Antimicrobial compounds and methods

Abstract

The present invention relates to compounds active as antibacterial agents. The compounds of the present invention are active against gram-positive and gram-negative bacteria and can be used to treat infections caused by gram-positive and gram-negative bacteria. Processes and intermediates for the preparation of the compounds are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 295,128, filed December 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] Statement regarding U.S. Federal Government Interests This invention was made with Government support under 1R44AI152665-01 awarded by the Federal Government agency administering the Small Business Innovation and Research (SBIR) Program. The Federal Government has certain rights in this invention.

[0003] The present disclosure relates to compounds that are active as antibacterial agents. The present disclosure also relates to methods of treating bacterial infections using the compounds. [Background technology]

[0004] Antibacterial resistance is a global problem. Both gram-positive and gram-negative bacteria are becoming increasingly resistant to antibiotics.

[0005] Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), are resistant to most antibiotics related to penicillin. MRSA strains are commonly associated with infections acquired in health care facilities and can cause infections in the larger community.

[0006] Gram-negative bacteria are thought to be more resistant to antibiotics than gram-positive bacteria because their cell walls are impermeable. According to the National Institutes of Health (NIH), gram-negative bacteria can cause many types of infections and spread to humans in a variety of ways. Some species, including Escherichia coli, are common causes of food-borne illnesses. Vibrio cholerae, the causative agent of cholera, is a waterborne pathogen. Gram-negative bacteria can also cause respiratory infections, such as certain types of pneumonia, and sexually transmitted diseases, including gonorrhea. Yersinia pestis, the causative agent of the plague, infects people through the bite of an infected insect or handling an infected animal. See www.niaid.nih.gov / research / gram-negative-bacteria (last accessed December 22, 2020).

[0007] Certain types of Gram-negative bacteria are becoming increasingly resistant to available antibiotics. Some strains are now resistant to many, most, or all available treatments, resulting in increased illness and death from bacterial infections and contributing to rising healthcare costs. Examples of Gram-negative bacteria that exhibit drug resistance include E. coli, which is responsible for the majority of urinary tract infections; Acinetobacter baumanii, which primarily causes disease in healthcare settings; Pseudomonas aeruginosa, which causes bloodstream infections and pneumonia in hospitalized patients and is a common cause of pneumonia in cystic fibrosis patients; Klebsiella pneumoniae, which causes many types of healthcare-associated infections, including pneumonia, urinary tract infections, and bloodstream infections; and Neisseria gonorrhoeae, which causes the sexually transmitted disease gonorrhea, the second most commonly reported infection in the United States. Consequently, new drugs are needed to combat Gram-positive and Gram-negative infections. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] www.niaid.nih.gov / research / gram-negative-bacteria Summary of the Invention

[0009] The present invention meets these and other needs and in one aspect provides a compound of formula I [ka] or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein: Ring A is a 3-8 membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, phenyl, OH, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and oxo; J is a C1-C6 alkylene or a C3-C8 cycloalkylene, any of which is optionally substituted with halo, OH, or C1-C6 alkoxy, and up to two methylene units of said C1-C6 alkylene are optionally and independently replaced with O, S, SO, SO2, or C=O; Y is a bond or a C1-C6 alkylene optionally substituted with OH, NH2, CN, halo, or C1-C6 alkoxy, wherein up to two methylene units of the C1-C6 alkylene are optionally independently selected from O, NH, N-(C1-C6 alkyl), N-(C1-C6 hydroxyalkyl), N-(C1-C6 haloalkyl), N-(C1-C6 alkoxy ... 1-6 Alkylene-C 3-8 Cycloalkyl), N-(C 3-8 Cycloalkyl), NH(C=O), N-(C 1-6 alkyl)(C=O), or replaced by (C=O); Ring B is a 3-8 membered monocyclic cycloalkylene, a 3-8 membered monocyclic heterocycloalkylene, a 6-12 membered bicyclic cycloalkylene, or a 6-12 membered bicyclic heterocycloalkylene, each of which is optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and C1-C6 hydroxyalkyl; L is a bond or a C1-C6 alkylene, wherein up to two methylene units of the C1-C6 alkylene are optionally independently selected from O, NH, (C=O), NH(C=O), N-(C 1-6 alkyl)(C=O), (C=NH), NH(C=N), or N-(C 1-6 alkyl); R 1 and R 2 are each independently selected from the group consisting of C1-C6 alkyl, halo, CN, OH, NH2, O(C1-C6 haloalkyl), NH(C1-C6 alkyl), N(C1-C6 alkyl)2, -COO(C1-C6 alkyl), CONH2, C1-C6 haloalkyl, C1-C6 alkoxy, and C1-C6 haloalkoxy; R x , R y , R x’ , and R y’ are each independently H, C1-C6 alkyl, or an amino protecting group; m and n are each independently 0, 1, 2, or 3; [ka] represents a single or double bond, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof.

[0010] In another aspect, the present invention provides a method of using a compound of formula I, or a pharma- ceutically acceptable salt thereof, for the treatment of a bacterial infection.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0012] In a further aspect, the present invention provides processes for the preparation of compounds of formula I, or pharma- ceutically acceptable salts thereof, and compounds intermediates used in such processes, as shown in the synthetic schemes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Similar or equivalent methods and materials to those described herein can be used in the practice or testing of the present invention, but suitable methods and materials are described below. In addition, the materials, methods, and examples are only illustrative and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, including U.S. Patent Publication No. 2013 / 0090326. In case of conflict, the present specification, including its definitions, will control.

[0014] As used herein, the terms "a," "an," and "the" include not only embodiments having one member, but also embodiments having more than one member.

[0015] As used herein, the term "about" means "approximately" and is used to modify a numerical value and indicates a range defined to be close to that value. When "X" is a relevant value, "about X" generally indicates a value between 0.95X and 1.05X. Reference to "about X" specifically indicates at least the values ​​X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" is intended to teach and provide support by the specification for a claim limitation of, for example, "0.98X". When the quantity "X" includes only integer values ​​(e.g., "X carbons"), "about X" indicates (X-1) to (X+1). In this case, as used herein, "about X" specifically indicates at least the values ​​X, X-1, and X+1.

[0016] When "about" is applied to the beginning of a numerical range, it applies to both ends of that range. Thus, "about 5% to 20%" is equivalent to "about 5% to about 20%." When "about" is applied to the first value in a set of values, it applies to every value in that set. Thus, "about 7, 9, or 11%" is equivalent to "about 7%, about 9%, or about 11%."

[0017] The symbol "-" means a single bond, "=" means a double bond, [ka] " means a triple bond, " [ka] " means a single bond or a double bond.

[0018] As used herein, wavy lines drawn in a structure may be used to indicate a point of attachment for that structure. For example, [ka] So, " [ka] " indicates the point of attachment.

[0019] An "*" in a chemical structure is used to identify a chiral center.

[0020] As used herein, the term "acyl" includes alkanoyl, aroyl, heterocycloyl, or heteroaroyl groups as defined herein. Examples of acyl groups include, but are not limited to, acetyl, benzoyl, and nicotinoyl.

[0021] As used herein, the term "alkanoyl" includes alkyl-C(O)- groups, where the alkyl group is as defined herein. Examples of alkanoyl groups include, but are not limited to, acetyl and propanoyl.

[0022] As used herein, the term "agent" includes a compound or mixture of compounds that, when added to a composition, tends to have a particular effect on the properties of the composition. For example, a composition that includes a thickening agent is likely to be more viscous than an otherwise identical comparative composition lacking the thickening agent.

[0023] As used herein, the term "alkyl" includes an aliphatic hydrocarbon chain that may be straight or branched. The chain may contain the indicated number of carbon atoms. For example, C1-C 10indicates that the group may contain 1 to 10 (inclusive) carbon atoms in it. Unless otherwise specified, alkyl groups contain 1 to about 20 carbon atoms. In some embodiments, alkyl groups have 1 to about 10 carbon atoms. In some embodiments, alkyl groups ("lower alkyl") have 1 to 8, 1 to 6, or 1 to 3 carbon atoms in the chain. Examples can include, but are not limited to, methyl, ethyl, propyl, isopropyl (iPr), 1-butyl, 2-butyl, isobutyl (iBu), tert-butyl, pentyl, 2-methylbutyl, 1,1-dimethylpropyl, hexyl, heptyl, octyl, nonyl, decyl, docecyl, cyclopentyl, or cyclohexyl.

[0024] The alkyl group may be an unsubstituted or optionally substituted alkyl group. When optionally substituted, one or more hydrogen atoms (e.g., 1-4, 1-2, or 1) of the alkyl group may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some embodiments, the alkyl group is an unsubstituted or not an optionally substituted alkyl group.

[0025] As used herein, "alkylene" includes di-substituted alkyl groups. Examples include methylene (-CH2-), propylene (-CH2CH2CH2-), and the like.

[0026] As used herein, the term "alkenyl" includes straight or branched chain hydrocarbons containing at least one carbon-carbon double bond. The chain may contain the number of carbon atoms indicated. For example, "C1-C 12 "Alkenyl" indicates that the group may have 1 to 12 (inclusive) carbon atoms and at least one carbon-carbon double bond. When the indicated number of carbon atoms is 1, C iThe alkenyl is double bonded to the carbon (i.e., the carbon corresponding to the oxo group). In certain embodiments, the chain contains 1 to 12, about 2 to 15, about 2 to 12, about 2 to 8, or about 2 to 6 carbon atoms. The alkenyl group may preferably be in one stereoisomer (i.e., cis- or, alternatively, trans-). Examples of alkenyl groups may include, but are not limited to, ethenyl (i.e., vinyl), aryl, propenyl, butenyl, crotyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, dodecenyl, cyclopentenyl, cyclohexenyl, 2-isopentenyl, allenyl, butadienyl, pentadienyl, 3-(1,4-pentadienyl), and hexadienyl.

[0027] The alkenyl group may be an unsubstituted alkenyl group or an optionally substituted alkenyl group. When optionally substituted, one or more hydrogen atoms (e.g., 1-4, 1-2, or 1) of the alkenyl group may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, but the hydrogen atom substituent of the carbon-carbon double bond is not replaced with a hydroxy, amino, or thio group. In some embodiments, the alkenyl group is an unsubstituted alkenyl group or is not an optionally substituted alkenyl group.

[0028] As used herein, "alkenylene" includes di-substituted alkenyl groups, such as, for example, but-2-enylene (-CHCH=CHCH-).

[0029] As used herein, the term "alkynyl" includes straight chain, branched, or cyclic hydrocarbons containing at least one carbon-carbon triple bond. Examples can include, but are not limited to, ethynyl, propargyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, or decynyl.

[0030] The alkynyl group may be an unsubstituted or optionally substituted alkynyl group. When optionally substituted, one or more hydrogen atoms (e.g., 1-4, 1-2, or 1) of the alkynyl group may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, but the sp hybridized hydrogen atom substituent is not replaced with a hydroxy, amino, or thio group. In some embodiments, the alkynyl group is an unsubstituted or optionally substituted alkynyl group.

[0031] As used herein, "alkynylene" includes di-substituted alkynyl groups, such as, for example, 2-butynylene (-CH2CCCH2-).

[0032] As used herein, the term "alkoxy" includes straight or branched chain saturated or unsaturated hydrocarbons containing at least one oxygen atom in an ether group (e.g., EtO-). The chain may contain the indicated number of carbon atoms. For example, "C1-C 12 "Alkoxy" indicates that the group may have 1 to 12 (inclusive) carbon atoms and at least one oxygen atom. 12 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, butoxy, n-pentoxy, isopentoxy, neopentoxy, and hexoxy.

[0033] The alkoxy group may be an unsubstituted or an optionally substituted alkoxy group. When optionally substituted, one or more hydrogen atoms (e.g., 1-4, 1-2, or 1) of the alkoxy group may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio, but the hydrogen atom alpha to the ether oxygen is not replaced with a hydroxy, amino, or thio group. In some embodiments, the alkoxy group is an unsubstituted or is not an optionally substituted alkoxy group.

[0034] As used herein, the term "aryl" includes cyclic aromatic carbocyclic ring systems containing 6 to 18 carbons. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetracenyl, biphenyl, and phenanthrenyl.

[0035] As used herein, the term "cycloalkyl" includes non-aromatic saturated monocyclic or polycyclic ring systems that may contain the indicated number of carbon atoms. For example, C3-C 12 indicates that the group may have 3 to 12 (inclusive) carbon atoms in it. Unless otherwise specified, cycloalkyl groups contain from about 3 to about 20 carbon atoms. In some embodiments, cycloalkyl groups have 3 to about 12 carbon atoms in the group. In some embodiments, cycloalkyl groups have 3 to about 7 carbon atoms in the group. Examples can include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4,4-dimethylcyclohexyl, and cycloheptyl. The term "cycloalkyl" also includes polycyclic rings, such as bicyclic cycloalkyls, or tricyclic cycloalkyls, which may be in fused, bridged, or spiro configurations.

[0036] As used herein, the term "cycloalkylene" includes di-substitued cycloalkyl groups.

[0037] The terms "disorder" and "disease" are used interchangeably herein with respect to the condition of a subject. A disorder is a disturbance or disruption that affects the normal functioning of the subject's body. A disease is a pathological condition of an organ, body part, or system that is characterized by a constellation of identifiable symptoms resulting from a variety of causes, such as infection, genetic defect, or environmental stress. A disorder or disease may also refer to a biofilm-associated disorder characterized by disease-associated bacterial growth or a disorder caused by a planktonic bacterial phenotype.

[0038] As used herein, the term "effective amount" or "effective dosage" includes an amount sufficient to achieve a desired result, and thus depends on the ingredient and the desired result, although once the desired effect has been ascertained, identifying an effective amount is within the skill of one of ordinary skill in the art.

[0039] As used herein, "fluoroalkyl" includes alkyl groups where the alkyl group contains one or more fluoro substituents. Examples include, but are not limited to, trifluoromethyl.

[0040] As used herein, "geminal" substitution includes two or more substituents directly attached to the same atom. An example is 3,3-dimethyl substitution on a cyclohexyl or spirocyclohexyl ring.

[0041] As used herein, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.

[0042] As used herein, a "heterocycloalkyl" includes a non-aromatic saturated ring of about 3 to about 12 ring atoms (e.g., 5 to about 10 ring atoms, 3 to about 8 ring atoms, or 3 to about 6 ring atoms), in which one or more atoms of the ring system is an element(s) other than carbon, such as nitrogen, oxygen, or sulfur. A heterocycloalkyl group optionally includes at least one sp 2In some embodiments, the nitrogen or sulfur atom of the heterocycloalkyl is optionally oxidized to the corresponding N-oxide, S-oxide, or S,S-dioxide. Monocyclic heterocycle refers to a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains 0 or 1 double bond and 1 heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. The 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycloalkyls include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, and pyrazolinyl. These include, but are not limited to, pyrazolidinyl, pyridazin-3(2H)-onyl, pyridin-2(1H)-onyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.

[0043] The term "heterocycloalkyl" also includes polycyclic rings, such as bicyclic or tricyclic heterocycles, which may be in fused, bridged or spiro configurations. A bicyclic heterocycle is a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or by an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, 3-azabicyclo[3.1.0]hexane, 3-azabicyclo[4.1.0]heptane, 3-azabicyclo[3.2.0]heptane, (3aR,6aS)-hexahydro-1H-2λ2-cyclopenta[c]pyrrole, (3aR,7aS)-octahydro-2λ2-isoindole.

[0044] A tricyclic heterocycle may be exemplified by a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or by an alkenylene bridge of 2, 3, or 4 carbon atoms.

[0045] The heterocycloalkyl group may be an unsubstituted or optionally substituted heterocycloalkyl group. When optionally substituted, one or more hydrogen atoms (e.g., 1-4, 1-2, or 1) of the group may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some embodiments, the substituted heterocyclyl group can incorporate an exocyclic or endocyclic alkene (e.g., cyclohex-2-en-1-yl). In some embodiments, the heterocycloalkyl group is an unsubstituted or not an optionally substituted heterocycloalkyl group.

[0046] The monocyclic, bicyclic, and tricyclic heterocycles can be connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring, and can be unsubstituted or substituted.

[0047] As used herein, the term "heterocycloalkylene" includes di-substitued heterocycloalkyl groups.

[0048] As used herein, the term "hydroxyalkyl" includes an alkyl group in which at least one hydrogen substituent is replaced with an alcohol (-OH) group. In certain embodiments, the hydroxyalkyl group has one alcohol group. In certain embodiments, the hydroxyalkyl group has one or two alcohol groups, each on a different carbon atom. In certain embodiments, the hydroxyalkyl group has 1, 2, 3, 4, 5, or 6 alcohol groups. Examples can include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 1-hydroxyethyl.

[0049] When any two substituents or any two instances of the same substituent from a list of alternatives are "independently selected," those groups may be the same or different. For example, R a and R bis independently selected from the group consisting of alkyl, fluoro, amino, and hydroxyalkyl, then two R a Group and two R b In a molecule having groups, all of the groups may be alkyl groups (e.g., four different alkyl groups). Alternatively, the first R a is alkyl, and the second R a is fluoro, and the first R b is hydroxyalkyl, and the second R b may be amino (or any other substituent from that group). Alternatively, both R a and the first R b is fluoro and the second R b may be alkyl (i.e., some pairs of substituents may be the same and other pairs may be different).

[0050] An "amino protecting group" is a protecting group suitable for preventing undesired reactions at an amino nitrogen. Representative amino protecting groups include, but are not limited to, formyl, acyl groups such as alkanoyl groups, e.g., acetyl and trifluoroacetyl, alkoxycarbonyl groups, e.g., tert-butoxycarbonyl (Boc), arylmethoxycarbonyl groups, e.g., benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc), arylmethyl groups, e.g., benzyl (Bn), trityl (Tr), and 1,1-di-(4'-methoxyphenyl)methyl.

[0051] A "hydroxyl protecting group" is a protecting group suitable for preventing undesired reactions at a hydroxyl oxygen. Representative hydroxy protecting groups include, but are not limited to, acyl groups, such as alkanoyl groups, e.g., acetyl, arylmethyl groups, e.g., benzyl (Bn), trityl (Tr), and 1,1-di-(4'-methoxyphenyl)methyl, silyl groups, e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), and the like.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid of amino groups, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed using other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Examples of the salts include phosphate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0053] "Pharmaceutically acceptable acid addition salts" refers to salts which retain the biological effectiveness of the free base and which are not biologically or otherwise objectionable, and are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, orotic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0054] "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Exemplary salts are ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (See, e.g., S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19, incorporated herein by reference.)

[0055] As used herein, "or" is generally to be interpreted non-exclusively. For example, an embodiment of "a composition comprising A or B" generally refers to an embodiment of a composition comprising both A and B. However, "or" should be interpreted to exclude those embodiments presented when combining is inconsistent (e.g., a composition with a pH of 9-10 or 7-8).

[0056] As used herein, "spirobibicyclic cycloalkyl" includes cycloalkyl in which geminal substituents on carbon atoms are replaced to participate in the formation of a 1,1-substituted ring. For example, but not limited to, -C(R 1 )(R 2 )-group, R 1 and R 2 But R 1 and R 2 is linked to form a cyclopropyl ring, incorporating the carbon to which it is attached, then it is a spiro bicyclic cycloalkyl group (ie, spirocyclopropyl).

[0057] As used herein, the term "spirobicyclic cycloalkylene" embraces spiro bicyclic cycloalkyl groups which are disubstituted.

[0058] As used herein, "spirobiicyclic heterocycloalkyl" includes heterocycloalkyl in which geminal substituents on carbon atoms are replaced to participate in the formation of a 1,1-substituted ring. For example, but not limited to, -C(R 1 )(R 2 )-group, R 1 and R 2 But R 1 and R 2 is linked to form a pyrrolidine ring, incorporating the carbon to which it is attached, then this is a spiro bicyclic heterocycloalkyl group.

[0059] As used herein, the term "spirobicyclic heterocycloalkylene" embraces 2-point substituted spiro bicyclic heterocycloalkyl groups.

[0060] Some of the compounds disclosed herein are characterized by the presence of an amino functionality, and therefore, those skilled in the art will understand that the compounds may be isolated as salts in which the nitrogen of that amino functionality has been quaternized.

[0061] As used herein, the terms "treat," "treating," or "treatment" include administering or applying a composition (e.g., a composition described herein) in an amount, manner (e.g., dosing schedule), and mode (e.g., route of administration) effective to ameliorate a disorder or a symptom thereof, or to prevent or slow the progression of a disorder or a symptom thereof. Such improvement can include, but is not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of a disease, stabilization of a disease condition (i.e., not worsening), delaying or slowing disease progression, improvement or palliation of a disease condition, reduction in recurrence of a disease, and remission, whether partial or complete, and whether detectable or undetectable.

[0062] Embodiment compound In a first aspect, the present disclosure provides a compound of formula I [ka] or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein: Ring A is a 3-8 membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, phenyl, OH, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and oxo; J is a C1-C6 alkylene or a C3-C8 cycloalkylene, any of which is optionally substituted with halo, OH, or C1-C6 alkoxy, and up to two methylene units of said C1-C6 alkylene are optionally and independently replaced with O, S, SO, SO2, or C=O; Y is a bond or a C1-C6 alkylene optionally substituted with OH, NH2, CN, halo, or C1-C6 alkoxy, wherein up to two methylene units of the C1-C6 alkylene are optionally independently selected from O, NH, N-(C1-C6 alkyl), N-(C1-C6 hydroxyalkyl), N-(C1-C6 haloalkyl), N-(C1-C6 alkoxy ... 1-6 Alkylene-C 3-8 Cycloalkyl), N-(C 3-8 Cycloalkyl), NH(C=O), N-(C 1-6 alkyl)(C=O), or replaced by (C=O); Ring B is a 3-8 membered monocyclic cycloalkylene, a 3-8 membered monocyclic heterocycloalkylene, a 6-12 membered bicyclic cycloalkylene, or a 6-12 membered bicyclic heterocycloalkylene, each of which is optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and C1-C6 hydroxyalkyl; L is a bond or a C1-C6 alkylene, wherein up to two methylene units of the C1-C6 alkylene are optionally independently selected from O, NH, (C=O), NH(C=O), N-(C 1-6 alkyl)(C=O), (C=NH), NH(C=N), or N-(C 1-6 alkyl); R 1 and R 2are each independently selected from the group consisting of C1-C6 alkyl, halo, CN, OH, NH2, O(C1-C6 haloalkyl), NH(C1-C6 alkyl), N(C1-C6 alkyl)2, -COO(C1-C6 alkyl), CONH2, C1-C6 haloalkyl, C1-C6 alkoxy, and C1-C6 haloalkoxy; R x , R y , R x’ , and R y’ are each independently H, C1-C6 alkyl, or an amino protecting group; m and n are each independently 0, 1, 2, or 3; [ka] represents a single or double bond, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof.

[0063] In one embodiment of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, ring A is a 5-6 membered monocyclic heterocycloalkylene optionally substituted with up to three substituents selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, phenyl, OH, NH2, and oxo.

[0064] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, ring A is [ka] where R 3 are each independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, phenyl, OH, NH2, and oxo; and q is 0, 1, or 2.

[0065] In other embodiments of the compounds of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, ring A is [ka] It is.

[0066] In other embodiments of a compound of Formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, J is a C-C alkylene optionally substituted with halo, OH, or C-C alkoxy, wherein up to two methylene units of the C-C alkylene are optionally and independently replaced with O, S, SO, SO, or C=O.

[0067] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, J is a C1-C6 alkylene optionally substituted with OH, and one methylene unit of the C1-C6 alkylene is optionally replaced with C=O.

[0068] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, J is C-C alkylene, and one methylene unit of the C-C alkylene is replaced with C=O.

[0069] In other embodiments of the compounds of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, J is [ka] It is.

[0070] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, Rx and R y is H. In other embodiments, R x is H and R y is C1-C6 alkyl. In another embodiment, R x is H and R y is an amino protecting group. x is H and R y is a Boc group. x and R y are each independently C1-C6 alkyl.

[0071] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, [ka] In another embodiment, [ka] It is.

[0072] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, [ka] It is.

[0073] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, [ka] where R 2are each independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, and CN; and n is 0, 1, or 2.

[0074] In another embodiment, [ka] where R 2 are each independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, and CN; and n is 0, 1, or 2.

[0075] In another embodiment, [ka] It is.

[0076] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, Y is C-C alkylene optionally substituted with OH, NH, CN, halo, or C-C alkoxy, wherein one methylene unit of the C-C alkylene is O, NH, N—(C-C alkyl), N—(C-C hydroxyalkyl), N—(C-C haloalkyl), N—(C 1-6 Alkylene-C 3-8 Cycloalkyl), N-(C 3-8 Cycloalkyl), NH(C=O), N-(C 1-6 alkyl)(C=O), or (C=O).

[0077] In other embodiments, Y is a C1-C3 alkylene optionally substituted with OH, NH2, halo, or C1-C6 alkoxy, wherein one methylene unit of the C1-C3 alkylene is selected from the group consisting of O, NH, N-(C1-C6 alkyl), N-(C1-C6 hydroxyalkyl), N-(C1-C6 haloalkyl), N-(C1-C6 alkoxy ... 3-8 Cycloalkyl), N-(C1-6 Alkylene-C 3-8 Cycloalkyl), NH(C=O), N-(C 1-6 alkyl)(C=O), or (C=O).

[0078] In other embodiments, Y is a C1-C3 alkylene, wherein one methylene unit of the C1-C3 alkylene is optionally replaced with NH, N-(C1-C6 haloalkyl), or N-(C1-C6 alkyl).

[0079] In other embodiments, Y is selected from the group consisting of -CH2-, -CH2NH-, -CH2NMe-, -CH2N(CH2CH2F)-, and -CH2NEt-.

[0080] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, [ka] is selected from the group consisting of:

[0081] In other embodiments of a compound of Formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, Ring B is a 3-8 membered monocyclic cycloalkylene, a 3-8 membered monocyclic heterocycloalkylene, or a 6-12 membered bicyclic heterocycloalkylene, each of which is optionally substituted with up to three substituents independently selected from the group consisting of C-C alkyl, C-C alkoxy, halo, CN, C-C haloalkyl, OH, COOH, COO(C-C alkyl), CONH, CONH(C-C alkyl), CON(C-C alkyl), and C-C hydroxyalkyl.

[0082] In other embodiments, Ring B is a 3-8 membered monocyclic cycloalkylene or a 3-8 membered monocyclic heterocycloalkylene, or a 6-12 membered bicyclic heterocycloalkylene, optionally substituted with C1-C6 alkyl.

[0083] In other embodiments, Ring B is a 5-6 membered monocyclic cycloalkylene, a 4-7 membered monocyclic heterocycloalkylene, or a 6-9 membered bicyclic heterocycloalkylene.

[0084] In other embodiments, Ring B is a 3-8 membered monocyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and C1-C6 hydroxyalkyl.

[0085] In other embodiments, Ring B is a 4-6 membered monocyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl.

[0086] In other embodiments, Ring B is a 5-6 membered monocyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl.

[0087] In another embodiment, ring B is a 4-6 membered monocyclic cycloalkylene.In another embodiment, ring B is cyclopentylene or cyclohexylene.

[0088] In other embodiments, Ring B is a 3-8 membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and C1-C6 hydroxyalkyl.

[0089] In other embodiments, Ring B is a 4-7 membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl, and Ring B contains up to two nitrogen atoms.

[0090] In another embodiment, Ring B is a 4-7 membered monocyclic heterocycloalkylene containing up to two heteroatoms selected from nitrogen and oxygen, and Ring B is optionally substituted with C1-C6 alkyl. In another embodiment, Ring B is a 4-7 membered monocyclic heterocycloalkylene containing one nitrogen atom, and Ring B is optionally substituted with C1-C6 alkyl.

[0091] In another embodiment, Ring B is a 6-10 membered bicyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl. In another embodiment, Ring B is a 6-9 membered bicyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl.

[0092] In another embodiment, Ring B is a 6-10 membered fused, spiro, or bridged bicyclic cycloalkylene. In another embodiment, Ring B is a 6-10 membered fused bicyclic cycloalkylene. In another embodiment, Ring B is a 6-10 membered bridged bicyclic cycloalkylene. In another embodiment, Ring B is a 6-10 membered spiro bicyclic cycloalkylene.

[0093] In other embodiments, Ring B is a 6-12 membered bicyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl.

[0094] In other embodiments, Ring B is a 6-10 membered bicyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl.

[0095] In other embodiments, Ring B is a 6-9 membered bicyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, C1-C6 haloalkyl, OH, and C1-C6 hydroxyalkyl.

[0096] In another embodiment, Ring B is a 6-10 membered fused, spiro, bridged bicyclic heterocycloalkylene containing up to two nitrogen atoms. In another embodiment, Ring B is a 6-9 membered fused, spiro, bridged bicyclic heterocycloalkylene containing up to two nitrogen atoms.

[0097] In another embodiment, ring B is a 6-10 membered fused, spiro, bridged bicyclic heterocycloalkylene containing one nitrogen atom. In another embodiment, ring B is a 6-9 membered fused bicyclic heterocycloalkylene containing one nitrogen atom. In another embodiment, ring B is a 6-9 membered spiro bicyclic heterocycloalkylene containing one nitrogen atom. In another embodiment, ring B is a 6-9 membered bridged bicyclic heterocycloalkylene containing one nitrogen atom.

[0098] In other embodiments, Ring B is [ka] is selected from the group consisting of:

[0099] In other embodiments of the compounds of Formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein L is a bond.

[0100] In other embodiments of the compound of Formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, L is a C1-C6 alkylene, and up to two methylene units of the C1-C6 alkylene are optionally independently selected from O, NH, (C=O), NH(C=O), N-(C 1-6 alkyl)(C=O), (C=NH), NH(C=N), or N-(C 1-6 It can be replaced by alkyl.

[0101] In other embodiments, L is a C1-C3 alkylene, and one methylene unit of the C1-C6 alkylene is optionally independently selected from O, NH, (C=O), NH(C=O), N-(C 1-6 alkyl)(C=O), (C=NH), NH(C=N), or N-(C 1-6 It can be replaced by alkyl.

[0102] In other embodiments, L is C1-C6 alkylene. In other embodiments, L is -CH2- or -CH2-CH2-.

[0103] In other embodiments, L is a bond or C1-C3 alkylene. In other embodiments, L is a bond, -CH2-, or -CH2-CH2-.

[0104] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, R x’ and R y’ are each independently H. In another embodiment, R x’ is H and R y’ is C1-C6 alkyl. In another embodiment, R x’ is H and R y’ is an amino protecting group. x’ is H and R y’ is a Boc group. x’ and R y’ are each independently C1-C6 alkyl.

[0105] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, Y is C1-C3 alkylene, in which one methylene unit is optionally replaced with NH, N-(C1-C6 haloalkyl), or N-(C1-C6 alkyl); Ring B is a 5-6 membered monocyclic cycloalkylene, a 4-7 membered monocyclic heterocycloalkylene, or a 6-9 membered bicyclic heterocycloalkylene, optionally substituted with C1-C6 alkyl; L is a bond or C1-C3 alkylene; R x’ and R y’ are each independently H or C1-C6 alkyl.

[0106] In other embodiments, Y is selected from the group consisting of -CH2-, -CH2NH-, -CH2NMe-, -CH2N(CH2CH2F)-, and -CH2NEt-; Ring B is [ka] L is a bond, -CH-, or -CH-CH-; R x’ and R y’ are each independently H.

[0107] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, [ka] is selected from the group consisting of:

[0108] In other embodiments of the compound of formula I, or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, R 1 and R 2 are each independently selected from the group consisting of C1-C6 alkyl, halo, C1-C6 haloalkyl, oxo, and C1-C6 alkoxy, and m and n are each independently 0, 1, or 2. 1 and R 2 are each independently C1-C6 alkyl, halo, oxo, or C1-C6 haloalkyl, and m and n are each independently 0 or 1. In other embodiments, m and n are 0.

[0109] In another embodiment, the compound of formula I is a compound of formula IA [ka] or a single stereoisomer thereof or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein: 2 , R x , R y , R x’ , R y’ , and n are as defined herein.

[0110] In other embodiments, the compound of formula I or IA is a compound of formula IA-1 [ka] or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, J, L, Y, R 2 , R x , R y , R x’ , R y’ and n is as defined herein; R 3 are each independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, NH2, and oxo; and q is 0, 1, 2, or 3.

[0111] In other embodiments, the compound of formula I, IA, or IA-1 is a compound of formula IA-2 [ka] or a single stereoisomer thereof or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, L, Y, R 2 , R x , R y , R x’ , R y’ and n are the same as defined herein; K is a C1-C4 alkylene optionally substituted with a halo, hydroxyl, or a C1-C6 alkoxy group.

[0112] In other embodiments, the compound of formula I, IA, IA-1, or IA-2 is a compound of formula IA-3 [ka] or a single stereoisomer thereof or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, L, Y, R 2 , R x’ , R y’ and n are as defined herein; and K is C1-C4 alkylene.

[0113] In other embodiments, the compound of formula I, IA, IA-1, IA-2, or IA-3 is a compound of formula IA-4 [ka] or a single stereoisomer thereof or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, L, Y, R 2 , R x’ , R y’ and n are as defined herein; and K is C1-C3 alkylene.

[0114] In other embodiments, the compound of formula I, IA, IA-1, IA-2, IA-3, or IA-4 is a compound of formula IA-5 [ka] or a single stereoisomer thereof or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, L, Y, K, R 2 , R x’ , R y’ , and n are as defined herein.

[0115] In other embodiments, the compound of formula I, IA, IA-1, IA-2, IA-3, IA-4, or IA-5 is a compound of formula IA-6 [ka] or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, L, Y, and K are as defined herein.

[0116] In other embodiments, a compound of formula I, IA, IA-1, IA-2, IA-3, IA-4, IA-5, or IA-6 is a compound of formula IA-7 [ka] or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein rings B, L, and Y are as defined herein.

[0117] In other embodiments, the compound of formula I, IA, IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, or IA-7 is a compound of formula IA-7a, formula IA-7b, formula IA-7c, formula IA-7d, IA-7e, IA-7f, or IA-7g. [ka] [ka] or a single stereoisomer or mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, wherein L and Y are as defined herein; each X1 is, independently, CH or N; and p is 1, 2, or 3.

[0118] In other embodiments, Y is C1-C3 alkylene, in which one methylene unit is optionally replaced with NH, N-(C1-C6 haloalkyl), or N-(C1-C6 alkyl); and L is a bond or C1-C3 alkylene.

[0119] In other embodiments, Y is C1-C3 alkylene, one methylene unit of which is optionally replaced with NH, N-(C1-C6 haloalkyl), or N-(C1-C6 alkyl); Ring B is a 5-6 membered monocyclic cycloalkylene, a 4-7 membered monocyclic heterocycloalkylene, or a 6-9 membered bicyclic heterocycloalkylene, optionally substituted with C1-C6 alkyl; L is a bond or C1-C3 alkylene; R x’ and R y’ are each independently H or C1-C6 alkyl.

[0120] In other embodiments, Y is C1-C3 alkylene, in which one methylene unit is optionally replaced with NH, N-(C1-C6 haloalkyl), or N-(C1-C6 alkyl); Ring B is a 5-6 membered monocyclic cycloalkylene, a 4-7 membered monocyclic heterocycloalkylene, or a 6-9 membered bicyclic heterocycloalkylene, optionally substituted with C1-C6 alkyl; and L is a bond or C1-C3 alkylene.

[0121] In other embodiments, Y is selected from the group consisting of -CH2-, -CH2NH-, -CH2NMe-, -CH2N(CH2CH2F)-, CH2NCH2F-, and -CH2NEt-; Ring B is [ka] L is a bond, -CH-, or -CH-CH-; R x’ and R y’ are each independently H.

[0122] In another aspect, the disclosure provides compounds, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, which are set forth in Table 1. Table 1 provides the free base and salt structures of the compounds. [Table 1-1] [Table 1-2]

[0123] In another aspect, the disclosure provides compounds, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, which are shown in Table 2. Table 2 shows the free base and salt structures of the compounds. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0124] Also, [ka] or a pharma- ceutically acceptable salt thereof.

[0125] In other embodiments, the compound of formula I, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharma- ceutically acceptable salt thereof, is selected from the compounds set forth in any of Tables 1 and 2.

[0126] In another aspect, the present disclosure provides a compound of formula E [ka] or a pharma- ceutically acceptable salt thereof, wherein rings A, J, R 1 , R 2 , R x , R y , m, and n are defined the same as in the preceding paragraph; and Y1 is a C1-C6 alkylene, wherein one methylene unit of the C1-C6 alkylene is replaced with (C=O).

[0127] In another embodiment of the compound of formula E or a pharma- ceutically acceptable salt thereof, Y1 is a C1-C3 linear alkylene, in which one methylene unit is replaced with (C=O).

[0128] In one embodiment, the compound of formula E or a pharma- ceutically acceptable salt thereof is [ka] is selected from the group consisting of:

[0129] Pharmaceutical Compositions and Administration The present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharma- ceutically acceptable excipient. In certain embodiments, the compound of the present invention is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

[0130] Pharmaceutically acceptable excipients include any solvent, diluent, or other liquid medium, dispersion agent, suspension aid, surfactant, isotonicity agent, thickening or emulsifying agent, preservative, solid binder, lubricant, etc., depending on the particular dosage form desired. General introductions to the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0131] The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology. In general, such methods of preparation include the step of bringing into association a compound of the present invention (the "active ingredient") with the carrier and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single or multi-dosage unit.

[0132] Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dose of the active ingredient administered to a subject, and / or a convenient fraction of such a dose, for example, half or a third of such a dose.

[0133] The relative amounts of the active ingredient, the pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as depending on the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

[0134] Pharmaceutically acceptable excipients used in the manufacture of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavorings, and perfumes may also be present in the composition.

[0135] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0136] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0137] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fats. Acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60], polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate [Span® 65], glyceryl monooleate, sorbitan monooleate [Span® 80]), polyoxyethylene Esters (e.g., polyoxyethylene monostearate [Myrj45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate,Potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0138] Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, and the like), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0139] Exemplary preservatives include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0140] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0141] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates: malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0142] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0143] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and phenylethyl alcohol.

[0144] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0145] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.In certain embodiments, the preservative is an antioxidant.In other embodiments, the preservative is a chelating agent.

[0146] Exemplary buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, hydroxyapatite, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0147] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0148] Exemplary natural oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, cade oil, chamomile oil, canola oil, caraway oil, carnauba oil, castor oil, cinnamon bark oil, cocoa butter, palm oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, kukui nut oil, lavandin oil, lavender oil, lemon oil, Litsea quinoa oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange roughy oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, sasquana oil, savory oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, camellia oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0149] Liquid dosage forms for oral and parenteral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, the oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and perfumes. In certain embodiments for parenteral administration, the conjugates of the invention are mixed with a solubilizing agent, such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[0150] Injectables, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectables can be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, 1,3-butanediol solutions. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending agents. For this purpose, any brand of fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0151] Sterile injectable compositions, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to techniques known in the art using suitable dispersants or wetting agents (such as Tween® 80) and suspending agents. The sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, 1,3-butanediol solutions. Among the acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending agents (for example, synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharma-ceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants. Other commonly used surfactants, such as Tween® or Span®, or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0152] In order to prolong the effect of a drug, it is often desirable to delay the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by using a suspension of crystalline or amorphous material that is poorly soluble in water. The rate of absorption of the drug therefore depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle.

[0153] Compositions for rectal or vaginal administration will usually be suppositories, which may be prepared by mixing the conjugate of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient.

[0154] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar, calcium carbonate, jasmine, and the like. Mixed with guinea pig starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain buffering agents.

[0155] Solid compositions of a similar type may be used as fillers for soft and hard gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols. Solid dosage forms tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, optionally delayed, in a certain part of the intestinal tract. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may be used as fillers for soft and hard gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0156] The active ingredient may be in microencapsulated form with one or more of the excipients mentioned above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. As is common practice, such dosage forms may contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may contain buffering agents. They may optionally contain opacifying agents, and may be of a composition that they release the active ingredient(s) only, or preferentially, optionally delayed, in a certain part of the intestinal tract. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0157] The dosage form for topical and / or transdermal administration of the compound of the present invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches.Generally, the active ingredient is mixed under sterile conditions with a pharma- ceutically acceptable carrier and / or any necessary preservatives and / or buffers as necessary.Furthermore, the present invention contemplates the use of transdermal patches, which often have the additional advantage of providing controlled delivery of active ingredient to the body.Such dosage forms can be prepared, for example, by dissolving and / or dispensing the active ingredient in a suitable medium.Alternatively or additionally, the rate can be controlled by providing a rate-limiting membrane and / or dispersing the active ingredient in a polymer matrix and / or gel.

[0158] Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Patent Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662. Intradermal compositions can be administered by devices that limit the effective penetration length of the needle into the skin, such as those described in PCT Publication No. WO99 / 34850 and those with comparable functionality. Liquid jet injectors that create a jet that penetrates the stratum corneum and reaches the dermis and / or jet injection devices that deliver liquid vaccines to the dermis through a needle are suitable. Jet injection devices are disclosed, for example, in U.S. Patent Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339, 163, 5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, and PCT Publication Nos. WO97 / 37705 and WO97 / 13537. A ballistic powder / particle delivery device is suitable, which uses compressed gas to advance the vaccine in powder form through the outer layer of the skin to the dermis. Alternatively or additionally, a conventional syringe can be used in the classical Mantoux method of intradermal administration.

[0159] The pharmaceutical compositions of the invention may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles, which comprise the active ingredient and have a diameter of about 0.5 to about 7 nanometers or about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir capable of directing a stream of propellant to disperse the powder, and / or using a device comprising a self-propelling solvent / powder dispensing vessel, e.g., a sealed vessel comprising the active ingredient dissolved and / or suspended in a low boiling propellant. Such powders comprise particles in which at least 98% of the particles by weight are greater than 0.5 nanometers in diameter and at least 95% of the particles by number are less than 7 nanometers in diameter. Alternatively, at least 95% of the particles by weight are greater than 1 nanometer in diameter and at least 90% of the particles by number are less than 6 nanometers in diameter. Dry powder compositions may comprise a solid fine powder diluent, e.g., sugar, and are conveniently provided in unit dose form.

[0160] Low boiling point propellants generally include liquid propellants having a boiling point below 65° F. at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional components, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size of the same order as the particles containing the active ingredient).

[0161] The pharmaceutical compositions of the present invention formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as optionally sterile aqueous and / or dilute alcoholic solutions and / or suspensions containing the active ingredient, and may be conveniently administered using any nebulizer and / or atomizer device. Such formulations may further include one or more additional ingredients, including, but not limited to, flavoring agents, such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives, such as methyl oxybenzoate. The droplets provided by this route of administration may have a mean diameter of about 0.1 to about 200 nanometers.

[0162] The formulations described herein as useful for pulmonary delivery are useful for intranasal delivery of the pharmaceutical compositions of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient, having an average particle size of about 0.2 to 500 micrometers. Such formulations are administered by rapid inhalation through the nasal passages from a container of the powder held close to the nostrils.

[0163] Formulations for nasal administration may, for example, contain as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein. Pharmaceutical compositions of the invention may be prepared, packaged, and / or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges manufactured using conventional methods and may contain, for example, 0.1 to 20% (w / w) of the active ingredient, the remainder comprising an orally dissolvable and / or disintegrable composition and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powders, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

[0164] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for administration to humans, one of skill in the art will understand that such compositions are generally suitable for administration to any animal. Modifications to pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or perform such modifications with routine experimentation.

[0165] The compound provided herein is usually formulated in dosage unit form for ease of administration and uniformity of dosage.However, it is understood that the total amount of the composition of the present invention to be used daily is determined by the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any specific subject or organism depends on various factors, including the disease, disorder or condition to be treated and the severity of said disorder, the activity of the specific active ingredient used, the specific composition used, the age, weight, general condition, sex and diet of said subject, the time of administration, route of administration and excretion rate of the specific active ingredient used, duration of treatment, the drug used in combination with or simultaneously with the specific active ingredient used, and similar factors well known in the medical field.

[0166] To carry out the method of the present invention, the compound or pharmaceutical composition thereof can be administered orally, parenterally, by inhalation spray, topically, rectally, bucally, vaginally, rectally, or via an implanted reservoir.As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.In general, the most suitable administration route depends on various factors, including the nature of the drug (e.g., its stability in the environment of the gastrointestinal tract) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration).

[0167] The exact amount of compound required to achieve an effective dose will vary between subjects, depending, for example, on the species, age, and general condition of the subject, the severity of the side effect or disorder, the identity of the particular compound(s), the method of administration, etc. The desired dose can be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations).

[0168] In certain embodiments, an effective amount of a compound for administration to a 70 kg adult, one or more times per day, may contain from about 0.0001 mg to about 3000 mg, from about 0.0001 mg to about 2000 mg, from about 0.0001 mg to about 1000 mg, from about 0.001 mg to about 1000 mg, from about 0.01 mg to about 1000 mg, from about 0.1 mg to about 1000 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 100 mg, from about 10 mg to about 1000 mg, or from about 100 mg to about 1000 mg of the compound, per unit dosage form.

[0169] In certain embodiments, the compounds of the invention may be administered orally or parenterally at a dosage level sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, and more preferably from about 1 mg / kg to about 25 mg / kg of the subject's body weight per day, one or more times per day to obtain the desired therapeutic effect.

[0170] It will be understood that the dosage ranges described herein provide guidance for administering the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent may be determined by a physician or by one skilled in the art and may be less than or the same as that administered to an adult.

[0171] It is also understood that the compounds or compositions described herein can be administered in combination with one or more additional therapeutically active agents.The compounds or compositions can be administered in combination with additional therapeutically active agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution in the body.It is also understood that the treatments used can achieve the desired effect on the same disorder and / or they can achieve different effects.

[0172] The compound or composition can be administered simultaneously with, prior to, or after one or more additional therapeutically active agents. Generally, each agent is administered at a dose and / or time schedule determined for that agent. It will be further understood that the additional therapeutically active agents used in this combination can be administered together in a single composition or separately in different compositions. The particular combination to use in a regimen will take into consideration the compatibility of the compound of the present invention with the additional therapeutically active agent and / or the desired therapeutic effect to be achieved. In general, the additional therapeutically active agents used in combination will be expected to be used at levels that do not exceed the levels at which they are used individually. In some embodiments, the levels used in combination are lower than the levels at which they are used individually. Additional therapeutically active agents include antibiotic agents, such as antibiotics useful in the treatment of tuberculosis. Exemplary antibiotics include, but are not limited to, isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin.

[0173] Kits (e.g., pharmaceutical packs) are also included in the present invention. The kits provided may include a pharmaceutical composition or compound of the present invention and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the kits provided may optionally further include a second container that includes a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound of the present invention. In some embodiments, the container and the pharmaceutical composition or compound of the present invention provided in the second container are mixed to form one unit dosage form.

[0174] Therapeutic Uses and Methods In another aspect, the present invention provides a method for treating bacterial infection in a patient who needs such treatment.The method comprises administering an effective amount of a compound of formula I or its pharmaceutically acceptable salt or a composition comprising a compound of formula I or its pharmaceutically acceptable salt.In certain embodiments, the effective amount is a therapeutically effective amount.In certain other embodiments, the effective amount is a prophylactically effective amount.

[0175] In some embodiments, the compounds of the present invention may have activity against a wide range of both gram-positive and gram-negative bacteria. In these and other embodiments, the compounds of the present invention may be used to treat infections and inhibit the growth of microorganisms. Thus, the compounds of the present invention can be used to treat humans and animals with a wide range of bacterial infections, such as impetigo, pneumonia, bronchitis, pharyngitis, endocarditis, urinary tract infections, diabetic foot ulcers, gastrointestinal infections and bacteremia. These bacterial infections may be caused by any of the following bacteria: Staphylococcus aureus, coagulase-negative staphylococci, methicillin-resistant Staphylococcus aureus, methicillin-resistant coagulase-negative staphylococci, enterococci, beta-hemolytic streptococci, viridans group streptococci, multi-resistant M. tuberculosis and other atypical mycobacteria such as M. intracellulare and M. avium, Bacillus mycobacterial infections, and emerging gram-negative pathogens such as Chryseobacterium meningosepticum, Chryseobacterium indologense and other gram-negative pathogens such as E. coli, Klebsiella, Proteus, Serratia, Citrobacter, Pseudomonas, Burkholderia, Brucella, Yersinia, Francisella, Coxiella, Chlamydia, Salmonella, Rickettsia, Shigella and Campylobacter.

[0176] In one embodiment, the bacterial infection is tuberculosis. In certain embodiments, the tuberculosis infection is a Mycobacterium tuberculosis infection. In certain embodiments, the tuberculosis infection is a multi-drug resistant tuberculosis (MDR-TB) infection, e.g., resistant to the first-line TB drugs rifampicin and / or isoniazid. In certain embodiments, the tuberculosis infection is an extensively drug resistant tuberculosis (XDR-TB) infection, e.g., resistant to three or more of six classes of second-line drugs (see, e.g., Centers for Disease Control and Prevention (CDC) (2006). "Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs worldwide, 2000-2004". MMWR Morb Mortal Wkly Rep 55(11):301-5).

[0177] Process In some embodiments, compounds and intermediates of the present disclosure can be prepared according to the following general synthetic scheme G-1. In scheme G-1, ring A, ring B, J, L, Y, R 1 , R 2 , R x , R y , R x’ , R y’, m, and n are defined the same as in the preceding paragraph; X is a leaving group such as halo, mesylate, tosylate, or triflate; Y is a C-C alkylene, one methylene unit of the C-C alkylene is optionally replaced with (C=O); Y is a C-C alkylene, one methylene unit of the C-C alkylene is optionally replaced with (C=O); P is a C-C alkyl or hydroxyl protecting group. In some embodiments, Y is (C-C alkylene)-C(=O)H. In some embodiments, Y is -CH-C(=O)H. In some embodiments, Y is a C-C alkylene, one methylene unit of the C-C alkylene is optionally replaced with (C=O). In some embodiments, Y3 is -CH2- or -C=O-. In some embodiments, -Y3-OP is C1-C6 alkylene-OTBS, (C1-C5 alkylene)-C(=O)O-(C1-C6 alkyl), or -COO(C1-C6 alkyl). In some embodiments, -Y3-OP is -CH2-OTBS or -COO(C1-C6 alkyl).

[0178] General synthetic scheme G-1 [ka] In step 1 of general synthetic scheme G-1, a compound of formula (a) is reacted with a boron reagent, e.g., bis(pinacolato)diboron (B2 pin2), in the presence of a phosphine ligand, e.g., [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (Pd(dppf)Cl2), a base, and a suitable solvent to form a boronate ester of the compound of formula (a). Bases include, but are not limited to, sodium bicarbonate, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, and cesium carbonate. Suitable solvents include aprotic solvents, e.g., dioxane, dichloromethane, dimethylformamide, acetonitrile, and the like. In several steps, 1.0 molar equivalent of a compound of formula (a) is synthesized with about 1.0-2.0 molar equivalents of a boron reagent, a base, and a phosphine ligand in a suitable solvent, e.g., dioxane. The mixture is then reacted at a temperature of about 0°C to 150°C for a sufficient time. For example, the temperature is about 25° C. to 130° C. or about 50° C. to 125° C., and the reaction time is about 1 to 24 hours, 2 to 24 hours, or about 10 to 24 hours.

[0179] In step 2 of general synthetic scheme G-1, this boronic ester is cross-coupled with cytosine in the presence of a base, such as a tertiary amine, and a copper reagent, such as a copper(II) reagent, to provide a compound of formula (b).

[0180] Alternatively, to produce a compound of formula (b), in step 1 of general synthetic scheme G-1, N 4 -benzoylcytosine can be used to react with the compound of formula (a) in the presence of 18-crown-6 and a base such as sodium bicarbonate, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, cesium carbonate, etc. The reaction can be carried out at a temperature of about 0° C. to 150° C. for a sufficient time. For example, the temperature is about 25° C. to 130° C. or about 50° C. to 125° C., and the reaction time is about 1 to 48 hours, or 2 to 36 hours, or about 10 to 30 hours. In step 2, the benzoyl group is then removed under acidic conditions to give the compound of formula (b).

[0181] In step 3 of the general synthesis scheme G-1, a compound of formula (b) and an iodide (c) are amide-coupled to obtain an intermediate (d). In some steps, 1 molar equivalent of a compound of formula (b) and about 1.1-2.0 molar equivalents of an iodide (c) are synthesized in a suitable solvent, such as a polar aprotic solvent. Examples of the polar aprotic solvent include dichloromethane, dimethylformamide, acetonitrile, and the like. The polar aprotic solvent containing the mixture is then reacted at a temperature of about 0° C. to 100° C. for a sufficient time. For example, the temperature is about 25° C. to 95° C. or about 50° C. to 95° C., and the reaction time is about 1-24 hours, or 2-20 hours, or about 5-18 hours.

[0182] In steps 4 and 5 of General Synthesis Scheme G-1, the compound of formula (d) is deprotected to produce a free alcohol (e), which is then oxidized to a ketone or aldehyde to give a compound of formula E. When -Y3-OP is (C1-C5 alkylene)-C(=O)O-(C1-C6 alkyl), the compound of formula (d) is reduced to the free alcohol (e), which is then oxidized to a ketone or aldehyde to give a compound of formula (E).

[0183] In step 6 of the general synthesis scheme G-1, a compound of formula E is reacted with an amine under reductive amination conditions to obtain a compound of formula I. This reductive amination can be carried out in the presence of a reducing agent and a suitable solvent. Suitable solvents include protic or aprotic solvents. Protic solvents include, but are not limited to, water and alcohols, such as methanol, ethanol, propanol, and the like. Aprotic solvents include, but are not limited to, solvents such as dichloromethane, dimethylformamide, acetonitrile, and the like. The suitable solvent can also be a combination of two or three solvents. Reducing agents include, but are not limited to, borohydride reagents or metal hydride reagents. Non-limiting examples include lithium borohydride, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

[0184] In one embodiment, the present disclosure provides a compound of formula I [ka] or a pharma- ceutically acceptable salt thereof, comprising: A compound of formula E [ka] Formula D [ka] or formula D' [ka] under reductive amination conditions to obtain said compound of formula I, In the formula, ring A, ring B, J, L, R 1 , R 2 , R x , R y , R x’ , R y’ , m, and n have the same definition as in the preceding paragraph; Ring B1 is a nitrogen-containing 3-8 membered monocyclic heterocycloalkylene or a nitrogen-containing 6-12 membered bicyclic heterocycloalkylene, each of which is optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and C1-C6 hydroxyalkyl; Y is a C1-C6 alkylene, wherein one methylene unit of the C1-C6 alkylene is optionally replaced with NH, N-(C1-C6 haloalkyl), or N-(C1-C6 alkyl); Y1 is a C1-C6 alkylene, in which one methylene unit is replaced by (C=O); R 4 is H or C1-C6 alkyl.

[0185] The procedures and conditions for carrying out the reductive amination of compounds of formula E are similar to step 6 of the general synthetic scheme.

[0186] In one embodiment, the process comprises: x , R y , R x’ , and R y’ When is an amino protecting group, the method further comprises the step of removing the amino protecting group.

[0187] In some embodiments, Ring B1 is a nitrogen-containing 4-7 membered monocyclic heterocycloalkylene or a nitrogen-containing 6-9 membered bicyclic heterocycloalkylene, each of which is optionally substituted with up to three substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, halo, CN, C1-C6 haloalkyl, OH, COOH, COO(C1-C6 alkyl), CONH2, CONH(C1-C6 alkyl), CON(C1-C6 alkyl)2, and C1-C6 hydroxyalkyl.

[0188] In some embodiments, ring B1 is a nitrogen-containing 4-7 membered monocyclic heterocycloalkylene or a nitrogen-containing 6-9 membered bicyclic heterocycloalkylene, the monocyclic and bicyclic heterocycloalkylene being optionally substituted with C1-C6 alkyl. In some embodiments, ring B1 is a nitrogen-containing 4-7 membered monocyclic heterocycloalkylene or a nitrogen-containing 6-9 membered bicyclic heterocycloalkylene.

[0189] In some embodiments, ring B in formula D is [ka] It is.

[0190] In some embodiments, ring B1 is [ka] is selected from the group consisting of:

[0191] In some embodiments, L is a bond, -CH-, or CH-CH-; R x’ and R y’ are each independently H or Boc.

[0192] In another embodiment, the compound of formula e is [ka] is selected from the group consisting of:

[0193] In other embodiments, the compound of formula E is [ka] is selected from the group consisting of:

[0194] Preparation of compounds The preparation of starting materials that are commercially available, described in the literature, or readily available to those skilled in the art is not described. Those skilled in the art will understand that when compounds are described as being prepared similarly to previous examples or intermediates, reaction times, equivalents of reagents, and temperatures may vary for each specific reaction, and that different work-up or purification techniques may be required or desirable to be used. When reactions are carried out using microwave irradiation, the microwave ovens used were either Biotage Initiator or CEM Discover System Model 908005. The actual power supplied during the reaction was varied to maintain a constant temperature.

[0195] Common methods Reactions requiring anhydrous conditions were carried out in oven-dried glassware under a positive pressure of either nitrogen or argon. Commercially available reagents were used as received or materials were otherwise purified according to Purification of Laboratory Chemicals. Dichloromethane (CH2Cl2), N,N'-dimethylformamide (DMF), toluene, and tetrahydrofuran (THF) were degassed with nitrogen and passed through a solvent purification system (Innovative Technologies Pure Solv). Dry 1,4-dioxane was purchased from Acros Organics in Acros Seal™ bottles. Triethylamine (Et3N) and N,N-diisopropylethylamine (DIPEA) were stored over 4 Å molecular sieves or distilled over 4 Å molecular sieves prior to use. Microwave reactions were performed on a CEM Discover System Model 908005. Reactions were monitored by TLC or LCMS, visualized with a dual shortwave / longwave UV lamp, and / or stained with ethanolic solutions of KMnO4, 12-phosphomolybdic acid, or other commonly used dyes. Flash chromatography was performed on silica gel Kieselgel 60 (230-400 mesh) from Merck from EM Science using the indicated HPLC grade solvents or an automated medium pressure column chromatography system (Teledyne ISCO CombiFlash® RF75 or CombiFlash® Rf+). Reverse phase HPLC was performed on a Waters HPLC Semi Prep 150B system using a Sunfire C18 Prep Column or an Atlantis T3 Prep Column, isocratically or gradient-based, with HO (0.1% TFA) and 10% HO:90CH3CN (0.1% TFA) as eluents.

[0196] Melting points were measured using a Mel-Temp® capillary melting point apparatus. Infrared spectra were obtained using a Nicolet 380-FT IR spectrometer equipped with a Smart Orbit sample system. Optical rotations were obtained at ambient temperature on a Perkin Elmer model 343 polarimeter (Na D line) using a microcell with a path length of 1 decimeter. Mass spectra measured by LCMS were collected on a Thermo Scientific™ UltiMate™ 3000UHPLC using an electrochemical detector with a fluorescence detector observed at 214 or 254 nm, or a Waters Aquity UPLC H-Class series equipped with a photodiode array detector and a QDa mass detector. 1 H NMR spectra were recorded at 500 MHz, 400 MHz, and 300 MHz. 13 C was recorded at 125 MHz. Proton resonances were reported using the chemical shift (δ) [multiplicity (s=singlet, br s=broad singlet, d=doublet, t=triplet, q=quartet, m=multiplet)] format for the deuterated solvent peaks: 7.27 ppm for CDCl3, 3.31 ppm for CD3OD (centerline signal), 2.50 for D6-DMSO, and 4.79 for DO. Carbon resonances were reported as chemical shifts in parts per million (δ) for 77.23 ppm for CDCl3 and 49.15 ppm for CD3OD at the centerline signal of the respective solvent peaks. Commercial chemicals are purchased from multiple vendors, including Sigma-Aldrich, Acros, Enamine, TCI America, Combi-Blocks, Alfa-Aesar, Angene, Ark Pharma, PharmaBlock, Strem Chemicals, Frontier Scientific, and AstaTech, Inc.

[0197] Liquid Chromatography Mass Spectrometry Liquid Chromatography Mass Spectrometry A

[0198] Total ion current (TIC) and DAD UV chromatographic traces as well as MS and UV spectra associated with those peaks were acquired on a UPLC / MS Acquity™ system equipped with a PDA detector coupled to a Waters single quadrupole mass spectrometer operated in alternating positive and negative electrospray ionization mode. [LC / MS-ES (+ / -): Analysis was performed on an Acquity UPLC™ CSH, C18 column (50×2.1 mm, 1.7 μm particle size), column temperature 40° C., mobile phase: A-water + 0.1% HCOOH / B-CH3CN + 0.1% HCOOH, flow rate: 1.0 mL / min, run time = 2.0 min, gradient: t = 0 min 3% B, t = 1.5 min 99.9% B, t = 1.9 min 99.9% B, t = 2.0 min 3% B, stop time 2.0 min. Positive ES100-1000, negative ES100-1000, UV detection DAD 210-350 nm were used.

[0199] Liquid Chromatography Mass Spectrometry B

[0200] Total ion current (TIC) and DAD UV chromatographic traces as well as MS and UV spectra associated with those peaks were acquired on a UPLC / MS Acquity™ system equipped with a PDA detector coupled to a Waters single quadrupole mass spectrometer operated in alternating positive and negative electrospray ionization mode. [LC / MS-ES (+ / -): Analysis was performed using an Acquity UPLC™ BEH, C18 column (50×2.1 mm, 1.7 μm particle size), column temperature 40° C., mobile phase: A-0.1% v / v aqueous ammonia (aq) pH 10 / B-CH3CN, flow rate: 1.0 mL / min, run time=2.0 min, gradient: t=0 min 3% B, t=1.5 min 99.9% B, t=1.9 min 99.9% B, t=2.0 min 3% B, stop time 2.0 min, positive ES 100-1000, negative ES 100-1000, UV detection DAD 210-350 nm.

[0201] Liquid Chromatography Mass Spectrometry C LC / MS-ES (+ / -): The analysis was performed using an AQUITY with PDA detector and QDA Performance, a C18 column (50 x 2.1 mm, particle size 1.6 μm), column temperature 35 °C, mobile phase: A-Milli Q water with 0.1% formic acid (pH = 2.70) / B-0.1% formic acid in water:acetonitrile (10:90), flow rate: 0.8-1.0 mL / min, run time = 4.0 min, gradient: t = 0 min 3% B, t = 2.7 min 98% B, t = 3.0 min 100% B, t = 3.51 min 3% B, stop time 4.0 min.

[0202] Liquid Chromatography Mass Spectrometry D

[0203] LC / MS-ES (+ / -): The analysis was performed using an AQUITY H-Class equipped with a PDA detector and a QDA, a C18 column (50 x 2.1 mm, particle size 1.6 μm), column temperature 35 °C, mobile phase: A-Milli Q water (pH = 2.70) containing 0.1% formic acid / B-0.1% formic acid in water:acetonitrile (10:90), flow rate: 0.8-1.0 mL / min, run time = 4.0 min, gradient: t = 0 min 3% B, t = 2.7 min 98% B, t = 3.0 min 100% B, t = 3.51 min 3% B, stop time 4.0 min.

[0204] Liquid Chromatography Mass Spectrometry

[0205] LC / MS-ES (+ / -): The analysis was performed using an AQUITY H-Class equipped with a PDA detector and a QDA, a C18 column (50 x 2.1 mm, 1.6 μm particle size), column temperature 35 °C, mobile phase: A-0.1% formic acid in water (pH = 2.70) / B-0.1% formic acid in water:acetonitrile (10:90), run time = 9.0 min, gradient: t = 0 min 1% B, t = 2.5 min 50% B, t = 4.5 min 97.5% B, t = 6.5 min 1% B, stop time 9.0 min.

[0206] Liquid Chromatography Mass Spectrometry F

[0207] LC / MS-ES (+ / -): Analysis was performed using an Agilent Infinity II G6125C LCMS, C18 column (50 x 4.6 mm, 3.5 μm particle size), column temperature 35 °C, mobile phase: A - Milli-Q water (pH = 7.35) containing 5 mM ammonium bicarbonate / B - methanol, run time = 7.0 min, gradient: t = 0 min 8% B, t = 3.0 min 70% B, t = 3.7 min 95% B, t = 4.2 min 100% B, t = 5.21 min 8% B, stop time 7.0 min.

[0208] Liquid Chromatography Mass Spectrometry G

[0209] LC / MS-ES (+ / -): The analysis was performed using a Waters Alliance 2690 and 996 PDA detector with a Micromass ZQ, a C18 column (150 x 4.6 mm, particle size 3.5 μm), column temperature 35 °C, mobile phase: A - 5 mM ammonium acetate + 0.1% FA in water / B - methanol, run time = 17.0 min, gradient: t = 0 min 10% B, t = 7.0 min 90% B, t = 9.0 min 100% B, t = 14.01 min 10% B, stop time 17.0 min.

[0210] Liquid Chromatography Mass Spectrometry

[0211] LC / MS-ES (+ / -): The analysis was performed using an AQUITY equipped with a PDA detector and QDA Performance, a C18 column (50 x 2.1 mm, 1.6 μm particle size), column temperature 35 °C, mobile phase: A-Milli Q water with 0.1% formic acid (pH = 2.70) / B-0.1% formic acid in water:acetonitrile (10:90), flow rate: 0.9 mL / min, run time = 3.0 min, gradient: t = 0 min 5% B, t = 1.8 min 98% B, t = 2.0 min 100% B, t = 2.51 min 5% B, stop time 17.0 min.

[0212] Analysis method 1 H nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Avance 400 instrument equipped with probe DUAL 400 MHz S1, probe 6 S1 400 MHz 5mm1 H- 13 Bruker Avance 400 instrument with C ID, Bruker Avance III 400 instrument with Nanobay with probe Broadband BBFO 5mm Direct ID, 400MHz Agilent Direct Drive instrument with AUTO-X PFG probe (all operating at 400MHz), or 5mm triple resonance 1 H{ 13 C / 15 The experiments were carried out using one of the following Agilent VNMRS500 Direct Drive instruments (operating at 500 MHz) equipped with a {N} cryoprobe. Spectra were acquired near room temperature in the solvents indicated unless otherwise stated. In all cases, the NMR data were consistent with the proposed structures. Characteristic chemical shifts (δ (ppm)) are given in parts per million using conventional abbreviations to designate the major peaks, e.g., s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), br (broad).

[0213] Thin-layer chromatography (TLC) refers to silica gel TLC using silica gel F254 (Merck) plates. Column chromatography was performed using an automated column chromatography (Biotage SP1 or Isolera) system with Biotage silica gel cartridges (KP-Sil or KP-NH) or, for reversed-phase chromatography, Biotage C18 cartridges (KP-C18).

[0214] Preparative HPLC was performed on a Shimadzu LC-20AP, Waters 2545 and Agilent 1260 infinity. Purity was measured with a Waters Alliance e2695-PDA detector 2998 and an Agilent 1260 Infinity-II. (Mobile phase: 0.05% HCl aqueous solution / methanol with gradient elution). [Table 3-1] [Table 3-2]

[0215] Synthesis of intermediates [ka] Ethyl trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate and Ethyl cis-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate Scheme I-1 [ka] Reagents: 1) NaBH4, MeOH, 0°C to room temperature, 16 hours 2) MsCl, Et3N, DCM, room temperature, 1.5 hours 3) N 4 -Benzoylcytosine, 18-crown-6, K2CO3, DMF, 100℃, 24 hours. Step 1: Ethyl 4-hydroxycyclohexane-1-carboxylate. To a solution of ethyl 4-oxocyclohexane-1-carboxylate (10 g, 58.8 mmol) in methanol (300 mL) was added sodium borohydride (4.56 g, 121 mmol) in portions at 0° C. and the mixture was allowed to warm to room temperature with stirring for 16 h. The reaction mixture was concentrated, taken up in EtOAc (1 L), washed with saturated aqueous NaHCO3 (2×500 mL) and brine (1×500 mL), dried over Na2SO4, filtered and concentrated to dryness to give the title compound (8.06 g).

[0216] Step 2: Ethyl 4-((methylsulfonyl)oxy)cyclohexane-1-carboxylate. To a solution of ethyl 4-hydroxycyclohexane-1-carboxylate (8.06 g, 46.8 mmol) and Et3N (9.8 mL, 70 mmol) in DCM (225 mL) was added methanesulfonyl chloride (4.4 mL, 56 mmol) dropwise and the mixture was stirred at room temperature for 1.5 h. The mixture was poured into saturated aqueous NaHCO3 (500 mL) and extracted with DCM (2 x 500 mL). The extract was dried over Na2SO4, filtered and concentrated to give the title compound (11.7 g).

[0217] Step 3: trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate and cis-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate 4 A mixture of 18-benzoylcytosine (201 mg, 0.93 mmol), ethyl 4-((methylsulfonyl)oxy)cyclohexane-1-carboxylate (280 mg, 1.12 mmol), 18-crown-6 (47 mg, 0.18 mmol), and K2CO3 (267 mg, 1.93 mmol) in DMF (5.0 mL) was stirred at 75 °C for 18 h and then at 100 °C for an additional 24 h. The mixture was cooled, diluted with EtOAc (75 mL), washed with saturated aqueous NaHCO3 (2 x 50 mL) and brine (2 x 50 mL), dried over Na2SO4, filtered through Celite®, and concentrated. The residue was purified by flash column chromatography (EtOAc / Hex) to give the title compound.

[0218] trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate ethyl (8.0 mg). 1H NMR(500 MHz, CDCl3)δ 8.98-8.53(m, 1H), 7.90(d, 2H), 7.68(d, 1H), 7.61(t, 1H), 7.56-7.44(m, 3H), 4.70(tt, 1H), 4.15(q, 2H), 2.32(tt, 1H), 2.24-2.16(m, 2H), 2.15-2.08(m, 2H), 1.77-1.63(m, 2H), 1.61-1.49(m, 2H), 1.29-1.23(m, 3H). LCMS[M+H]370.3.

[0219] cis-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate ethyl (35 mg). 1 H NMR(500 MHz, CDCl3)δ 8.84-8.54(m, 1H), 7.88(d, 2H), 7.67(d, 1H), 7.59(t, 1H), 7.53-7.42(m, 3H), 4.78-4.67(m, 1H), 4.19(q, 2H), 2.74-2.68(m, 1H), 2.35-2.25(m, 2H), 1.95-1.86(m, 2H), 1.78-1.63(m, 4H), 1.28(t, 3H). LCMS[M+H]370.3.

[0220] Alternatively, ethyl trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate can be prepared as shown in Scheme I-2.

[0221] [ka] Ethyl trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate Scheme I-2 [ka] Reagents: 1) L-Selectride, THF, -78°C, 2 hours 2) MsCl, Et3N, DCM, room temperature, 22 hours 3) N 4 -Benzoylcytosine, 18-crown-6, K2CO3, DMF, 100℃, 4 days. Step 1: cis-Ethyl 4-hydroxycyclohexane-1-carboxylate. To a solution of ethyl 4-oxocyclohexane-1-carboxylate (14.3 g, 84 mmol) in THF (500 mL) was added dropwise 1M L-Selectride in THF (100 mL) at -78 °C and the mixture was stirred for 2 h. Saturated NaHCO3 (100 mL) was added dropwise, the mixture was warmed to 0 °C, 30% H2O2 (25 mL) was added and the mixture was stirred at room temperature overnight. EtOAc (2 L) was added, the layers were separated and washed with saturated aqueous NaHCO3 (1 L) and brine (1 L), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (EtOAc / hexane) to give the title compound (11.96 g). 1 H NMR (500 MHz, CDCl3) δ 4.14 (q, 2H), 3.93-3.85 (m, 1H), 2.37 (tt, 1H), 2.02-1.90 (m, 2H), 1.74-1.60 (m, 6H), 1.26 (t, 3H). The minor diastereomer, ethyl trans-4-hydroxycyclohexane-1-carboxylate (1.06 g), was also recovered.

[0222] Step 2: Ethyl cis-4-((methylsulfonyl)oxy)cyclohexane-1-carboxylate. To a solution of ethyl cis-4-hydroxycyclohexane-1-carboxylate (11.9 g, 69 mmol) and Et3N (14.5 mL, 104 mmol) in DCM (350 mL) was added methanesulfonyl chloride (6.5 mL, 83 mmol) dropwise at 0° C. The mixture was allowed to warm to room temperature and stirred for 3 h. An additional amount of MsCl (1.3 mL, 17 mmol) was added and the mixture was stirred for a further 19 h. The crude material was purified by column chromatography (Hex / DCM / CH3CN) to give the title compound (14.6 g).

[0223] Step 3: Ethyl trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate N 4 A mixture of 15.1 g, 70 mmol), cis-benzoylcytosine (15.1 g, 70 mmol), ethyl cis-4-((methylsulfonyl)oxy)cyclohexane-1-carboxylate (14.6 g, 58 mmol), 18-crown-6 (3.1 g, 11.7 mmol), and K2CO3 (16.1 g, 116 mmol) in DMF (285 mL) was stirred at 100 °C for 4 days. The mixture was cooled and poured into water (2.5 L) and the precipitate was collected by vacuum filtration. The product was purified by column chromatography (Hex / EtOAc / MeOH) to give the title compound (1.40 g). 1 H NMR(500 MHz, CDCl3)δ 7.94(d, 2H), 7.70(d, 1H), 7.62(t, 1H), 7.58-7.47(m, 3H), 4.69(tt, 1H), 4.15(q, 2H), 2.33(tt, 1H), 2.25-2.17(m, 2H), 2.16-2.08(m, 2H), 1.70(qd, 2H), 1.56(qd, 2H), 1.27(t, 3H). LCMS[M+H]370.3.

[0224] [ka] 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide Scheme I-3 [ka] Reagents: 1) 2-(Boc-amino)isobutyric acid, EDC, HOBt, DIPEA, DMF, rt, 44 h 2) H2, 10% Pd / C, EtOH, rt, 24 h 3) CDI, DCM, rt, 22 h 4) CH3I, CH3CN, rt, 2 days. Step 1: Benzyl 4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxylate. To a mixture of 2-(boc-amino)isobutyric acid (22.7 g, 111.5 mmol), EDC (23.3 g, 122 mmol), HOBt (19.4 g, 85%, 128 mmol), and DIPEA (35 mL, 200 mmol) in DMF (200 mL) was added benzyl piperazine-1-carboxylate (19.5 mL, 101 mmol) and the mixture was stirred at room temperature for 44 h. Water (1 L) was added, forming a gummy precipitate and the liquid was poured off. EtOAc (1 L) was added and the solution was washed with saturated aqueous NaHCO3 (2 x 500 mL) and brine (1 x 500 mL), dried over Na2SO4, filtered and concentrated to give the title compound (30.7 g) as a white foam.

[0225] Step 2: tert-Butyl (2-methyl-1-oxo-1-(piperazin-1-yl)propan-2-yl)carbamate. A mixture of benzyl 4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxylate (30.7 g, 76 mmol) and 10 wt% Pd / C (ca. 50% H2O, 4.58 g) in EtOH (350 mL) was stirred under H2 (atmosphere) for 24 h. The mixture was filtered through a Celite® pad and the filtrate was concentrated to dryness and dried with toluene followed by Et2O to give the title compound (20.4 g).

[0226] Step 3: tert-Butyl (1-(4-(1H-imidazole-1-carbonyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. A mixture of tert-butyl (2-methyl-1-oxo-1-(piperazin-1-yl)propan-2-yl)carbamate (21.85 g, 77 mmol) and CDI (16.7 g, 93 mmol) in DCM (300 mL) was stirred at room temperature for 22 h. The mixture was diluted with DCM (1 L), washed with saturated aqueous NaHCO3 (2 x 500 mL) and brine (1 x 500 mL), dried over Na2SO4, filtered and concentrated to dryness to give the title compound (29.3 g).

[0227] Step 4: 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide. To a suspension of tert-butyl (1-(4-(1H-imidazole-1-carbonyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (29.3 g, 80 mmol) in CH3CN (375 mL) was added methyl iodide (25 mL, 403 mmol) and the mixture was stirred at room temperature under N2 for 2 days. The reaction mixture was concentrated to a total volume of 100 mL and Et2O (350 mL) was added. The precipitate was collected by vacuum filtration and rinsed with additional Et2O to give the title compound (38.8 g). 1 H NMR(500 MHz, DMSO-d6)δ 9.54(s, 1H), 8.01(s, 1H), 7.87-7.82(m, 1H), 7.39-7.31(m, 1H), 3.91(s, 3H), 3.86-3.57(m, 4H), 3.55-3.43(m, 4H), 1.39(s, 9H), 1.31(s, 6H).

[0228] [ka] (trans-4-Formylcyclohexyl)carbamic acid benzyl ester Scheme I-4 [ka] Reagents: 1) Cbz-Cl, 1M NaOH, dioxane, room temperature, 16 hours. 2) SO3·pyridine, DIPEA, DMSO, 0℃ to room temperature, 19 hours. Step 1: (trans-4-(hydroxymethyl)cyclohexyl)benzylcarbamate. A mixture of (trans-4-aminocyclohexyl)methanol (25 g, 190 mmol), 1 M NaOH (580 mL, 580 mmol), and Cbz-Cl (33 mL, 230 mmol) in dioxane (300 mL) was stirred at room temperature for 16 h. The mixture was neutralized with 1 M HCl (600 mL) to pH ∼4-7, and the precipitate was collected by vacuum filtration to give the title compound (46 g). 1 H NMR(500 MHz, CDCl3)δ 7.37-7.28(m, 5H), 5.08(s, 2H), 4.68(s, 1H), 3.50-3.39(m, 3H), 2.09-1.98(m, 2H), 1.86-1.71(m, 3H), 1.48-1.38(m, 1H), 1.18-0.98(m, 4H).

[0229] Step 2: Benzyl (trans-4-formylcyclohexyl)carbamate To a vigorously stirred mixture of benzyl (trans-4-(hydroxymethyl)cyclohexyl)carbamate (11.6 g, 44 mmol) and DIPEA (75 mL, 430 mmol) in DMSO (75 mL) was added dropwise a solution of SO3·pyridine (21 g, 132 mmol) in DMSO (75 mL, 1.1 mol) at 0 °C and the mixture was allowed to warm to room temperature and stirred for 19 h. 10% citric acid (500 mL) was then added dropwise. Water (750 mL) was added and the mixture was stirred for 1 h. The precipitate was collected by vacuum filtration. The product was taken up in Et2O (1 L) and washed with 5% citric acid (600 mL), saturated aqueous NaHCO3 (600 mL), brine (600 mL), dried (Na2SO4), filtered and concentrated in vacuo to give the title compound (12.0 g). 1 H NMR(500 MHz, CDCl3)δ 9.62(s, 1H), 7.41-7.27(m, 5H), 5.09(s, 2H), 4.68-4.57(m, 1H), 3.55-3.41(m, 1H), 2.22-2.09(m, 3H), 2.08-1.98(m, 2H), 1.47-1.33(m, 2H), 1.23-1.12(m, 2H).

[0230] [ka] tert-Butyl ((trans-4-aminocyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate Scheme I-5 [ka] Reagents: 1) tert-Butyl (trans-4-aminocyclohexyl)carbamate, NaBH(OAc)3, DCE, rt, 18 hrs. 2) Boc2O, 1M NaOH, dioxane, rt, 24 hrs. 3) H2, 10% Pd / C, MeOH, rt, 20 hrs. Step 1: Benzyl (trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohexyl)carbamate. To a mixture of benzyl (trans-4-formylcyclohexyl)carbamate (5.96 g, 23 mmol) and tert-butyl (trans-4-aminocyclohexyl)carbamate (5.86 g, 27 mmol) in dichloroethane (DCE, 250 mL) was added sodium triacetoxyborohydride (9.7 g, 46 mmol) and the mixture was stirred at room temperature for 18 h. 1M K2CO3 (50 mL) was added and the reaction mixture was stirred vigorously for 2 h. The mixture was poured into saturated aqueous NaHCO3 (500 mL) and extracted with DCM (2 x 250 mL). The extract was dried over Na2SO4, filtered and concentrated to give the title compound (10.7 g).

[0231] Step 2: tert-Butyl ((trans-4-(((benzyloxy)carbonyl)amino)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. A mixture of benzyl (trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohexyl)carbamate (10.5 g, 22.8 mmol), Boc2O (6.8 mL, 30 mmol), and 1 M NaOH (69 mL, 69 mmol) in dioxane (100 mL) was stirred at room temperature for 24 h. Additional portions each of 1 M NaOH (35 mL, 35 mmol) and Boc2O (3.4 mL, 15 mmol) were added and the reaction mixture was stirred for an additional 20 h. Water (350 mL) was added and the mixture was brought to neutral pH with 1 M HCl. The mixture was poured into EtOAc (500 mL) and the organic layer was washed with brine (1×500 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (EtOAc / Hexanes) to give the title compound (7.12 g) as a cream foam. 1 H NMR(500 MHz, MeOD)δ 7.39-7.24(m, 5H), 5.05(s, 2H), 3.39-3.20(m, 3H), 3.02(d, 2H), 2.00-1.89(m, 4H), 1.81-1.64(m, 6H), 1.58-1.48(m, 1H), 1.45(s, 9H), 1.43(s, 9H), 1.32-1.13(m, 4H), 1.10-0.97(m, 2H).

[0232] Step 3: tert-Butyl ((trans-4-aminocyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate A mixture of tert-butyl ((trans-4-(((benzyloxy)carbonyl)amino)cyclohexyl)methyl)(trans-4-(((tert-butoxycarbonyl)amino)cyclohexyl)carbamate (7.12 g, 12.7 mmol) and 10% Pd / C (ca. 50% in water, 720 mg) in MeOH (200 mL) was stirred under an atmosphere of H for 20 h. The mixture was filtered through a Celite® pad and the filtrate was concentrated in vacuo and then reconcentrated from toluene to give the title compound (5.24 g).

[0233] [ka] tert-Butyl (trans-4-(((trans-4-aminocyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate Scheme I-6 [ka] Reagents: 1) Acetaldehyde, NaBH(OAc)3, AcOH, DCE, rt, 22 h 2) H2, 10% Pd / C, 7M NH3 in MeOH, MeOH, rt, 20 h. Step 1: Benzyl (trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)methyl)cyclohexyl)carbamate. To a mixture of (trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohexyl)carbamate (2.05 g, 4.46 mmol), acetaldehyde (2.5 mL, 45 mmol) and acetic acid (0.41 mL, 7.2 mmol) in DCE (50 mL) was added NaBH(OAc)3 (1.9 g, 9.0 mmol) and the mixture was stirred at room temperature for 22 h. The mixture was poured into saturated aqueous NaHCO3 (125 mL) and extracted with DCM (2 x 125 mL). The extract was dried (Na2SO4), filtered and concentrated to dryness to give the title compound (2.07 g). 1 H NMR(500 MHz, DMSO-d6)δ 7.40-7.27(m, 5H), 7.13(d, 1H), 6.63(d, 1H), 4.98(s, 2H), 3.26-3.16(m, 1H), 3.16-3.05(m, 1H), 2.39(q, 2H), 2.35-2.25(m, 1H), 2.13(d, 2H), 1.85-1.71(m, 6H), 1.66-1.57(m, 2H), 1.36(s, 9H), 1.27-1.04(m, 7H), 0.91(t, 3H), 0.86-0.73(m, 2H). LCMS [M+H] 488.3.

[0234] Step 2: tert-Butyl (trans-4-(((trans-4-aminocyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate A mixture of benzyl (trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)(ethyl)amino)methyl)cyclohexyl)carbamate (1.96 g, 4.02 mmol) and 10% Pd / C (ca. 50% in water, 193 mg) in MeOH (80 mL) and 7M NH in MeOH (4 mL) was stirred under an atmosphere of H for 20 h. The mixture was filtered through a Celite® pad and the filtrate was concentrated in vacuo and then reconcentrated from toluene to give the title compound (1.42 g). 1 H NMR(500 MHz, MeOD)δ 3.26-3.15(m, 1H), 2.59(tt, 1H), 2.51(q, 2H), 2.48-2.41(m, 1H), 2.22(d, 2H), 1.98-1.82(m, 6H), 1.81-1.72(m, 2H), 1.43(s, 9H), 1.38-1.25(m, 3H), 1.24-1.06(m, 4H), 1.00(t, 3H), 0.95-0.83(m, 2H).

[0235] [ka] tert-Butyl (7-((trans-4-aminocyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate Scheme I-7 [ka] Reagents: 1) tert-Butyl (7-azaspiro[3.5]nonan-2-yl)carbamate, NaBH(OAc)3, DCE, room temperature, 3 days. 2) H2, 5% Pd / C, 7M NH3 in MeOH, MeOH, room temperature, 3 hours. Step 1: (trans-4-((2-((tert-butoxycarbonyl)amino)-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)benzylcarbamate. To a mixture of benzyl (trans-4-formylcyclohexyl)carbamate (1.43 g, 5.47 mmol) and tert-butyl (7-azaspiro[3.5]nonan-2-yl)carbamate (1.2 g, 5.0 mmol) in DCE (50 mL) was added NaBH(OAc)3 (2.35 g, 11.1 mmol) and the mixture was stirred at room temperature for 3 days. The reaction was quenched with 1 M K2CO3 (35 mL) and stirred vigorously for 1 h. The mixture was then poured into saturated aqueous NaHCO3 (75 mL) and extracted with DCM (2 x 75 mL). The extract was dried (Na2SO4), decanted and concentrated. The residue was purified by column chromatography (Hex / EtOAc / MeOH / NH4OH) to give the title compound. 1 H NMR(500 MHz, MeOD)δ 7.38-7.24(m, 5H), 5.05(s, 2H), 4.00-3.91(m, 1H), 3.39-3.32(m, 1H), 2.48-2.21(m, 4H), 2.21-2.14(m, 2H), 2.12(d, 2H), 1.98-1.89(m, 2H), 1.88-1.79(m, 2H), 1.68-1.53(m, 6H), 1.53-1.37(m, 10H), 1.27-1.15(m, 2H), 1.06-0.92(m, 2H).

[0236] Step 2: tert-Butyl (7-((trans-4-aminocyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate A mixture of benzyl (trans-4-((2-((tert-butoxycarbonyl)amino)-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)carbamate (2.31 g, 4.76 mmol) and 5% Pd / C (323 mg) in MeOH (60 mL) and 7M NH in MeOH (4 mL) was stirred under an atmosphere of H for 3 h. The mixture was filtered through a Celite® pad and the filtrate was concentrated in vacuo and then reconcentrated from toluene / DCM to give the title compound (1.67 g).

[0237] Compound synthesis [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 1) Scheme C-1 [ka] Reagents: 1) TBSCl, imidazole, DMF, 16 hours, room temperature, 2) 2,6-di-tert-butyl-4-methylpyridine, Tf2O, CH2Cl2, 0°C to room temperature, 8 hours, 3) Pd(dppf)Cl2·CH2Cl2, KOAc, B2pin2, 1,4-dioxane, 105°C, 16 hours, 4) cytosine, Cu(OAc)2·H2O, TMEDA, MeOH:H2O (4:1), room temperature, 48 hours, 5) 1-(4-(2-((tert-butyl) (trans-4-aminocyclohexyl)carbamate, Na(OAc)3BH, DCE, room temperature, 16 hours, 9) HCl / MeOH, room temperature, 4 hours. Step 1: 4-(((tert-Butyldimethylsilyl)oxy)methyl)cyclohexan-1-one. To a solution of 4-(hydroxymethyl)cyclohexan-1-one (1.5 g, 12 mmol) in DMF (25 mL) was added TBSCl (2.6 g, 18 mmol) and imidazole (1.6 g, 23 mmol). The reaction was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (150 mL) and washed with saturated aqueous LiCl (3×150 mL). The organic layer was concentrated under reduced pressure and purified by column chromatography (Hex:EtOAc) to give the desired product (56%).

[0238] Step 2: 4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohex-1-en-1-yl trifluoromethanesulfonate 4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohexan-1-one (1.6 g, 6.6 mmol) and 2,6-di-tert-butyl-4-methylpyridine (1.76 g, 8.6 mmol) in CHCl (40 mL) was added TfO (1.34 mL, 7.9 mmol) dropwise over 10 min at 0 °C. The reaction was allowed to warm to room temperature and stirred for 8 h. The reaction was quenched by the addition of saturated aqueous NaHCO (40 mL) and the organic layer was separated, concentrated under reduced pressure and purified by column chromatography (Hex: EtOAc) to give the desired product as an amber oil.

[0239] Step 3: tert-Butyldimethyl((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)methoxy)silane. A flask containing 4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohex-1-en-1-yl trifluoromethanesulfonate (2.25 g, 6.0 mmol), Pd(dppf)Cl2·CH2Cl2 (245 mg, 0.3 mmol), KOAc (1.77 g, 18 mmol), and B2pin2 (1.68 g, 6.6 mmol) was evacuated and flushed with N2. 1,4-Dioxane (100 mL) was added and the reaction mixture was degassed and heated to 105 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (250 mL), and filtered through a Celite® bed. The crude reaction mixture was purified by flash chromatography (EtOAc:Hex) to give the desired product as a brown oil. 1 H NMR(500 MHz, CDCl3)δ 6.56(s, 1H), 3.47(d, 2H), 2.29-2.04(m, 2H), 1.88-1.66(m, 3H), 1.54(s, 1H), 1.26(s, 12H), 1.23-1.16(m, 1H), 0.89(s, 9H)0.04(s, 6H).

[0240] Step 4: 4-Amino-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohex-1-en-1-yl)pyrimidin-2(1H)-one. A suspension of tert-butyldimethyl((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)methoxy)silane (1.00 g, 2.84 mmol) and cytosine (315 mg, 2.84 mmol) in MeOH:H2 (4:1, 125 mL) was stirred at room temperature for 30 min. Cu(OAc)2·H2O (567 mg, 2.8 mmol) and TMEDA (0.51 mL, 3.4 mmol) were added and the reaction was stirred at room temperature for 48 h. The reaction mixture was concentrated under reduced pressure and H2O (150 mL) was added. The solid was filtered and washed with Et2O (25 mL) and cold H2O (25 mL) to give the desired product. LCMS [M+H] 336.3.

[0241] Step 5: tert-Butyl (1-(4-((1-(4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a solution of 4-amino-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohex-1-en-1-yl)pyrimidin-2(1H)-one (200 mg, 0.6 mmol) in MeCN (50 mL) was added 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (363 mg, 0.7 mmol). The reaction mixture was heated to 85 °C for 16 h. The volatiles were removed under reduced pressure and the crude solid was dissolved in CHCl (100 mL) and the organic layer was washed with H0 (1 x 100 mL) and concentrated under reduced pressure. The crude solid was purified by column chromatography (CHCl:MeOH) to give the desired title compound. LCMS [M+H] 633.4.

[0242] Step 6: tert-Butyl (1-(4-((1-(4-(hydroxymethyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of tert-butyl (1-(4-((1-(4-(((tert-butyldimethylsilyl)oxy)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (290 mg, 0.46 mmol) in MeOH (10 mL) was added TsOH (174 mg, 0.92 mmol) at room temperature. The reaction was maintained at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and the crude solid was dissolved in CH2Cl2 (25 mL). The organic layer was washed with saturated aqueous NaHCO3 (1 x 25 mL), dried over Na2SO4 and purified by column chromatography (CH2Cl2:MeOH) to give the desired product. LCMS [M+H] 519.3.

[0243] Step 7: tert-Butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of tert-butyl (1-(4-((1-(4-(hydroxymethyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (50 mg, 0.096 mmol) in DCM (25 mL) was added Dess-Martin periodinane (82 mg, 0.2 mmol). The solution was stirred at room temperature for 2 h. The crude reaction mixture was dissolved in CH2Cl2 (20 mL) and washed with aqueous NaHCO3 / Na2S2O3 (1 x 20 mL). The aqueous layer was extracted with CH2Cl2 (1 x 10 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the desired product. LCMS [M+H] 517.3.

[0244] Step 8: tert-Butyl (1-(4-((1-(4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (25 mg, 0.05 mmol) in 1,2-dichloroethane (5 mL) was added tert-butyl (trans-4-aminocyclohexyl)carbamate (10 mg, 0.05 mmol) and Na(OAc)3BH (15 mg, 0.07 mmol). The reaction was stirred at room temperature for 16 h. The reaction mixture was diluted with CHCl2 and washed with saturated NaHCO3 (1×15 mL). The aqueous layer was extracted with CHCl2 (2×20 mL). The combined organics were dried over Na2SO4 and concentrated under reduced pressure to give the desired product.

[0245] Step 9: 4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride. (1-(4-((1-(4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)tert-butyl carbamate was dissolved in HCl / MeOH (5 mL) and stirred for 4 h. The reaction mixture was concentrated under reduced pressure and the crude solid was purified by reverse phase HPLC (HO:CHCN:TFA). The desired fractions were concentrated under reduced pressure, HCl / MeOH was added (3×15 mL) and evaporated under reduced pressure to give the desired product. 1 H NMR(400 MHz, D2O)δ 7.94(d, 1H), 6.75(d, 1H), 5.99(s, 1H), 3.78(br. s, 3H), 3.72(br. s, 5H), 3.32-3.20(m, 2H), 3.13(d, 2H), 2.51-2.36(m, 3H), 2.36-1.97(m, 7H), 1.74(s, 6H), 1.67-1.48(m, 5H). LCMS[(M+2H) / 2]258.2.

[0246] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((exo-6-(aminomethyl)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (compound 2) The compound was prepared in a similar manner to Scheme C-1 from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl ((exo-3-azabicyclo[3.1.0]hexan-6-yl)methyl)carbamate. 1 H NMR(400 MHz, D2O)δ 7.84(d, 1H), 6.77(d, 1H), 5.95(s, 1H), 3.88(d, 2H), 3.78(br. s, 3H),3.72(br. s, 5H), 3.53(d, 2H), 3.27(d, 2H), 3.01-2.93(m, 2H), 2.53-2.28(m, 3H), 2.26-1.93(m, 5H), 1.74(s, 6H), 1.69-1.40(m, 2H). LCMS[M+H]513.3.

[0247] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((((1S,3S)-3-aminocyclopentyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (compound 3) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate in a similar manner to Scheme C-1. 1H NMR(500 MHz, D2O)δ 7.91(d, 1H), 6.79(d, 1H), 6.00(s, 1H), 3.99-3.87(m, 2H), 3.80(s, 3H), 3.74(s, 5H), 3.20-3.09(m, 2H), 2.52-2.28(m, 7H), 2.22-2.01(m, 3H), 1.91-1.79(m, 2H), 1.77(s, 6H), 1.68-1.59(m, 1H). LCMS[M+H]501.3.

[0248] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-aminoazepan-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 4) Similarly to Scheme C-1, (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)tert-butyl carbamate and tert-butyl azepan-4-ylcarbamate. 1 H NMR(500 MHz, D2O)δ 7.96(d, 1H), 6.77(d, 1H), 6.03(d, 1H), 3.81(s, 3H), 3.75(s, 5H), 3.66-3.53(m, 3H), 3.47-3.38(m, 1H), 3.34-3.16(m, 3H), 2.55-2.22(m, 7H), 2.22-1.99(m, 4H), 1.97-1.86(m, 1H), 1.77(s, 6H), 1.69-1.59(m, 1H). LCMS[(M+2H) / 2]258.3.

[0249] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((exo-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (compound 5) Scheme C-2 [ka] Reagents: 1) 1M HCl in EtOH, 75°C, 16 hours 2) 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide, Et3N, CH3CN, heat, 17 hours 3) DIBAL (25% by weight in toluene), THF, 0°C, 4 hours 4) DMP, DCM, room temperature, 2 hours 5) tert-Butyl (exo-3-azabicyclo[3.1.0]hexan-6-yl)carbamate, NaBH(OAc)3, DIPEA, DCE, CH3CN, room temperature, 20 hours 6) 2M HCl in MeOH, room temperature, 18 hours. Step 1: cis-ethyl 4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate. A mixture of cis-ethyl 4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (984 mg, 2.66 mmol) and 1M HCl in EtOH (25 mL, 25 mmol) was stirred at 7° C. for 16 h. The mixture was concentrated to dryness to give the title compound (1.03 g). LCMS [M+H] 266.2.

[0250] Step 2: cis-Ethyl 4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate. A mixture of ethyl cis-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (984 mg, 2.66 mmol), EtN (0.52 mL, 3.7 mmol), and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (1.88 g, 3.7 mmol) in acetonitrile (25 mL) was stirred at reflux for 17 h. The mixture was cooled, Et3N (0.52 mL) was added, and the mixture was concentrated in vacuo. The residue was purified by flash chromatography (MeOH / EtOAc / Hexanes) to give the title compound (1.25 g) as a pale yellow oil. 1 H NMR(500 MHz, CDCl3)δ 13.08-12.57(m, 1H), 7.24-7.09(m, 1H), 5.90-5.66(m, 1H), 4.95-4.69(m, 1H), 4.58-4.40(m, 1H), 4.17(q, 2H), 3.91-3.64(m, 6H), 3.63-3.49(m, 2H), 2.73-2.65(m, 1H), 2.34-2.21(m, 2H), 1.84-1.75(m, 2H), 1.74-1.60(m, 4H), 1.49(s, 6H), 1.41(s, 9H), 1.30-1.26(m, 3H). LCMS[M+H]563.4.

[0251] Step 3: tert-Butyl (1-(4-((1-(cis-4-(hydroxymethyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a solution of ethyl cis-4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (513 mg, 0.912 mmol) in THF (15 mL) was added DIBAL (25 wt% in toluene, 3.1 mL, 4.6 mmol) at 0 °C under N2 and the mixture was stirred at °C for 4 h. The mixture was quenched with aqueous potassium sodium tartrate (15 mL), stirred vigorously for 30 min, poured into additional potassium sodium tartrate (35 mL) and extracted with DCM (3×50 mL). The extracts were washed with brine (35 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound (419 mg, 0.81 mmol). 1 H NMR(500 MHz, CDCl3)δ 12.90-12.73(m, 1H), 7.17(d, 1H), 5.71(d, 1H), 4.92-4.81(m, 1H), 4.48-4.37(m, 1H), 3.89-3.53(m, 10H), 1.99-1.87(m, 3H), 1.78-1.55(m, 6H), 1.50(s, 6H), 1.42(s, 9H). LCMS[M+H]521.3.

[0252] Step 4: tert-Butyl (1-(4-((1-(cis-4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. A mixture of tert-butyl (1-(4-((1-(cis-4-(hydroxymethyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (134 mg, 0.26 mmol) and DMP (327 mg, 0.77 mmol) in DCM (6 mL) was stirred at room temperature for 2 h. The mixture was poured into 1:1 aqueous Na2S2O3 and saturated aqueous NaHCO3 (20 mL) and extracted with DCM (3 x 20 mL). The extracts were dried over Na2SO4, filtered and concentrated to give the crude title compound as an off-white foam.

[0253] Step 5: tert-Butyl (1-(4-((1-(4-((exo-6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a mixture of tert-butyl (1-(4-((1-(cis-4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (133 mg, 0.26 mmol), tert-butyl (exo-3-azabicyclo[3.1.0]hexan-6-yl)carbamate (64 mg, 0.32 mmol) and DIPEA (90 μL, 0.52 mmol) in DCE (4 mL) and CH3CN (2 mL) was added sodium triacetoxyborohydride (166 mg, 0.78 mmol) and the mixture was stirred at room temperature for 20 h. The mixture was poured into a saturated aqueous solution of NaHCO3 (20 mL) and extracted with DCM (3×15 mL). The extract was dried over Na2SO4, decanted and concentrated. The residue was purified twice by flash chromatography (MeOH / EtOAc / Hexanes followed by DCM / MeOH / NH4OH) to give the title compound (61 mg, 0.09 mmol). LCMS [M+H] 701.6.

[0254] Step 6: tert-Butyl 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((exo-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (1-(4-((1-(4-((exo-6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (61 mg, 0.09 mmol) and 2M A mixture of HCl in MeOH (3 mL) was stirred at room temperature for 18 h. The mixture was concentrated, EtO (5 mL) was added, the mixture was sonicated, and the precipitate was collected by vacuum filtration to give the title compound (44 mg, 0.07 mmol). 1 H NMR(500 MHz, D2O)δ 8.14(d, 1H), 6.75(d, 1H), 4.52-4.43(m, 1H), 4.30-4.19(m, 1H), 4.02-3.92(m, 1H), 3.88-3.69(m, 8H), 3.67-3.57(m, 1H), 3.24-3.16(m, 1H), 3.15-2.92(m, 3H), 2.56-2.48(m, 1H), 2.48-2.41(m, 1H), 2.10-1.95(m, 3H), 1.92-1.82(m, 2H), 1.80-1.68(m, 8H), 1.38‐1.26(m, 2H). LCMS[M+H]501.3.

[0255] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-((exo-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 7) Scheme C-3 [ka] Reagents: 1) Column chromatography 2) 2M HCl in MeOH, room temperature, 18 hours. Step 1: tert-Butyl (1-(4-((1-(trans-4-((exo-6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. The mixture of cis and trans isomers of tert-butyl (1-(4-((1-(4-((exo-6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3)-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate was purified by column chromatography (DCM / MeOH / NH4OH followed by DCM / MeOH) to give the title compound (94.0 mg). 1 H NMR(500 MHz, DMSO-d6)δ 7.95-7.77(m, 1H), 7.39(s, 1H), 6.94-6.86(m, 1H), 4.36-4.24(m, 1H), 3.80-3.40(m, 8H), 2.92(d, 2H), 2.62-2.56(m, 1H), 2.21(d, 2H), 2.16(d, 2H), 1.83(d, 2H), 1.77-1.69(m, 2H), 1.65-1.53(m, 2H), 1.43-1.33(m, 18H), 1.29(s, 9H), 1.00-0.88(m, 2H). LCMS [M+H] 701.7.

[0256] Step 2: 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-((exo-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride. Tert-butyl (1-(4-((1-(trans-4-((exo-6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (94 mg, 134 μmol) and 2M HCl in MeOH (3.3 mL, 6.7 mmol) were stirred at room temperature for 18 h. IPA (3 mL) was added and the precipitate was collected by vacuum filtration and washed with IPA and Et2O. The filtrate was concentrated and purified by reverse phase preparative HPLC (CH3CN / H2O w / 0.1% TFA). The product fractions were collected, concentrated, and 2M HCl in MeOH was added. The solvent was removed under reduced pressure and the solid was combined with the original precipitate to give the title compound (71 mg). 1 H NMR(500 MHz, D2O)δ 8.18(d, 1H), 6.74(d, 1H), 4.53-4.43(m, 1H), 4.29-4.20(m, 1H), 4.00-3.93(m, 1H), 3.87-3.70(m, 8H), 3.66-3.56(m, 1H), 3.20(d, 1H), 3.16-3.01(m, 2H), 2.99-2.92(m, 1H), 2.56-2.48(m, 1H), 2.48-2.41(m, 1H), 2.11-1.95(m, 4H), 1.95-1.81(m, 2H), 1.80-1.68(m, 7H), 1.38-1.25(m, 2H). LCMS[M+H]501.4.

[0257] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(cis-4-((exo-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 6) The compound was prepared from a mixture of cis and trans isomers of tert-butyl (1-(4-((1-(4-((exo-6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate in a similar manner to Scheme C-3. 1 H NMR(500 MHz, D2O)δ 8.14(d, 1H), 6.75(d, 1H), 4.47(t, 1H), 4.30-4.18(m, 1H), 3.96(d, 1H), 3.89-3.67(m, 8H), 3.66-3.57(m, 1H), 3.24-3.15(m, 1H), 3.14-2.99(m, 2H), 2.98-2.90(m, 1H), 2.57-2.49(m, 1H), 2.48-2.40(m, 1H), 2.10-1.95(m, 3H), 1.94-1.80(m, 2H), 1.80-1.68(m, 8H), 1.38-1.24(m, 2H). LCMS[M+H]501.4.

[0258] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((exo-6-(aminomethyl)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 8) The compound was prepared from tert-butyl (1-(4-((1-(cis-4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl ((exo-3-azabicyclo[3.1.0]hexan-6-yl)methyl)carbamate in a similar manner to Scheme C-2. 1 H NMR(500 MHz, D2O)δ 8.15(d, 1H), 6.75(d, 1H), 4.52-4.43(m, 1H), 4.20-4.11(m, 1H), 3.93-3.84(m, 1H), 3.83-3.68(m, 8H), 3.57-3.47(m, 2H), 3.45-3.40(m, 1H), 3.18(d, 1H), 3.04-2.93(m, 2H), 2.16-2.09(m, 1H), 2.09-1.95(m, 4H), 1.94-1.80(m, 3H), 1.80-1.67(m, 7H), 1.52-1.41(m, 1H), 1.37-1.25(m, 2H). LCMS[M+H]515.3.

[0259] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-aminoazepan-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 9) The compound was prepared from tert-butyl (1-(4-((1-(cis-4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl azepan-4-ylcarbamate in a similar manner to Scheme C-2. 1H NMR(500 MHz, D2O)δ 8.05(dd, 1H), 6.78(d, 1H), 4.52-4.42(m, 1H), 3.91-3.67(m, 8H), 3.65-3.49(m, 3H), 3.44-3.34(m, 1H), 3.28-3.11(m, 3H), 2.42-2.28(m, 2H), 2.28-2.20(m, 1H), 2.20-2.08(m, 2H), 2.08-1.94(m, 4H), 1.93-1.84(m, 2H), 1.82-1.68(m, 8H), 1.41-1.27(m, 2H). LCMS [M+H] 517.4.

[0260] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-aminoazetidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 10) The compound was prepared from tert-butyl (1-(4-((1-(cis-4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl azetidin-3-ylcarbamate in a similar manner to Scheme C-2. 1 H NMR(500 MHz, D2O)δ 8.14(d, 1H), 6.75(d, 1H), 4.63-4.36(m, 5H), 3.88-3.68(m, 8H), 3.37-3.31(m, 1H), 2.11-2.02(m, 2H), 1.99-1.92(m, 2H), 1.92-1.80(m, 2H), 1.79-1.68(m, 7H), 1.40-1.29(m, 2H). LCMS[M+H]475.3.

[0261] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 11) Scheme C-4 [ka] Reagents: 1) tert-Butyl (trans-4-aminocyclohexyl)carbamate, NaBH3CN, MeOH, rt, 20 h. 2) Boc2O, saturated NaHCO3, THF, rt, 19 h. 3) 2M HCl in MeOH, rt, 19 h. Step 1: tert-Butyl (1-(4-((1-(4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a mixture of tert-butyl (1-(4-((1-(cis-4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (61 mg, 0.12 mmol) and tert-butyl (trans-4-aminocyclohexyl)carbamate (32 mg, 0.15 mmol) in MeOH (2 mL) was added sodium cyanoborohydride (25 mg, 0.4 mmol) and the mixture was stirred at room temperature for 20 h. The reaction mixture was purified by flash chromatography (DCM / MeOH / NH4OH) to give the title compound (50 mg) as a mixture of cis / trans diastereomers. LCMS [M+H] 717.6.

[0262] Step 2: tert-Butyl ((4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. A mixture of (1-(4-((1-(4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)tert-butyl carbamate (50 mg, 0.07 mmol) and di-tert-butyl dicarbonate (30 mg, 0.14 mmol) in THF (0.75 mL) and saturated sodium bicarbonate (0.75 mL) was stirred at room temperature for 19 h. The mixture was diluted with EtOAc (10 mL), washed with saturated NaHCO3 (2 x 10 mL) and brine (1 x 10 mL), dried over Na2SO4, decanted, and concentrated. The residue was purified by flash chromatography (MeOH / EtOAc / hexanes) to give the title compound (31 mg) as a mixture of cis / trans diastereomers. LCMS [M+H] 817.7.

[0263] Step 3: A mixture of 4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride ((4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)tert-butylcarbamate (31 mg, 0.04 mmol) and 2M HCl in MeOH (1.5 mL) was stirred at room temperature for 19 h. The mixture was concentrated in vacuo and dried with EtOH and Et2O to give the title compound (24 mg). 1H NMR(500 MHz, D2O)δ 8.26-8.15(m, 1H), 6.79-6.70(m, 1H), 4.54-4.44(m, 1H), 3.93-3.66(m, 8H), 3.33-3.19(m, 3H), 3.09-3.01(m, 1H), 2.39-2.27(m, 2H), 2.26-2.18(m, 2H), 2.11-1.98(m, 2H), 1.94-1.81(m, 4H), 1.80-1.69(m, 7H), 1.66-1.51(m, 4H), 1.39-1.22(m, 2H). LCMS [M+H] 517.4.

[0264] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((1R,5S,6s)-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (compound 12) Scheme C-5 [ka] Reagents: Step 1) 2,6-Di-tert-butyl-4-methylpyridine, Tf2O, DCM, rt, 8 h 2) PdCl2(dppf)·DCM, KOAc, B2pin2, dioxane, 100 °C, 2 h 3) Cytosine, Cu(OAc)2·H2O, TMEDA, MeOH, water, rt, 72 h 4) ACN, 80 °C, 16 h 5) LiBH4, THF, rt, 18 h 6) DMP, DCM, rt, 2 h 7) exo-tert-butyl (3-azabicyclo[3.1.0]hexan-6-yl)carbamate, NaBH(OAc)3, DCM, rt, 2 h 8) 4M HCl in dioxane, DCM, rt, 2 h. Step 1: Ethyl 4-(((trifluoromethyl)sulfonyl)oxy)cyclohex-3-ene-1-carboxylate. To a stirred solution of ethyl 4-oxocyclohexane-1-carboxylate (1.0 g, 5.8 mmol) and 2,6-di-tert-butyl-4-methylpyridine (1.5 g, 7.6 mmol) in DCM (25 mL) was added Tf2O (1.14 mL, 7.0 mmol) dropwise at 0 °C. The resulting reaction mixture was stirred at room temperature for 8 h. The reaction mixture was poured into NaHCO3 solution (100 mL) and extracted with DCM (2 x 100 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by column chromatography (5% EtOAc in hexane) to give the title compound (1.2 g) as a colorless liquid. 1 H NMR(400 MHz, CDCl3): δ5.78(s, 1H), 4.20-4.14(m, 2H), 2.63-2.57(m, 1H), 2.47-2.45(m, 2H), 2.43-2.42(m, 2H), 2.18-2.11(m, 1H), 1.98-1.90(m, 1H), 1.29(t, 3H).

[0265] Step 2: Ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-ene-1-carboxylate. To a stirred solution of ethyl 4-(((trifluoromethyl)sulfonyl)oxy)cyclohex-3-ene-1-carboxylate (0.75 g, 2.4 mmol) in dioxane (10 mL) was added KOAc (0.73 g, 7.4 mmol) and B2Pin2 (0.69 g, 2.7 mmol), then the reaction mixture was purged with N2 for 30 min. PdCl2(dppf)·DCM (0.1 g, 0.12 mmol) was added at room temperature. The reaction mixture was heated at 100 °C for 2 h. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by column chromatography (EtOAc / Hexanes) to afford the title compound (1.5 g) as a pale yellow liquid.

[0266] Step 3: Ethyl 4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohex-3-ene-1-carboxylate. To a stirred solution of ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-ene-1-carboxylate (1.5 g, 5.3 mmol) in methanol:HO (40 mL:10 mL) was added cytosine (0.59 g, 5.3 mmol) at room temperature in air. After 30 min, Cu(OAc)2·HO (1.06 g, 5.3 mmol) and TMEDA (1.0 mL, 6.4 mmol) were added. The resulting reaction mixture was stirred at room temperature for 72 h. The reaction mixture was concentrated under reduced pressure and diluted with HO (50 mL). The precipitated solid was filtered and washed with H2O (3 x 50 mL) and hexanes (20 mL) to give the title compound (0.26 g). 1 H NMR(400 MHz, DMSO-d6): δ 7.34(d, 1H), 7.13(s, 1H), 7.04(s, 1H), 5.62(t, 2H), 4.11-4.06(m, 2H), 2.57(m, 1H), 2.32-2.30(m, 4H), 2.00-1.96(m, 1H), 1.71-1.65(m, 1H), 1.21(t, 3H). LCMS[M+H]264.1.

[0267] Step 4: Ethyl 4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohex-3-ene-1-carboxylate. To a stirred solution of ethyl 4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohex-3-ene-1-carboxylate (0.25 g, 0.95 mmol) and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (0.73 g, 1.42 mmol) in ACN (15 mL). The reaction mixture was heated to 80° C. for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give the crude material, which was purified by column chromatography (7% MeOH:MDC) to give the title compound (0.36 g, 67%) in pale yellow color. LCMS [M+H] 561.31.

[0268] Step 5: tert-Butyl (1-(4-((1-(4-(hydroxymethyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of ethyl 4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohex-3-ene-1-carboxylate (0.35 g, 0.62 mmol) in THF (10 mL) was added LiBH4 (0.07 g, 3.12 mmol) at room temperature. The resulting reaction mixture was stirred at room temperature for 18 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into saturated NH4Cl solution (50 mL) and extracted with MDC:IPA (7:3) (2 x 50 mL), the combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to give the crude material, which was purified by column chromatography (15% MeOH:MDC) to give the title compound (0.23 g, 70%). LCMS [M+H] 519.37.

[0269] Step 6: tert-Butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of tert-butyl (1-(4-((1-(4-(hydroxymethyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (0.23 g, 0.44 mmol) in DCM (10 mL) was added DMP (0.38 g, 0.88 mmol) at room temperature. The resulting reaction mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with DCM (100 mL) and washed with saturated NaHCO3 solution (2 x 50 mL). The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude material, which was purified by trituration (diethyl ether) to give the title compound (0.2 g, 80%). LCMS [M+H] 517.22.

[0270] Step 7: exo-tert-butyl (1-(4-((1-(4-((6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)tert-butyl carbamate (0.2 g, 0.38 mmol) and (3-azabicyclo[3.1.0]hexan-6-yl)carbamate (0.08 g, 0.38 mmol) in DCE (5 mL) was added NaBH(OAc)3 (0.12 g, 0.58 mmol) after 15 min. The reaction mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the resulting reaction mixture was poured into saturated NaHCO3 solution (50 mL) and extracted with DCM (2 x 50 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude material which was purified by column chromatography (8% MeOH:MDC) to give the title compound (0.12 g, 25%). LCMS [M+H] 699.4.

[0271] Step 8: exo-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide. To a stirred solution of exo-tert-butyl (1-(4-((1-(4-((6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (0.12 g, 0.11 mmol) in DCM (20 mL) was added 4N HCl in dioxane (0.5 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (0.01% HCl in H2O:MeOH) to give the title compound (0.06 g, 50%). 1 H NMR(400 MHz, D2O): δ 7.75(d, 1H), 6.61(d, 1H), 5.82(s, 1H), 4.10(bs, 1H), 3.84-3.81(m, 2H), 3.36-3.57(m, 8H), 3.49-3.41(m, 1H), 3.15-3.05(m, 2H), 2.93-2.81(m, 2H), 2.29-2.18(m, 4H), 2.10-2.03(m, 1H), 1.94-1.85(m, 2H), 1.58(s, 6H), 1.43-1.35(m, 1H). LCMS[M+H]499.4.

[0272] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((((1S,3S)-3-aminocyclopentyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 13) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate in a similar manner to Scheme C-5. 1 H NMR(500 MHz, D2O)δ 8.21-8.14(m, 1H), 6.78-6.72(m, 1H), 4.54-4.44(m, 1H), 3.97-3.85(m, 2H), 3.85-3.69(m, 8H), 3.30-3.21(m, 1H), 3.08-2.99(m, 1H), 2.49-2.35(m, 2H), 2.35-2.26(m, 2H), 2.26-2.19(m, 1H), 2.10-1.98(m, 2H), 1.93-1.78(m, 6H), 1.76(s, 6H), 1.40-1.28(m, 2H). LCMS [M+H] 503.3.

[0273] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((3-(aminomethyl)azetidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 14) Prepared as a mixture of diastereomers from tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (azetidin-3-ylmethyl)carbamate in a similar manner to Scheme C-2. 1H NMR(500 MHz, D2O)δ 8.14-8.07(m, 1H), 6.80-6.73(m, 1H), 4.55-4.40(m, 2H), 4.37-4.25(m, 2H), 4.13-4.03(m, 1H), 3.88-3.66(m, 8H), 3.56-3.42(m, 2H), 3.40-3.32(m, 2H), 3.31-3.19(m, 1H), 2.19-2.00(m, 2H), 1.98-1.79(m, 4H), 1.79-1.67(m, 7H), 1.40-1.26(m, 2H). LCMS [M+H] 489.2.

[0274] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-amino-4-methylpiperidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 15) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (4-methylpiperidin-4-yl)carbamate in a similar manner to Scheme C-2. 1 H NMR(500 MHz, D2O)δ 8.19-8.11(m, 1H), 6.79-6.73(m, 1H), 4.54-4.44(m, 1H), 3.91-3.62(m, 9H), 3.45-3.36(m, 2H), 3.36-3.22(m, 2H), 3.22-3.12(m, 1H), 2.43-2.35(m, 1H), 2.33-2.12(m, 4H), 2.11-1.97(m, 2H), 1.96-1.85(m, 3H), 1.84-1.70(m, 7H), 1.62-1.52(m, 3H), 1.42-1.29(m, 2H). LCMS[M+H]517.3.

[0275] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((2-amino-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 16) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (7-azaspiro[3.5]nonan-2-yl)carbamate in a similar manner to Scheme C-2. 1 H NMR(500 MHz, D2O)δ 8.21-8.13(m, 1H), 6.78-6.72(m, 1H), 4.54-4.43(m, 1H), 3.92(p, 1H), 3.86-3.68(m, 8H), 3.64-3.47(m, 2H), 3.30(d, 1H), 3.12-2.94(m, 3H), 2.58-2.48(m, 1H), 2.41-2.31(m, 2H), 2.18-2.03(m, 4H), 2.03-1.84(m, 7H), 1.83-1.69(m, 8H), 1.40-1.27(m, 1H). LCMS [M+H] 543.5.

[0276] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(trans-4-(((cis-4-aminocyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 17) Prepared in a similar manner to Scheme C-3 from tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (cis-4-aminocyclohexyl)carbamate. 1 H NMR(400 MHz, D2O)δ 8.04(d, 1H), 6.60(d, 1H), 4.42-4.30(m, 1H), 3.80-3.53(m, 8H), 3.51-3.40(m, 1H), 3.29-3.16(m, 1H), 2.93(d, 2H), 2.03-1.78(m, 10H), 1.78-1.53(m, 11H), 1.29-1.11(m, 2H). LCMS[M+H]517.9.

[0277] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(trans-4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 18) Scheme C-6 [ka] Reagents: 1) DIBAL, THF, 0°C, 2 hours 2) MsCl, Et3N, DCM, room temperature, 2.5 hours 3) tert-butyl (trans-4-aminocyclohexyl)carbamate, K2CO3, NaI, CH3CN, 92°C, 42 hours 4) Boc2O, dioxane, saturated aqueous NaHCO3, room temperature, 16 hours 5) NH4OH, MeOH, 50°C, 2.5 hours 6) 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide, CH3CN, □, 22 hours 7) 2M HCl in MeOH, room temperature, 17 hours. Step 1: N-(1-(trans-4-(hydroxymethyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide. To a solution of ethyl trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (1.13 g, 3.06 mmol) in THF (30 mL) was added DIBAL (25 wt% in toluene, 8.3 mL) dropwise at 0° C. and the mixture was stirred at 0° C. for 2 h. 1M HCl (50 mL) was added to the mixture and extracted with EtOAc (3×50 mL). The extract was washed with brine (75 mL). The biphasic mixture was filtered. The filtrate was separated and extracted with EtOAc (75 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated. The precipitate and the EtOAc extract were combined to give the title compound (1.0 g). LCMS [M+H] 328.1.

[0278] Step 2: (trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl methanesulfonate. To a mixture of N-(1-(trans-4-(hydroxymethyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (1.0 g, 3.0 mmol) and Et3N (0.85 mL, 6.1 mmol) in DCM (60 mL) was added MsCl (0.36 mL, 4.6 mmol) dropwise and the mixture was stirred at room temperature for 2.5 h. The mixture was purified by column chromatography (Hex / EtOAc / MeOH followed by MeOH / DCM) to give the title compound (1.57 g). LCMS [M+H] 406.2.

[0279] Step 3: tert-Butyl (trans-4-(((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)amino)cyclohexyl)carbamate. A mixture of (trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl methanesulfonate (943 mg, 1.86 mmol), tert-butyl (trans-4-aminocyclohexyl)carbamate (797 mg, 3.72 mmol), KCO (514 mg, 3.72 mmol) and NaI (55.8 mg, 372 μmol) in CHCN (25 mL) was stirred at 92 °C for 42 h. The mixture was cooled, diluted with MeOH (50 mL), concentrated and purified by column chromatography (DCM / MeOH / NH4OH) to give the title compound (409 mg). LCMS [M+H] 524.3.

[0280] Step 4: tert-Butyl ((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. A mixture of tert-butyl (trans-4-(((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)amino)cyclohexyl)carbamate (306 mg, 584 μmol) and di-tert-butyl dicarbonate (0.32 mL, 1.4 mmol) in dioxane (12 mL) and saturated aqueous NaHCO3 (6.0 mL) was stirred at room temperature for 16 h. It was diluted with EtOAc (65 mL), washed with saturated aqueous NaHCO3 (1 x 50 mL) and brine (1 x 50 mL), dried (Na2SO4), decanted and concentrated. The residue was purified by column chromatography (Hex / EtOAc / MeOH) to give the title compound (129 mg). 1H NMR(500 MHz, CDCl3)δ 7.89(d, 2H), 7.69(d, 1H), 7.61(t, 1H), 7.55-7.42(m, 3H), 4.73-4.61(m, 1H), 4.42-4.30(m, 2H), 3.45-3.31(m, 2H), 3.02(d, 2H), 2.12-1.96(m, 6H), 1.89(d, 2H), 1.82-1.73(m, 1H), 1.70-1.55(m, 2H), 1.50-1.38(m, 18H), 1.28-1.12(m, 6H). LCMS[M+H]624.4.

[0281] Step 5: tert-Butyl ((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. A mixture of tert-butyl ((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate (129 mg, 207 μmol) and 29 wt % NH4OH (0.71 mL) in MeOH (3 mL) was stirred at 50° C. for 2.5 h. The mixture was concentrated in vacuo and then concentrated with MeOH and then with CH3CN to give the title compound. LCMS [M+H] 520.3.

[0282] Step 6: tert-Butyl ((trans-4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. A mixture of tert-butyl ((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate (207 μmol) and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (191 mg, 376 μmol) in CH3CN (5.0 mL) was stirred at reflux for 22 h. The mixture was cooled, Et3N (0.1 mL) was added and the mixture was concentrated. The residue was purified by column chromatography (Hex / EtOAc / MeOH) to give the title compound (116 mg). LCMS [M+H] 817.6.

[0283] Step 7: A mixture of 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride ((trans-4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)tert-butylcarbamate (116 mg, 142 μmol) and 2M HCl in MeOH (2.8 mL, 5.5 mmol) was stirred at room temperature for 17 h. IPA (3 mL) was added to the mixture and the precipitate was collected by vacuum filtration, rinsing with additional IPA and Et2O to give the title compound (88.3 mg). 1H NMR(500 MHz, D2O)δ 8.14(d, 1H), 6.75(d, 1H), 4.53-4.44(m, 1H), 3.93-3.65(m, 8H), 3.32-3.18(m, 2H), 3.05(d, 2H), 2.35-2.17(m, 4H), 2.10-1.96(m, 4H), 1.91-1.80(m, 1H), 1.79-1.67(m, 8H), 1.64-1.50(m, 4H), 1.39-1.26(m, 2H). LCMS[M+H]517.3.

[0284] Alternatively, compound 18 may be prepared according to Scheme C-7. Scheme C-7 [ka]

[0285] Reagents: Step 1) 3-Ethoxyacryloyl chloride, silver cyanate, toluene, THF, rt, 16 hrs. 2) 2M HCl, MeOH, 80°C, 12 hrs. 3) 1,2,4-Triazole, POCl3, TEA, ACN, 20°C, 6 hrs. 28% NH4OH solution, 50°C, 16 hrs. 4) ACN, 80°C, 16 hrs. 5) LiBH4, THF, rt, 16 hrs. 6) DMP, DCM, rt, 2 hrs. 7) tert-Butyl (4-aminocyclohexyl)carbamate, NaBH(OAc)3, DCE, rt, 2 hrs. 8) 4M HCl in dioxane, DCM, rt, 2 hrs.

[0286] Step 1: (E)-trans-methyl 4-(3-(3-ethoxyacryloyl)ureido)cyclohexane-1-carboxylate. To a stirred solution of silver cyanate (2.3 g, 15.48 mmol) in toluene (30 mL) was added 3-ethoxyacryloyl chloride (2.08 g, 15.46 mmol) dropwise at room temperature. The resulting reaction mixture was stirred at 80° C. for 30 min, cooled to room temperature, and trans-methyl-4-aminocyclohexane-1-carboxylic acid HCl salt (1.0 g, 5.16 mmol) in THF (40 mL) was added at −30° C. The reaction mixture was stirred for 16 h. The reaction mixture was filtered through Celite® and washed with ethyl acetate (100 mL). The organic layer was concentrated under reduced pressure and purified by column chromatography (40% EtOAc in hexane) to give the title compound (3.7 g). LCMS [M+H] 298.7.

[0287] Step 2: trans-methyl 4-(4-hydroxy-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate. To a stirred solution of trans-methyl (E)-4-(3-(3-ethoxyacryloyl)ureido)cyclohexane-1-carboxylate (0.5 g, 1.84 mmol) in MeOH (10 mL) was added 2M HCl solution (4.5 mL, 9.0 mmol). The resulting reaction mixture was heated at 80° C. for 12 h. The reaction mixture was poured into NaHCO3 solution (200 mL) and extracted with DCM (2×200 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to give the title compound (1.3 g, 30% for two steps). LCMS [M+H] 252.8.

[0288] Step 3: trans-Methyl 4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate. To a mixture of 1,2,4-triazole (4.8 g, 69 mmol) and TEA (11.2 mL, 77.7 mmol) in ACN (5 mL) was added POCl3 (0.9 mL, 9.7 mmol) dropwise at 0° C. The reaction mixture was stirred at 0° C. for 20 min. trans-Methyl 4-(4-hydroxy-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (0.7 g, 2.8 mmol) in ACN (10 mL) was added, then the reaction mixture was warmed to 20° C. and stirred for 6 h. Ammonium hydroxide solution (19 mL) was added to the reaction mixture and stirred at 50° C. for 16 h. The reaction mixture was concentrated under reduced pressure and purified by reverse phase chromatography (40% MeOH in H2O) to give the title compound (0.35 g, 65%). 1 H NMR(400 MHz, D2O): δ 7.60(d, 1H), 7.00-6.91(m, 2H), 5.66(d, 1H), 4.36-4.30(m, 1H), 3.58(s, 3H), 2.35-2.29(m, 1H), 2.02-1.99(m, 2H), 1.74-1.70(m, 2H), 1.63-1.56(m, 2H), 1.54-1.47(m, 2H). LCMS[M+1]251.9.

[0289] Step 4: trans-methyl 4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate. To a stirred solution of trans-methyl 4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (0.45 g, 1.8 mmol) and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (1.36 g, 2.68 mmol) in ACN (20 mL). The reaction mixture was heated at 80° C. for 16 h. The reaction mixture was concentrated under reduced pressure and purified by flash column chromatography (9% MeOH:DCM) to give the title compound (0.9 g, 90%). LCMS [M+H] 549.1.

[0290] Step 5: trans-tert-butyl (1-(4-((1-(4-(hydroxymethyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of trans-methyl 4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohexane-1-carboxylate (0.9 g, 1.64 mmol) in THF (30 mL) was added LiBH4 (0.36 g, 16.4 mmol) at room temperature. The reaction mixture was poured into ice water (100 mL) and extracted with DCM (2 x 100 mL), and the combined organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by reverse phase chromatography (25% MeOH in H2O) to give the title compound (0.55 g, 75%). LCMS [M+H] 521.3.

[0291] Step 6: trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. To a stirred solution of trans-tert-butyl (1-(4-((1-(4-(hydroxymethyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (0.55 g, 1.05 mmol) in DCM (20 mL) was added DMP (0.89 g, 2.1 mmol) at room temperature. The resulting reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with DCM (100 mL) and washed with saturated NaHCO3 solution (3 x 100 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by trituration with diethyl ether (10 mL) to give the title compound (0.5 g, 80%).

[0292] Step 7: trans-trans-tert-butyl (1-(4-((1-(4-((4-((tert-butoxycarbonyl)amino)cyclohexylamino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopr opan-2-yl)carbamate. To a stirred solution of trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (0.5 g, 0.96 mmol) and trans-tert-butyl (4-aminocyclohexyl)carbamate (0.3 g, 1.4 mmol) in DCE (15 mL) was added NaBH(OAc)3 (1.0 g, 4.8 mmol) at room temperature. The reaction mixture was poured into saturated NaHCO3 (50 mL) and extracted with DCM (2 x 100 mL). The organic layer was washed with NH4Cl solution (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by reverse phase chromatography (80% MeOH in H2O) to give the title compound (0.13 g, 17%). LCMS [M+H] 717.5.

[0293] Step 8: trans-trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-aminocyclohexylamino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride. To a stirred solution of trans-trans-tert-butyl (1-(4-((1-(4-((4-((tert-butoxycarbonyl)amino)cyclohexylamino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (0.12 g, 0.16 mmol) in DCM (20 mL) was added 4N HCl in dioxane (1.0 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC purification (0.01% HCl in H2O / MeOH) to give the title compound (0.035 g, 35%). 1H NMR (400 MHz, D2O): δ 7.98 (d, 1H), 6.62 (d, 1H), 4.34 (t, 1H), 3.65-3.59 (m, 8H), 3.23-3.02 (m, 2H), 2.90 (d, 2H), 2.61 (s, 1H), 2.14-2.09 (m, 4H), 1.89 (t, 4H), 1.78-1.69 (m, 1H), 1.61 (s, 7H), 1.57-1.43 (m, 4H), 1.23-1.17 (m, 2H). LCMS[M+1]517.8.

[0294] Alternatively, compound 18 may be prepared according to Scheme C-8. Scheme C-8 [ka] Reagents: 1) i) 3-ethoxyacryloyl chloride, AgOCN, toluene, □, 4 h, ii) DCM, 0°C to rt, 18 h. 2) i) TFA, rt, 3 h, ii) Boc2O, saturated aqueous NaHCO3, dioxane, 4 h. 3) POCl3, 1,2,4-triazole, Et3N, CH3CN, rt, 3 h, then NH4OH, 50°C, 18 h. 4) CH3CN, heat, 17 h. 5) 2M HCl / MeOH, rt, 18 h.

[0295] Step 1: tert-Butyl (trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans-4-(3-(3-ethoxyacryloyl)ureido)cyclohexyl)methyl)carbamate. A solution of 3-ethoxyacryloyl chloride (3.15 g, 23 mmol) in toluene (67 mL) was added dropwise to a suspension of silver cyanate (3.87 g, 26 mmol) in toluene (50 mL) and the mixture was stirred at reflux for 4 h. The reaction mixture was cooled to room temperature and allowed to settle. A portion of the formed solution (82 mL, 0.16.4 mmol) was transferred via cannula to an addition funnel and added dropwise to a cooled (-78 °C) solution of tert-butyl ((trans-4-aminocyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate (5 g, 12 mmol) in dry DCM (100 mL) under N2. The mixture was allowed to warm to room temperature and stirred for 18 h. MeOH (50 mL) was added and the mixture was concentrated to dryness. The residue was purified by column chromatography (EtOAc / Hexanes) to give the title compound (5.90 g) as a pale yellow foam. 1H NMR(500 MHz, MeOD)δ 7.64(d, 1H), 5.41(d, 1H), 3.97(q, 2H), 3.70-3.51(m, 2H), 3.30-3.21(m, 1H), 3.04(d, 2H), 2.08-1.99(m, 2H), 1.99-1.91(m, 2H), 1.85-1.65(m, 6H), 1.63-1.51(m, 1H), 1.46(s, 9H), 1.43(s, 9H), 1.33(t, 3H), 1.30-1.17(m, 4H), 1.14-1.02(m, 2H). LCMS [M-2(t-Bu)+H] 455.2.

[0296] Step 2: tert-Butyl (trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclohexyl)methyl)carbamate. A mixture of tert-butyl (trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans-4-(3-(3-ethoxyacryloyl)ureido)cyclohexyl)methyl)carbamate (5.9 g, 10.4 mmol) and TFA (40 mL, 520 mmol) was stirred at room temperature for 3 h and then concentrated to dryness. Dioxane (100 mL) and saturated NaHCO3 (100 mL) were added. 2M NaOH (20 mL) was added until pH 8. Di-tert-butyl dicarbonate (4.8 mL, 21 mmol) was added and the mixture was stirred vigorously for 3 days. Additional di-tert-butyl dicarbonate (2.4 mL, 11 mmol) was added and the reaction mixture was stirred for 1 day. The mixture was diluted with EtOAc (500 mL), washed with saturated aqueous NaHCO3 (2 x 500 mL) and brine (1 x 500 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (EtOAc / Hex) to give the title compound (5.30 g) as a white foam. 1H NMR(500 MHz, MeOD)δ 7.69(d, 1H), 5.66(d, 1H), 4.36(tt, 1H), 3.34-3.22(m, 2H), 3.07(d, 2H), 2.00-1.93(m, 2H), 1.93-1.84(m, 4H), 1.84-1.69(m, 4H), 1.69-1.58(m, 3H), 1.47(s, 9H), 1.43(s, 9H), 1.34-1.22(m, 2H), 1.22-1.10(m, 2H). LCMS[M-2(t-Bu)+H]409.1.

[0297] Step 3: tert-Butyl ((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. POCl3 (2.85 mL, 30.5 mmol) was added dropwise to a mixture of 1,2,4-triazole (17 g, 244 mmol) and Et3N (34.1 mL, 244 mmol) in CH3CN (50 mL) at 0 °C, and the mixture was stirred at 0 °C for 15 min. A solution of tert-butyl (trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)((trans-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclohexyl)methyl)carbamate (5.3 g, 10 mmol) in CH3CN (50 mL) was added dropwise and the mixture was allowed to warm to room temperature and stirred for 3 h. 28 wt % NH4OH (72 mL, 509 mmol) was added and the mixture was stirred at 50 °C for 18 h. Water and ice (500 mL) were added and the mixture was stirred for 30 min. The precipitate was collected by vacuum filtration, added CH3CN (200 mL), concentrated under reduced pressure, and dried under vacuum to give the title compound (4.66 g). 1H NMR(500 MHz, MeOD)δ 7.66(d, 1H), 5.87(d, 1H), 4.45(tt, 1H), 3.73-3.59(m, 1H), 3.31-3.23(m, 1H), 3.08(d, 2H), 2.00-1.92(m, 2H), 1.92-1.83(m, 4H), 1.83-1.68(m, 4H), 1.68-1.53(m, 3H), 1.47(s, 9H), 1.43(s, 9H), 1.34-1.11(m, 4H). LCMS[M+H]520.2.

[0298] Step 4: tert-Butyl ((trans-4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate. A mixture of tert-butyl ((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate (4.66 g, 8.97 mmol) and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (8.24 g, 16 mmol) in CH3CN (90 mL) was stirred at reflux for 17 h. The mixture was cooled, diluted with EtOAc (500 mL), washed with saturated NaHCO3 (2 x 500 mL) and brine (500 mL), dried over Na2SO4, decanted, and concentrated. The residue was purified by column chromatography (Hex / EtOAc / MeOH) to give the title compound (5.91 g). LCMS [M+H] 817.6.

[0299] Step 5: 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-(((trans-4-aminocyclohexyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride. A mixture of tert-butyl ((trans-4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamide)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)carbamate (5.9 g, 7.2 mmol) and 2M HCl in MeOH (150 mL, 300 mmol) was stirred at room temperature for 18 h. IPA (150 mL) was added, the mixture was stirred for 1 h, and the precipitate was collected by vacuum filtration and washed with IPA and Et 2 O. The solid was lyophilized from water to give the title compound (4.50 g). 1 H NMR(500 MHz, D2O)δ 8.20(d, 1H), 6.74(d, 1H), 4.50(tt, 1H), 3.94-3.66(m, 8H), 3.32-3.18(m, 2H), 3.05(d, 2H), 2.35-2.18(m, 4H), 2.11-1.97(m, 4H), 1.91-1.80(m, 1H), 1.80-1.68(m, 8H), 1.64-1.50(m, 4H), 1.40-1.27(m, 2H). LCMS[M+H]517.3.

[0300] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((3-aminocyclobutyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 43) The compound was prepared from trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and trans-tert-butyl (3-aminocyclobutyl)carbamate in a similar manner to Scheme C-7. LCMS [M+H] 489.6. [ka] cis-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((3-aminocyclobutyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 44)

[0301] The compound was prepared from trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and cis-tert-butyl (3-aminocyclobutyl)carbamate in a similar manner to Scheme C-7. LCMS [M+H] 489.6. [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((6-amino-3-azabicyclo[4.1.0]heptan-3-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (compound 45)

[0302] The compound was prepared in a similar manner to Scheme C-7 from trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (3-azabicyclo[4.1.0]heptan-6-yl)carbamate. LCMS [M+H] 515.8. [ka] (S)-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-(aminomethyl)pyrrolidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 46)

[0303] The compound was prepared in a similar manner to Scheme C-7 from trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and (S)-tert-butyl (pyrrolidin-3-ylmethyl)carbamate. 1 H NMR:(400 MHz, D2O): δ 7.92(d, 1H), 6.63(d, 1H), 4.32(t, 2H), 3.84-3.81(m, 1H), 3.64-3.57(m, 8H), 3.39(d, 1H), 3.12-3.02(m, 6H), 2.84-2.81(m, 1H), 2.70-2.60(m, 1H), 2.91-2.80(m, 1H), 2.78-2.68(m, 1H), 1.91-1.86(m, 4H), 1.85-1.76(m, 1H), 1.60(s, 6H), 1.24-1.18(m, 2H). LCMS [M+H] 503.7.

[0304] [ka] (R)-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-(aminomethyl)pyrrolidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 47) The compound was prepared in a similar manner to Scheme C-7 using trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and (R)-tert-butyl (pyrrolidin-3-ylmethyl)carbamate. LCMS [M+H] 503.4. [ka] trans-cis-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(2-fluoroethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 48)

[0305] The compound was prepared from (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)trans-tert-butyl carbamate and ((trans)-4-((2-fluoroethyl)amino)cyclohexyl)carbamate-tert-butyl in a manner similar to that of Scheme C-7. TLC and 1A cis / trans mixture of tert-butyl (1-(4-((1-((trans)-4-((((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-fluoroethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate was observed by H NMR. Purification of each isomer was obtained using a Waters SFC 350 with Column Chiralpack® IA 250×50 mm, 5 micron, 2489 UV detector, liquid CO2 mobile phase using 0.1% triethylamine in isopropyl alcohol: tert-butyl methyl ether: acetonitrile (50:25:25), isogradient (55% A, 45% B), flow rate 180 mL / min, sample load 20 mg, run for 32 min. Deprotection as in scheme C-7 gave the desired product. First fraction: 1 H NMR:(400 MHz, D2O): δ 7.93(d, 1H), 6.65(d, 1H), 4.62-4.59(m, 1H), 4.39-4.28(m, 1H), 3.72-3.59(m, 8H), 3.49-3.43(m, 2H), 3.42-3.37(m, 1H), 3.24-3.17(m, 3H), 2.23-2.08(m, 5H), 1.77-1.72(m, 6H), 1.69-1.66(m, 4H), 1.62.(s, 6H), 1.54-1.45(m, 3H). LCMS[M+H]563.6.

[0306] [ka] trans-trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(2-fluoroethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 49) In a similar manner, trans-cis-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(2-fluoroethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride was prepared. Second fraction. 1 H NMR:(400 MHz, D2O): δ 7.92(d, 1H), 6.64(d, 1H), 4.89-4.84(m, 1H), 4.46-4.32(m, 1H), 3.83-3.59(m, 8H), 3.50-3.39(m, 2H), 3.30-3.04(m, 4H), 2.25-2.04(m, 4H), 2.02-1.89(m, 4H), 1.56(s, 13H), 1.24-1.13(m, 2H). LCMS[M+H]563.6.

[0307] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((6-aminospiro[3.3]heptan-2-yl)(ethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 50) In a similar manner to Scheme C-7, trans-tert-butyl (6-(((4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)amino)spiro[3.3]heptan-2-yl)carbamate and acetaldehyde were prepared. TLC and 1A cis / trans mixture of (1-(4-((1-((trans)-4-(((6-((tert-butoxycarbonyl)amino)spiro[3.3]heptan-2-yl)(ethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)tert-butylcarbamate was observed by H NMR. Purification of the isomers was obtained using a Waters SFC 350 with a Chromegachiral™ column 250×20 mm, 5 micron, 2489 UV detector, liquid CO2 mobile phase (A), (B) 0.1% ammonia·methanol in methanol:acetonitrile (50:50), 90 ml / min, 6 min run time, and 10 mg sample load. The peak was isolated and subjected to deprotection as in Scheme C-7 to give the desired product. 1 H NMR:(400 MHz, D2O): δ 7.97(d, 1H), 6.63(d, 1H), 4.48-4.28(m, 1H), 3.78-3.74(m, 3H), 3.72-3.60(m, 8H), 3.12-3.05(m, 2H), 2.89-2.81(m, 2H), 2.48-2.46(m, 2H), 2.35-2.26(m, 3H), 2.24-2.15(m, 3H), 1.93-1.84(m, 4H), 1.81-1.72(m, 1H), 1.66-1.58(m, 9H), 1.23-1.33(m, 3H). LCMS [M+H] 557.4.

[0308] [ka] (R)-Trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-(aminomethyl)pyrrolidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 51) The compound was prepared in a similar manner to Scheme C-7 using trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and (R)-tert-butyl (pyrrolidin-3-ylmethyl)carbamate. TLC and 1 A cis / trans mixture of tert-butyl (2-methyl-1-(4-((1-((trans)-4-(((R)-3-((methylamino)methyl)pyrrolidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-1-oxopropan-2-yl)carbamate was observed by H NMR. Purification of the major isomer was obtained using a Waters SFC 350 Chromegachiral™ column 250×20 mm, 5 micron, 2489 UV detector, liquid CO2 mobile phase (A), (B) 0.1% ammonia·methanol in methanol:acetonitrile (50:50), 90 ml / min, 7 min run time, and 15 mg sample loading. The peak was isolated and subjected to deprotection as in Scheme C-7 to give the desired product. 1 H NMR:(400 MHz, D2O): δ 7.92(d, 1H), 6.65(d, 1H), 4.34(t, 1H), 3.87-3.82(m, 1H), 3.79-3.51(m, 8H), 3.40(d, 1H), 3.22-3.20(m, 1H), 3.13-3.01(m, 4H), 2.87-2.82(m, 1H), 2.71-2.60(m, 1H), 2.42-2.31(m, 1H), 2.30-2.18(m, 1H), 1.97-1.83(m, 4H), 1.81-1.75(m, 2H), 1.62(s, 6H), 1.59-1.54(m, 1H), 1.25-1.16(m, 2H). LCMS[M+H]503.4.

[0309] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-aminoazetidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 52) The compound was prepared in a similar manner to Scheme C-7 using trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl azetidin-3-ylcarbamate. Purification of each isomer of tert-butyl (1-(4-((1-((cis / trans)-4-((3-((tert-butoxycarbonyl)amino)azetidin-1-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate was obtained using a Waters SFC 350 with a Chiralpack® IA 250×50 mm, 5 micron, 2489 UV detector, liquid CO2 mobile phase using 0.1% ammonia:methanol in isopropyl alcohol:acetonitrile (70:30), isogradient (55% A, 45% B), flow rate 150 mL / min, sample load 10 mg, run for 12 min. Deprotection as in Scheme C-7 gave the major product. 1 H NMR:(400 MHz, D2O): δ 7.99(d, 1H), 6.62(d, 1H), 4.78-4.75(m, 2H), 4.62-4.55(m, 1H), 4.45-4.32(m, 2H), 4.31-4.19(m, 1H), 3.65-3.53(m, 8H), 3.30-3.17(m, 2H), 3.15-2.97(m, 1H), 1.93-1.79(m, 4H), 1.72-1.66(m, 1H), 1.62(s, 6H), 1.58-1.54(m, 1H), 1.25-1.19(m, 2H). LCMS [M+H] 475.6.

[0310] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-(((6-aminospiro[3.3]heptan-2-yl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 53) The compound was prepared in a similar manner from trans-tert-butyl (1-(4-((1-(4-formylcyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (6-aminospiro[3.3]heptan-2-yl)carbamate. LCM [M+H] 529.6. [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-(((trans-4-aminocyclohexyl)(methyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 19) Scheme C-9 [ka] Reagents: 1) Formaldehyde, NaBH3CN, MeOH, rt, 24 h 2) NH4OH, MeOH, 50°C, 3 h 3) CH3CN, heat, 21 h 4) 2M HCl in MeOH, rt, 16 h.

[0311] Step 1: tert-Butyl (trans-4-(((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(methyl)amino)cyclohexyl)carbamate. To a mixture of tert-butyl (trans-4-(((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)amino)cyclohexyl)carbamate (51 mg, 97 μmol) and 37 wt% formaldehyde (40 μL, 0.54 mmol) in MeOH (1 mL) was added NaBH3CN (19 mg, 307 μmol) and the mixture was stirred at room temperature for 24 h. The crude material was purified by column chromatography (DCM / MeOH / NH4OH) to give the title compound (6.7 mg). LCMS [M+H] 538.4.

[0312] Step 2: tert-Butyl (trans-4-(((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(methyl)amino)cyclohexyl)carbamate. A mixture of tert-butyl (trans-4-(((trans-4-(4-benzamido-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(methyl)amino)cyclohexyl)carbamate (6.7 mg, 12 μmol) and 29 wt % NH4OH (0.050 mL, 0.36 mmol) in MeOH (1 mL) was stirred at 50° C. for 3 h and then concentrated to dryness to give the title compound. LCMS [M+H] 434.3.

[0313] Step 3: tert-Butyl (1-(4-((1-(trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)(methyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. A mixture of tert-butyl (trans-4-(((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(methyl)amino)cyclohexyl)carbamate (5.2 mg, 12 μmol) and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (12.1 mg, 24 μmol) in HCN (1 mL) was stirred at 85° C. for 21 h. The mixture was cooled, EtN (2 drops) was added and the mixture was concentrated. The residue was purified by column chromatography (DCM / MeOH / NHOH) to give the title compound (5.6 mg). LCMS [M+H] 731.7.

[0314] Step 4: 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-(((trans-4-aminocyclohexyl)(methyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride. A mixture of tert-butyl (1-(4-((1-(trans-4-(((trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)(methyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (5.6 mg, 7.7 μmol) and 2M HCl in MeOH (0.50 mL, 1.0 mmol) was stirred at room temperature for 16 h. The reaction mixture was concentrated to give the title compound (5.0 mg). 1H NMR(500 MHz, D2O)δ 8.14(d, 1H), 6.75(d, 1H), 4.54-4.45(m, 1H), 3.88-3.68(m, 8H), 3.44-3.36(m, 1H), 3.32-3.17(m, 2H), 3.10-3.03(m, 1H), 2.88(s, 3H), 2.32-2.15(m, 4H), 2.12-2.03(m, 3H), 2.03-1.89(m, 2H), 1.83-1.69(m, 9H), 1.67-1.55(m, 2H), 1.43-1.27(m, 3H). LCMS [M+H] 531.3.

[0315] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-((2-amino-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 20) Scheme C-10 [ka] Reagents: 1) i) 3-ethoxyacryloyl chloride, AgOCN, toluene, □, 3 hrs, ii) amine, DCM, -78°C to rt, 20 hrs 2) i) TFA, rt, 2.5 hrs, ii) Boc2O, 2M NaOH, THF, rt, 20 hrs 3) i) POCl3, 1,2,4-triazole, Et3N, CH3CN, rt, 17 hrs, ii) NH4OH, CH3CN, 50°C, 48 hrs 4) CH3CN, heat, 17 hrs 5) 2M HCl / MeOH, rt, 22 hrs. Step 1: tert-Butyl (7-((trans-4-(3-(3-ethoxyacryloyl)ureido)cyclohexyl)methyl)-7-azaspiro[3.5]non-2-yl)carbamate. A solution of 3-ethoxyacryloyl chloride (2.0 g, 15 mmol) in toluene (36 mL) was added dropwise to a suspension of silver cyanate (2.5 g, 16.7 mmol) in toluene (40 mL) and the mixture was stirred at reflux for 3 h, cooled and allowed to settle. A portion of the solution (44.4 mL, 8.9 mmol) was filtered through a syringe filter and added to a solution of tert-butyl (7-((trans-4-aminocyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate (2.6 g, 7.4 mmol) in dry DCM (74 mL) cooled to -78 °C under N2. The mixture was allowed to warm to room temperature and stirred for 20 h. MeOH (50 mL) was added, the solution concentrated, Et2O (350 mL) was added and extracted with 5% citric acid (2 x 325 mL). The aqueous extract was brought to pH 12 with NaOH (20 g) and then extracted with DCM (2 x 350 mL). The extract was dried over Na2SO4, filtered and concentrated to dryness to give the title compound (3.39 g). LCMS [M+H] 493.3.

[0316] Step 2: tert-Butyl (7-((trans-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate. A mixture of tert-butyl (7-((trans-4-(3-(3-ethoxyacryloyl)ureido)cyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate (3.39 g, 6.88 mmol) and TFA (26.5 mL, 344 mmol) was stirred at room temperature for 2.5 h. The mixture was concentrated to dryness. The residue was dissolved in THF (70 mL) and 2M NaOH (51.6 mL, 103 mmol). Di-tert-butyl dicarbonate (3.16 mL, 13.8 mmol) was added and the mixture was stirred vigorously at room temperature for 20 h. The mixture was poured into saturated aqueous NaHCO3 (350 mL) and extracted with EtOAc (2 x 350 mL). The extract was washed with brine (350 mL), dried (Na2SO4), decanted and concentrated. The residue was purified by column chromatography (DCM / MeOH / NH4OH) to give the title compound (2.60 g). 1 H NMR(500 MHz, MeOD)δ 7.67(d, 1H), 5.66(d, 1H), 4.36(tt, 1H), 4.03-3.90(m, 1H), 2.50-2.23(m, 4H), 2.23-2.11(m, 4H), 2.03-1.93(m, 2H), 1.93-1.81(m, 3H), 1.72-1.54(m, 8H), 1.42(s, 9H), 1.20-1.06(m, 2H). LCMS[M+H]447.3.

[0317] Step 3: tert-Butyl (7-((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate. To a mixture of 1,2,4-triazole (3.7 g, 54 mmol) and EtN (7.5 mL, 54 mmol) in CHCN (25 mL) was added POCl (0.63 mL, 6.8 mmol) dropwise at 0 °C and the mixture was stirred at 0 °C for 10 min. Meanwhile, to tert-butyl (7-((trans-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate (1.01 g, 2.26 mmol) was added CH3CN (25 mL). The POCl3 mixture was then poured into the suspension and the mixture was stirred at room temperature for 17 h. Water (150 mL) was added and the precipitate was collected by vacuum filtration to give the intermediate (821 mg, 1.65 mmol). LCMS [M+H] 498.3. CH3CN (20 mL) and 28 wt% NH4OH (11 mL, 80 mmol) were added and the reaction mixture was stirred at 50 °C for 48 h. The mixture was cooled, diluted with water (50 mL) and the precipitate was collected by vacuum filtration and dried under vacuum to give the title compound (681 mg). 1 H NMR(500 MHz, MeOD)δ 7.64(d, 1H), 5.87(d, 1H), 4.44(tt, 1H), 4.02-3.90(m, 1H), 2.50-2.23(m, 4H), 2.23-2.10(m, 4H), 2.01-1.92(m, 2H), 1.92-1.82(m, 2H), 1.70-1.52(m, 9H), 1.42(s, 9H), 1.20-1.06(m, 2H). LCMS[M+H]446.3.

[0318] Step 4: tert-Butyl (1-(4-((1-(trans-4-((2-((tert-butoxycarbonyl)amino)-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate. A mixture of tert-butyl (7-((trans-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)-7-azaspiro[3.5]nonan-2-yl)carbamate (593 mg, 1.33 mmol) and 1-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carbonyl)-3-methyl-1H-imidazol-3-ium iodide (1.17 g, 2.3 mmol) in CH3CN (12 mL) was stirred at reflux for 17 h. The mixture was cooled, diluted with EtOAc (150 mL), washed with saturated aqueous NaHCO3 (2 x 125 mL) and brine (125 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (DCM / MeOH / NH4OH) to give the title compound (691 mg). LCMS [M+H] 743.6.

[0319] Step 5: 4-(2-amino-2-methylpropanoyl)-N-(1-(trans-4-((2-amino-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride. A mixture of tert-butyl (1-(4-((1-(trans-4-((2-((tert-butoxycarbonyl)amino)-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate (691 mg, 0.93 mmol) and 2M HCl in MeOH (20 mL, 40 mmol) was stirred at room temperature for 22 h. IPA was added and the precipitate was collected by vacuum filtration, washed with IPA and Et2O, and dried under vacuum to give the title compound (573 mg). 1 H NMR(500 MHz, D2O)δ 8.06(d, 1H), 6.77(d, 1H), 4.52-4.42(m, 1H), 3.91(p, 1H), 3.87-3.65(m, 8H), 3.62-3.46(m, 2H), 3.12-2.92(m, 4H), 2.58-2.47(m, 1H), 2.40-2.30(m, 1H), 2.19-1.86(m, 11H), 1.82-1.66(m, 8H), 1.40-1.26(m, 2H). LCMS[M+H]543.4.

[0320] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(trans-4-(((trans-4-aminocyclohexyl)(ethyl)amino)methyl)cyclohexyl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 21) The compound was prepared from tert-butyl ((trans)-4-((((trans)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate in a similar manner to Scheme C-10. 1 H NMR (500 MHz, D2O) δ 8.12 (d, 1H), 6.76 (d, 1H), 4.49 (tt, 1H), 3.93-3.65 (m, 8H), 3.45 (tt, 1H), 3.39-3.23 (m, 3H), 3.20 (dd, 1H), 3.06 (dd, 1H), 2.32-2.14 (m, 4H), 2.12-1.98 (m, 4H), 1.96-1.85 (m, 1H), 1.85-1.68 (m, 10H), 1.68-1.55 (m, 2H), 1.44-1.29 (m, 5H). LCMS[M+H]545.4.

[0321] [ka] endo-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (compound 22) The compound was prepared in a similar manner to Scheme C-5 from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and endo-tert-butyl (3-azabicyclo[3.1.0]hexan-6-yl)carbamate. 1H NMR:(400 MHz, D2O): δ 7.73(d, 1H), 6.61(d, 1H), 5.81(s, 1H), 4.21-4.02(m, 1H), 3.82(d, 1H), 3.62-3.56(m, 8H), 3.49-3.47(m, 2H), 3.51-3.13(m, 1H), 3.11-3.02(m, 1H), 2.93-2.85(m, 1H), 2.85-2.75(m, 1H), 2.35-2.22(m, 4H), 2.11-2.01(m, 1H), 1.94-1.80(m, 2H), 1.59(s, 6H), 1.49-1.35(m, 1H). LCMS[M+H]499.4.

[0322] [ka] (S)-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-(aminomethyl)pyrrolidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 23) The compound was prepared in a similar manner to Scheme C-5 from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and (S)-tert-butyl (pyrrolidin-3-ylmethyl)carbamate. 1H NMR:(400 MHz, D2O): δ 7.79(d, 1H), 6.60(d, 1H), 5.85(s, 1H), 3.87-3.83(m, 1H), 3.76-3.58(m, 8H), 3.41(d, 1H), 3.21-3.16(m, 3H), 3.11-3.00(m, 2H), 2.85-2.81(m, 1H), 2.66-2.62(m, 1H), 2.33-2.20(m, 4H), 2.09(bs, 1H), 1.94-1.89(m, 2H), 1.69-1.66(m, 1H), 1.59(s, 6H), 1.47-1.45(m, 1H). LCMS[M+H]501.5.

[0323] [ka] (R)-4-(2-amino-2-methylpropanoyl)-N-(1-(4-((3-(aminomethyl)pyrrolidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 24) The compound was prepared in a similar manner to Scheme C-5 from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and (R)-tert-butyl (pyrrolidin-3-ylmethyl)carbamate. 1H NMR:(400 MHz, D2O): δ 7.71(d, 1H), 6.63(d, 1H), 5.82(s, 1H), 3.85-3.83(m, 1H), 3.81-3.55(m, 8H), 3.43(d, 1H), 3.21-3.16(m, 3H), 3.11-3.00(m, 2H), 2.85-2.83(m, 1H), 2.71-2.62(m, 1H), 2.33-2.23(m, 4H), 2.09(bs, 1H), 1.94-1.88(m, 2H), 1.75-1.66(m, 1H), 1.59(s, 6H), 1.47-1.44(m, 1H). LCMS[M+H]501.6.

[0324] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((2-amino-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexan-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 25) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (7-azaspiro[3.5]nonan-2-yl)carbamate in a manner similar to that of Scheme C-5. 1 H NMR:(400 MHz, D2O): δ 7.74(d, 1H), 6.63(d, 1H), 5.84(s, 1H), 3.77(t, 1H), 3.74-3.58(m, 8H), 3.46-3.39(m, 2H), 3.02(d, 2H), 2.96-2.90(m, 2H), 2.49-2.39(m, 1H), 2.32-2.21(m, 5H), 2.01-1.77(m, 8H), 1.61(s, 6H), 1.47-1.45(m, 1H). LCMS[M+H]541.5.

[0325] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-(2-aminoethyl)piperidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 26) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (2-(piperidin-4-yl)ethyl)carbamate in a manner similar to that of Scheme C-5. 1 H NMR:(400 MHz, D2O): δ 7.75-7.73(m, 1H), 6.62-6.60(m, 1H), 5.83(s, 1H), 4.64-4.61(m, 1H), 3.63-3.51(m, 9H), 3.25-3.11(m, 1H), 3.01-3.00(m, 2H), 2.92-2.83(m, 4H), 2.30-2.23(m, 3H), 2.21-2.09(m, 1H), 1.90-1.87(m, 4H), 1.59-1.54(m, 8H), 1.45-1.35(m, 3H). LCMS [M+H] 529.6.

[0326] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-amino-4-methylpiperidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 27) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (4-methylpiperidin-4-yl)carbamate in a manner similar to that of Scheme C-5. 1 H NMR:(400 MHz, D2O): δ 7.71(d, 1H), 6.64(d, 1H), 5.83(s, 1H), 3.62-3.58(m, 10H), 3.14-3.12(m, 4H), 2.30-2.20(m, 4H), 2.19-2.06(m, 4H), 1.90-1.85(m, 2H), 1.60(s, 6H), 1.44-1.40(m, 4H). LCMS[M+H]515.6.

[0327] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-aminopiperidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 28) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl piperidin-4-ylcarbamate in a manner similar to that of Scheme C-5. 1H NMR:(400 MHz, D2O): δ 7.69(d, 1H), 6.62(d, 1H), 5.81(s, 1H), 3.63-3.55(m, 8H), 3.44-3.35(m, 2H), 3.07-2.98(m, 4H), 2.41-2.30(m, 3H), 2.22-2.19(m, 4H), 1.88-1.70(m, 4H), 1.58(s, 6H), 1.51-1.45(m, 1H). LCMS[M / 2+H]251.2.

[0328] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((1-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 29) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl (3-azabicyclo[3.1.0]hexan-1-yl)carbamate in a manner similar to that of Scheme C-5. 1 H NMR:(400 MHz, D2O): δ 7.78-7.76(d, 1H), 6.63-6.61(d, 1H), 5.84(s, 1H), 4.04(bs, 1H), 3.64-3.58(m, 9H), 3.24(m, 2H), 2.29-2.24(m, 4H), 2.07(m, 3H), 1.95-1.89(m, 6H), 1.60(s, 2H), 1.44-1.30(m, 3H). LCMS[M+H]499.4.

[0329] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(methyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 30) The compound was prepared in a similar manner to Scheme C-5 from trans-tert-butyl (1-(4-((1-(4-(((4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and formaldehyde. 1 H NMR:(400 MHz, D2O): δ 7.67(d, 1H), 6.62(d, 1H), 5.80(s, 1H), 3.61-3.55(m, 8H), 3.26-3.24(m, 1H), 3.17-3.07(m, 2H), 3.02-2.91(m, 1H), 2.73(s, 3H), 2.33-2.18(m, 3H), 2.08-2.02(m, 5H), 1.91-1.80(m, 3H), 1.57(s, 6H), 1.48-1.42(m, 4H). LCMS[M+H]529.6.

[0330] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(methyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (enantiomer 1) (compound 32) The compound was prepared by chiral chromatography of trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(methyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide according to the following parameters: Instrument: Agilent 1260 infinity; Column: Chiralpack® IA 250×50 mm, 5 micron, DAD detector, Mobile phase (A) 0.1% TEA in EtOH (B) ACN; Method: Isocratic (97% A, 3% B); Flow rate 20 ml / min; Run time 45 min; Sample load 5 mg. 1 H NMR:(400 MHz, D2O): δ 7.69(d, 1H), 6.68(d, 1H), 5.85(s, 1H), 3.73-3.53(m, 8H), 3.42-3.25(m, 2H), 3.22-3.12(m, 2H), 3.07-2.99(m, 1H), 2.79(d, 3H), 2.34-2.22(m, 3H), 2.16-2.11(m, 5H), 1.99-1.86(m, 2H), 1.67(s, 6H), 1.53-1.38(m, 4H). LCMS[M+H]529.6.

[0331] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)(ethyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Compound 31) The compound was prepared from trans-tert-butyl (1-(4-((1-(4-(((4-((tert-butoxycarbonyl)amino)cyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and acetaldehyde in a similar manner to Scheme C-5. 1 H NMR:(400 MHz, D2O): δ 7.32(d, 1H), 6.35-6.22(m, 1H), 5.69(s, 1H), 3.60-3.47(m, 8H), 3.19(s, 2H), 2.71(d, 4H), 2.55(bs, 2H), 2.25-1.15(m, 3H), 1.88-1.75(m, 6H), 1.35-1.33(m, 2H), 1.27(s, 6H), 1.76-1.11(m, 2H), 0.99(t, 3H). LCMS[M+H]543.0.

[0332] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((4-aminoazepan-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Enantiomer 1) (Compound 41) 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((4-aminoazepan-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Enantiomer 2) (Compound 42) The compound was prepared from tert-butyl (1-(4-((1-(4-formylcyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate and tert-butyl azepan-4-ylcarbamate in a similar manner to Scheme C-5. Chiral chromatography of 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((4-aminoazepan-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide was carried out according to the following parameters: Instrument: Agilent 1260 infinity; Column: Daicel Chiralpack®-AD250×30 mm, 10 micron; DAD detector; Mobile phase: (A) 0.1% triethylamine in acetonitrile; (B) 0.1% triethylamine in ethanol; Isocratic method (40% A, 60% B): flow rate 25 ml / min; run time 48 min; sample load 25 mg. Fraction 1 1 H NMR:(400 MHz, D2O): δ 7.67(d, 1H), 6.70(d, 1H), 5.85(s, 1H), 3.67-3.59(m, 8H), 3.56-3.45(m, 3H), 3.28-3.26(m, 1H), 3.19-3.13(m, 3H), 2.35-2.31(m, 3H), 2.27-2.23(m, 3H), 2.19-2.15(m, 1H), 2.05-1.99(m, 3H), 1.96-1.88(m, 2H), 1.64(s, 6H), 1.54-1.45(m, 1H). LCMS[M+H]515.6. fraction 2 1H NMR:(400 MHz, D2O): δ 7.67(d, 1H), 6.70(d, 1H), 5.84(s, 1H), 3.67-3.59(m, 8H), 3.54-3.45(m, 3H), 3.33-3.28(m, 1H), 3.18-3.08(m, 3H), 2.35-2.31(m, 2H), 2.28-2.23(m, 3H), 2.21-2.11(m, 2H), 2.09-1.98(m, 3H), 1.97-1.87(m, 2H), 1.64(s, 6H), 1.53-1.48(m, 1H). LCMS [M+H] 515.6.

[0333] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-(((exo)-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Enantiomer 1) (Compound 33) 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-(((exo)-6-amino-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (Enantiomer 2) (Compound 34) Chiral chromatography of exo-tert-butyl (1-(4-((1-(4-((6-((tert-butoxycarbonyl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate was carried out according to the following parameters, followed by HCl deprotection of the Boc groups of each isomer in a similar manner to Scheme C-5. Chiral Chromatography Parameters: Instrument: Agilent 1260 infinity; Column: Chiralpack® IA 250×50 mm, 5 micron; Detector: DAD; Mobile phase: (A) EtOH with 0.1% TEA (B) ACN; Method: Isocratic (97% A, 3% B); Flow rate: 20 ml / min; Run time: 45 min; Sample load: 5 mg. 1 H NMR(400 MHz, D2O): δ 7.81(d, 1H), 6.64(d, 1H), 5.87(s, 1H), 4.22-4.11(m, 1H), 3.87(d, 1H), 3.78-3.62(m, 8H), 3.54-3.51(m, 1H), 3.22-3.15(m, 1H), 3.13-3.04(m, 1H), 3.02-2.89(m, 1H), 2.88-2.82(m, 1H), 2.51-2.31(m, 4H), 2.30-2.18(m, 1H), 2.17-2.07(m, 1H), 1.97-1.85(m, 2H), 1.63(s, 6H), 1.49-1.47(m, 1H). LCMS[M+1]499.6.

[0334] [ka] 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((2-amino-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexan-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide (enantiomer 1) (compound 35) 4-(2-amino-2-methylpropanoyl)-N-(1-(4-((2-amino-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexan-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide (enantiomer 2) (compound 36) Chiral chromatography of tert-butyl (1-(4-((1-(4-((2-((tert-butoxycarbonyl)amino)-7-azaspiro[3.5]nonan-7-yl)methyl)cyclohexan-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate was carried out according to the following parameters, followed by HCl deprotection of the Boc groups of each isomer in a similar manner to Scheme C-1. Chiral Chromatography Parameters: Instrument: Waters SFC350; Column: Chiralpack® IA 250×50 mm, 5 micron; Detector: 2489 UV detector; Mobile phase: (A) liquid CO2, (B) 0.1% TEA: tert-butyl methyl ether: CAN (50:25:25) in IPA; Method: Isocratic (55% A, 45% B), Flow rate: 180 ml / min, Run time: 32 min; Sample load: 20 mg. 1 H NMR:(400 MHz, D2O): δ 7.75(d, 1H), 6.66(d, 1H), 5.86(s, 1H), 3.81-3.77(m, 1H), 3.75-3.61(m, 8H), 3.60-3.47(m, 3H), 3.03(d, 2H), 2.98-2.85(m, 2H), 2.44-2.20(m, 6H), 2.03-1.79(m, 7H), 1.63(s, 6H), 1.51-1.47(m, 1H). LCMS[M+H]541.7.

[0335] [ka] 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((4-amino-4-methylpiperidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (enantiomer 1) (compound 37) 4-(2-Amino-2-methylpropanoyl)-N-(1-(4-((4-amino-4-methylpiperidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (enantiomer 2) (compound 38) Chiral chromatography of tert-butyl (1-(4-((1-(4-((4-((tert-butoxycarbonyl)amino)-4-methylpiperidin-1-yl)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)piperazin-1-yl)-2-methyl-1-oxopropan-2-yl)carbamate was carried out according to the following parameters, followed by HCl deprotection of the Boc group of each isomer in a similar manner as in Scheme C-1. Chiral Chromatography Parameters: Instrument: Agilent 1260 infinity; Column: Daicel Chiralpack®-AD 250×30 mm, 10 micron; Detector: DAD detector; Mobile phase: (A) 0.1% triethylamine in n-heptane, (B) IPA:MeOH (96:04); Method: Isocratic (75% A, 25% B), Flow rate: 35 ml / min; Run time: 50 min; Sample load: 20 mg. 1 H NMR:(400 MHz, D2O): δ 7.77(d, 1H), 6.62(d, 1H), 5.85(s, 1H), 3.71-3.51(m, 11H), 3.17-3.10(m, 4H), 2.33-2.30(m, 2H), 2.25-2.15(m, 2H), 2.13-2.02(m, 4H), 1.98-1.85(m, 2H), 1.60(s, 6H), 1.48-1.41(m, 3H). LCMS[M+H]515.6.

[0336] [ka] trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (enantiomer 1) (compound 39) trans-4-(2-amino-2-methylpropanoyl)-N-(1-(4-(((4-aminocyclohexyl)amino)methyl)cyclohex-1-en-1-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)piperazine-1-carboxamide hydrochloride (enantiomer 2) (compound 40) Preparation was carried out by chiral chromatography of ((4-(4-(4-(2-((tert-butoxycarbonyl)amino)-2-methylpropanoyl)piperazine-1-carboxamido)-2-oxopyrimidin-1(2H)-yl)cyclohex-3-en-1-yl)methyl)((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)benzyl carbamate, followed by deprotection of the Boc / Cbz groups of each isomer in a manner similar to that in Scheme C-1. Chiral chromatography parameters: Instrument: Agilent 1260 infinity; Column: Chiralpack® IA 250×50 mm, 5 μM; Detector: DAD detector; Mobile phase: (A) EtOH with 0.1% TEA (B) ACN; Method: Isocratic (97% A, 3% B); Flow rate: 20 ml / min; Run time: 45 min; Sample load: 5 mg. 1 H NMR:(400 MHz, D2O): δ 7.63(d, 1H), 6.66(d, 1H), 5.80(s, 1H), 3.71-3.51(m, 8H), 3.11-3.10(m, 2H), 2.99(d, 2H), 2.31-2.23(m, 3H), 2.16-2.14(m, 2H), 2.11-2.05(m, 2H), 1.92-1.86(m, 2H), 1.60(s, 6H), 1.51-1.45(m, 6H). LCMS[M+H]515.6.

[0337] [ka] 4-Amino-1-((trans)-4-((((trans)-4-aminocyclohexyl)(ethyl)amino)methyl)cyclohexyl)pyrimidin-2(1H)-one Scheme C-11 (Compound 54) [ka] Reagents: 1) (E)-3-ethoxyacryloyl chloride, AgOCN, toluene, □, 3.5h; DCM, rt, 16h 2) TFA, rt, 2h 3) POCl3, Et3N, ACN, 0°C to rt, 4h; NH4OH, 50°C, 18h 10) 2M HCl, MeOH, rt, 18h. Step 1: tert-Butyl ((trans)-4-((((trans)-4-(3-((E)-3-ethoxyacryloyl)ureido)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate. To a suspension of silver cyanate (1.2 g, 8.1 mmol) in toluene (6 mL) was added dropwise a solution of (E)-3-ethoxyacryloyl chloride (934 mg, 6.9 mmol) in toluene (6 mL) and the mixture was stirred at 110° C. for 3.5 h and then cooled without stirring. A solution of tert-butyl ((trans)-4-((((trans)-4-aminocyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate (818 mg, 2.3 mmol) in dry DCM (10 mL) was cooled at 0° C. under N2. 10 mL of the clear acyl isocyanate solution formed was drawn up via syringe and added dropwise to the cooled mixture. The reaction was allowed to warm to room temperature and stirred for 4 h. The mixture was quenched with MeOH (5 mL), concentrated, dissolved in Et2O (100 mL) and extracted with 5% citric acid (2 x 100 mL). The aqueous layer was brought to approximately pH 12 with NaOH and then extracted with DCM (2 x 100 mL). The extracts were dried (Na2SO4), filtered and concentrated to give the desired product (1.21 g, quantitative). 1H NMR(500 MHz, MeOD)δ 7.63(d, 1H), 5.41(d, 1H), 3.97(q, 2H), 3.55(tt, 1H), 3.27-3.17(m, 1H), 2.51(q, 2H), 2.48-2.41(m, 1H), 2.24(d, 2H), 2.05-1.98(m, 2H), 1.97-1.85(m, 4H), 1.82-1.73(m, 2H), 1.43(s, 9H), 1.38-1.28(m, 6H), 1.26-1.14(m, 4H), 1.05-0.89(m, 5H). LCMS [M+H] 495.2.

[0338] Step 2: tert-Butyl ((trans)-4-((((trans)-4-(3-((E)-3-ethoxyacryloyl)ureido)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate. A mixture of tert-butyl ((trans)-4-((((trans)-4-(3-((E)-3-ethoxyacryloyl)ureido)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate (0.8 g, 1.6 mmol) and TFA (6 mL) was stirred at 20° C. for 2 h. The residue was dissolved in 1,4-dioxane (16 mL) and 1 M saturated NaHCO3 (16 mL, 16 mmol) and di-tert-butyl dicarbonate (743 μL, 3.23 mmol) were added. The mixture was stirred vigorously for 18 h. It was cooled, diluted with EtOAc (100 mL) and washed with saturated NaHCO3 (x75 mL) and brine (1x75 mL). The aqueous layer was extracted with DCM (100 mL), combined with the EtOAc layer, dried over Na2SO4, decanted and concentrated. The residue was purified by flash chromatography (0-40% [80 / 20 / 2 DCM / MeOH / NH4OH] / DCM) to give the desired product (469 mg, 64.6%) as a foam. 1H NMR(500 MHz, MeOD)δ 7.67(d, 1H), 5.66(d, 1H), 4.36(tt, 1H), 3.28-3.17(m, 1H), 2.58-2.44(m, 3H), 2.28(d, 2H), 2.05-1.98(m, 2H), 1.98-1.91(m, 2H), 1.91-1.83(m, 2H), 1.83-1.74(m, 2H), 1.69-1.58(m, 2H), 1.48-1.27(m, 12H), 1.26-1.13(m, 2H), 1.11-0.98(m, 5H). LCMS [M+H] 449.3.

[0339] Step 3: tert-Butyl ((trans)-4-((((trans)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate. To a mixture of 1,2,4-triazole (1.97 g, 28.5 mmol) and triethylamine (3.97 mL, 28.5 mmol) in CH3CN (6 mL) was added POCl3 (332 μL, 3.56 mmol) dropwise at 0 °C and the mixture was stirred at 0 °C for 15 min. A solution of tert-butyl ((trans)-4-((((trans)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate (533 mg, 1.2 mmol) in CH3CN (6 mL) was added dropwise and the mixture was allowed to warm to room temperature and stirred for 4 h. NH4OH (3.6 mL, 89 mmol) was added and the mixture was stirred at 50 °C for 18 h. The mixture was concentrated until a fine precipitate formed which was collected by vacuum filtration to give the desired product (0.25 g, 47.0%). 1H NMR(500 MHz, MeOD)δ 7.65(d, 1H), 5.87(d, 1H), 4.44(tt, 1H), 3.28-3.17(m, 1H), 2.59-2.42(m, 3H), 2.28(d, 2H), 2.05-1.97(m, 2H), 1.97-1.91(m, 2H), 1.91-1.83(m, 2H), 1.83-1.75(m, 2H), 1.63-1.52(m, 2H), 1.51-1.26(m, 12H), 1.26-1.13(m, 2H), 1.13-0.98(m, 5H). LCMS [M+H] 448.3.

[0340] Step 4: A mixture of tert-butyl ((trans)-4-((((trans)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)pyrimidin-2(1H)-one ((trans)-4-((((trans)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)cyclohexyl)methyl)(ethyl)amino)cyclohexyl)carbamate (200 mg, 0.45 mmol) and 2M HCl in MeOH (7.6 mL) was stirred at room temperature for 18 h. The precipitate was collected by vacuum filtration to give the desired product (131 mg, 84.9%). 1 H NMR(500 MHz, D2O)δ 7.65(d, 1H), 6.05-5.91(m, 1H), 4.35-4.16(m, 1H), 3.38-3.25(m, 1H), 3.25-2.98(m, 3H), 2.98-2.84(m, 1H), 2.20-1.79(m, 8H), 1.79-1.38(m, 4H), 1.29-1.10(m, 4H). LCMS[M+H]348.3.

[0341] Biological Examples Standard microbiological effects: A certified BSL-2 laboratory was used for testing. Compounds were evaluated against S. aureus (Sa), E. coli (Ec), K. pneumoniae (Kp), A. baumannii (Ab), E. faecalis (Ef), and P. aeruginosa (Pa) using broth microdilution minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays as defined by the Clinical and Laboratory Standards Institute (CLSI) M26-A guidelines.

[0342] E. coli S30 Extract: Inhibition of bacterial protein synthesis was measured using the E. coli S30 Extract System for Circular DNA (Promega Cat. No. L-2010) and Luciferase Assay Reagent (Promega Cat. No. E1500) with minor modifications to published protocols. Fyfe, C., Sutcliffe, JA and Grossman, TH (2012) “Development and characterization of a Pseudomonas aeruginosa in vitro coupled transcription-translation assay system for evaluation of translation inhibitors” J. Microbiol. Methods 90(3), 256-261.

[0343] Compounds were serially diluted in 0.5 mL microcentrifuge tubes by mixing and transferring 50 μL from the highest concentration to 50 μL water, and mixing and transferring 50 μL of this 2-fold dilution to 50 μL water. This mixing and transferring was repeated so that there were a total of 8 tubes with compounds serially diluted at 10-fold the desired screening concentration. These were finally diluted to 1-fold by the addition of S30 luciferase synthesis mix. Compound serial dilutions (2 μL) were added to wells of a black round-bottom 96-well plate. Water (2 μL) was used as a "no inhibitor" control, 4 wells / plate. A control reaction mix without DNA (20 μL, see below) was used as a control for background luminescence, 4 wells / plate. S30 luciferase synthesis mix (18 μL, see below) was added to wells with compound or water mix and incubated at 37° C. for 1 hour. The reaction was stopped by transferring to a 4° C. refrigerator for 5 min, after which 25 μL of luciferase activity mix was added. Luminescence was measured using a BioTek Synergy HTX plate reader. Percent inhibition was determined relative to a no inhibitor control. S30 Luciferase Synthesis Mix: 445 μL of S30 extract, circular 712 μL S30 Premix without amino acids 4.45 μL of pBESTluc™ DNA (1 μg / μL) 78 μL complete amino acid mixture 267 μL water No DNA control: 20 μL of S30 extract, circular 32 μL of S30 premix (no amino acids) 7 μL complete amino acid mixture 21 μL water

[0344] Rabbit reticulocyte lysate Inhibition of eukaryotic protein synthesis was measured using the Rabbit Reticulocyte Lysate System, Nuclease-Treated from Promega (cat. no. L-4960) with minor modifications to the manufacturer's protocol. Compounds were serially diluted in 0.5 mL microcentrifuge tubes by mixing and transferring 50 μL from the highest concentration to 50 μL water, and mixing and transferring 50 μL of this 2-fold dilution to 50 μL water. This mixing and transfer was repeated so that there were a total of eight tubes with compounds serially diluted at 10-fold the desired screening concentration. These were finally diluted to 1-fold by the addition of rabbit reticulocyte luciferase synthesis mix. Serial dilutions of compounds (2.5 μL) were added to wells of a black round-bottom 96-well plate. Water (2.5 μL) was used as a "no inhibitor" control, 4 wells / plate. For background luminescence, a control reaction mixture without RNA (2 μL, see below) was used as a control with 4 wells / plate. Rabbit reticulocyte luciferase synthesis mixture (22.5 μL, see below) was added to wells with compound or water mixture and incubated at 30° C. for 90 minutes. Luciferase assay reagent (25 μL) was added and luminescence was measured using a BioTek Synergy HTX plate reader. % inhibition was determined relative to the no inhibitor control. Rabbit reticulocyte luciferase synthetic mixture: 1,000μL of rabbit reticulocyte lysate 5.7 μL of luciferase control RNA (1 μg / μL) 26 μL complete amino acid mixture 395 μL water No RNA control 70μL of rabbit reticulocyte lysate 2 μL of complete amino acid mixture 28 μL water

[0345] Minimum inhibitory concentration (MIC) MICs were determined using a slightly modified broth microdilution method from the Clinical Laboratory and Standards Institute (CLSI). Clinical and Laboratory Standards Institute (2012). "Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 9th ed. M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA." Serial two-fold dilutions of compounds are prepared in sterile clear round-bottom 96-well plates.

[0346] To prepare the microdilution tray, two-fold dilutions of antimicrobial agents are prepared in growth medium: Cation-Adjusted Mueller-Hinton Broth (CAMHB), or CAMHB supplemented with sodium bicarbonate (final concentration 25 mM prepared from a 1.0 M stock solution) or CAMHB supplemented with 0-50% heat-inactivated human serum (Fisher catalog number BP2657100) by adding 200 μL of the highest concentration to be tested (e.g., 64 μg / mL) to row A, mixing and transferring 100 μL from row A to 100 μL of growth medium in row B, then repeating this mixing and transferring process up to row H of the 96-well plate, discarding the remaining excess 100 μL. This slight modification to the CLSI protocol allows for evaluation of MICs in triplicate on one plate for three compounds, despite only eight compound dilutions (CLSI protocol allows two compounds in triplicate with 10 dilutions). For each bacterium to be evaluated, a 0.5 McFarland suspension (1 × 10 8 Add 5 µL of a 1:10 dilution of the bacterial suspension (CFU / mL) to obtain a final concentration of 5 x 10 4CFU / well. Bacterial suspensions were prepared using the growth method described by CLSI. Using a sterile loop, well-isolated colonies (3-5 from the agar plate) were selected and used to inoculate tubes containing 4 mL of CAMHB. These cultures were cultured in a 100 mL tube containing 100 mL of CAMHB. 600nm Incubate at 35 ± 2°C (usually 2-6 hours) until the turbidity reaches or exceeds that of a 0.5 McFarland standard, as determined by measuring the turbidity of the 0.5 McFarland standard. If growth exceeds that of a 0.5 McFarland standard, adjust the turbidity with medium until it is equivalent to that of a 0.5 McFarland standard.

[0347] Compound data are shown in Tables 4 and 5. IC ≥ 1 μM (% inhibition ≤ 50% at 1 μM) 50 Values ​​(μM) are indicated with "+". IC values ​​are 0.5 μM or more and less than 1 μM. 50 Values ​​(% inhibition >50% and ≦90% at 1 μM) are indicated with "++". IC less than 0.5 μM 50 Values ​​(inhibition %>90% at 1 μM) are indicated with "+++". MIC values ​​(μg / mL) ≥ 32 μg / mL are indicated with "+". MIC values ​​(μg / mL) ≥ 8 μg / mL and < 32 μg / mL are indicated with "++". MIC values ​​(μg / mL) < 8 μg / mL are indicated with "+++". "NA" means not applicable. [Table 4] [Table 5-1] [Table 5-2]

[0348] Other embodiments The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with reference to various specific and preferred embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be apparent to those skilled in the art that changes and modifications may be practiced within the scope of the appended claims. It is therefore to be understood that the above description is intended to be illustrative, rather than limiting. The scope of the invention should therefore be determined not with reference to the above description, but instead with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. Compounds of Formula I 【Chemical 119】 or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein: Ring A is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, phenyl, OH, NH ２ , NH(C １ -C ６ alkyl), N(C １ -C ６ alkyl) ２ , COOH, COO(C １ -C ６ alkyl), CONH ２ , CONH(C １ -C ６ alkyl), CON(C １ -C ６ alkyl) ２ and oxo; J is C １ -C ６ alkylene or C ３ -C ８ cycloalkylene, any of which is halo, OH, or C １ -C ６ Optionally substituted with alkoxy, １ -C ６ Up to two methylene units of the alkylene are optionally independently selected from O, S, SO, SO ２ or replaced by C=O; Y is a bond, or OH, NH ２ , CN, halo, or C １ -C ６ C optionally substituted with alkoxy １ -C ６ alkylene, １ -C ６ Up to two methylene units of the alkylene are optionally independently selected from O, NH, N—(C １ -C ６ alkyl), N-(C １ -C ６ hydroxyalkyl), N-(C １ -C ６ haloalkyl), N-(C １－６ Alkylene-C ３－８ cycloalkyl), N-(C ３－８ cycloalkyl), NH(C=O), N-(C １－６ alkyl)(C=O), or (C=O); Ring B is a 3- to 8-membered monocyclic cycloalkylene, a 3- to 8-membered monocyclic heterocycloalkylene, a 6- to 12-membered bicyclic cycloalkylene, or a 6- to 12-membered bicyclic heterocycloalkylene, each of which is selected from the group consisting of C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, COOH, COO(C １ -C ６ alkyl), CONH ２ , CONH(C １ -C ６ alkyl), CON(C １ -C ６ alkyl) ２ , and C １ -C ６ optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl; L is a bond or C １ -C ６ alkylene, １ -C ６ Up to two methylene units of the alkylene are independently O, NH, (C=O), NH(C=O), N-(C １－６ alkyl)(C═O), (C═NH), NH(C═N), or N—(C １－６ alkyl); R １ and R ２ are each independently C １ -C ６ Alkyl, halo, CN, OH, NH ２ , O(C １ -C ６ haloalkyl), NH(C １ -C ６ alkyl), N(C １ -C ６ alkyl) ２ , -COO(C １ -C ６ alkyl), CONH ２ , C １ -C ６ Haloalkyl, C １ -C ６ Alkoxy, and C １ -C ６ haloalkoxy; R ｘ , R ｙ , R ｘ’ , and R ｙ’ are each independently H, C １ -C ６ alkyl, or amino protecting groups; m and n are each independently 0, 1, 2, or 3; 【Chemical 120】 represents a single bond or a double bond, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

2. Ring A is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, phenyl, OH, NH ２ 10. The compound of claim 1, wherein R is a 5- to 6-membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of: , and oxo, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

3. Ring A is 【Chemistry 121】 wherein R ３ are each independently C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, phenyl, OH, NH ２ and oxo, and q is 0, 1, or 2, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

4. Ring A is 【Chemistry 122】 4. The compound of claim 3, wherein:

5. J is halo, OH, or C １ -C ６ C optionally substituted with alkoxy １ -C ６ alkylene, １ -C ６ Up to two methylene units of the alkylene are optionally independently selected from O, S, SO, SO ２ or C=O, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

6. J is C １ -C ６ alkylene, １ -C ６ 6. The compound of claim 5, wherein one methylene unit of the alkylene is replaced by C=O, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

7. J is 【Chemical 123】 7. The compound of claim 6, wherein:

8. R ｘ and R ｙ is each independently H, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Request Item 9】 【Chemistry 124】 2. The compound of claim 1, wherein: 【Request Item 10】 【Chemistry 125】 10. The compound of claim 9, wherein: 【Request Item 11】 【Chemistry 126】 wherein R ２ are each independently C １ -C ６ Alkyl, C １ -C ６ 2. The compound of claim 1, wherein n is selected from the group consisting of alkoxy, halo, and CN; and n is 0, 1, or 2, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Request Item 12】 【Chemistry 127】 wherein R ２ are each independently C １ -C ６ Alkyl, C １ -C ６ 12. The compound of claim 11, wherein n is selected from the group consisting of alkoxy, halo, and CN; and n is 0, 1, or 2, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Request Item 13】 【Chemistry 128】 13. The compound of claim 12, wherein:

14. Y is OH, NH ２ , halo, or C １ -C ６ C optionally substituted with alkoxy １ -C ３ alkylene, １ -C ３ One methylene unit of alkylene is O, NH, N—(C １ -C ６ alkyl), N-(C １ -C ６ hydroxyalkyl), N-(C １ -C ６ haloalkyl), N-(C ３－８ cycloalkyl), N-(C １－６ Alkylene-C ３－８ cycloalkyl), NH(C=O), N-(C １－６ 10. The compound of claim 1, wherein the compound is optionally substituted with (C═O) (alkyl), (C═O), or (C═O), or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

15. Y is C １ -C ３ alkylene, １ -C ３ One methylene unit of alkylene is NH, N—(C １ -C ６ haloalkyl), or N—(C １ -C ６ 15. The compound of claim 14, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, optionally substituted with (alkyl).

16. Y is -CH ２ -, -CH ２ NH-, -CH ２ NMe-, -CH ２ N (CH ２ CH ２ F) - and -CH ２ 15. The compound of claim 14, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: NEt-; 【Request Item 17】 【Chemistry 129】 10. The compound of claim 1, selected from the group consisting of: or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

18. Ring B is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, COOH, COO(C １ -C ６ alkyl), CONH ２ , CONH(C １ -C ６ alkyl), CON(C １ -C ６ alkyl) ２ , and C １ -C ６ 10. The compound of claim 1, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is a 3- to 8-membered monocyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl.

19. Ring B is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, and C １ -C ６ 19. The compound of claim 18, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, which is a 5-6 membered monocyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl.

20. Ring B is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, COOH, COO(C １ -C ６ alkyl), CONH ２ , CONH(C １ -C ６ alkyl), CON(C １ -C ６ alkyl) ２ , and C １ -C ６ 10. The compound of claim 1, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is a 3- to 8-membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl.

21. Ring B is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, and C １ -C ６ 21. The compound of claim 20, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein R is a 4- to 7-membered monocyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl, and Ring B contains up to two nitrogen atoms.

22. Ring B is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, Halo, C １ -C ６ Haloalkyl, OH, and C １ -C ６ 10. The compound of claim 1, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is a 6- to 10-membered bicyclic cycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl.

23. 23. The compound of claim 22, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein Ring B is a 6-10 membered fused, spiro, or bridged bicyclic cycloalkylene.

24. 24. The compound of claim 23, wherein Ring B is a 6- to 10-membered fused bicyclic cycloalkylene, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

25. 24. The compound of claim 23, wherein Ring B is a 6- to 10-membered bridged bicyclic cycloalkylene, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

26. 24. The compound of claim 23, wherein Ring B is a 6-10 membered spiro bicyclic cycloalkylene, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

27. Ring B is C １ -C ６ Alkyl, C １ -C ６ Alkoxy, Halo, C １ -C ６ Haloalkyl, OH, and C １ -C ６ 10. The compound of claim 1, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is a 6- to 12-membered bicyclic heterocycloalkylene optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl.

28. 28. The compound of claim 27, wherein Ring B is a fused, spiro, or bridged bicyclic heterocycloalkylene containing up to two nitrogen atoms, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

29. 29. The compound of claim 28, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein Ring B is a fused bicyclic heterocycloalkylene containing up to two nitrogen atoms.

30. 29. The compound of claim 28, wherein Ring B is a spiro bicyclic heterocycloalkylene containing up to two nitrogen atoms, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

31. 29. The compound of claim 28, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein Ring B is a bridged bicyclic heterocycloalkylene containing up to two nitrogen atoms.

32. Ring B is 【Chemistry 130】 10. The compound of claim 1, selected from the group consisting of: or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

33. 2. The compound of claim 1, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein L is a bond.

34. L is C １ -C ６ alkylene, １ -C ６ Up to two methylene units of the alkylene are optionally independently selected from O, NH, (C=O), NH(C=O), N—(C １－６ alkyl)(C═O), (C═NH), NH(C═N), or N—(C １－６ 10. The compound of claim 1, wherein R is substituted with R(R) or R(R) ...

35. L is C １ -C ６ 35. The compound of claim 34, which is alkylene, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

36. L is -CH ２ - or -CH ２ -CH ２ 36. The compound of claim 35, wherein: -, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

37. R ｘ’ and R ｙ’ is H, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Request Item 38】 【Chemistry 131】 10. The compound of claim 1, selected from the group consisting of: or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

39. R １ and R ２ are each independently C １ -C ６ Alkyl, halo, C １ -C ６ Haloalkyl, oxo, and C １ -C ６ alkoxy; and m and n are each independently 0, 1, or 2, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

40. R １ and R ２ are each independently C １ -C ６ Alkyl, halo, oxo, or C １ -C ６ 40. The compound of claim 39, wherein m is haloalkyl and m and n are each independently 0 or 1, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

41. 10. The compound of claim 1, which is a compound of formula IA, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Chemistry 132】

42. A compound of formula IA-1, 【Chemistry 133】 In the formula, R ３ are each independently H, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, NH ２ and oxo, wherein q is 0, 1, 2, or 3, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

43. A compound of formula IA-2, 【Chemistry 134】 wherein K is halo, hydroxyl, or C １ -C ６ C optionally substituted with an alkoxy group １ -C ４ 43. The compound of claim 42, wherein said compound is alkylene, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

44. A compound of formula IA-3, 【Chemistry 135】 In the formula, K is C １ -C ３ 44. The compound of claim 43, wherein said compound is alkylene, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

45. 45. The compound of claim 44, which is a compound of formula IA-4, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Transformation 136】

46. 46. ​​The compound of claim 45, which is a compound of formula IA-5, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Chemistry 137】

47. 47. The compound of claim 46, which is a compound of formula IA-6, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Chemistry 138】

48. 48. The compound of claim 47, which is a compound of formula IA-7, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof. 【Chemistry 139】

49. A compound of formula IA-7a, IA-7b, IA-7c, IA-7d, IA-7e, IA-7f, or IA-7g, [Chemistry 140] 【Chemistry 141】 In the formula, X １ are each independently CH or N, and p is 1, 2, or 3, or a single stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

50. Y is -CH ２ -, -CH ２ NH-, -CH ２ NMe-, -CH ２ NCH ２ CH ２ F- and -CH ２ 50. The compound of any one of claims 41 to 49, selected from the group consisting of: NEt-, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

51. Ring B is 【Chemistry 142】 49. The compound of any one of claims 41 to 48, selected from the group consisting of: or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

52. L is a bond or C １ -C ３ 50. The compound of any one of claims 41 to 49, which is alkylene, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

53. A compound shown in Table 1 and Table 2, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

54. A compound shown in Table 2, or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

55. 10. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

56. A composition comprising a compound according to any one of claims 1 to 49, 53, 54, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 55, for the treatment of a bacterial infection in a patient in need thereof.

57. 57. The composition or pharmaceutical composition of claim 56, wherein the bacterial infection is caused by bacteria, including gram-positive and gram-negative bacteria.

58. The bacteria are Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Bacillus anthracis, Yersinia pestis, Salmonella, Clostridium 58. The composition or pharmaceutical composition of claim 57, comprising Bacillus subtilis, Bacillus difficile, Citrobacter, Enterobacter, Burkholderia, cepacia, Mycobacterium, Proteus, Streptococcus, Serratia, Enterobacteriaceae, Escherichia, Klebsiella, Pseudomonas, and Acinitobacter.

59. Compounds of Formula I 【Chemistry 143】 or a single stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, comprising: a compound of formula E; 【Chemistry 144】 Formula D 【Chemistry 145】 or formula D' 【Chemistry 146】 under reductive amination conditions to obtain the compound of formula I; In the formula, ring A, ring B, J, L, R １ , R ２ , R ｘ , R ｙ , R ｘ’ , R ｙ’ , m, and n are defined as in claim 1; Ring B １ is a nitrogen-containing 3- to 8-membered monocyclic heterocycloalkylene or a nitrogen-containing 6- to 12-membered bicyclic heterocycloalkylene, each of which is １ -C ６ Alkyl, C １ -C ６ Alkoxy, halo, CN, C １ -C ６ Haloalkyl, OH, COOH, COO(C １ -C ６ alkyl), CONH ２ , CONH(C １ -C ６ alkyl), CON(C １ -C ６ alkyl) ２ , and C １ -C ６ optionally substituted with up to three substituents independently selected from the group consisting of hydroxyalkyl; Y is C １ -C ６ alkylene, １ -C ６ One methylene unit of alkylene is NH or N—(C １ -C ６ alkyl); Y １ is C １ -C ６ alkylene, １ -C ６ one methylene unit of the alkylene is replaced with (C=O); R ４ is H or C １ -C ６ The above step, wherein the alkyl is alkyl.

60. Compound of Formula E 【Chemistry 147】 or a pharmaceutically acceptable salt thereof, wherein rings A, J, R １ , R ２ , R ｘ , R ｙ , m, and n are defined as in claim 1; Y １ is C １ -C ６ alkylene, １ -C ６ The compound or a pharmaceutically acceptable salt thereof, wherein one methylene unit of the alkylene is replaced with (C=O).

61. The compound is 【Chemistry 148】 61. The compound of claim 60, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

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