A (125.5 mg). ESI (M+H) + = 428.1.

[0571] General method 14 3-Methyl-1-(6-(((1S,3S)-3-((5-(Methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)imidazolidine-2,4-dione (463) A suspension of 463A (65.5 mg, 0.153 mmol, 1.0 equiv), 3-methylimidazolidine-2,4-dione (35.0 mg, 0.307 mmol, 2.0 equiv), N1,N2-dimethylcyclohexane-1,2-diamine (10.9 mg, 0.077 mmol, 0.5 equiv), CuI (14.6 mg, 0.077 mmol, 0.5 equiv) and K3PO4 (97.6 mg, 0.460 mmol, 3.0 equiv) in i-PrOH (3 mL) was purged with N2. The reaction mixture was stirred at 110 °C for 4 h under microwave irradiation. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM:MeOH = 20:1) to afford Example 463 (20 mg) as a white powder. 1 H NMR (400 MHz, TFA-d4) δ 8.8 (brs, 2H), 8.40 (d, J = 2.4 Hz, 1H), 8.25 (dd, J = 9.8, 2.3 Hz, 1H), 7.23 (d, J = 9.8 Hz, 1H), 4.83 - 4.78 (m, 1H), 4.68 (s, 2H), 4.56 - 4.39 (m, 1H), 3.28 (s, 3H), 2.62 - 2.49 (m, 2H), 2.54 (s, 3H), 2.45 - 2.30 (m, 2H), 1.96 - 1.88 (m, 2H). ESI(M+H) + = 414.3.

[0572] The following examples were synthesized using the procedure above.

[0573]

Table 79

[0574]

Table 80

[0575] Scheme 79

Chem.

[0576] (1S,3S)-N1 -(5-(2-Chlorophenyl)pyridin-2-yl)-N 3 -(5-(Methylthio)pyrimidin-2-yl)cyclopentane-1,3-diamine (Example 467) The title compound was obtained from starting material 463A and (2-chlorophenyl)boronic acid in a similar manner to that described in General Method 4. 1 H NMR (400 MHz, CDCl3) δ 10.20 (s, 1H), 8.53 (brs, 1H), 8.45 (s, 2H), 7.95 (dd, J = 9.2, 1.4 Hz, 1H), 7.83 (s, 1H), 7.56 - 7.47 (m, 1H), 7.36 (dd, J = 5.9, 3.4 Hz, 2H), 7.25 - 7.27 (m, 1H), 7.13 (d, J = 9.3 Hz, 1H), 4.59 (brs, 1H), 4.24 (brs, 1H), 2.52 - 2.29 (m, 5H), 2.20 (ddt, J = 20.7, 13.8, 7.0 Hz, 2H), 1.99 - 1.86 (m, 1H), 1.74 (dt, J = 13.0, 6.6 Hz, 1H). ESI(M + H) + = 412.3.

[0577] Scheme 80

Chemical formula

[0578] (1S,3S)-N 1 -(5-Cyclopropylpyrimidin-2-yl)-N 3 -(5-Iodopyridin-2-yl)cyclopentane-1,3-diamine (468A) From starting material 425C (44.5 g, 208.7 mmol, 1.0 equivalent), 425D (24.7 g) was obtained as a pale solid in a similar manner to the above. LCMS[M + H] + = 422.

[0579] 2-(6-(((1S,3S)-3-((5-Cyclopropylpyrimidin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)pyridazin-3(2H)-one (Example 468) 468A (18.7 g, 44.4 mmol, 1.0 equiv) in DMSO (150 mL), pyridazin-3(2H)-one (8.53 g, 88.8 mmol, 2.0 equiv), (1S,2S)-N 1 ,N 2 -dimethylcyclohexane-1,2-diamine (1.26 g, 8.88 mmol, 0.2 equiv) and CuI (0.85 g, 4.44 mmol, 0.1 equiv), K2CO3 (18.5 g, 133.2 mmol, 3.0 equiv) was added to the solution, and the resulting system was stirred at 135 °C for 12 h under N2. The reaction mixture was cooled to room temperature, poured into water (1 L), and extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. The ethyl acetate phase was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with DCM:MeOH = 100:1 to 50:1) to obtain Example 468 (25.3 g) as a pale yellow solid. 1 1H NMR (400 MHz, CD3OD) δ 8.15 (d, J = 2.5 Hz, 1H), 8.07 (s, 2H), 8.02 (dd, J = 3.9, 1.6 Hz, 2H), 7.60 (dd, J = 9.0, 2.6 Hz, 1H), 7.46 (dd, J = 9.4, 3.9 Hz, 1H), 7.06 (dd, J = 9.5, 1.6 Hz, 1H), 6.59 (d, J = 9.0 Hz, 1H), 4.42 - 4.27 (m, 2H), 2.34 - 2.18 (m, 2H), 1.99 (t, J = 6.8 Hz, 2H), 1.81 - 1.70 (m, 1H), 1.65 - 1.53 (m, 2H), 0.95 - 0.85 (m, 2H), 0.66 - 0.54 (m, 2H). LCMS [M+H] + = 390.

[0580] The following examples were synthesized using the above procedure.

[0581]

Table 81

[0582]

Table 82

[0583]

Table 83

[0584]

Table 84

[0585] Scheme 74

Chem.

[0586] 2-Oxo-1,2-dihydroquinoline-5-carboxylic acid (478A) A suspension of 2-chloroquinoline-5-carboxylic acid (300 mg, 1.45 mmol, 1.0 eq) in AcOH (5 mL) and H2O (2 mL) was stirred at 130 °C overnight. The reaction mixture was cooled to 0 °C and stirred for 0.5 h. The precipitate was collected by filtration and the solid was dried in vacuo to give 478A (250 mg).

[0587] 1-(6-(((1S,3S)-3-((5-Cyclopropylpyrimidin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)-2-oxo-1,2-dihydroquinoline-5-carboxylic acid (Example 478) A suspension of 478A (18 mg, 0.1 mmol, 1.0 eq), (1S,3S)-N1-(5-cyclopropylpyrimidin-2-yl)-N3-(5-iodopyridin-2-yl)cyclopentane-1,3-diamine (40 mg, 0.1 mmol, 1.0 eq), quinolin-8-ol (3 mg, 0.02 mmol, 0.2 eq), CuI (4 mg, 0.02 mmol, 0.2 eq), and K2CO3 (20 mg, 0.15 mmol, 1.5 eq) in DMSO (3 mL) was purged with N2. Next, the reaction mixture was stirred at 120 °C for 2 h under microwave irradiation. The reaction mixture was filtered, and the filtrate was partitioned between ethyl acetate (5 mL) and water (10 mL). After separation, the aqueous phase was concentrated under reduced pressure. The crude residue was purified by preparative HPLC to give Example 478 (4.7 mg). 1 H NMR (400 MHz, CD3OD) δ 9.11 (d, J = 10.1 Hz, 1H), 8.25 (s, 2H), 8.05 (s, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.79 (d, J = 9.4 Hz, 1H), 7.67 - 7.50 (m, 1H), 7.27 - 7.15 (m, 2H), 6.84 (d, J = 10.1 Hz, 1H), 4.58 - 4.45 (m, 1H), 4.38 - 4.28 (m, 1H), 2.53 - 2.28 (m, 2H), 2.27 - 2.06 (m, 2H), 1.91 - 1.68 (m, 3H), 1.03 - 0.92 (m, 2H), 0.73 - 0.63 (d, J = 4.5 Hz, 2H). ESI(M + H) + = 483.

[0588] Using the above procedure, the following examples were synthesized.

[0589]

Table 85

[0590] Scheme 75

Chemical Structure

[0591] General Method 15 6'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-4-(2H-tetrazol-5-yl)-2H-[1,3'-bipyridin]-2-one (Example 481) A mixture of 451 (35 mg, 0.083 mmol, 1.0 equiv), NH4Cl (44 mg, 0.83 mmol, 10.0 equiv), and NaN3 (54 mg, 0.83 mmol, 10.0 equiv) in DMF (2 mL) was stirred at 100 °C for 5 h. The mixture was cooled to room temperature and filtered. The filtrate was purified by preparative HPLC to give Example 481 (13 mg, 33.7%) as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ ppm: 8.37 (s, 2H), 8.15 (d, J = 2.0 Hz, 1H), 7.98 (dd, J = 2.1 Hz, 9.6 Hz, 1H), 7.83 (dd, J = 0.4 Hz, 7.2 Hz, 1H), 7.34 (d, J = 1.2 Hz, 1H), 7.10 - 7.15 (m, 2H), 4.46 - 4.50 (m, 1H), 4.27 - 4.31 (m, 1H), 2.43 - 2.43 (m, 5H), 2.11 - 2.18 (m, 2H), 1.71 - 1.80 (m, 2H). LCMS [M+H] + = 463.4.

[0592] Using the above procedure, the following examples were synthesized.

[0593]

Table 86

[0594] Scheme 76

Chemical Structure

[0595] Methyl 1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (484A) A solution of methyl 2-oxo-2H-pyran-5-carboxylate (3 g, 19.465 mmol, 1.0 eq) and methylamine (33% in EtOH, 1.904 g, 20.439 mmol, 1.05 eq) in EtOH (10 mL) was stirred at 60 °C for 18 h in a sealed tube. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 1:2) to give 484A (393.6 mg) as a yellow solid. ESI(M+H) + =168.1

[0596] Methyl 5-bromo-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (484B) A solution of 484A (393.6 mg, 2.355 mmol, 1 eq) and NBS (628.6 mg, 3.532 mmol, 1.5 eq) in AcOH (16 mL) was stirred at 80 °C for 2 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 1:1) to give 484B (377.3 mg) as a white solid. 1 H NMR(400MHz,CDCl3)δ 8.26(d,J=2.3Hz,1H),8.18(d,J=2.3Hz,1H),3.90(s,3H),3.68(s,3H).ESI(M+H) + =246.0.

[0597] Methyl 6’-chloro-1-methyl-2-oxo-1,2-dihydro-[3,3’-bipyridine]-5-carboxylate (484C) 484C was obtained from the starting material 484B and (6-chloropyridin-3-yl)boronic acid in the same manner as above. 1 H NMR(400MHz,CDCl3)δ 8.68(d,J=2.2Hz,1H),8.27(d,J=2.3Hz,1H),8.21-8.13(m,1H),8.08(d,J=2.4Hz,1H),7.39(t,J=11.7Hz,1H),3.91(s,3H),3.69(s,3H).ESI(M+H) + =279.2

[0598] Methyl 6'-(((1S,3S)-3-((tert-butoxycarbonyl)amino)cyclopentyl)amino)-1-methyl-2-oxo-1,2-dihydro-[3,3'-bipyridine]-5-carboxylate (484D) 484D was obtained from the starting material 484C and tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate in the same manner as described above. ESI(M+H) = 443.3

[0599] Methyl 6'-(((1S,3S)-3-aminocyclopentyl)amino)-1-methyl-2-oxo-1,2-dihydro-[3,3'-bipyridine]-5-carboxylate (484E) To a solution of 484D (35 mg) in DCM (1 mL) was added dropwise TFA (1 mL). The reaction mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was dissolved in MeOH and then ion exchange resin (Ambersep® 900 OH - type) was added to adjust the pH level to 8. The mixture was filtered and the filtrate was concentrated under reduced pressure to give 484E (27 mg) 8 as a yellow oil.

[0600] Methyl 1-methyl-6'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2-oxo-1,2-dihydro-[3,3'-bipyridine]-5-carboxylate (484F) 484F was obtained from the starting material 484E and 2-chloro-5-(methylthio)pyrimidine in the same manner as described above. ESI(M+H) + = 467.2.

[0601] 1-Methyl-6'-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-2-oxo-1,2-dihydro-[3,3'-bipyridine]-5-carboxylic acid (Example 484) Example 484 was obtained from the starting material 484F in the same manner as described above. 11H NMR (400 MHz, CD3OD) δ 8.51 (dd, J = 14.0, 2.1 Hz, 2H), 8.35 (s, 2H), 8.25 (dd, J = 10.7, 2.2 Hz, 2H), 7.10 (d, J = 9.5 Hz, 1H), 4.51 - 4.41 (m, 1H), 4.31 - 4.20 (m, 1H), 3.69 (s, 3H), 2.49 - 2.39 (m, 1H), 2.35 (d, J = 7.6 Hz, 3H), 2.33 - 2.27 (m, 1H), 2.22 - 2.07 (m, 2H), 1.84 - 1.65 (m, 2H). ESI(M+H) + = 453.3.

[0602] Scheme 77

Chem.

[0603] 6'-(((1S,3S)-3-Aminocyclopentyl)amino)-2H-[1,3'-bipyridin]-2-one (485) 485 was obtained from the starting material 6'-chloro-2H-[1,3'-bipyridin]-2-one by a method similar to those described in General Method 2 and General Method 6. m / z 271.0 [M+H] + .

[0604] The following examples were synthesized using the above procedure.

[0605]

Table 87

[0606] Scheme 78

Chem.

[0607] 6'-(((1S,3S)-3-(Thieno[3,2-d]pyrimidin-2-ylamino)cyclopentyl)amino)-2H-[1,3'-bipyridin]-2-one (487) A solution of 485 (150 mg, 0.405 mmol, 1.0 equiv), DIPEA (260 mg, 2.0 mmol, 5.0 equiv) and 2-chlorothieno[3,2-d]pyrimidine (69 mg, 0.405 mmol, 1.0 equiv) in DMA (2 mL) was stirred at 150 °C for 30 minutes. The resulting mixture was diluted with ethyl acetate (10 mL), washed with water (2 × 10 mL), and the aqueous phase was extracted with ethyl acetate (4 × 20 mL). The combined organic layers were concentrated under vacuum and purified by preparative HPLC to give 487 (12 mg) as a white solid. LCMS m / z 405 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 10.73 (brs, 1H), 10.21 (brs, 1H), 9.03 (brs, 1H), 8.19 (d, J = 5.3 Hz, 1H), 7.92 (dd, J = 9.4, 2.3 Hz, 1H), 7.83 (d, J = 2.3 Hz, 1H), 7.54 - 7.43 (m, 2H), 7.41 (d, J = 5.5 Hz, 1H), 7.26 - 7.21 (m, 1H), 6.69 (dd, J = 9.3, 1.2 Hz, 1H), 6.38 - 6.29 (m, 1H), 4.81 - 4.59 (m, 1H), 4.53 - 4.25 (m, 1H), 2.52 - 2.31 (m, 3H), 2.08 (dt, J = 14.3, 7.8 Hz, 1H), 1.98 - 1.77 (m, 2H).

[0608] The following examples were synthesized using the procedure above.

[0609]

Table 88

[0610] Scheme 79

Chemical Structure

[0611] 2-(6-(((1S,3S)-3-(Thieno[2,3-d]pyrimidin-2-ylamino)cyclopentyl)amino)pyridin-3-yl)pyridazin-3(2H)-one (489) A solution of 486 (50 mg, 0.18 mmol), 5-chlorothiazolo[4,5-d]pyrimidine (30.5 mg, 0.18 mmol), and DIPEA (70 mg, 0.54 mmol) in DMSO (5 mL) was stirred at 100 °C overnight under N2. The reaction mixture was extracted with EA and water. The organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by preparative TLC and preparative HPLC to give 2.6 mg of 489 (2.3 mg). ESI(M+H) + =406.48; 1 H NMR(400MHz,CD3OD)δ 8.76(s,1H),8.39(d,J=2.2Hz,1H),8.22(d,J=9.7Hz,1H),8.10(d,J=3.8,1.6Hz,1H),7.52(dd,J=9.5,3.9Hz,1H),7.22(d,J=6.0Hz,2H),7.16 - 7.06(m,2H),4.67 - 4.48(m,1H),4.36 - 4.23(m,1H),2.52 - 2.31(m,2H),2.30 - 2.12(m,2H),1.86 - 1.69(m,2H).

[0612] The following examples were synthesized using the procedure above.

[0613]

Table 89

[0614]

Table 90

[0615] Scheme 80

Chemical formula

[0616] 2-Chloro-7-methylthieno[3,2-d]pyrimidine (494A) A suspension of 2,4-dichloro-7-methylthieno[3,2-d]pyrimidine (500 mg, 2.3 mmol, 1 equiv), Pd(OH)₂ (20% on carbon, 200 mg, 0.14 mmol, 0.55 equiv), and NaOAc (400 mg, 4.8 mmol, 2.0 equiv) in EA (8 mL) and i-PrOH (1 mL) was stirred at room temperature for 18 h under a H₂ atmosphere (50 psi) in a Prr apparatus. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (PE:EA = 4:1) to afford 494A (320 mg) as a white powder. ESI(M+H) + =185.0; 1 H NMR (400 MHz, CDCl₃) δ 9.09 (s, 1H), 7.73 (q, J = 1.1 Hz, 1H), 2.51 (d, J = 1.2 Hz, 3H).

[0617] 6'-(((1S,3S)-3-((7-Methylthieno[3,2-d]pyrimidin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridin]-2-one (494) A suspension of 494A (28.45 mg, 0.1541 mmol, 1.0 equiv), 485 (50 mg, 0.185 mmol, 1.2 equiv), and K₂CO₃ (63.9 mg, 0.462 mmol, 3.0 equiv) in DMSO (5 mL) was stirred at 140 °C for 16 h under N₂. The reaction mixture was extracted with DCM and water. The organic phase was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (DCM:MeOH = 20:1) to afford 494 (10.4 mg) as a white powder. 11H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 2.6 Hz, 1H), 7.60 (dd, J = 6.8, 1.8 Hz, 1H), 7.47 (ddd, J = 9.0, 6.6, 2.1 Hz, 1H), 7.39 (dd, J = 8.9, 2.7 Hz, 1H), 7.21 (d, J = 7.2 Hz, 1H), 6.92 (d, J = 7.0 Hz, 1H), 6.53 (d, J = 9.1 Hz, 1H), 6.44 (d, J = 8.9 Hz, 1H), 6.27 (td, J = 6.7, 1.3 Hz, 1H), 4.52 - 4.39 (m, 1H), 4.38 - 4.26 (m, 1H), 2.27 (d, J = 0.9 Hz, 3H), 2.16 (dd, J = 9.8, 5.4 Hz, 2H), 2.03 - 1.86 (m, 3H), 1.63 - 1.47 (m, 1H). ESI (M + H) + = 419.3

[0618] The following examples were synthesized using the above procedure.

[0619]

Table 91

[0620] Scheme 81

Chemical Structure

[0621] 1-(2-Chloropyrimidin-5-yl)ethan-1-ol (496) To a solution of 1-(2-chloropyrimidin-5-yl)ethan-1-one (500 mg, 3.2 mmol, 1.0 equiv) in MeOH (15 mL) was added NaBH4 (240 mg, 6.4 mmol, 2.0 equiv) at 0 °C. After stirring at room temperature for 2 h, water (5 mL) was added to the above mixture to quench the reaction. The resulting mixture was concentrated under reduced pressure, and the crude residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1 to ethyl acetate) to afford 496 (100 mg) as a pale solid. ESI [M + H] + = 159.1.

[0622] Scheme 82

Chem.

[0623] 2-(6-(((1S,3S)-3-((5-(methylthio)pyridin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)pyridazin-3(2H)-one (497) 497 was obtained from starting materials 416 and 486 by a method similar to that described in General Method 2. 1 H NMR (400 MHz, CD3OD) δ 8.28 (d, J = 2.3 Hz, 1H), 8.04 (dd, J = 9.6, 2.3 Hz, 1H), 7.97 (dd, J = 3.8, 1.5 Hz, 1H), 7.82 (dd, J = 9.4, 2.2 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 9.5, 3.9 Hz, 1H), 7.00 (ddd, J = 9.6, 6.5, 5.0 Hz, 3H), 4.37 - 4.20 (m, 2H), 2.39 (s, 3H), 2.37 - 2.29 (m, 2H), 2.17 (t, J = 6.4 Hz, 2H), 1.80 - 1.67 (m, 2H). ESI(M + H) + = 395.3

[0624] The following examples were synthesized using the above procedure.

[0625]

Table 92

[0626] Scheme 83

Chem.

[0627] General Procedure 16 6’-(((1S,3S)-3-((5-(methylthio)pyrimidin-2-yl)amino)cyclopentyl)amino)-[3,3’-bipyridin]-4-ol (Example 499) To a solution of Compound 402 (40 mg, 0.1 mmol, 1 equiv) in NMP (3 mL) were added LiCl (42 mg, 1 mmol, 10 equiv) and p-toluenesulfonic acid (172 mg, 1 mmol, 10 equiv). The mixture was stirred at 180 °C for 4 h and cooled to room temperature. The mixture was diluted with water (10 mL), followed by basification with saturated NaHCO3 to pH = 10. The resulting mixture was extracted with ethyl acetate (10 mL × 6), and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The obtained residue was purified by preparative HPLC to give Example 499 (10 mg, 26%) as a yellow solid. LCMS [M+H] + = 395; 1 H NMR (400 MHz, CD3OD) δ: 8.33 (s, 2H), 8.18 (s, 1H), 7.88 (s, 1H), 7.77 - 7.72 (m, 1H), 6.63 (d, J = 9.2 Hz, 1H), 6.52 (d, J = 9.2 Hz, 1H), 4.45 - 4.35 (m, 2H), 2.35 (s, 3H), 2.30 - 2.24 (m, 2H), 2.04 - 1.98 (m, 2H), 1.63 - 1.60 (m, 2H).

[0628] The following examples were synthesized using the above procedure.

[0629]

Table 93

[0630] Scheme 84

Chemical Structure

[0631] (1S,3S)-N 1 -(5-(Difluoromethoxy)pyrimidin-2-yl)-N3-(5-nitropyridin-2-yl)cyclopentane-1,3-diamine (500A) A suspension of 411 (43 g, 176 mmol, 1.0 eq), 2-chloro-5-nitropyridine (27.9 g, 176 mmol, 1.0 eq) and K2CO3 (48.6 g, 382 mmol, 2.0 eq) in DMSO (500 mL) was stirred at 80 °C for 16 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was partitioned between EA (400 mL) and water (400 mL); the aqueous phase was extracted with EA (300 mL). The combined organic phases were washed with brine (400 mL) and then concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (PE:EA = 3:1) to afford 500A (52 g).

[0632] N 2 -((1S,3S)-3-((5-(Difluoromethoxy)pyrimidin-2-yl)amino)cyclopentyl)pyridine-2,5-diamine (500B) To a solution of 500A (45 g, 123 mmol, 1.0 eq) in MeOH (450 mL) was added 10% Pd / C (4.5 g). Next, the reaction mixture was degassed with H2 three times and stirred at room temperature under H2 for 8 h. The reaction mixture was filtered through Celite. The filtrate was concentrated to remove the solvent and the residue was purified by column chromatography on silica gel (EA:MeOH = 30:1) to afford 500B (31 g).

[0633] 3-(6-(((1S,3S)-3-((5-(Difluoromethoxy)pyrimidin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)-1-methylimidazolidine-2,4-dione (Example 500) A solution of 500B (27.2 g, 81 mmol, 1.0 equiv) and 4-nitrophenyl carbonochloridate (16.3 g, 81 mmol, 1.0 equiv) in acetonitrile (280 mL) was stirred at room temperature for 1 hour. Methyl glycinate methyl hydrochloride (11.8 g, 84 mmol, 1.1 equiv) and DIPEA (31.3 g, 242 mmol, 3.0 equiv) were added to the reaction and stirring was continued for 16 hours. The reaction mixture was concentrated to remove most of the solvent and the residue was partitioned between DCM (70 mL) and water (70 mL). The organic phase was separated and concentrated. The resulting residue was purified by column chromatography on silica gel (DCM:MeOH = 40:1) to give the crude product as a slurry. The compound was further purified by trituration in PE / EA (4:1, 40 mL) and decolorized with activated carbon in MeOH to give Example 500 (25 g). 1 H NMR (400 MHz, CD3OD) δ 8.16 (s, 2H), 7.91 (d, J = 2.1 Hz, 1H), 7.36 (dd, J = 9.0, 2.6 Hz, 1H), 6.93 - 6.43 (m, 2H), 4.47 - 4.25 (m, 2H), 4.08 (s, 2H), 3.02 (s, 3H), 2.35 - 2.19 (m, 2H), 2.12 - 1.87 (m, 2H), 1.69 - 1.46 (m, 2H).

[0634] Scheme 85

Chemical Structure

[0635] 2-Chloro-6-methylthieno[3,2-d]pyrimidine (501A) A solution of 2,4-dichloro-6-methylthieno[3,2-d]pyrimidine (1.5 g, 6.85 mmol, 1.0 eq), zinc (1.8 g, 27.39 mmol, 4.0 eq) and acetic acid (2.4 mL, 41.08 mmol, 6.0 eq) in methanol ...

Claims

1. A therapeutic pharmaceutical composition comprising an inhibitor of PCSK9 and a pharmaceutically acceptable excipient, wherein the inhibitor of PCSK9 binds to a binding pocket in the C-terminal domain of human PCSK9 formed between amino acid residues 558-590 and amino acid residues 631-650, and the inhibitor of PCSK9 comprises a 2-NH-pyrimidinyl moiety.

2. A pharmaceutical composition for treating or preventing cardiovascular disease, comprising an inhibitor of PCSK9 and a pharmaceutically acceptable excipient, wherein the inhibitor of PCSK9 binds to a binding pocket in the C-terminal domain of human PCSK9 formed between amino acid residues 558-590 and amino acid residues 631-650, and the inhibitor of PCSK9 comprises a 2-NH-pyrimidinyl moiety.

3. A medicament for treating a disease or disorder mediated by PCSK9, the medicament comprising an inhibitor of PCSK9 that binds to a binding pocket in the C-terminal domain of human PCSK9 formed between amino acid residues 558-590 and amino acid residues 631-650, and the inhibitor of PCSK9 comprises a 2-NH-pyrimidinyl moiety.

4. The inhibitor further comprises: a) an H-bond acceptor moiety positioned to bind to amino acid residue Ser642, His643, or Val644; b) an H-bond donor moiety positioned to bind to amino acid residue Ala637 or Thr641; and c) one or more of the cation-π stacking interacting moieties arranged to bind to amino acid residues Arg495 or His591; The pharmaceutical composition or medicament according to any one of claims 1 to 3.

5. 5. The pharmaceutical composition or medicament of any one of claims 1 to 4, wherein the inhibitor further comprises an H-bond acceptor moiety positioned to bind to amino acid residue Glu612.

6. 6. The pharmaceutical composition or medicament according to any one of claims 1 to 5, wherein the inhibitor interacts with at least one residue in the M2 C-terminal domain, at least one residue in the M3 C-terminal domain, and at least one residue in the M1 C-terminal domain.

7. 7. The pharmaceutical composition or medicament according to any one of claims 1 to 6, wherein the inhibitor interacts with at least one residue in each of beta strands 3 and 5 of the M2 C-terminal domain and beta strands 3 and 4 of the M3 C-terminal domain.

8. The pharmaceutical composition or medicament according to claim 7, wherein the binding pocket in PCSK9 interacts with a pocket in PCSK9 generated among amino acid residues 558 to 566 in beta strand 3 of the M2 C-terminal domain, amino acid residues 587 to 590 in beta strand 5 of the M2 C-terminal domain, amino acid residues 631 to 637 in beta strand 3 of the M3 C-terminal domain, and amino acid residues 644 to 650 in beta strand 4 of the M3 C-terminal domain.

9. 10. An in vitro or ex vivo method of inhibiting PCSK9, comprising contacting PCSK9 with an inhibitor of PCSK9 according to any one of claims 1 to 8.

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