Inhibitors of plasma kallikrein

Abstract

The present disclosure provides compounds of formula (IA) and pharmaceutical compositions thereof that are useful for inhibiting the activity of plasma kallikrein, and are useful in methods of therapy and treatment of diseases and conditions, such as ocular disorders. TIFF2025526464000125.tif37165

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 393,427, filed July 29, 2022, and 63 / 460,204, filed April 18, 2023, the entireties of which are incorporated as if fully set forth herein. [Background technology]

[0002] background Plasma kallikrein is a serine protease that circulates in the blood as an inactive precursor, prekallikrein, and participates in the surface-mediated defense system via high molecular weight kininogen (HK), which is involved in the activation and signal transduction of factor XII. Elements of the kallikrein-kinin system (KKS) are involved in activities such as surface-mediated defense responses, regulation of blood flow, fibrin deposition, blood pressure, smooth muscle contractility, nociception, electrolyte transport, and mediator release. See Donald H. Miller, Harry S. Margolius, Chapter 19 The kallikrein-kinin-kininogen system, Editor(s): E. Edward Bittar, Neville Bittar, Principles of Medical Biology, Elsevier, Volume 8, 1997, Pages 363-384 (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Donald H. Miller, Harry S. Margolius, Chapter 19 The kallikrein-kinin-kininogen system, Editor(s): E. Edward Bittar, Neville Bittar, Principles of Medical Biology, Elsevier, Volume 8, 1997, Pages 363-384 Summary of the Invention

[0004] overview The present disclosure provides small molecule inhibitors of plasma kallikrein and methods of using such inhibitors to treat diseases. Accordingly, in various embodiments, the present disclosure provides a compound of formula (IA) or a pharmaceutically acceptable salt thereof: TIFF2025526464000002.tif37165

[0005] In formula (IA), D 1 is N or CR 1 and D 2 is N or CR 2 and D 3 is N or CR 3 and D 4 is N or CR 4 Furthermore, D 1 , D 2 , D 3 , and D 4 Not more than three of these are simultaneously N.

[0006] Substituent R 1 , R 2 , R 3 , and R 4 are independently H, C2-C6-alkenyl, C1-C6-haloalkyl, halo, NR a R b , OR a , -NR a C(O)R b , -C(O)R a , -C(O)halo, -OC(O)R a , -OC(O)ORa , -C(O)OR a , C6~C 10 -aryl, CN, -S(O) 0~2 R a , -S(O)2OR a and NO2.

[0007] Each R a and R b is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0008] Substituent R c1 , R c2 , and R c3 is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0009] Q1, Q2, and Q3 are independently CR 5 , N, O, and S.

[0010] R 5 is H, C1-C6 alkyl, OR a , -(C1-C6-alkyl)OR a , and C3~C 10 -cycloalkyl.

[0011] The ring member P is C or N.

[0012] L 1 is -SO2- or -C1-C8-alkylene-.

[0013] portion TIFF2025526464000003.tif6165 is C3~C 10 -Cycloalkyl, C6-C 10- a divalent monocyclic or bicyclic moiety selected from the group consisting of aryl, 3-10 membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof.

[0014] L 2 represents a bond; -C1-C8-alkylene; -C2-C8-alkenylene; -C2-C8-alkynylene; and C3-C 10 -Cycloalkyl, C3-C 10 -Cycloalkenyl, C6-C 10 -aryl, 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), (C1-C8-alkyl)C3-C 10 -Cycloalkyl, (C1-C8-alkyl)C3-C 10 -Cycloalkenyl, (C1-C8-alkyl)C6-C 10 -a divalent moiety selected from the group consisting of aryl, and (C1-C8-alkyl) 5-10 membered heteroaryl, where 1-4 heteroaryl members are independently selected from N, O, and S. 2 In the formula (I), any cycloalkyl, cycloalkenyl, aryl, and heteroaryl are monocyclic or bicyclic. 2 may be substituted with 1 to 3 substituents selected from the group consisting of C1-C6-alkyl, C1-C6-haloalkyl, and CN.

[0015] Z is -OR c , -OC(O)R c , -OC(O)NR c R d , -S(O) 0~2 R c , -CN, -C(O)R c , -C(O)OR c , -C(O)NR c R d , -C(S)R c , -NRc R d , =NR c , -NR c C(O)NR c R d , -NR c CO2R d , -NR c -NO, -NO2, -NR c -OR d , -N=C=O, -N=C=S, and -NR c -NR c R d selected from the group consisting of R c and R d Each example is independently H, C1-C6-alkyl, C1-C6-haloalkyl, and C6-C 10 -aryl.

[0016] In another embodiment, the compound is one of Formula I. TIFF2025526464000004.tif37165

[0017] In Formula I, the moiety TIFF2025526464000005.tif6165 is i) a cyclic hydrocarbon, bicyclic hydrocarbon, or heterocycle containing up to 10 atoms consisting of C or N, or R is ii) selected from the group consisting of cyclopentyl, cyclohexyl, phenyl, naphthyl, pyridine, pyrimidine, indene, 2,3-dihydro-1H-indene, or any saturated and unsaturated cyclic hydrocarbon or heterocycle thereof.

[0018] Z is -OH, -OR', -OC(O)H, -OC(O)R', -OC(O)NH2, -OC(O)NHR', -OC(O)NH(R')2, -SH, -SR', -S(O)R', -S(O)2R', -CN, -C( O)H, -C(O)R', -C(O)OH, -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)NH(R')2, -C(S)R', -NH2, -NH2R', -NR'2, =NH, =NR', - and -NHC(O)NH, -NR'C(O)NH, -NR'C(O)NHR', -NR'C(O)N(R'), -NHCOH, -NHCOR', -NR'COR', -NH-NO, -NR'-NO, -NO, -NH-OH, -OH, -NR'-OR', -N=C=O, -N=C=S, -NH-NH, and -NH-NHR', wherein each R' is independently alkyl or alkyl halide. In some embodiments, Z is not halogen or hydrogen.

[0019] Q1, Q2 and Q3 are selected from the group consisting of C, N, O or S.

[0020] P is selected from the group consisting of C or N.

[0021] L1 is a linking group selected from the group consisting of a C1-C8 alkyl linker or SO2.

[0022] L2 is selected from: i) a hydrocarbon that does not contain a double bond to O or S, ii) a hydrocarbon that does not contain heteroatoms such as O, N, or S, or iii) a linking group selected from the group consisting of C1-C8 alkyl linkers including saturated hydrocarbons, unsaturated hydrocarbons, branched hydrocarbons, cyclic hydrocarbons, and combinations thereof, wherein the cyclic hydrocarbon is selected from the group consisting of cyclopentyl, cyclohexyl, phenyl, naphthyl, indene, 2,3-dihydro-1H-indene, or any saturated or unsaturated cyclic hydrocarbon thereof. In some embodiments, L2 is a hydrocarbon that contains a heteroatom such as O, N, or S.

[0023] In some embodiments, the compositions comprise a compound of formula I and a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description The present disclosure provides compounds, compositions, and methods for inhibiting plasma kallikrein ("PKal"), which, in exemplary embodiments, are useful in treating inflammatory and ocular disorders.

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly requires otherwise. Thus, for example, a reference to "an agent" includes a plurality of such agents, a reference to "the cell" includes a reference to one or more cells (or cells) and equivalents thereof known to those skilled in the art, and so forth.

[0027] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. When referring to a number or range of numbers, the term "about" means that the referenced number or range of numbers is an approximation within experimental variation (or within statistical experimental error), and thus, in some cases, the number or range of numbers varies by 1% to 15% of the stated number or range of numbers.

[0028] In this disclosure, the number of atoms of a particular element in a substituent is generally given as a range (e.g., alkyl groups containing 1 to 4 carbon atoms or C1~4 Reference to such a range is intended to include specific reference to groups having each integer number of atoms within the specified range. For example, an alkyl group of 1 to 4 carbon atoms includes each of C1, C2, C3, and C4. For example, C 1~12 Heteroalkyl includes 1 to 12 carbon atoms in addition to one or more heteroatoms. Other numbers of atoms and types of atoms can be indicated in a similar manner.

[0029] In other specific embodiments, the term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude embodiments, such as, for example, any compositions of matter, compositions, methods, or processes described herein, from "consisting of" or "consisting essentially of" the recited features.

[0030] "Alkyl" refers to a straight or branched chain hydrocarbyl containing from 1 to about 20 carbon atoms. For example, an alkyl can have from 1 to 10 carbon atoms or from 1 to 6 carbon atoms. Exemplary alkyls include straight chain alkyl groups such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like, as well as branched chain isomers of straight chain alkyl groups (for example, but not limited to, -CH(CH3)2, -CH(CH3)(CH2CH3), -CH(CH2CH3)2, -C(CH3)3, -C(CH2CH3)3, -CH2CH(CH3)2, -CH2CH(CH3)(C H2CH3), -CH2CH(CH2CH3), -CH2C(CH3), -CH2C(CH2CH3), -CH(CH3)CH(CH3)(CH2CH3), -CH2CH2CH(CH3)2, -CH2CH2CH(CH3)(CH2CH3), -CH2CH2CH(CH2CH3), -CH2CH2CH(CH2CH3), -CH2CH2C(CH3), -CH2CH2C(CH2CH3), -CH(CH3)CH2CH(CH3)2, -CH(CH3)CH(CH3)CH(CH3)2, etc.). Thus, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. Alkyl groups can be unsubstituted or optionally substituted with one or more substituents described herein, such as, for example, halogen(s).

[0031] The terms "halogen," "halide," and "halo" refer to -F or fluoro, -Cl or chloro, -Br or bromo, or -I or iodo, respectively.

[0032] The term "alkenyl" refers to a straight- or branched-chain hydrocarbyl group containing 2 to about 20 carbon atoms and having 1 to 3, 1 to 2, or at least one carbon-carbon double bond. Alkenyl groups can be unsubstituted or optionally substituted with one or more substituents described herein.

[0033] "Alkyne" or "alkynyl" refers to a straight- or branched-chain unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. Examples of (C2-C8)alkynyl groups include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. Alkynyl groups can be unsubstituted or optionally substituted with one or more substituents described herein.

[0034] The term "cycloalkyl" refers to a saturated monocyclic, bicyclic, tricyclic, or polycyclic 3- to 14-membered ring system (e.g., C3-C8 cycloalkyl). The cycloalkyl can be attached via any atom. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be unsubstituted or optionally substituted with one or more substituents described herein.

[0035] Embodiments of plasma kallikrein inhibitors include compounds comprising a C1-C8 alkyl linker. 1~8 "Alkyl" can be characterized by a branched or unbranched hydrocarbon group having 1 to 8 carbon atoms. 1~8 Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, and cyclopentyl.

[0036] "Aryl" (Ar), used alone or as part of another term, means a carbocyclic aromatic group, whether fused or not, having the specified number of carbon atoms, or up to 14 carbon atoms if no number is specified (C6-C8). 10 -Aryl or C6-C 14-aryl, etc.). In embodiments, Ar may be characterized by an aromatic group having a ring system made up of carbon atoms with conjugated π electrons (e.g., phenyl). The term includes aryl groups having 6 to 12 carbon atoms. Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, each ring having 5 or 6 members. Examples of aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, etc. (See, e.g., Lang's Handbook of Chemistry (Dean, JA, ed.) 13 th ed. Table 7-2

[1985] ). "Aryl" also contemplates aryl rings that are part of a fused polycyclic system, such as an aryl fused to a cycloalkyl, as defined herein. An exemplary aryl is phenyl. Aryl groups can be unsubstituted or optionally substituted with one or more substituents described herein.

[0037] The term "heteroatom" refers to N, O, and S. Compounds of the present disclosure containing an N or S atom can be optionally oxidized to the corresponding N-oxide, sulfoxide, or sulfone compound.

[0038] "Heteroaryl," alone or in combination with any other moiety described herein, is a monocyclic aromatic ring structure containing 5 to 10 (e.g., 5 or 6) ring atoms or a bicyclic aromatic group having 8 to 10 atoms, containing one or more (e.g., 1 to 4, 1 to 3, or 1 to 2) heteroatoms independently selected from the group consisting of O, S, and N. Heteroaryl is also intended to include oxidized S or N (such as sulfinyl, sulfonyl, and N-oxide of a tertiary ring nitrogen). A carbon or heteroatom is the point of attachment of the heteroaryl ring structure such that a stable compound results. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrazinyl, quinaoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, and indolyl. Heteroaryl groups can be unsubstituted or optionally substituted with one or more substituents described herein.

[0039] A "heterocycloalkyl" is a saturated or partially unsaturated non-aromatic monocyclic, bicyclic, tricyclic, or polycyclic ring system having 3 to 14 (e.g., 3 to 6) atoms, in which 1 to 3 carbon atoms in the ring are replaced by heteroatoms O, S, or N. Ring heteroatoms can also include oxidized S or N (e.g., sulfinyl, sulfonyl, and N-oxide of a tertiary ring nitrogen). A heterocycloalkyl can be fused to another ring system (e.g., to a 5- or 6-membered aryl or heteroaryl). The point of attachment of the heterocycloalkyl ring is on a carbon or heteroatom such that a stable ring is maintained. Examples of heterocycloalkyl groups include, but are not limited to, morpholino, tetrahydrofuranyl, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydrobenzofuryl, and dihydroindolyl. A heterocycloalkyl group can be unsubstituted or optionally substituted with one or more substituents described herein.

[0040] The terms "nitrile" or "cyano" may be used interchangeably and refer to the group --CN.

[0041] The compounds described herein may exist in various isomeric forms, including configurational isomers, geometric isomers, and conformational isomers (e.g., including cis- or trans-conformations). Compounds may also exist in one or more tautomeric forms, including both single tautomers and tautomeric mixtures. The term "isomer" is intended to encompass all isomeric forms of the disclosed compounds, including tautomeric forms. The disclosed compounds may also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms may result from loss of water. The specific composition of the open-chain and cyclized forms may depend on how the compound is isolated, stored, or administered. For example, a compound may exist predominantly in an open-chain form under acidic conditions, but may cyclize under neutral conditions. All forms are included in the present disclosure.

[0042] Some compounds described herein may have asymmetric centers and therefore may exist in various enantiomeric and diastereomeric forms. The compounds described herein may be in the form of optical isomers or diastereoisomers. Thus, the present disclosure encompasses the compounds described herein and their use in the form of optical isomers, diastereoisomers, and mixtures thereof (including racemic mixtures). Optical isomers of the compounds of the present disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology, or by chemical separation of stereoisomers using optically active resolving agents.

[0043] Unless otherwise indicated, the term "stereoisomer" means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of that compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereoisomers of that compound. A typical stereoisomerically pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of the other stereoisomer of the compound (e.g., more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomer of the compound, or more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomer of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomer of the compound, or more than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomer of the compound). The above stereoisomers can be considered as compositions containing two stereoisomers present in the respective weight percentages described herein.

[0044] In the event of a discrepancy between a depicted structure and the name given to that structure, the depicted structure shall prevail. Additionally, if the stereochemistry of a structure or portion of a structure is not indicated, for example, by a bold or dashed line, that structure or portion of a structure shall be interpreted as encompassing all stereoisomers thereof. However, in some cases, when two or more chiral centers are present, the structure and name may be expressed as a single enantiomer to facilitate the description of the relative stereochemistry. Those skilled in the art of organic synthesis will know whether a compound is prepared as a single enantiomer from the method used to prepare the compound.

[0045] As used herein, unless otherwise specified to the contrary, the term "compound" is inclusive in that it encompasses a compound, or a pharmaceutically acceptable salt, stereoisomer, isotopic substitution, and / or tautomer thereof. Thus, a compound includes, for example, pharmaceutically acceptable salts of tautomers of the compound. Similarly, a compound includes pharmaceutically acceptable salts of isotopic substitutions of the compound.

[0046] In the present disclosure, a "pharmaceutically acceptable salt" is a pharmaceutically acceptable organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, for example, alkali metal salts, alkaline earth salts, ammonium salts, water soluble and water insoluble salts, such as acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxybenzoate ... salts such as hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, embonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate. A pharmaceutically acceptable salt may have more than one charged atom in its structure. In this case, the pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0047] The terms "treat," "treating," and "treatment" refer to the amelioration or eradication of a disease or symptoms associated with a disease. In various embodiments, these terms refer to the minimization or slowing of the spread, progression, or worsening of a disease resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient with such a disease.

[0048] The terms "prevent," "preventing," and "prevention" refer to preventing the onset, recurrence, or spread of disease in a patient due to the administration of a compound described herein.

[0049] The term "effective amount" refers to an amount of a compound or other active ingredient described herein sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease, or sufficient to delay or minimize symptoms associated with a disease. Furthermore, a therapeutically effective amount with respect to a compound described herein means an amount of a therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. The term, when used in connection with a compound described herein, can encompass an amount that improves overall treatment, an amount that reduces or avoids the symptoms or causes of a disease, or an amount that enhances the therapeutic effect of or is synergistic with another therapeutic agent.

[0050] A "patient" or "subject" includes animals such as humans, cows, horses, sheep, lambs, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs. According to some embodiments, the animals are mammals, such as non-primates and primates (e.g., monkeys and humans). In one embodiment, the patient is a human, such as a human infant, child, adolescent, or adult. In this disclosure, the terms "patient" and "subject" are used interchangeably.

[0051] Plasma kallikrein inhibitors Compounds of Formula IA In some embodiments, the inhibitor is a compound of formula (IA) or a pharmaceutically acceptable salt thereof. TIFF2025526464000006.tif37165

[0052] In formula (IA), D 1 is N or CR 1 and D 2 is N or CR 2 and D 3 is N or CR 3 and D 4 is N or CR 4 Furthermore, D 1 , D 2 , D 3 , and D 4 Not more than three of these are simultaneously N.

[0053] Substituent R 1 , R 2 , R 3 , and R 4 are independently H, C2-C6-alkenyl, C1-C6-haloalkyl, halo, NR a R b , OR a , -NR a C(O)R b , -C(O)R a , -C(O)halo, -OC(O)R a , -OC(O)OR a , -C(O)OR a , C6~C 10 -aryl, CN, -S(O) 0~2 R a , -S(O)2OR a and NO2.

[0054] Each R a and R b is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0055] Substituent R c1 , R c2 , and R c3is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0056] Q1, Q2, and Q3 are independently CR 5 , N, O, and S.

[0057] R 5 is H, C1-C6 alkyl, OR a , -(C1-C6-alkyl)OR a , and C3~C 10 -cycloalkyl.

[0058] The ring member P is C or N.

[0059] L 1 is -SO2- or -C1-C8-alkylene-.

[0060] portion TIFF2025526464000007.tif6165 is C3~C 10 -Cycloalkyl, C6-C 10 - a divalent monocyclic or bicyclic moiety selected from the group consisting of aryl, 3-10 membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof.

[0061] L 2 represents a bond; -C1-C8-alkylene; -C2-C8-alkenylene; -C2-C8-alkynylene; and C3-C 10 -Cycloalkyl, C3-C 10 -Cycloalkenyl, C6-C 10 -aryl, 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), (C1-C8-alkyl)C3-C 10 -Cycloalkyl, (C1-C8-alkyl)C3-C10 -Cycloalkenyl, (C1-C8-alkyl)C6-C 10 -a divalent moiety selected from the group consisting of aryl, and (C1-C8-alkyl) 5-10 membered heteroaryl, where 1-4 heteroaryl members are independently selected from N, O, and S. 2 In the formula (I), any cycloalkyl, cycloalkenyl, aryl, and heteroaryl are monocyclic or bicyclic. 2 may be substituted with 1 to 3 substituents selected from the group consisting of C1-C6-alkyl, C1-C6-haloalkyl, and CN.

[0062] Z is -OR c , -OC(O)R c , -OC(O)NR c R d , -S(O) 0~2 R c , -CN, -C(O)R c , -C(O)OR c , -C(O)NR c R d , -C(S)R c , -NR c R d , =NR c , -NR c C(O)NR c R d , -NR c CO2R d , -NR c -NO, -NO2, -NR c -OR d , -N=C=O, -N=C=S, and -NR c -NR c R d selected from the group consisting of R c and R d Each example is independently H, C1-C6-alkyl, C1-C6-haloalkyl, and C6-C 10 -aryl.

[0063] In some embodiments, D 1 , D 2, D 3 , and D 4 In another embodiment, three of the D 1 , D 2 , D 3 , and D 4 and two of are N. In yet another embodiment, D 1 , D 2 , D 3 , and D 4 is N. In still further embodiments, D 1 , D 2 , D 3 , and D 4 At most one of is N. In an exemplary embodiment, D 1 , D 2 , D 3 , and D 4 The ring containing: TIFF2025526464000008.tif32165.

[0064] In certain embodiments, D 1 , D 2 , D 3 , and D 4 The ring containing: TIFF2025526464000009.tif32165.

[0065] In various embodiments, R 1 , R 2 , R 3 , and R 4 are independently H, OR a In some embodiments, R is selected from the group consisting of , halo, and CN. 1 , R 2 , R 3 , and R 4 is independently selected from the group consisting of H and halo. For example, in some embodiments, R 1 , R 2 , R 3 , and R 4 In another embodiment, at least one of R 1 , R 2 , R3 , and R 4 Each of is H.

[0066] According to various embodiments, some compounds of formula (IA) may comprise R c1 , R c2 , and R c3 is H. Thus, for example, some embodiments include 1 , D 2 , D 3 , and D 4 The ring containing: The compound is provided as TIFF2025526464000010.tif32165.

[0067] In various embodiments, Q 1 is C and Q 2 and Q 3 Each of these is independently CR 5 , N, O, and S, and P is selected from the group consisting of C and N. In one embodiment, P is N. In a further embodiment, Q 2 is CR 5 and Q 3 is N or Q 2 is N and Q 3 is CR 5 In an exemplary embodiment, P, Q 1 , Q 2 , and Q 3 The ring containing: The compound is TIFF2025526464000011.tif32165.

[0068] In additional embodiments, L 1 is -C1-C8-alkylene- (such as -C1-C3-alkylene-). In an exemplary embodiment, L 1 is methylene.

[0069] In other embodiments, the moiety TIFF2025526464000012.tif6165 is a divalent monocyclic C6-C 10In an exemplary embodiment, TIFF2025526464000013.tif6165 is TIFF2025526464000014.tif22165.

[0070] The present disclosure provides, in additional embodiments, compounds of formula (IA), wherein L 2 -C1~C8-alkylene, C6~C 10 -aryl, and -(C1-C8-alkyl)C6-C 10 -aryl, any of which may be substituted as described herein. In some embodiments, L 2 is -C1-C8-alkylene (such as -C1-C3-alkylene). As generally described herein, the alkyl (or alkylene) moiety can be linear or branched. In other embodiments, L 2 C6~C 10 -aryl (including phenyl).

[0071] In some embodiments, the moiety Z is -OR c , CN, -C(O)OR c , and -C(O)NR c R d In various embodiments, each R c and R d are independently H or C1-C6-alkyl. Thus, for example, Z can be selected from the group consisting of OH, OCH3, -COOH, -C(O)NH2, and -C(O)NHCH3. In another embodiment, Z is CN.

[0072] Compounds of Formula I In some embodiments, the present disclosure provides PKal inhibitors of Formula I: TIFF2025526464000015.tif37165 wherein R is i) a cyclic hydrocarbon, bicyclic hydrocarbon, or heterocycle containing up to 10 atoms consisting of C or N, or R is ii) selected from the group consisting of cyclopentyl, cyclohexyl, phenyl, naphthyl, pyridine, pyrimidine, indene, 2,3-dihydro-1H-indene, or any saturated and unsaturated cyclic hydrocarbon or heterocycle thereof.

[0073] In some embodiments, Z is -OH, -OR', -OC(O)H, -OC(O)R', -OC(O)NH2, -OC(O)NHR', -OC(O)NH(R')2, -SH, -SR', -S(O)R', -S(O)2R', -CN, -C(O)H, -C(O)R', -C(O)OH, -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)NH(R')2, -C(S)R', -NH2, -NH2R', -NR'2, ═NH, ═NR′, —NHC(O)NH, —NR′C(O)NH, —NR′C(O)NHR′, —NR′C(O)N(R′), —NHCOH, —NHCOR′, —NR′COR′, —NH—NO, —NR′—NO, —NO, —NH—OH, —OH, —NR′—OR′, —N═C═O, —N═C═S, —NH—NH, and —NH—NHR′, where each R′ is independently alkyl or alkyl halide. In some embodiments, Z is not halogen or hydrogen.

[0074] In some embodiments, Q1, Q2, and Q3 are selected from the group consisting of C, N, O, or S.

[0075] In some embodiments, P is selected from the group consisting of C or N.

[0076] In some embodiments, L1 is a linking group selected from the group consisting of a C1-C8 alkyl linker or SO2.

[0077] In some embodiments, L2 is selected from: i) a hydrocarbon that does not contain a double bond to O or a double bond to S, ii) a hydrocarbon that does not contain a heteroatom such as O, N, or S, or iii) a linking group selected from the group consisting of a C1-C8 alkyl linker comprising a saturated hydrocarbon, an unsaturated hydrocarbon, a branched hydrocarbon, a cyclic hydrocarbon, and combinations thereof, wherein the cyclic hydrocarbon is selected from the group consisting of cyclopentyl, cyclohexyl, phenyl, naphthyl, indene, 2,3-dihydro-1H-indene, or any saturated or unsaturated cyclic hydrocarbon thereof. In some embodiments, L2 is a hydrocarbon that contains a heteroatom such as O, N, or S.

[0078] In some embodiments, the compositions comprise a compound of formula I and a pharmaceutically acceptable salt thereof.

[0079] In some embodiments, the compounds include subspecies where Q1, Q2, and Q3 are selected from the group consisting of C and N, P is N, L1 is CH2, R is a para-substituted benzene, L2 is a methylene or ethylene alkyl linker in the para position of the R group, and Z is selected from the group consisting of CN and OH.

[0080] Additional embodiments include those listed below: Q1 is C, Q2 is C, Q3 is N, L2 is a methylene linker and Z is CN, Q1 is C, Q2 is C, Q3 is N, L2 is a methylene linker, and Z is OH; Q1 is C, Q2 is C, Q3 is N, L2 is an ethylene linker and Z is CN, Q1 is C, Q2 is N, Q3 is C, L2 is an ethylene linker and Z is CN, Q1, Q2, and Q3 are selected from the group consisting of C or N, P is N, L1 is CH2, R is benzene, L2 is selected from the group consisting of a branched ethyl linker and an isopropyl linker in the para position of the R group, and Z is a nitrile group (CN); Q1 is C, Q2 is C, Q3 is N, and L2 is a branched ethyl linker; Q1 is C, Q2 is N, Q3 is C, and L2 is a branched ethyl linker; Q1 is C, Q2 is C, Q3 is N, and L2 is an isopropyl linker; Q1 is C, Q2 is N, Q3 is C and L2 is an isopropyl linker; Q1 is C, Q2 is selected from the group consisting of N, O or S, Q3 is selected from the group consisting of C or N, L1 is a methylene or ethylene linker, R is benzene, L2 is a methylene linker in the para position of the R group, and Z is CN; Q2 is N, Q3 is C, P is N and L1 is a methylene linker, Q2 is O, Q3 is N, P is C and L1 is a methylene linker, Q2 is S, Q3 is N, P is C and L1 is a methylene linker; Q2 is C, Q3 is N, P is N and L1 is an ethylene linker, Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is a phenyl linker in the para position of the R group, and Z is CN; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is a benzyl linker in the para position of the R group, and Z is CN in the para position; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is a methylene or ethylene linker in the para position of the R group, and Z is selected from the group consisting of a methoxy group, a carboxylic acid, an amide, and an amide substituted with a C1 alkyl (methyl) group bonded to N, Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is an ethylene linker in the para position of the R group, and Z is a methoxy group; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is a methylene linker in the para position of the R group, and Z is a carboxylic acid; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is a methylene linker in the para position of the R group, and Z is an amide; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is a methylene linker in the para position of the R group, and Z is an amide substituted with a C1 alkyl (methyl) group attached to N; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is cyclopentadiene or contains a methylene linker at the 1-position of the cyclopentadiene attached to the para-position of the R group, and Z is CN attached to the 3-position of the cyclopentadiene; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is cyclopentadiene attached to the para position of the R group, and Z is CN attached to the 3 position of the cyclopentadiene; Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, R is benzene, L2 is cyclopentadiene with a methylene linker at the 1-position attached to the para-position of the R group, and Z is CN attached to the 3-position of the cyclopentadiene.

[0081] Additional embodiments include those listed below: Q1 is C, Q2 is N, Q3 is C, P is N, L1 is CH2, B is 1H-indene and Z is CN, Q1 is C, Q2 is C, Q3 is N, P is N, L1 is CH2, B is 2,3-dihydro-1H-indene and Z is CN.

[0082] Compound of Formula IB In an additional embodiment, optionally in combination with any other embodiment described herein, the present disclosure provides a compound of formula (IB): or a pharmaceutically acceptable salt thereof: TIFF2025526464000016.tif42165

[0083] Q1, Q2, and Q3 are independently CR 5 , N, O, and S.

[0084] R 5 is H, C1-C6 alkyl, OR a , -(C1-C6-alkyl)OR a , and C3~C 10 -cycloalkyl, wherein each R a is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0085] P is C or N.

[0086] L 1 is -SO2- or -C1-C8-alkylene-.

[0087] TIFF2025526464000017.tif6165 is C3~C 10 -Cycloalkyl, C6-C 10 - a divalent monocyclic or bicyclic moiety selected from the group consisting of aryl, 3-10 membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof.

[0088] L 2 represents a bond; -C1-C8-alkylene; -C2-C8-alkenylene; -C2-C8-alkynylene; and C3-C 10 -Cycloalkyl, C3-C 10 -Cycloalkenyl, C6-C 10 -aryl, 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), (C1-C8-alkyl)C3-C 10 -Cycloalkyl, (C1-C8-alkyl)C3-C 10 -Cycloalkenyl, (C1-C8-alkyl)C6-C 10 -a divalent moiety selected from the group consisting of cycloalkyl, cycloalkenyl, aryl, and (C1-C8-alkyl) 5-10 membered heteroaryl, where 1-4 heteroaryl members are independently selected from N, O, and S; wherein cycloalkyl, cycloalkenyl, aryl, and heteroaryl are monocyclic or bicyclic.

[0089] L 2 may be substituted with 1 to 3 substituents selected from the group consisting of C1-C6-alkyl, C1-C6-haloalkyl, and CN.

[0090] Z is -OR c , -OC(O)R c , -OC(O)NR c R d , -S(O) 0~2 Rc , -CN, -C(O)R c , -C(O)OR c , -C(O)NR c R d , -C(S)R c , -NR c R d , =NR c , -NR c C(O)NR c R d , -NR c CO2R d , -NR c -NO, -NO2, -NR c -OR d , -N=C=O, -N=C=S, and -NR c -NR c R d is selected from the group consisting of:

[0091] R c and R d Each example is independently H, C1-C6-alkyl, C1-C6-haloalkyl, and C6-C 10 -aryl.

[0092] Compound of formula IB-1 In an additional embodiment, optionally in combination with any other embodiment described herein, the present disclosure provides a compound of formula (IB-1) or a pharmaceutically acceptable salt thereof: TIFF2025526464000018.tif58165

[0093] Q1, Q2, and Q3 are independently CR 5 , N, O, and S.

[0094] R 5 is H, C1-C6 alkyl, OR a , -(C1-C6-alkyl)OR a , and C3~C 10 -cycloalkyl, wherein each R ais independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0095] P is C or N.

[0096] R e and R f are each independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0097] Compound of formula IC In an additional embodiment, optionally in combination with any other embodiment described herein, the present disclosure provides a compound of formula (IC) or a pharmaceutically acceptable salt thereof: TIFF2025526464000019.tif37165

[0098] D 1 is N or CR 1 and D 2 is N or CR 2 and D 3 is N or CR 3 and D 4 is N or CR 4 where D 1 , D 2 , D 3 , and D 4 Not more than three of these are simultaneously N.

[0099] R 1 , R 2 , R 3 , and R 4 are independently H, C2-C6-alkenyl, C1-C6-haloalkyl, halo, NR a R b , OR a , -NR a C(O)R b , -C(O)R a , -C(O)halo, -OC(O)R a , -OC(O)OR a , -C(O)OR a , C6~C 10-aryl, CN, -S(O) 0~2 R a , -S(O)2OR a and NO2.

[0100] Each R a and R b is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0101] R c1 , R c2 , and R c3 is independently selected from the group consisting of H, C1-C6-alkyl, and C1-C6-haloalkyl.

[0102] Q1, Q2, and Q3 are independently CR 5 , N, O, and S.

[0103] R 5 is H, C1-C6 alkyl, OR a , -(C1-C6-alkyl)OR a , and C3~C 10 -cycloalkyl.

[0104] P is C or N.

[0105] L 1 is -SO2- or -C1-C8-alkylene-.

[0106] TIFF2025526464000020.tif6165 is C3~C 10 -Cycloalkyl, C6-C 10 - a divalent monocyclic or bicyclic moiety selected from the group consisting of aryl, 3-10 membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof.

[0107] L 2 represents a bond; -C1-C8-alkylene; -C2-C8-alkenylene; -C2-C8-alkynylene; and C3-C 10 -Cycloalkyl, C3-C 10 -Cycloalkenyl, C6-C 10 -aryl, 5-10 membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), (C1-C8-alkyl)C3-C 10 -Cycloalkyl, (C1-C8-alkyl)C3-C 10 -Cycloalkenyl, (C1-C8-alkyl)C6-C 10 -a divalent moiety selected from the group consisting of cycloalkyl, cycloalkenyl, aryl, and (C1-C8-alkyl) 5-10 membered heteroaryl, where 1-4 heteroaryl members are independently selected from N, O, and S; wherein cycloalkyl, cycloalkenyl, aryl, and heteroaryl are monocyclic or bicyclic.

[0108] L 2 may be substituted with 1 to 3 substituents selected from the group consisting of C1-C6-alkyl, C1-C6-haloalkyl, and CN.

[0109] Z is -OR c , -OC(O)R c , -OC(O)NR c R d , -S(O) 0~2 R c , -CN, -C(O)R c , -C(O)OR c , -C(O)NR c R d , -C(S)R c , -NR c R d , =NR c , -NR c C(O)NR c R d , -NR c CO2R d , -NR c -NO, -NO2, -NR c-OR d , -N=C=O, -N=C=S, and -NR c -NR c R d is selected from the group consisting of:

[0110] R c and R d Each example is independently H, C1-C6-alkyl, C1-C6-haloalkyl, and C6-C 10 -aryl.

[0111] D 1 , D 2 , D 3 , or D 4 At least one of is other than CH, or R c1 , R c2 , or R c3 At least one of is other than H.

[0112] Exemplary embodiments of the present disclosure pertain to the specific compounds presented in the tables and examples described herein.

[0113] Pharmaceutical Compositions The present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds described herein, or pharmaceutically acceptable salts, stereoisomers, and / or tautomers thereof, in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavoring agents, in accordance with accepted practices in pharmaceutical compounding. The pharmaceutical composition may be administered by any suitable means that results in a concentration of the compound in the subject effective to treat a disease or condition suitable for treatment with the disclosed compounds.

[0114] In some embodiments, the compound is present in an amount of 1-95% by weight of the total weight of the composition. The "therapeutically effective amount" of the compound or its pharmaceutically acceptable salt, stereoisomer, and / or tautomer administered is governed by such considerations and is the minimum amount necessary to induce inhibition of PKal. Such an amount should be below an amount that is toxic to normal cells or the subject as a whole. Generally, the initial therapeutically effective amount of a compound of the present disclosure (or its pharmaceutically acceptable salt, stereoisomer, or tautomer) administered is within the range of about 0.001 to about 200 mg / kg or about 0.1 to about 20 mg / kg of patient body weight per day, with a typical initial range being about 0.3 to about 15 mg / kg / day. Oral unit dosage forms, such as tablets and capsules, can contain about 0.1 mg to about 1000 mg of a compound of the present disclosure (or its pharmaceutically acceptable salt, stereoisomer, or tautomer). In another embodiment, such dosage forms contain about 50 mg to about 500 mg of a compound of the present disclosure (or a pharmaceutically acceptable salt, stereoisomer, or tautomer). In yet another embodiment, such dosage forms contain about 25 mg to about 200 mg of a compound of the present disclosure (or a pharmaceutically acceptable salt, stereoisomer, or tautomer). In yet another embodiment, such dosage forms contain about 10 mg to about 100 mg of a compound of the present disclosure (or a pharmaceutically acceptable salt, stereoisomer, or tautomer). In a further embodiment, such dosage forms contain about 5 mg to about 50 mg of a compound of the present disclosure (or a pharmaceutically acceptable salt, stereoisomer, or tautomer). In other embodiments, the compound is processed into a dosage form for administration into or around the eye as described herein, and for this dosage form, the therapeutically effective amount of the compound may range from about 0.0005 mg / kg to about 0.005 mg / kg, about 0.0007 mg / kg to about 0.004 mg / kg, or about 0.001 mg / kg to about 0.003 mg / kg of patient body weight. In any of the foregoing embodiments, the dosage form may be administered once daily or twice daily.

[0115] While the attending physician will ultimately determine the appropriate amount and dosing regimen, in additional embodiments, a therapeutically effective amount of a compound described herein may be, for example, in the range of 0.0035 μg to 20 μg / kg body weight / day or 0.010 μg to 140 μg / kg body weight / week. In some embodiments, a therapeutically effective amount is in the range of 0.025 μg to 10 μg / kg, e.g., at least 0.025, 0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 μg / kg body weight administered daily, every other day, or twice weekly. In some embodiments, the therapeutically effective amount can be in the range of 0.05 μg to 20 μg / kg, e.g., at least 0.05, 0.7, 0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0, or 18.0 μg / kg body weight administered weekly, biweekly, or monthly. In some embodiments, the therapeutically effective amount of the compound can be, e.g., 100 μg / m 2 ~100,000μg / m 2 (with respect to the subject's body surface area) and is administered daily, every other day, once a week, or every other week. In some embodiments, the therapeutically effective amount is 1000 μg / m 2 ~20,000μg / m 2 For example, the compound is at least 1000, 1500, 4000, or 14,000 μg / m 2 It may be administered daily, every other day, twice a week, weekly, or every other week.

[0116] In some embodiments, the compounds of the disclosure are administered in a dose of about 0.01 mg to 1000 mg (e.g., 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 10 , 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg).

[0117] In certain embodiments, the compounds described herein or pharmaceutically acceptable salts or solvates thereof are substantially pure in that they contain less than about 5%, or less than about 2%, or less than about 1%, or less than about 0.5%, or less than about 0.1% of unreacted intermediates or other small organic molecules, such as, for example, synthetic by-products created in one or more of the steps of the synthetic method.

[0118] In various embodiments, the composition is provided in a dosage form suitable for oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, intraarterial), buccal, sublingual, rectal, cutaneous, nasal, vaginal, intranasal, inhalation, transdermal, ocular, intraosseous, otic, or intracranial routes of administration. Thus, in some embodiments, the composition is in a dosage form selected from a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel, including hydrogel, paste, patch, ointment, cream, plaster, drench, osmotic delivery device, suppository, enema, injection, implant, spray, and aerosol. Pharmaceutical compositions are formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A.R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York).

[0119] Pharmaceutical compositions can be formulated to release the active compound immediately upon administration or at any predetermined time or period after administration (eg, sustained release formulations). Examples of sustained release formulations include: (i) formulations that provide a substantially constant concentration of an agent(s) of the present disclosure in the body over an extended period of time; (ii) formulations that provide a substantially constant concentration of an agent(s) of the present disclosure in the body over an extended period of time after a predetermined lag time; (iii) formulations that sustain the action of an agent(s) over a predetermined period of time by maintaining a relatively constant effective level of the agent(s) in the body while minimizing undesirable side effects associated with fluctuations in the plasma levels of the agent(s) (sawtooth kinetics pattern); (iv) formulations that localize the action of the agent(s) (e.g., a sustained release composition spatially adjacent to or within an affected tissue or organ); (v) formulations that allow for convenience of administration (e.g., administering a composition once a week or once every two weeks); and (vi) formulations that target the action of an agent(s) by using carriers or chemical derivatives to deliver the compound to a specific target cell type. In some embodiments, for compounds that have a limited opportunity for absorption in the gastrointestinal tract or a relatively short biological half-life, it may be desirable to administer the compound in the form of a sustained release formulation.

[0120] In some embodiments, sustained release is achieved by appropriate selection of various formulation parameters and components (including, for example, various types of sustained release compositions and coatings). In some embodiments, the compound is formulated with appropriate excipients into a pharmaceutical composition that releases the compound in a controlled manner upon administration. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.

[0121] Pharmaceutical compositions containing the compounds described herein can be administered parenterally by injection, infusion, or implantation (e.g., intraocular, subcutaneous, intravenous, intramuscular, intraperitoneal) in dosage forms, formulations, or by suitable delivery devices or implants containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation.

[0122] Suitable oral compositions described herein include, but are not limited to, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs.

[0123] In another embodiment, also encompassed is a pharmaceutical composition suitable for a single unit dose comprising a compound of the present disclosure or a pharmaceutically acceptable stereoisomer, salt, or tautomer thereof and a pharmaceutically acceptable carrier.

[0124] The composition of the present disclosure suitable for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions.For example, in order to provide the pharmaceutically palatable preparation of the compound of the present disclosure, the liquid formulation of the compound of the present disclosure contains one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives.

[0125] For tablet compositions, the compounds of the present disclosure mixed with non-toxic pharmaceutically acceptable excipients are used to manufacture tablets. Examples of such excipients include, but are not limited to, inert diluents (such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate), granulating and disintegrating agents (such as corn starch or alginic acid), binders (such as starch, gelatin, or gum arabic), and lubricants (such as magnesium stearate, stearic acid, or talc). Tablets may be uncoated or may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract, thereby maintaining the therapeutic effect for a desired period of time. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate may be used.

[0126] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0127] For aqueous suspensions, the compounds of the present disclosure are mixed with excipients suitable for maintaining a stable suspension, examples of which include, but are not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia.

[0128] Oral suspensions may also contain a dispersing or wetting agent (such as a naturally occurring phosphatide (e.g., lecithin), or a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), or a condensation product of ethylene oxide with a long-chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyethylene sorbitan monooleate)). Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents (such as sucrose or saccharin).

[0129] Oily suspensions can be formulated by suspending the compounds of the present disclosure in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or in a mineral oil (such as liquid paraffin). Oily suspensions can contain a thickening agent (e.g., beeswax, hard paraffin, or cetyl alcohol).

[0130] Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0131] In dispersible powders and granules suitable for preparation of an aqueous suspension by adding water, the compounds of the present disclosure are mixed with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients (for example, sweeteners, flavoring agents, and coloring agents) may also be present.

[0132] The pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil (e.g., olive oil or peanut oil), a mineral oil (e.g., liquid paraffin), or a mixture thereof. Suitable emulsifiers may be naturally occurring gums (e.g., gum arabic or gum tragacanth), naturally occurring phosphatides (e.g., soybean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate)), and condensation products of such partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsion may also contain sweeteners and flavoring agents.

[0133] Syrups and elixirs may be formulated with sweetening agents (e.g., glycerol, propylene glycol, sorbitol, or sucrose). Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. These suspensions may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents, as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally-acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland, fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, have found use in injectable preparations.

[0134] The compounds of the present disclosure can be administered in the form of suppositories for rectal administration of drugs. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such excipients include cocoa butter and polyethylene glycol.

[0135] The composition for parenteral administration is administered in a sterile medium.Depending on the vehicle used and the concentration of the drug in the preparation, the parenteral preparation can be either a suspension or a solution containing the dissolved drug.Adjuvants such as local anesthetics, preservatives and buffers can also be added to the parenteral composition.

[0136] In some embodiments, the composition is particularly suitable for administration into the eye or around the eye.For example, the composition can be suitable for use as eye drops or for injection into the eye (for example, by periocular injection or intravitreal injection).Such composition must be sterile, substantially free of endotoxin, and within an acceptable pH range.In some embodiments, preservative-free formulations are used. Formulation of ophthalmic drugs is known in the art, see, for example, Ocular Therapeutics and Drug Delivery: A Multi-Disciplinary Approach, Reddy, Ed. (CRC Press 1995), Kaur and Kanwar, Drug Dev Ind Pharm. 2002 May;28(5):473-93, Clinical Ocular Pharmacology, Bartlett et al. (Butterworth-Heinemann; 4th edition (Mar. 15, 2001)), and Ophthalmic Drug Delivery Systems (Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs), Mitra (Marcel Dekker; 2nd Rev&Ex edition (Mar. 1, 2003)).

[0137] Compositions for parenteral use may be provided in unit dosage form (e.g., single-dose ampoules) or vials containing several doses, to which suitable preservatives may be added (see below). The compositions may be in the form of solutions, suspensions, emulsions, injection devices, or delivery devices for implantation, or may be presented as dry powders to be reconstituted with water or another suitable vehicle before use. In addition to the active agent(s), the compositions may contain suitable parenterally acceptable carriers and / or excipients. The active agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for sustained release. Additionally, the compositions may contain suspending agents, solubilizing agents, stabilizing agents, pH adjusters, osmotic adjusters, and / or dispersing agents.

[0138] In some embodiments, pharmaceutical compositions of the present disclosure are in a form suitable for sterile injection. To prepare such compositions, the active agent(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH (by adding an appropriate amount of hydrochloric acid, sodium hydroxide, or a suitable buffer), 1,3-butanediol, Ringer's solution, dextrose solution, and isotonic sodium chloride solution. Aqueous formulations may also contain one or more preservatives (e.g., methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, or n-propyl p-hydroxybenzoate). If the compound has limited solubility in water, a solubility enhancer or solubilizer may be added, or the solvent may contain 10-60% w / w propylene glycol.

[0139] The pharmaceutical composition can be administered to a subject in a single dose or multiple doses. For example, the compounds described herein can be administered once a week, or for 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It should be understood that for any particular subject, the specific dosing regimen should be adjusted over time according to the individual need and the professional judgment of the healthcare provider administering or supervising the administration of the compound. For example, the dosage of the compound can be increased when a low dose does not provide sufficient biological activity (e.g., in the treatment of a disease or condition described herein). Conversely, the dosage of the compound can be decreased, for example, when a disease or condition is alleviated or eliminated, or to reduce undesirable side effects.

[0140] Methods of Use and Treatment As an advantage of the present disclosure, the compounds described herein are potent inhibitors of plasma kallikrein, i.e., the compounds are capable of reducing the activity of plasma kallikrein. In various embodiments, the compounds described herein have an inhibition constant, IC, of 500 nM or less (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 10, 1, or 0.1 nM). 50 (half maximal inhibitory concentration).

[0141] In certain embodiments, the present disclosure provides methods for inhibiting plasma kallikrein, hi some embodiments, the methods comprise contacting PKal with a compound of the present disclosure in an amount effective to inhibit the activity of PKal. In some embodiments, compounds of the disclosure have an IC in the range of 0.1 to 500 nM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nM). 50In some embodiments, compounds of the present disclosure have an IC50 of 500 nM or less (e.g., 500, 450, 400, 350, 300, 250, 200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM or less). 50 inhibits PKal activity.

[0142] In some embodiments, the method includes administering to a subject an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes administering to a subject an IC500 or IC5000 in the range of 0.1 to 500 nM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nM). 50 In some embodiments, the method inhibits PKal activity in vivo with an IC50 value greater than 100 nM (e.g., 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM). 50 and inhibits PKal activity in vivo.

[0143] In additional embodiments, the present disclosure provides a method for treating a subject suffering from a disease or condition. The method comprises administering to the subject an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the compound is selected from the group consisting of any of the compounds in Table 1, combinations thereof, and pharmaceutically acceptable salts thereof.

[0144] In various embodiments, the disease or condition is ischemic stroke, hemorrhagic stroke, hypertension, retinopathy, diabetic retinopathy, nephropathy, cerebral edema, pulmonary hypertension, inflammation, acute myocardial infarction, deep vein thrombosis, complications of fibrinolytic treatment, stroke, angina pectoris, angioedema, sepsis, arthritis, complications of cardiopulmonary bypass, capillary leak syndrome, inflammatory bowel disease, vascular complications of diabetes, diabetic macular edema, macular degeneration, neuropathy, age-related macular degeneration, retinal vein occlusion, cerebral edema, edema), ischemia-reperfusion injury, angiogenesis, asthma, anaphylaxis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, glioblastoma multiforme, complications of fibrinolytic treatment, increased albumin excretion, macroalbuminuria, pain, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, epilepsy, brain trauma, high altitude cerebral edema, cancer, disseminated intravascular coagulation, pancreatitis, inflammation, shock, hereditary angioedema (HAE), uveitis, polyangiitis, acute respiratory distress syndrome (ARDS), thrombosis, vasculitis, Crohn's disease, ulcerative colitis, enteritis, arteritis, glomerulonephritis, psoriasis, intrauterine The disease is selected from the group consisting of membranous vasculitis, pre-eclampsia, malaria, arthritis, periodic and recurrent fever, Chagas' disease, Raynaud's disease, systemic sclerosis, granulomatosis with polyangiitis, small-vessel vasculitis, medium-vessel vasculitis, large-vessel vasculitis, panvasculitis, systemic autoinflammatory diseases, renal failure, cerebral malaria, Clarkson's disease (systemic vascular leak syndrome), hantavirus infection, hantavirus renal syndrome, hantavirus pulmonary syndrome, virus-associated inflammatory diseases, retinal vasculitis, uveitis, Eales' disease, Behcet's disease, sarcoidosis, whooping cough, coronavirus infection, and non-infectious posterior uveitis.

[0145] In some embodiments, a subject is an animal, such as a human or non-human animal (e.g., a mammal), and is used interchangeably with "patient" when the subject is receiving medical treatment by a health care provider.

[0146] Combination therapy The present disclosure also provides a pharmaceutical composition combination comprising (a) at least one compound disclosed herein or a pharmaceutically acceptable salt thereof, and (b) at least one inflammation, pain, or edema inhibitor. In some embodiments, an oral delivery pharmaceutical composition is provided comprising the pharmaceutical composition combination of the present disclosure. The present disclosure further provides tablets, capsules, oral delivery particles, injectable suspensions and solutions, and pulmonary or nasal delivery compositions comprising the pharmaceutical composition combination.

[0147] kit In another embodiment, the present disclosure provides a kit comprising a compound described herein or a pharmaceutically acceptable salt thereof, the kit also comprising instructions for a healthcare provider to administer the compound to a patient. [Example]

[0148] The following examples provide further embodiments of the present disclosure. The examples are illustrative and non-limiting. Although any compositions, compounds, and methods essentially similar to those described herein can be used in practicing or testing the present disclosure, only exemplary compositions, compounds, and methods are described.

[0149] Compound synthesis As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. While the general methods are representative of the synthesis of certain specific compounds of the invention, it will be understood that the following general methods, and other methods known to those of skill in the art, can be applied to all compounds described herein and to each subclass and species of these compounds. Additional compounds of the invention are prepared by methods substantially similar to those described herein in the Examples, known to those of skill in the art.

[0150] Preparation of intermediate compounds Example 1A: Preparation of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (7_Int-5) TIFF2025526464000021.tif48165 Step-1 Synthesis of 2-methyl-2-(p-tolyl)propanenitrile (7_Int-2) Sodium tert-butoxide (586.07 g, 6098 mmol, 4.0 eq) was added portionwise to a solution of 2-(p-tolyl)acetonitrile (200 g, 1524 mmol, 1.0 eq) in N-methyl-2-pyrrolidone (1000 mL, 5V) and tetrahydrofuran (1000 mL, 5V) under a nitrogen atmosphere at 0°C to 5°C. The reaction mixture (RM) was stirred at 0°C to 5°C for 30 min. Iodomethane (865.62 g, 6098 mmol, 4.0 eq) was added dropwise to the RM at 0°C to 5°C. The RM was stirred at 0°C to room temperature for 2.5 h. After completion of the reaction, the RM was transferred to deionized water (400 mL) and the product was extracted with ethyl acetate (2000 mL). The combined organic fractions were washed with cold water (4x500 mL) to remove N-methyl-2-pyrrolidone, concentrated, and then purified by column chromatography (2-5% EtOAc in hexanes) to give compound 7_Int-2. 1 H NMR (400 MHz, DMSO-d6) δ 1.651(s, 6H), 2.298 (s, 3H), 7.226 d, J=8 Hz, 2H), 7.392 (d, J=6.8 Hz, 2H).

[0151] Step-2 Synthesis of 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (7_Int-3) A 10,000 mL 4N round-bottom flask (RBF) equipped with a mechanical stirrer and condenser was charged with 2-methyl-2-(p-tolyl)propanenitrile (170 g, 1067 mmol, 1.0 eq) and carbon tetrachloride (3400 mL, 20 V) at room temperature. AIBN (17.53 g, 106 mmol, 0.1 eq) was added to the RM. N-Bromosuccinimide (209.04 g, 1174 mmol, 1.1 eq) was added portionwise to the RM at room temperature. The resulting RM was heated to 90 °C and stirred for 2 h. Note: After completion of the reaction, the reaction mixture was cooled to room temperature. The RM was quenched with DM water (3400 mL) and the product was extracted with DCM (2 x 2000 mL). The combined organic fractions were concentrated and then purified by column chromatography (7% EtOAc in hexanes) to give compound (7_Int-3). 1 H NMR (400 MHz, DMSO-d6) δ 1.654 (s, 6H), 4.714 (s, 2H), 7.507 (s, 4H).

[0152] Step-3 Synthesis of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (7_Int-5) A 3000 mL 4N RBF equipped with a mechanical stirrer and condenser was charged with ethyl 1H-pyrazole-4-carboxylate (70 g, 499 mmol, 1.0 eq), 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (130.84 g, 549 mmol, 1.1 eq), and acetone (700 mL, 10 V) at room temperature. CsCO (390.8 g, 1198 mmol, 2.4 eq) was added to the RM. The RM was heated to 60-65 °C and stirred for 6 h. After completion of the reaction, the reaction mixture was cooled to room temperature. The RM was filtered to remove CsCO and washed with EtOAc. The filtrate was concentrated and then purified by column chromatography (15% ethyl acetate in hexane) to give compound (7_Int-5). 1H NMR (400 MHz, DMSO-d6) δ 1.656 (s, 6H), 4.204 (q, J=6.8 Hz, 2H), 5.372 (s, 2H), 7.328 (d, J=8 Hz, 2H), 7.498 (d, J=8 Hz, 2H), 8.053 (s, 1H), 8.482 (s, 1H).

[0153] Example 2A: Preparation of ethyl 1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_Int-4) TIFF2025526464000022.tif42165 Step-1 Synthesis of ethyl 1-(4-methylbenzyl)-1H-pyrazole-4-carboxylate (1_Int-2) A 10,000 mL 4N RBF equipped with a mechanical stirrer and condenser was charged with ethyl 1H-pyrazole-4-carboxylate (250 g, 1783 mmol, 1.0 eq), 1-(bromomethyl)-4-methylbenzene (363.1 g, 1962 mmol, 1.1 eq), and acetone (6250 mL, 25 V) at room temperature. CsCO (390.8 g, 1198 mmol, 2.4 eq) was added to the RM. The RM was heated to 60-65 °C and stirred for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature. The RM was filtered to remove CsCO and washed with EtOAc. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by trituration in hexane to give compound (1_Int-2). 1 H NMR (400 MHz, DMSO-d6) δ 1.227 (t, J=6.8 Hz, 3H), 2.247 (s, 3H), 4.176 (q, J=6.8 Hz, 2H), 5.282 (s, 2H), 7.144-7.136 (m, 4H), 7.828 (s, 1H), 8.390 (s, 1H).

[0154] Step-2 Synthesis of ethyl 1-(4-(bromomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_Int-3) A 10,000 mL 4N RBF equipped with a mechanical stirrer and condenser was charged with ethyl 1-(4-methylbenzyl)-1H-pyrazole-4-carboxylate (300 g, 1228 mmol, 1.0 eq) and 1,2-dichloroethane (6000 mL, 20 V) at room temperature. Benzoyl peroxide (29.71 g, 122.8 mmol, 0.1 eq) was added to the RM. N-Bromosuccinimide (240.4 g, 1350 mmol, 1.1 eq) was added portionwise to the RM at room temperature. The resulting RM was heated to 90 °C and stirred for 4 h. After completion of the reaction, the reaction mixture was cooled to room temperature. The RM was diluted with DCM (2000 mL). The RM was washed with saturated Na2SO4 solution (2 x 4000 mL) and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (17-25% EtOAc in hexanes) to give compound (1_Int-3). 1 H NMR (400 MHz, DMSO-d6) δ 1.270-1.235 (m, 3H), 4.207 (q, J=6.8 Hz, 2H), 4.682 (s, 2H), 5.364 (s, 2H), 7.243 (d, J=8.4 Hz, 4H), 7.421 (d, J=8.4Hz, 2H), 7.868 (s, 1H), 8.478 (s, 1H).

[0155] Step-3 Synthesis of ethyl 1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_Int-4) A 5000 mL 4N RBF equipped with a mechanical stirrer and condenser was charged with ethyl 1-(4-(bromomethyl)benzyl)-1H-pyrazole-4-carboxylate (242 g, 499748 mmol, 1.0 eq), ACN (2420 mL, 10 V), and CsCO (488 g, 1497 mmol, 2.0 eq) at room temperature, followed by the slow addition of TMSCN (334 g, 3369 mmol, 4.5 eq) at room temperature. The RM was heated to 80-85 °C and stirred for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature. The RM was filtered to remove CsCO and washed with EtOAc. The solid residue was discarded, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (12-15% EtOAc in hexanes) to give compound (1_Int-4). 1 H NMR (400 MHz, DMSO-d6) δ 1.282-1.234 (m, 3H), 4.016 (s, 2H), 4.20 (q, J=7.2 Hz, 2H), 5.363 (s, 2H), 7.339-7.280 (m, 4H), 8.051 (s, 1H), 8.468 (s, 1H).

[0156] Example 3A: Preparation of ethyl 1-(4-((3-cyano-1H-pyrrol-1-yl)methyl)benzyl)-1H-pyrazole-4-carboxylate (6_Int-1) TIFF2025526464000023.tif42165 Step-1 Synthesis of ethyl 1-(4-((3-cyano-1H-pyrrol-1-yl)methyl)benzyl)-1H-pyrazole-4-carboxylate (6_Int-1) A stirred solution of 1H-pyrrole-3-carbonitrile (0.2 g, 2.16 mmol, 1.0 eq) in DMF (2 mL, 10 V) was prepared, and NaH (60% in mineral oil) (0.156 g, 3.24 mmol, 1.5 eq) was added at 0 °C. The RM was stirred at 0 °C for 30 min. At 0 °C, 1-(4-(bromomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_Int-3) (0.772 g, 2.39 mmol, 1.1 eq) was added to the RM. The RM was warmed to room temperature and stirred for 3 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by column chromatography (20% ethyl acetate in hexane) to give compound (6_Int-1). MS (ES): 335.30m / z [M+H]+, LCMS purity: 95.09%, 1 H NMR (400 MHz, DMSO-d6) δ 1.246 (t, J=8 Hz, 3H), 4.194 (q, J=7.2 Hz, 2H), 5.336 (s, 2H), 6.987 (t, J=2.4 Hz, 1H), 7.267-7.208 (m, 4H), 7.699 (t, J=2 Hz, 1H), 7.845 (s, 1H), 8.448 (s, 1H).

[0157] Example 4A: Preparation of 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) TIFF2025526464000024.tif58165 Step-1 Synthesis of tert-butyl (4-cyano-2-fluorobenzyl)carbamate (43_Int-2) A stirred solution of 4-(aminomethyl)-2-fluorobenzonitrile (43_Int-1) (3 g, 19.97 mmol, 1.0 eq) in 1,4 dioxane (36 mL, 12 V) and 2N NaOH (18 mL, 6 V) was prepared, and Boc anhydride (4.79 g, 21.97 mmol, 1.1 eq) was added at 0 °C. The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (43_Int-2). MS (ES): 251.00 m / z [M+H]+, LCMS purity: 100%, 1 H NMR (400 MHz, DMSO-d6) δ 1.393 (s, 9H), 4.216 (d, J=5.2 Hz, 2H), 7.281 (t, J=7.5 Hz, 2H), 7.551 (s, 1H), 7.885 (t, J=8 Hz, 1H).

[0158] Step-2 Synthesis of tert-butyl (3-fluoro-4-(N-hydroxycarbamimidoyl)benzyl)carbamate (43_Int-3) A stirred solution of tert-butyl (4-cyano-3-fluorobenzyl)carbamate (43_Int-2) (3.4 g, 13.58 mmol, 1.0 eq) and methanol (34 mL, 10 V) was prepared, and hydroxylamine hydrochloride (1.604 g, 23.09 mmol, 1.7 eq) and DIPEA (3.95 g, 23.09 mmol, 1.7 eq) were added at room temperature. The RM was stirred at 70 °C for 16 h. After completion of the reaction, the RM was evaporated, and the residue was quenched with water and extracted with 10% methanol in DCM. The combined organic fractions were dried over Na SO and concentrated to give compound (43_Int-3). MS (ES): 284.10 m / z [M+H] LCMS purity: 81.45%, 1H NMR (400 MHz, DMSO-d6) δ 1.393 (s, 9H), 4.133 (d, J=5.2 Hz, 2H), 7.101-70.31 (m, 2H), 7.125 (s, 1H), 7.483-7.417 (m, 2H), 7.611(t, J=8 Hz, 1H), 9.593 (s, 1H).

[0159] Step-3 Synthesis of tert-butyl (4-carbamimidoyl-3-fluorobenzyl)carbamate (43_Int-4) A stirred solution of tert-butyl (3-fluoro-4-(N-hydroxycarbamimidoyl)benzyl)carbamate (43_Int-3) (3.5 g, 12.35 mmol, 1.0 eq) and methanol (35 mL, 10 V) was prepared, and ammonium formate (2.34 g, 37.06 mmol, 3.0 eq) and 10% Pd / C (0.175 g, 0.065% w / w) were added at room temperature. The RM was stirred in an autoclave under 20 kg H pressure at 70 °C for 16 h. After completion of the reaction, the RM was filtered through a celite bed, and the filtrate was concentrated to give compound (43_Int-4). MS (ES): 267.80 m / z [M+H]+ LCMS purity: 96.46%, 1 H NMR (400 MHz, DMSO-d6) δ 1.394 (s, 9H), 4.195 (d, J=5.6 Hz, 2H), 7.244 (d, J=8 Hz, 2H), 7.254-7.233 (m, 3H), 7.639-7.580 (m, 3H), 8.415 (s, 1H).

[0160] Step-4 Synthesis of 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) A stirred solution of tert-butyl (4-carbamimidoyl-3-fluorobenzyl)carbamate (43_Int-4) (4.8 g, 16.83 mmol, 1.0 eq) and water (52 mL, 11 V) was prepared, and concentrated HCl (16 mL, 3.3 V) was added at room temperature. The RM was stirred at room temperature for 3 hours. After completion of the reaction, the RM was concentrated and triturated with methanol to give compound (43_Int-5). MS (ES): 168.13 m / z [M+H]+, 1 H NMR (400 MHz, DMSO-d6) δ 4.141 (d, J=4 Hz, 2H), 7.658-7.155(m, 3H), 7.737-7.686 (m, 2H), 8.772 (s, 1H), 9.564-9.440 (m, 2H).

[0161] Example 5A: Preparation of 4-(aminomethyl)-3-fluorobenzimidamide dihydrochloride (45_Int-5) TIFF2025526464000025.tif68165 Step-1 Synthesis of tert-butyl (4-cyano-2-fluorobenzyl)carbamate (45_Int-2) A stirred solution of 4-(aminomethyl)-3-fluorobenzonitrile (45_Int-1) (1 g, 6.65 mmol, 1.0 eq) in 1,4 dioxane (12 mL, 12 V) and 2 N NaOH (6 mL, 6 V) was prepared, and Boc anhydride (1.59 g, 7.32 mmol, 1.1 eq) was added at 0 °C. The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (45_Int-2). 1 H NMR (400 MHz, DMSO-d6) δ 1.146 (s, 9H), 4.220 (d, J=5.2 Hz, 2H), 7.442 (t, J=7.5 Hz, 2H), 7.525 (s, 1H), 7.812 (t, J=8 Hz, 2H).

[0162] Step-2 Synthesis of tert-butyl (2-fluoro-4-(N-hydroxycarbamimidoyl)benzyl)carbamate (45_Int-3) A stirred solution of tert-butyl (4-cyano-2-fluorobenzyl)carbamate (45_Int-2) (5.3 g, 21.17 mmol, 1.0 eq) and methanol (53 mL, 10 V) was prepared, and hydroxylamine hydrochloride (2.5 g, 36.0 mmol, 1.7 eq) and DIPEA (4.64 g, 36.0 mmol, 1.7 eq) were added at room temperature. The RM was stirred at 70 °C for 16 h. After completion of the reaction, the RM was evaporated, and the residue was quenched with water and extracted with 10% methanol in DCM. The combined organic fractions were dried over Na SO and concentrated to give compound (45_Int-3). MS (ES): 284.21 m / z [M+H].

[0163] Step-3 Synthesis of tert-butyl (4-carbamimidoyl-2-fluorobenzyl)carbamate (45_Int-4) A stirred solution of tert-butyl (2-fluoro-4-(N-hydroxycarbamimidoyl)benzyl)carbamate (45_Int-3) (2.4 g, 8.47 mmol, 1.0 eq) and methanol (24 mL, 10 V) was prepared, and ammonium formate (1.6 g, 25.41 mmol, 3.0 eq) and 10% Pd / C (0.16 g, 0.065% w / w) were added at room temperature. The RM was stirred in an autoclave under 20 kg H pressure at 70 °C for 16 h. After completion of the reaction, the RM was filtered through a celite bed, and the filtrate was concentrated to give compound (45_Int-4). MS (ES): 268.18 m / z [M+H]+.

[0164] Step-4 Synthesis of 4-(aminomethyl)-3-fluorobenzimidamide dihydrochloride (45_Int-5) A stirred solution of tert-butyl (4-carbamimidoyl-2-fluorobenzyl)carbamate (45_Int-4) (2.0 g, 7.48 mmol, 1.0 eq) and water (22 mL, 11 V) was prepared, and concentrated HCl (6.6 mL, 3.3 V) was added at room temperature. The RM was stirred at room temperature for 3 hours. After completion of the reaction, the RM was concentrated and triturated with methanol to give compound (45_Int-5). MS (ES): 168.13 m / z [M+H]+, 1 H NMR (400 MHz, DMSO-d6) δ 4.145 (s, 2H), 7.210 (s, 1H), 7.336 (s, 1H), 7.463 (s, 1H), 7.878-7.765 (m, 2H), 8.816 (s, 2H), 9.442 (s, 1H), 9.634 (s, 1H).

[0165] Example 6A: Preparation of ethyl 1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-4) TIFF2025526464000026.tif48165 Step-1 Synthesis of ethyl 1-(4-chlorobenzyl)-1H-pyrazole-4-carboxylate (28_Int-2) A stirred solution of 1H-ethyl pyrazole-4-carboxylate (1 g, 7.14 mmol, 1 eq) and 1-(bromomethyl)-4-chlorobenzene (28_int-1) (1.5 g, 7.85 mmol, 1.1 eq) in acetone (10 mL, 10 V) was prepared, and Cs2CO3 (5.53 g, 17.14 mmol, 2.4 eq) was added at room temperature. The RM was heated to 65 °C and stirred for 4 h. After completion of the reaction, the RM was cooled at room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (50% ethyl acetate in hexane) to give compound (28_Int-2). MS (ES): 265.30 m / z [M+], 266.30 [M+2]+, LCMS purity: 63%, 1H NMR (400 MHz, DMSO-d6) δ 400 MHz, DMSO-d6: δ 1.26 (t, J = 6.8 Hz, 3H), 4.28-4.210 (m, 2H), 5.38 (s, 2H), 7.30 (d, J = 8.40 Hz, 2H), 7.43 (d, J = 8.40 Hz, 2H), 7.88 (s, 1H), 8.49 (s, 1H).

[0166] Step-2 Synthesis of ethyl 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-3) A stirred solution of ethyl 1-(4-chlorobenzyl)-1H-pyrazole-4-carboxylate (28_Int-2) (1.0 g, 3.78 mmol, 1.0 eq) in 1,4-dioxane (5 mL, 5 V) was prepared, and bis(pinacolato)diboron (1.14 g, 4.54 mmol, 1.2 eq), KOAc (1.11 g, 11.36 mmol, 3 eq), XPhosPdG2 (0.350 g, 0.37 mmol, 0.1 eq), and water (0.5 mL, 0.5 V) were added at room temperature. The RM was heated to 100 °C and stirred for 16 h. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (20% ethyl acetate in hexane) to give compound (28_Int-3). MS (ES): 357.51 m / z [M+1]+, LCMS purity: 70%, 1 H NMR (400 MHz, DMSO-d6) δ 1.26 (s, 12H), 1.26 (t, 3H), 4.228-4.210 (m, 2H), 5.38 (d, J = 12.80 Hz, 2H), 7.25 (d, J = 8.00 Hz, 2H), 7.65 (d, J = 8.00 Hz, 1H), 7.87 (s, 1H), 0.00 (s, 1H), 8.47 (s, 1H).

[0167] Step-3 Synthesis of ethyl 1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-4) A stirred solution of ethyl 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-3) (0.300 g, 0.84 mmol, 1.0 eq) in 1,4-dioxane (3 ml, 10 V) was prepared, and 3-(bromomethyl)benzonitrile (0.181 g, 0.92 mmol, 1.1 eq), KPO (0.535 g, 3.51 mmol, 3 eq), Pd(dppf)Cl, DCM (0.068 g, 0.11 mol, 0.1 eq), and water (1.5 ml, 5 V) were added at room temperature. The RM was heated to 100 °C and stirred for 6 h. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (30% ethyl acetate in hexane) to give compound (28_Int-4). MS (ES): 346.39 m / z [M+1]+, LCMS purity: 100%, 1 H NMR (400 MHz, DMSO-d6) δ 1.22 (s, 3H), 3.98 (s, 2H), 4.20 (q, J = 6.8 Hz, 2H), 5.31 (s, 2H), 7.254-7.193 (m, 4H), 7.49 (t, J = 8.00 Hz, 1H), 7.56 (d, J = 8.00 Hz, 1H), 0.00 (d, J = 7.60 Hz, 1H), 7.72 (s, 1H), 7.84 (s, 1H), 8.434 (s, 1H).

[0168] Example 7A: Preparation of 4-(aminomethyl)-2,6-difluorobenzimidamide dihydrochloride (44_Int-8) TIFF2025526464000027.tif99165 Step-1 Synthesis of 2,6-difluoro-4-(hydroxymethyl)benzonitrile (44_Int-2) A stirred solution of 2,6-difluoro-4-formylbenzonitrile (44_Int-1) (8 g, 0.047 mmol, 1.0 eq) in methanol (160 mL, 20 V) was prepared, and NaBH (2.21 g, 0.047 mmol, 1 eq) was added at 0 °C. The RM was stirred at room temperature for 1 h. After completion of the reaction, the RM was quenched with water and extracted with DCM. The combined organic fractions were dried over NaSO and concentrated to give compound (44_Int-2). H NMR (400 MHz, DMSO-d) δ 4.60 (d, J = 8 Hz, 2H), 5.72 (t, J = 9.1 Hz, 1H), 7.34 (d, J = 8 Hz, 2H).

[0169] Step-2 Synthesis of 2,6-difluoro-4-(hydroxymethyl)benzonitrile (44_Int-3) A stirred solution of 2,6-difluoro-4-(hydroxymethyl)benzonitrile (44_Int-2) (7.5 g, 0.0443 mmol, 1.0 eq) in MTBE (75 mL, 10 V) was prepared, and PBr (14.37 g, 0.0532 mmol, 1.2 eq) was added at 0 °C. The RM was stirred at room temperature for 2 h. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over NaSO and concentrated to give compound (44_Int-3). 1H NMR (400 MHz, DMSO-d6) δ 4.721 (s, 2H), 7.564 (d, J = 8.8 Hz, 2H).

[0170] Step-3 Synthesis of 4-(aminomethyl)-2,6-difluorobenzonitrile (44_Int-4) Ammonia gas was purged into methanol at 0° C. for 30 minutes. A solution of 2,6-difluoro-4-(hydroxymethyl)benzonitrile (44_Int-3) (9.0 g, 0.06 mmol, 1.0 eq) in MeOH (40 mL, 10 V) was added to the RM and stirred at 0° C. for 4 hours. After completion of the reaction, the RM was concentrated to give compound (44_Int-4). MS (ES): 169 m / z [M+1]+, LCMS purity: 84%.

[0171] Step-4 Synthesis of tert-butyl (4-cyano-3,5-difluorobenzyl)carbamate (44_Int-5) A stirred solution of 4-(aminomethyl)-2,6-difluorobenzonitrile (44_Int-4) (10 g, 59.5238 mmol, 1.0 eq) in 1,4 dioxane (120 mL, 12 V) was prepared. 2N NaOH (60 ml, 6 V) and Boc anhydride (14.27 gm, 0.065 mmol, 1.1 eq) were added at room temperature and stirred for 5 hours. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (44_Int-5). 1H NMR (400 MHz, DMSO-d6) δ 1.39 (s, 9H), 4.52 (s, 2H), 7.27 (d, J = 8.8 Hz, 2H).

[0172] Step-5 Synthesis of tert-butyl (3,5-difluoro-4-(N-hydroxycarbamimidoyl)benzyl)carbamate (44_Int-6) A stirred solution of tert-butyl (4-cyano-3,5-difluorobenzyl)carbamate (44_Int-5) (6 g, 22.388 mmol, 1.0 eq) in MeOH (60 mL, 10 V) was prepared. Hydroxylamine hydrochloride (2.6 gm, 38.0596 mmol, 1.7 eq) and DIPEA (6.5 ml, 38.0596 mmol, 1.7 eq) were added at room temperature and stirred at 70 °C for 16 hours. After completion of the reaction, the RM was cooled to room temperature. The RM was then quenched with water and extracted with DCM. The combined organic fractions were dried over Na SO and concentrated to give compound (44_Int-6). MS (ES): 302 m / z [M+1], LCMS purity: 65%.

[0173] Step-6 Synthesis of tert-butyl (4-carbamimidoyl-3,5-difluorobenzyl)carbamate (44_Int-7) A stirred solution of tert-butyl (3,5-difluoro-4-(N-hydroxycarbamimidoyl)benzyl)carbamate (44_Int-6) (6 g, 19.933 mmol, 1.0 eq) in MeOH (60 mL, 10 V) was prepared. Ammonium chloride (5.38 g, 99.66 mmol, 5.0 eq) and iron (5.40 g, 99.66 mmol, 5.0 eq) were added at room temperature. The RM was cooled to 0 °C, followed by the dropwise addition of acetic acid (30 mL, 5 V). The RM was then heated to 70 °C and stirred for 16 h. After completion of the reaction, the RM was cooled to room temperature. The RM was concentrated, quenched with cold water, and slowly basified to pH 10. The solid was isolated via filtration and extracted with DCM. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (44_Int-7). MS (ES): 286 m / z [M+1]+, LCMS purity: 65%.

[0174] Step-7 Synthesis of 4-(aminomethyl)-2,6-difluorobenzimidamide (44_Int-8) A stirred solution of tert-butyl (4-carbamimidoyl-3,5-difluorobenzyl)carbamate (44_Int-7) (2.2 g, 25.473 mmol, 1.0 eq) in water (24 mL, 11 V) was prepared, and concentrated HCl (7.26 mL, 3.3 V) was added at room temperature and stirred for 3 h. After completion of the reaction, the RM was concentrated and triturated in methanol to give compound (44_Int-8). MS (ES): 231 m / z [M+1]+, LCMS purity: 67%.

[0175] Example 9A: Preparation of methyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (82_Int-3) TIFF2025526464000028.tif48165 Step-1 Synthesis of methyl 5-(methoxymethyl)-1H-pyrazole-4-carboxylate (82_Int-2) A solution of methyl 4-methoxy-3-oxobutanoate (82_Int-1) (15 g, 102 mmol, 1.0 eq) and dimethylformamide dimethyl acetal (12.23 g, 102 mmol, 1.0 eq) was prepared at 110 °C and stirred for 1 h. The RM was cooled to room temperature, followed by the addition of ethanol (150 mL, 10 V) and hydrazine hydrate (99%) (5.13 g, 102 mmol, 1.0 eq). The RM was heated to 70 °C and stirred for 2 h. After completion of the reaction, the RM was evaporated and then purified by column chromatography (25-30% ethyl acetate in hexane) to give compound (82_Int-2). 1H NMR (400 MHz, DMSO-d6) δ 3.258 (s, 3H), 3.758 (s, 3H), 4.702(s, 2H), 7.837 (s, 1H), 8.281 (s, 1H).

[0176] Step-2 Synthesis of methyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (82_Int-3) A stirred solution of methyl 5-(methoxymethyl)-1H-pyrazole-4-carboxylate (82_Int-2) (1.0 g, 5.87 mmol, 1.0 eq) in DMF (10 mL, 10 V) was prepared, and NaHMDS (1 M in THF) (5.8 mL, 5.87 mmol, 1.0 eq) was added at 0 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (7_Int-3) (1.39 g, 5.87 mmol, 1.0 eq) was then added to the RM at 0 °C and stirred for an additional 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over NaSO, concentrated, and then purified by flash column chromatography (15% ethyl acetate in hexane) to give 82_Int-3. 2D NMR (ROE analysis) confirmed the reaction. MS (ES): 328 m / z, LCMS purity: 99.11%,1H 400 MHz, DMSO-d6: δ 1.654 (s, 6H), 3.258 (s, 3H), 3.758 (s, 3H), 4.797 (s, 2H), 5.394 (s, 2H), 7.258 (d, J= 8Hz, 2H), 7.478 (d, J=8 Hz, 2H), 7.903 (s,1H).

[0177] Example 10A: Preparation of methyl 1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (85_Int-3) TIFF2025526464000029.tif53165 Step-1 Synthesis of methyl 1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (85_Int-3) A stirred solution of methyl 5-(methoxymethyl)-1H-pyrazole-4-carboxylate (82_Int-2) (1.0 g, 5.87 mmol, 1.0 eq) in DMF (10 mL, 10 V) was prepared, and NaHMDS (1 M in THF) (5.8 mL, 5.87 mmol, 1.0 eq) was added at 0 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)acetonitrile (1.4 g, 7.05 mmol, 1.2 eq) was then added to the RM at 0 °C and stirred for an additional 16 h at room temperature. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (25–30% ethyl acetate in hexane) to give 85_Int-3. 2D NMR (ROE analysis) confirmed the reaction. MS (ES): 300 m / z, 1H 400 MHz, DMSO-d6: δ 3.233 (s, 3H), 3.745 (s, 3H), 4.029 (s, 2H), 4.514 (s, 2H), 5.352 (s, 2H), 7.356-7.301 (m, 4H), 8.046 (s, 1H).

[0178] Example 11A: Preparation of ethyl 1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (33_Int-3) TIFF2025526464000030.tif68165 Step-1 Synthesis of 2-(4-(bromomethyl)phenyl)-N-methylacetamide (33_Int-2) A stirred solution of 2-(4-(bromomethyl)phenyl)acetic acid (33_Int-1) (1.0 g, 4.36 mmol, 1.0 eq) in toluene (10 mL, 10 V) was prepared. SOCl (0.54 g, 4.53 mmol, 1.04 eq) and DMF (0.051 g, 0.69 mmol, 0.16 eq) were added at room temperature and stirred at 85 °C for 3 h. The RM was cooled to 0 °C, followed by the addition of dimethylamine (2 M in THF) (2 mL, 2 V). The RM was allowed to reach room temperature and stirred for 6 h. After completion of the reaction, the RM was quenched with saturated NaHCO solution and extracted with ethyl acetate. The combined organic fractions were dried over NaSO and concentrated to give compound (33_Int-2). MS (ES): 256.6 m / z [M]+ 258.1.m / z [M+2]+, LCMS purity: 98.96%, 1H NMR (400 MHz, DMSO-d6) δ 2.826 (s, 3H), 2.997 (s, 3H), 3.694 (s, 2H), 4.706 (s, 3H), 7.210 (d, J=8Hz, 2H), 7.385 (d, J=8Hz, 2H).

[0179] Step-2 Synthesis of ethyl 1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (33_Int-3) A stirred solution of ethyl 1H-pyrazole-4-carboxylate (0.3 g, 2.14 mmol, 1.0 eq) was prepared in acetone (6 mL, 20 V). 2-(4-(bromomethyl)phenyl)-N,N-dimethylacetamide (33_Int-2) (0.657 g, 2.56 mmol, 1.2 eq) and CsCO (1.67 g, 5.13 mmol, 2.4 eq) were added at room temperature. The RM was then heated to 65 °C and stirred for 16 h. After the reaction was complete, the RM was cooled to room temperature. The RM was filtered to remove CsCO and washed with ethyl acetate. The solid residue was discarded, and the filtrate was concentrated and then purified by column chromatography (25-30% ethyl acetate in hexane) to give compound 33_Int-3. MS (ES): 316.3 m / z [M+1]+, LCMS purity: 100%, 1H NMR (400 MHz, DMSO-d6) δ 1.252 (t, J= 2.8Hz, 3H), 2.802 (s, 3H), 2.974 (s, 3H), 3.396-3.295 (m, 2H), 4.188 (s, 2H), 5.324 (s, 2H), 7.189 (s, 4H), 7.852 (s, 1H), 8.466 (s, 1H).

[0180] Preparation of final compounds Example 1: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (7) TIFF2025526464000031.tif53165 A 3000 mL 4N RBF equipped with a mechanical stirrer and condenser was charged with ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (7_Int5) (120 g, 403 mmol, 1.0 eq), 4-(aminomethyl)benzimidamide dihydrochloride (134.4 g, 605 mmol, 1.5 eq), and toluene (1200 mL, 10V) at room temperature. DIPEA (130.14 g, 1008 mmol, 2.5 eq) was added to the RM. The RM was cooled to 0°C-5°C and stirred for 20 min. TMA (2 M in toluene) (605.3 mL, 121 mmol, 3.0 eq) was added dropwise to the RM. The RM was heated to 95°C and stirred for 16 hours. After completion of the reaction, the reaction mixture was cooled to room temperature. The RM was slowly quenched with DM water (151 mL) and evaporated to give a residue. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified three times by column chromatography using 60-120 mesh silica. The product was eluted with 10% MeOH in DCM to give a solid, which was dissolved in 4V methanol and stirred at 70°C for 2 hours. EtOAc was added at 60°C until a cloudy solution was observed. The solution was slowly cooled to room temperature and stirred for 16 hours until a solid precipitate was obtained, which was collected by filtration. The filtrate was evaporated and purified by column chromatography to give compound (7). MS (ES): 473.87 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 95.07% 1 H NMR (400 MHz, DMSO-d6) δ 1.682 (s, 6H), 4.470 (s, 2H), 5.362 (s, 2H), 7.326 (d, J=8.4 Hz, 2H), 7.515-7.419 (m, 4H), 7.772 (d, J=8.4 Hz, 2H), 7.939 (s, 1H), 8.316 (s, 1H), 8.865-8.835 (m, 1H), 9.123 (s, 2H), 9.303 (s, 2H).

[0181] Example 2: N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanoethyl)benzyl)-1H-pyrazole-4-carboxamide (3) TIFF2025526464000032.tif89165 Step-1 Synthesis of ethyl 1-(4-iodobenzyl)-1H-pyrazole-4-carboxylate (3_Int-2) A stirred solution of 1H-pyrazole-4-carboxylate (0.5 g, 3.56 mmol, 1.0 eq) in DMF (5 mL, 10 V) was prepared, and NaH (60% in mineral oil) (0.213 g, 5.35 mmol, 1.5 eq) was added at 0 °C. The RM was stirred at 0 °C for 30 min. 1-(Bromomethyl)-4-iodobenzene (1.05 g, 3.56 mmol, 1.0 eq) was added at 0 °C, and the RM was allowed to warm to room temperature and stirred for 2 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (12–15% ethyl acetate in hexanes) to give compound (3_Int-2). MS (ES): 357.1.m / z [M+H]+, LCMS purity: 100%, 1H 400 MHz, DMSO-d6: δ 1.25 (t, J = 7.20 Hz, 3H), 4.20 (d, J = 7.20 Hz, 2H), 5.33 (s, 2H), 7.066 (d, J = 7.60 Hz, 2H), 7.715 (d, J = 8.00 Hz, 2H), 7.87 (s, 1H), 8.47 (s, 1H).

[0182] Step-2 Synthesis of (E)-1-(4-(2-cyanovinyl)benzyl)-1H-pyrazole-4-carboxylate (3_Int-3) A stirred solution of ethyl 1-(4-iodobenzyl)-1H-pyrazole-4-carboxylate (3_Int-2) (0.55 g, 1.54 mmol, 1.0 eq) and 1,4-dioxane (11 mL, 20 V) was prepared, and acrylonitrile (0.08 g, 1.54 mmol, 1 eq) and TEA (0.390 g, 3.86 mmol, 2.5 eq) were added at room temperature. N was purged into the RM at room temperature for 10 minutes. Palladium diacetate (0.034 g, 0.15 mmol, 0.1 eq) and JohnPhos (0.092 g, 0.30 mmol, 0.2 eq) were added, and the RM was heated to 110 °C and stirred for 3 hours. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (22-25% ethyl acetate in hexanes) to give compound (3_Int-3). MS (ES): 282.30.m / z [M+H]+, LCMS purity: 99.41%, 1H 400 MHz, DMSO-d6: δ 1.29 (d, J = 5.6 Hz, 3H), 2.231 (d, J = 5.6 Hz, 2H), 5.441 (d, J = 7.2 Hz, 2H), 5.92 (d, J = 12 Hz, 1H), 7.444-7.328 (m, Hz, 3H), 7.799 (d, J = 5.6 Hz, 2H), 7.907 (s, Hz, 1H), 8.533 (s, 1H).

[0183] Step-3 Synthesis of ethyl 1-(4-(2-cyanoethyl)benzyl)-1H-pyrazole-4-carboxylate (3_Int-4) A stirred solution of (E)-1-(4-(2-cyanovinyl)benzyl)-1H-pyrazole-4-carboxylate (3_Int-3) (0.32 g, 1.13 mmol, 1.0 eq) in THF (6 mL, 20 V) was prepared, and 5% Pd / C was added at room temperature. The RM was stirred at room temperature under an H atmosphere for 8 h. The RM was filtered through a Celite bed and washed with ethyl acetate. The filtrate was concentrated and then purified by column chromatography (20-22% EtOAc in hexanes) to give compound (3_Int-4). MS (ES): 284.30.m / z [M+H]+, LCMS purity: 65.00%, 1H 400 MHz, DMSO-d6: δ 1.32 (t, J = 6.8 Hz, 3H), 2.81 (d, J = 6.00 Hz, 2H), 2.86 (d, J = 6.00 Hz, 2H), 4.22 (d, J = 6.80 Hz, 2H), 5.36 (s, 2H), 7.27 (d, J = 7.60 Hz, 2H), 7.66 (d, J = 8.80 Hz, 2H), 7.87 (s, 1H), 8.50 (s, 1H).

[0184] Step-4 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanoethyl)benzyl)-1H-pyrazole-4-carboxamide (3) A stirred solution of ethyl 1-(4-(2-cyanoethyl)benzyl)-1H-pyrazole-4-carboxylate (3_Int-4) (0.2 g, 7.05 mmol, 1.0 eq) in toluene (2 mL, 10 V) was prepared, and 4-(aminomethyl)benzimidamide dihydrochloride (0.15 g, 10.58 mmol, 1.5 eq) and DIPEA (0.23 g, 17.64 mmol, 2.5 eq) were added at room temperature. After cooling to 0 °C, TMA (2.0 M in toluene) (1 mL, 2.11 mmol, 3.0 eq) was added, and the RM was heated to 100 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with 1 V of water and evaporated. The residue was washed with 20% methanol in dichloromethane and filtered. The filtrate was concentrated and purified by preparative HPLC ((A) 0.1% TFA in water (B) 100% MeCN)) to give compound (3). MS (ES): 387.29.m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ 2.80 (d, J = 6.00 Hz, 2H), 2.84 (d, J = 6.00 Hz, 2H), 4.48 (d, J = 5.60 Hz, 2H), 5.33 (s, 2H), 7.25 (q, J = 27.20 Hz, 3H), 7.49 (d, J = 8.00 Hz, 1H), 7.74 (d, J = 8.00 Hz, 1H), 0.00 (s, 1H), 8.27 (s, 1H), 8.78 (d, J = 6.00 Hz, 1H), 8.95 (s, 2H), 9.25 (s, 2H).

[0185] Example 3: N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanoethyl)benzyl)-1H-imidazole-4-carboxamide (4) TIFF2025526464000033.tif84165 Step-1 Synthesis of ethyl 1-(4-iodobenzyl)-1H-imidazole-4-carboxylate (4_Int-2) A stirred solution of ethyl 1H-imidazole-4-carboxylate (0.5 g, 3.56 mmol, 1.0 eq) in DMF (5 mL, 10 V) was prepared, and NaHMDS (1 M in THF) (3.5 mL, 3.56 mmol, 1.0 eq) was added at 0 °C and stirred for 30 min. 1-(Bromomethyl)-4-iodobenzene (0.105 g, 3.56 mmol, 1.0 eq) was added to the RM at 0 °C, brought to room temperature, and stirred for 2 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (35-38% ethyl acetate in hexane) to give compound (4_Int-2). MS (ES): 357.20.m / z [M+H]+, LCMS purity: 99.05%, 1H 400 MHz, DMSO-d6: δ 1.24 (t, J = 7.2 Hz, 3H), 4.19 (q, J = 7.2 Hz, 2H), 5.20 (s, 2H), 7.12 (d, J = 8.00 Hz, 2H), 7.74 (d, J = 8.00 Hz, 2H), 7.88 (s, 1H), 7.94 (s, 1H).

[0186] Step-2 Synthesis of (E)-1-(4-(2-cyanovinyl)benzyl)-1H-pyrazole-4-carboxylate (4_Int-3) A stirred solution of ethyl 1-(4-iodobenzyl)-1H-imidazole-4-carboxylate (4_Int-2) (0.7 g, 1.96 mmol, 1.0 eq) and 1,4-dioxane (14 mL, 20 V) was prepared, and acrylonitrile (0.104 g, 1.96 mmol, 1 eq) and TEA (0.497 g, 4.91 mmol, 2.5 eq) were added at room temperature. N was purged into the RM at room temperature for 10 minutes. Palladium diacetate (0.044 g, 0.19 mmol, 0.1 eq) and JohnPhos (0.117 g, 0.39 mmol, 0.2 eq) were added to the RM. The RM was heated to 110 °C and stirred for 3 hours. The RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (22-25% ethyl acetate in hexanes) to give compound (3_Int-3). MS (ES): 282.28.m / z [M+H]+, LCMS purity: 89.04%, 1H 400 MHz, DMSO-d6: δ 1.23 (t, J = 7.2 Hz, 3H), 4.21 (q, J = 5.60 Hz, 2H), 5.22 (s, 2H), 6.49 (d, J = 16.80 Hz, 1H), 7.41 (t, J = 6.8 Hz, 3H), 7.66 (d, J = 6 Hz, 1H), 7.82 (d, J = 7.60 Hz, 2H), 7.99 (s, 1H).

[0187] Step-3 Synthesis of ethyl 1-(4-(2-cyanoethyl)benzyl)-1H-imidazole-4-carboxylate (4_Int-4) A stirred solution of (E)-1-(4-(2-cyanovinyl)benzyl)-1H-imidazole-4-carboxylate (4_Int-3) (0.4 g, 1.42 mmol, 1.0 eq) in THF (5 mL, 12 V) and ethanol (5 mL, 12 V) was prepared, and 5% Pd / C was added at room temperature. The RM was stirred at room temperature under a hydrogen atmosphere for 16 h. After completion of the reaction, the RM was filtered through a celite bed and washed with ethyl acetate. The filtrate was concentrated and then purified by column chromatography (38-40% EtOAc in hexanes) to give compound (3_Int-4). MS (ES): 284.22 m / z [M+H]+, LCMS purity: 69.16.

[0188] Step-4 N-(4-Carbamimidoylbenzyl)-1-(4-(2-cyanoethyl)benzyl)-1H-pyrazole-4-carboxamide (4) was prepared from ethyl 1-(4-(2-cyanoethyl)benzyl)-1H-imidazole-4-carboxylate (4_Int-4) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2(4). MS (ES): 387.30.m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ 2.80 (d, J = 6.40 Hz, 2H), 2.85 (d, J = 6.00 Hz, 2H), 4.47 (d, J = 5.60 Hz, 2H), 5.23 (s, 2H), 7.30 (s, 3H), 7.46 (d, J = 28.00 Hz, 2H), 7.74 (t, J = 8 Hz, 3H), 8.00 (s, 1H), 8.74 (s, 1H), 8.95 (s, 2H), 9.24 (s, 2H).

[0189] Example 4 Preparation of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (43) N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (301) was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (7_Int-5) and 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) in a manner similar to that described in Example 2(3). MS (ES): 419.20. m / z [M+H]+, LCMS purity: 100%, HPLC purity: 10% 1 H NMR (400 MHz, DMSO-d6) δ 1.667 (s, 6H), 2.504 (s, 2H), 4.771 (d, J=4.2 Hz, 2H), 5.368 (s, 2H), 7.343-7.324 (m, 4H), 7.511 (d, J=8 Hz, 2H), 7.625 (t, J=8 Hz, 1H), 7.919 (s, 1H), 8.295 (s, 1H), 8.795 (t, J=6 Hz, 1H), 9.184 (s, 2H), 9.369 (s, 2H).

[0190] Example 5: N-(4-Carbamimidoylbenzyl)-1-(4-(cyanomethyl)phenethyl)-1H-pyrazole-4-carboxamide (12) TIFF2025526464000035.tif125165 Step-1 Synthesis of ethyl 1-(4-methylphenethyl)-1H-pyrazole-4-carboxylate (12_Int-2) A stirred solution of ethyl 1H-pyrazole-4-carboxylate (2 g, 14.28 mmol, 1.0 eq) in acetone (20 mL, 10 V) was prepared, and 1-(2-bromoethyl)-4-methylbenzene (304 g, 17.14 mmol, 1.2 eq) and CsCO (11.14 g, 34.28 mmol, 2.4 eq) were added at room temperature. The RM was heated to 60-65 °C, stirred for 16 h, and cooled to room temperature. The RM was filtered, washed with EtOAc, and purified by column chromatography (12-15% ethyl acetate in hexanes) to give compound 12_Int-2. MS (ES): 259.23 m / z [M+H].

[0191] Step-2 Synthesis of ethyl 1-(4-(bromomethyl)phenethyl)-1H-pyrazole-4-carboxylate (12_Int-3) A stirred solution of ethyl 1-(4-methylphenethyl)-1H-pyrazole-4-carboxylate (12_Int-2) (1.7 g, 6.58 mmol, 1.0 eq) in carbon tetrachloride (34 mL, 20 V) was prepared, and benzoyl peroxide (0.159 g, 0.65 mmol, 0.1 eq) and N-bromosuccinimide (1.40 g, 7.89 mmol, 1.2 eq) were added at room temperature. The RM was stirred at room temperature for 4 h, quenched with water, and extracted with DCM. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (10-12% ethyl acetate in hexanes) to give compound (12_Int-3). MS (ES): 337.4 m / z [M]+ 339.4 [M+2]+, LCMS purity: 75.91%, 1 H NMR (400 MHz, DMSO-d6) δ 1.239 (t, J=7.2 Hz, 3H), 3.111 (t, J=7.2 Hz, 2H), 4.179 (q, J=7.2 Hz, 2H), 4.385 (t, J=7.2 Hz, 2H), 4.664 (s, 2H), 7.148-7.127 (m, 2H), 7.350-7.330 (m, 2H), 7.841 (s, 1H), 8.220 (s, 1H).

[0192] Step-3 Synthesis of ethyl 1-(4-(cyanomethyl)phenethyl)-1H-pyrazole-4-carboxylate (12_Int-4) A stirred solution of ethyl 1-(4-(bromomethyl)phenethyl)-1H-pyrazole-4-carboxylate (12_Int-3) (0.750 g, 2.22 mmol, 1.0 eq) in MeCN (6 mL, 20 V) was prepared at room temperature. Tetrabutylammonium cyanide (2.38 g, 8.89 mmol, 4.0 eq) was added, and the RM was stirred at room temperature for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (7-10% ethyl acetate in hexanes) to give compound (12_Int-4). MS (ES): 284.28 m / z [M+H]+, LCMS purity: 93.34%, 1 H NMR (400 MHz, DMSO-d6) δ 1.239 (t, J=7.2 Hz, 3H), 3.112 (t, J=7.2 Hz, 2H), 3.975 (s, 2H), 4.188 (q, J=7.2 Hz, 2H), 4.383 (t, J=7.2 Hz, 2H), 7.164 (d, J=8 Hz, 2H), 7.238 (d, J=8 Hz, 2H), 7.839 (s, 1H), 8.216 (s, 1H).

[0193] Step-4 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(cyanomethyl)phenethyl)-1H-pyrazole-4-carboxamide (12) The title compound was prepared from ethyl 1-(4-(cyanomethyl)phenethyl)-1H-pyrazole-4-carboxylate (12_Int-4) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 387.37 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 95.02% 1H NMR (400 MHz, DMSO-d6) δ 3.110 (t, J=7.2 Hz, 2H), 3.979 (s, 2H), 4.367 (t, J=7.2 Hz, 2H), 4.771 (d, J=6 Hz, 2H), 7.182 (d, J=8 Hz, 2H), 7.244 (d, J=8 Hz, 2H), 7.485 (d, J=8 Hz, 2H), 7.751 (d, J=8 Hz, 2H), 7.900 (s, 1H), 8.086 (s, 1H), 8.720 (t, J=6 Hz, 1H), 8.974 (s, 2H), 9.248 (s, 2H).

[0194] Example 6: Preparation of 2-(4-((4-((4-carbamimidoylbenzyl)carbamoyl)-1H-pyrazol-1-yl)methyl)phenyl)acetic acid (16) TIFF2025526464000036.tif48165 Step-1 Synthesis of 2-(4-((4-(ethoxycarbonyl)-1H-pyrazol-1-yl)methyl)phenyl)acetic acid (16_Int-2) A stirred solution of 1H-pyrazole-4-carboxylic acid salt (0.5 g, 3.56 mmol, 1.0 eq) in DMF (5 mL, 10 V) was prepared, and NaH (60% in mineral oil) (0.214 g, 5.35 mmol, 1.5 eq) was added at 0 °C. The RM was stirred at 0 °C for 30 min. 2-(4-(bromomethyl)phenyl)acetic acid (0.980 g, 4.28 mmol, 1.2 eq) was added to the RM at 0 °C and stirred at room temperature for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (5-7% methanol in DCM) to give compound (16_Int-2). MS (ES): 289.3 m / z [M+H]+, LCMS purity: 47.20%, 1H NMR (400 MHz, DMSO-d6) δ 1.252 (t, J=7.2 Hz, 3H), 3.542 (s, 2H), 4.201 (q, J=7.2 Hz, 2H), 5.335 (s, 2H), 7.222 (s, 4H), 7.853 (s, 1H), 8.451 (s, 1H), 12.321 (s, 1H).

[0195] Step-2 2-(4-((4-((4-carbamimidoylbenzyl)carbamoyl)-1H-pyrazol-1-yl)methyl)phenyl)acetic acid (16) was prepared from 2-(4-((4-(ethoxycarbonyl)-1H-pyrazol-1-yl)methyl)phenyl)acetic acid (16_Int-2) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2 (16). MS (ES): 392.50 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 99.29% 1 H NMR (400 MHz, DMSO-d6) δ 1.841 (s, 2H), 3.503 (s, 2H), 4.785 (s, 2H), 7.073-7.051 (m, 4H), 7.297 (d, J=8 Hz, 2H), 7.504 (d, J=8 Hz, 2H), 7.794 (s, 1H), 8.065 (s, 1H).

[0196] Example 7: Preparation of 1-(4-(2-amino-2-oxoethyl)benzyl)-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide (18) TIFF2025526464000037.tif42165 Step-1 Synthesis of 2-(4-(bromomethyl)phenyl)acetamide (18_Int-2) A stirred solution of 2-(4-(bromomethyl)phenyl)acetic acid (0.5 g, 2.18 mmol, 1.0 eq) in toluene (5 mL, 10 V) was prepared, and SOCl (0.270 g, 2.26 mmol, 1.04 eq) and DMF (0.025 g, 0.34 mmol, 0.16 eq) were added at room temperature. The RM was heated to 80-85 °C for 3 h, followed by purging with ammonia gas at 0 °C for 3 h. The RM was filtered, and the solid was washed with EtOAc to give compound (18_Int-2). MS (ES): 228.30 m / z [M]+, 230.12 m / z [M+2]+.

[0197] Step-2 Synthesis of ethyl 1-(4-(2-amino-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (18_Int-3) A stirred solution of 1H-pyrazole-4-carboxylate (18_Int-2) (0.5 g, 3.56 mmol, 1.0 eq) in DMF (5 mL, 10 V) was prepared, and NaH (60% in mineral oil) (0.214 g, 5.35 mmol, 1.5 eq) was added at 0 °C. The RM was stirred at 0 °C for 30 min. 2-(4-(bromomethyl)phenyl)acetamide (0.976 g, 4.28 mmol, 1.2 eq) was added to the RM at 0 °C and stirred at room temperature for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (40-42% ethyl acetate in hexane) to give compound (18_Int-3). MS (ES): 288.23.m / z [M+H]+, LCMS purity: 97.01%, 1 H NMR (400 MHz, DMSO-d6) δ 1.244 (t, J=7.2 Hz, 3H), 3.231 (s, 3H), 4.201 (q, J=7.2 Hz, 2H), 4.210 (s, 2H), 5.320 (s, 2H), 7.210 (s, 4H), 7.917-7.848 (m, 2H), 8.436 (s, 1H).

[0198] Step-3 Synthesis of 1-(4-(2-amino-2-oxoethyl)benzyl)-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide (18) The title compound was prepared from ethyl 1-(4-(2-amino-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (18_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 391.32 m / z [M+H]+, LCMS purity: 99.23%, HPLC purity: 98.73% 1 H NMR (400 MHz, DMSO-d6) δ 3.321 (d, J=3.2 Hz, 2H), 4.479 (d, J=6 Hz, 2H), 5.313 (s, 2H), 6.489 (s, 1H), 7.247-7.193 (m, 4H), 7.505-7.449 (m, 3H), 7.748 (d, J=8 Hz, 1H), 7.902 (s, 1H), 8.253 (s, 1H), 8.752 (t, J=6 Hz, 1H), 8.967 (s, 2H), 9.244 (s, 2H).

[0199] Example 8: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (19) TIFF2025526464000038.tif42165 Step-1 Synthesis of 2-(4-(bromomethyl)phenyl)-N-methylacetamide (19_Int-2) A stirred solution of 2-(4-(bromomethyl)phenyl)acetic acid (1.0 g, 4.36 mmol, 1.0 eq) in toluene (5 mL, 10 V) was prepared at room temperature. SOCl2 (0.54 g, 4.53 mmol, 1.04 eq) and DMF (0.051 g, 0.69 mmol, 0.16 eq) were added at room temperature, followed by heating to 80-85 °C for 3 h. The solution was cooled to 0 °C, and methylamine (2 mL) was added to the RM and stirred at room temperature for 6 h. The RM was filtered, and the solid was washed with EtOAc to give compound (19_Int-2). MS (ES): 242.1 m / z [M]+ 244.1 m / z [M+2]+, LCMS purity: 96.6%, 1 H NMR (400 MHz, DMSO-d6) δ 3.389 (s, 2H), 4.726 (s, 2H), 7.245 (d, J=8 Hz, 2H), 7.344 (d, J=8 Hz, 2H), 8.019 (s, 1H).

[0200] Step-2 Synthesis of ethyl 1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (19_Int-3) A stirred solution of ethyl 1H-pyrazole-4-carboxylate (0.5 g, 3.56 mmol, 1.0 eq) in acetone (10 mL, 20 V) was prepared, and 2-(4-(bromomethyl)phenyl)-N-methylacetamide (19_Int-2) (1.03 g, 4.28 mmol, 1.2 eq) and s2CO3 (2.78 g, 8.56 mmol, 2.4 eq) were added at room temperature. The RM was heated to 60-65 °C and stirred for 16 h. After completion of the reaction, the RM was cooled to room temperature. The RM was filtered and washed with EtOAc. The solid residue was discarded, and the filtrate was concentrated and purified by column chromatography (12-15% ethyl acetate in hexanes) to give compound (19_Int-3). MS (ES): 302.40 m / z [M+H]+, LCMS purity: 86.48%, 1H NMR (400 MHz, DMSO-d6) δ 1.658 (s, 6H), 4.556 (s, 2H), 5.366 (s, 2H), 7.331 (d, J=6 Hz, 2H), 7.505 (d, J=8 Hz, 4H), 7.983-7.934 (m, 2H), 8.314 (s, 1H), 8.595 (s, 1H), 8.813 (s, 1H).

[0201] Step-3 N-(4-carbamimidoylbenzyl)-1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (19) was prepared from ethyl 1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (19_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 405.55 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR (400 MHz, DMSO-d6) δ 2.545 (d, J=4.8 Hz, 3H), 3.361 (s, 2H), 4.476 (d, J=6 Hz, 2H), 5.306 (s, 2H), 7.134-7.186 (m, 4H), 7.492 (d, J=8 Hz, 2H), 7.743 (d, J=8 Hz, 2H), 7.916-7.896 (m, 2H), 8.246 (s, 1H), 8.748 (t, J=6 Hz, 1H), 8.896 (s, 2H), 9.234 (s, 2H).

[0202] Example 9: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-((3-cyano-1H-pyrrol-1-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (26) TIFF2025526464000039.tif68165 N-(4-carbamimidoylbenzyl)-1-(4-((3-cyano-1H-pyrrol-1-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (26) was prepared from ethyl 1-(4-((3-cyano-1H-pyrrol-1-yl)methyl)benzyl)-1H-pyrazole-4-carboxylate (6_Int-1) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in the Examples. MS (ES): 43841. m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR (400 MHz, DMSO-d6) δ 4.482 (d, J=6 Hz, 2H), 5.156 (s, 2H), 5.335 (s, 2H), 6.467 (s, 2H), 7.001 (s, 1H), 7.254-7.250 (m, 4H), 7.496 (d, J=8 Hz, 2H), 7.711 (s, 1H), 7.754 (d, J=8 Hz, 2H), 7.909 (s, 1H), 8.270 (s, 1H), 8.772 (t, J=6 Hz, 1H), 9.012 (s, 2H), 9.258 (s, 2H).

[0203] Example 10: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(1-cyanoethyl)benzyl)-1H-pyrazole-4-carboxamide (5) TIFF2025526464000040.tif48165 Step-1 Synthesis of ethyl 1-(4-(1-cyanoethyl)benzyl)-1H-pyrazole-4-carboxylate (5_Int-1) A stirred solution of ethyl 1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_int-4) (0.3 g, 1.11 mmol, 1.0 eq) in THF (6 mL, 20 V) was prepared, and LiHMDS (1 M in hexane) (1.23 mL, 1.22 mmol, 1.1 eq) was added at -70 °C. The RM was stirred at -70 °C for 30 min. Methyl iodide (0.2 g, 1.44 mmol, 1.3 eq) was added to the RM at -70 °C. The RM was warmed to room temperature and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (12-15% ethyl acetate in hexane) to give compound (5_Int-1). MS (ES): 284.28m / z [M+H]+, LCMS purity: 46.39%.

[0204] Step-2 N-(4-Carbamimidoylbenzyl)-1-(4-(1-cyanoethyl)benzyl)-1H-pyrazole-4-carboxamide (45) was prepared from ethyl 1-(4-(1-cyanoethyl)benzyl)-1H-pyrazole-4-carboxylate (5-Int-1) in a manner similar to that described in Example 2. MS (ES): 387.33.m / z [M+H]+, LCMS purity: 94.00%.

[0205] Example 11: Preparation of N-(4-carbamimidoyl-2-fluorobenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (45) N-(4-carbamimidoyl-2-fluorobenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (45) was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (4-Int-5) and 4-(aminomethyl)-3-fluorobenzimidamide dihydrochloride (45_Int-5) in a manner similar to that described in Example 2. MS (ES): 419.4 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR (400 MHz, DMSO-d6) δ 1.664 (s, 6H), 4.499 (d, J=6.4 Hz, 2H), 5.366 (s, 2H), 7.331 (d, J=8 Hz, 2H), 7.554-7.500 (m, 3H), 7.608 (d, J=8 Hz, 1H), 7.771 (d, J=8 Hz, 1H), 7.925 (s, 1H), 8.303 (s, 1H), 8.776 (t. J=8.8 Hz, 1H), 9.115 (s, 2H), 9.334(s, 2H), 19 F NMR (400 MHz, DMSO-d6) δ -73-659 (1F), -116.871 (0.35F).

[0206] Example 12: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxamide (1) TIFF2025526464000042.tif63165 A 3000 mL 4N RBF equipped with a mechanical stirrer and condenser was charged with ethyl 1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_Int4) (140 g, 517 mmol, 1.0 eq), 4-(aminomethyl)benzimidamide dihydrochloride (127 g, 571 mmol, 1.1 eq), and toluene (1400 mL, 10 V) at room temperature. DIPEA (266.5 mL, 1559 mmol, 3.0 eq) was added to the RM. The RM was cooled to 0°C-5°C and stirred for 20 min. TMA (2 M in toluene) (649.8 mL, 1299 mmol, 2.5 eq) was added dropwise to the RM. The RM was heated to 95°C and stirred for 16 hours. The RM was cooled to room temperature, slowly quenched with DM water (151 mL), and evaporated to give a residue. The solid residue was washed with 50% methanol in DCM (3x1000 mL), and the filtrate was discarded. The solid residue was washed again with 20% MeOH in DCM (2x2000 mL) and then discarded. The filtrate was concentrated and purified by flash column chromatography (12-15% methanol in DCM) to give compound (1). MS (ES): 373.35m / z [M+H]+, LCMS purity: 96.80%, HPLC purity: 95.11% 1 H NMR (400 MHz, DMSO-d6) δ 3.805 (s, 2H), 4.455 (s, 2H), 5.314 (s, 2H), 7.304-7.289 (m, 4H), 7.469 (d, J=8 Hz 2H), 7.710 (d, J=8 Hz, 2H), 7.897 (s, 1H), 8.246 (s, 1H).

[0207] Example 13: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(hydroxymethyl)benzyl)-1H-pyrazole-4-carboxamide (2) TIFF2025526464000043.tif42165 Step-1 Synthesis of ethyl 1-(4-(hydroxymethyl)benzyl)-1H-pyrazole-4-carboxylate (2_Int-2) A stirred solution of ethyl 1H-pyrazole-4-carboxylate (2_int-1) (0.5 g, 3.56 mmol, 1.0 eq) in acetone (5 mL, 10 V) was prepared, and (4-(bromomethyl)phenyl)methanol (0.86 g, 4.28 mmol, 1.2 eq) and Cs2CO3 (3.25 g, 9.98 mmol, 2.8 eq) were added at room temperature. The RM was heated to 60 °C and stirred for 3 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (12-15% ethyl acetate in hexane) to give compound (2_Int-2). MS (ES): 261.30 m / z [M+H]+, LCMS purity: 97.45%, 1 H NMR (400 MHz, DMSO-d6) δ 1.255 (s, 3H), 4.203 (d, J=6.4 Hz, 2H), 4.469 (s, 2H), 5.187(s, 1H), 5.340 (s, 2H), 7.269 (m, 4H), 7.860 (s, 1H), 8.444 (s, 1H).

[0208] Step-2 N-(4-carbamimidoylbenzyl)-1-(4-(hydroxymethyl)benzyl)-1H-pyrazole-4-carboxamide (2) was prepared from ethyl 1-(4-(hydroxymethyl)benzyl)-1H-pyrazole-4-carboxylate (2_Int-2) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 364.28 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 95.74% 1H NMR (400 MHz, DMSO-d6) δ 4.469 (s, 1H), 5.199 (s, 1H), 5.321 (s, 2H), 7.282-7.223 (m, 4H), 7.485 (d, J=6.4 Hz, 2H), 7.741 (d, J=6.4 Hz, 2H), 7.904 (s, 1H), 8.251 (s, 1H), 8.776 (s, 1H), 8.995 (s, 2H), 9.250 (s, 2H), 19 F NMR (400 MHz, DMSO-d6) δ -73.575 (1F).

[0209] Example 14: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-imidazole-4-carboxamide (8) TIFF2025526464000044.tif42165 Step-1 Synthesis of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-imidazole-4-carboxylate (8_Int-1) A stirred solution of ethyl 1H-imidazole-4-carboxylate (0.45 g, 3.21 mmol, 1.0 eq) in DMF (4.5 mL, 10 V) was prepared, and LiHMDS (1 M in THF) (3.2 mL, 3.21 mmol, 1.0 eq) was added at -78 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (7_int-3) (0.91 g, 3.85 mmol, 1.2 eq) was added to the RM at -78 °C. The RM was cooled to room temperature and stirred at room temperature. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (1.2-1.5% methanol in DCM) to give compound (8_Int-1). MS (ES): 297.85m / z [M+H]+, LCMS purity: 86.41%, 1H NMR (400 MHz, DMSO-d6) δ 1.246 (t, J=5.2 Hz, 3H), 1.649 (s, 6H), 4.220-4.185 (m, 2H), 5.256 (s, 2H), 7.381 (d, J=8 Hz, 2H), 7.524 (d, J=8 Hz, 2H), 7.895 (d, J=2 Hz, 1H), 7.952 (d, J=2 Hz, 1H).

[0210] Step-2 N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-imidazole-4-carboxamide (8) was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-imidazole-4-carboxylate (8_Int-1) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 401.25 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 99.63% 1 H NMR (400 MHz, DMSO-d6) δ 1.662 (s, 6H), 4.772 (d, J=6 Hz, 2H), 5.257 (s, 2H), 7.395 (d, J=8 Hz, 2H), 7.536-7.473 (m, 4H), 7.759-7.717 (m, 2H), 8.717 (s, 1H), 8.873 (s, 2H), 9.231 (s, 2H).

[0211] Example 15: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(cyanomethyl)benzyl)-1H-imidazole-4-carboxamide (9) TIFF2025526464000045.tif94165 Step-1 Synthesis of 2-(4-(bromomethyl)phenyl)acetonitrile (9_Int-2) A stirred solution of 2-(p-tolyl)acetonitrile (2 g, 15.24 mmol, 1.0 eq) and carbon tetrachloride (40 mL, 20 V) was prepared at room temperature. AIBN (0.12 g, 0.76 mmol, 0.1 eq) and N-bromosuccinimide (2.24 g, 1.67 mmol, 1.1 eq) were added at room temperature, and the RM was then heated to 90 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with DCM. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (7-10% ethyl acetate in hexanes) to give compound (9_Int-2). 1 H NMR (400 MHz, DMSO-d6) δ 4.092 (s, 2H), 4.705 (s, 2H), 7.335 (d, J=8 Hz, 2H), 7.469 (d, J=8 Hz, 2H).

[0212] Step-2 Synthesis of ethyl 1-(4-(cyanomethyl)benzyl)-1H-imidazole-4-carboxylate (9_Int-3) A stirred solution of ethyl 1H-imidazole-4-carboxylate (1.13 g, 8.09 mmol, 1.0 eq) in DMF (11.3 mL, 10 V) was prepared, and LiHMDS (1 M in THF) (8.9 mL, 8.86 mmol, 1.1 eq) was added at −78 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)acetonitrile (9_Int-2) (1.7 g, 8.09 mmol, 1.0 eq) was added to the RM at −78 °C. The RM was allowed to reach room temperature and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (1.2–2.0% methanol in DCM) to give compound (9_Int-3). MS (ES): 270.23m / z [M+H]+, LCMS purity: 77.67%, 1H NMR (400 MHz, DMSO-d6) δ 1.239 (t, J=7.2 Hz, 3H), 4.020 (s, 2H), 4.186 (q, J=7.2 Hz, 2H), 5.240 (s, 2H), 7.348 (s, 4H), 7.877 (s, 1H), 7.929 (s, 1H).

[0213] Step-3 N-(4-carbamimidoylbenzyl)-1-(4-(cyanomethyl)benzyl)-1H-imidazole-4-carboxamide (9) was prepared from ethyl 1-(4-(cyanomethyl)benzyl)-1H-imidazole-4-carboxylate (9_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 373.29 m / z [M+H]+, LCMS purity: 98.06%, HPLC purity: 98.03% 1 H NMR (400 MHz, DMSO-d6) δ 4.023 (s, 2H), 4.470 (s, 2H), 5.250 (s, 2H), 7.354 (s, 4H), 7.481 (d, J=6.0 Hz, 2H), 7.755-7.718 (m, 2H), 7.974 (s, 1H), 8.739 (s, 1H), 8.929 (s, 2H), 9.237 (s, 2H).

[0214] Example 16: Preparation of N-(4-carbamimidoylbenzyl)-3-(4-(cyanomethyl)benzyl)isoxazole-5-carboxamide (10) TIFF2025526464000046.tif145165 Step-1 Synthesis of (E)-2-(p-tolyl)acetaldehyde oxime (10_Int-2) A stirred solution of 2-(p-tolyl)acetaldehyde (2.2 g, 16.39 mmol, 1.0 eq) in DCM (44 mL, 20 V) was prepared at room temperature. Hydroxylamine hydrochloride (2.3 g, 32.79 mmol, 2.0 eq) and TEA (7 g, 68.86 mmol, 4.2 eq) were added at 0 °C. The RM was then allowed to reach room temperature and stirred for 16 h. After completion of the reaction, the reaction mixture was diluted with DCM and washed with 1N HCl in saturated NaHCO3 and brine solution. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (10_Int-2). MS (ES): 150.10 m / z [M+H]+.

[0215] Step-2 Synthesis of (Z)-N-hydroxy-2-(p-tolyl)acetimidoyl chloride (10_Int-3) A stirred solution of (£)-2-(p-tolyl)acetaldehyde oxime (10_Int-2) (4 g, 26.84 mmol, 1.0 eq) in DMF (80 mL, 20 V) was prepared, and a solution of NCS (3.6 g, 26.84 mmol, 1.0 eq) was added at 0 °C. The RM was heated to 50 °C and stirred for 4 h. After completion of the reaction, the reaction mixture was evaporated. The remaining residue was quenched with NaSO solution and extracted with ethyl acetate. The combined organic fractions were dried over NaSO and concentrated to give compound (10_Int-3). MS (ES): 184.16 m / z [M+H], LCMS purity: 60.43%.

[0216] Step-3 Synthesis of ethyl 3-(4-methylbenzyl)isoxazole-5-carboxylate (10_Int-4) A stirred solution of (Z)-N-hydroxy-2-(p-tolyl)acetimidoyl chloride (10_Int-3) (4 g, 21.78 mmol, 1.0 eq) in diethyl ether (80 mL, 20 V) was prepared, and TEA (3.3 g, 32.67 mmol, 1.5 eq) followed by ethyl propiolate (2.14 g, 21.78 mmol, 1.0 eq) was added at 0 °C and stirred for 4 h. After completion of the reaction, the RM was filtered. The filtrate was concentrated and then purified by flash column chromatography (1.2-2.0% ethyl acetate in hexane) to give compound (10_Int-4). MS (ES): 246.24 m / z [M+H]+, LCMS purity: 100%, 1 H NMR (400 MHz, DMSO-d6) δ 1.419-1.384 (m, 3H), 2.386 (s, 3H), 4.089 (s, 2H), 4.439-4.403 (m, 2H), 7.211 (s, 4H), 7.410 (s, 1H).

[0217] Step-4 Synthesis of ethyl 3-(4-(bromomethyl)benzyl)isoxazole-5-carboxylate (10_Int-5) A stirred solution of ethyl 3-(4-methylbenzyl)isoxazole-5-carboxylate (10_Int-4) (1.8 g, 7.33 mmol, 1.0 eq) was prepared, and 1,2-dichloroethane (54 mL, 30 V), benzoyl peroxide (0.18 g, 0.73 mmol, 0.1 eq), and N-bromosuccinimide (1.3 g, 1.3 mmol, 1.0 eq) were added at room temperature. The RM was heated to 90 °C and stirred for 6 h. After completion of the reaction, the reaction mixture was quenched with NaSO solution and extracted with DCM. The combined organic fractions were dried over NaSO, concentrated, and then purified by flash column chromatography (10–12% ethyl acetate in hexane) to give compound (10_Int-5). MS (ES): 324.2m / z [M]+ 326.2 [M+2]+, LCMS purity: 89.94%, 1H NMR (400 MHz, DMSO-d6) δ 1.286 (t, J=7.2 Hz, 3H), 4.070 (s, 2H), 4.325 (q, J=7.2 Hz, 2H), 4.685 (s, 2H), 7.155 (s, 1H), 7.290 (d, J=8 Hz, 2H), 7.401 (d, J=8 Hz, 2H).

[0218] Step-5 Synthesis of ethyl 3-(4-(cyanomethyl)benzyl)isoxazole-5-carboxylate (10_Int-6) A stirred solution of ethyl 3-(4-(bromomethyl)benzyl)isoxazole-5-carboxylate (10_Int-5) (0.3 g, 0.92 mmol, 1.0 eq) and acetone (6 mL, 20 V) was prepared, and tetrabutylammonium cyanide (1 g, 3.7 mmol, 4 eq) was added under a nitrogen atmosphere and stirred at room temperature for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (17-20% ethyl acetate in hexane) to give compound (10_Int-6). MS (ES): 242.42 m / z [M-42]+, LCMS purity: 100%, 1 H NMR (400 MHz, DMSO-d6) δ 1.287 (t, J=7.2 Hz, 3H), 4.004 (s, 2H), 4.072 (s, 2H), 4.327 (q, J=7.2 Hz, 2H), 7.133 (s, 1H), 3.343-7.291 (m, 4H).

[0219] Step-6 N-(4-Carbamimidoylbenzyl)-3-(4-(cyanomethyl)benzyl)isoxazole-5-carboxamide (10) was prepared in a manner similar to that described in Example 2 from ethyl 3-(4-(cyanomethyl)benzyl)isoxazole-5-carboxylate (10_Int-6) and 4-(aminomethyl)benzimidamide dihydrochloride. MS (ES): 374.41 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 99.64% 1 H NMR (400 MHz, DMSO-d6) δ 3.991 (s, 2H), 4.060 (s, 2H), 4.500 (s, 2H), 6.929 (s, 1H), 7.316 (s, 4H), 7.497 (d, J=7.2 Hz, 2H), 7.739 (d, J=7.2 Hz, 2H).

[0220] Example 17: Preparation of N-(4-carbamimidoylbenzyl)-1-((4'-cyano-[1,1'-biphenyl]-4-yl)methyl)-1H-pyrazole-4-carboxamide (13) TIFF2025526464000047.tif94165 Step-1 Synthesis of ethyl 3-(4-(bromomethyl)benzyl)isoxazole-5-carboxylate (13_Int-2) A stirred solution of 4'-methyl-[1,1'-biphenyl]-4-carbonitrile (13_Int-1) (0.1 g, 0.51 mmol, 1.0 eq) and carbon tetrachloride (2 mL, 20 V) was prepared. Benzoyl peroxide (0.013 g, 0.05 mmol, 0.1 eq) and N-bromosuccinimide (0.1 g, 0.56 mmol, 1.1 eq) were then added at room temperature. The RM was heated to 90 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with Na2SO3 solution and extracted with DCM. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (2-5% ethyl acetate in hexane) to give compound (13_Int-2). 1H NMR (400 MHz, DMSO-d6) δ 4.776 (s,2H), 7.585 (d, J=8 Hz, 2H), 7.845 (d, J=8 Hz, 2H), 7.947 (s, 4H).

[0221] Step-2 Synthesis of ethyl 1-((4'-cyano-[1,1'-biphenyl]-4-yl)methyl)-1H-pyrazole-4-carboxylate (13_Int-3) A stirred solution of ethyl 1H-imidazole-4-carboxylate (0.25 g, 1.83 mmol, 1.0 eq) in acetone (10 mL, 20 V) was prepared. 4'-(Bromomethyl)-[1,1'-biphenyl]-4-carbonitrile (0.5 g, 1.89 mmol, 1.0 eq) and Cs2CO3 (1.43 g, 1.40 mmol, 2.4 eq) were added at room temperature. The RM was heated to 65 °C and stirred for 16 h. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (15-20% ethyl acetate in hexanes) to give compound (13_Int-3). MS (ES): 332.23 m / z [M+H]+.

[0222] Step-3 N-(4-Carbamimidoylbenzyl)-1-((4'-cyano-[1,1'-biphenyl]-4-yl)methyl)-1H-pyrazole-4-carboxamide (13) was prepared from ethyl 1-((4'-cyano-[1,1'-biphenyl]-4-yl)methyl)-1H-pyrazole-4-carboxylate (13_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 435.37 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 99.83% 1H NMR (400 MHz, DMSO-d6) δ 4.485 (d, J=6 Hz, 2H), 5.424 (s, 2H), 7.340 (d, J=8 Hz, 2H), 7.495 (d, J=8 Hz, 2H), 7.758-7.739 (m, 4H), 7.858 (s, 1H), 7.879 (s, 1H), 7.940-7.920 (m, 3H), 8.329 (s, 1H), 8.794 (t, J=6 Hz, 1H), 8.984 (s, 1H), 9.248 (s, 2H).

[0223] Example 18: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (28) TIFF2025526464000048.tif63165 Step-1 N-(4-Carbamimidoylbenzyl)-1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (28) was synthesized from ethyl 1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-4) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 449.34 m / z [M+1]+, LCMS purity: 99.33%, HPLC purity: 100% 1 H NMR (400 MHz, DMSO-d6) δ 3.98 (s, 2H), 4.47 (d, J = 6.40 Hz, 2H), 5.30 (s, 2H), 7.23 (q, J = 8.8 Hz, 4H), 7.50-7.47 (m, 2H), 7.56 (d, J = Hz, 1H), 7.66 (d, J = 7.60 Hz, 1H), 0.00 (d, J = 8.4 Hz, 2H), 7.89 (s, 1H), 8.25 (s, 1H), 8.75 (s, 2H), 8.90 (s, 2H), 9.24 (s, 2H).

[0224] Example 19: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(3-cyano-1H-pyrrol-1-yl)benzyl)-1H-pyrazole-4-carboxamide (25) TIFF2025526464000049.tif130165 Step-1 Synthesis of ethyl 1-(4-iodobenzyl)-1H-pyrazole-4-carboxylate (25_Int-2) A stirred solution of 1H-ethyl pyrazole-4-carboxylate (1.3 g, 9.21 mmol, 1 eq) and 1-(bromomethyl)-4-iodobenzene (25_int-1) (3 g, 10.2 mmol, 1.1 eq) in acetone (13 mL, 10 V) was prepared, and Cs2CO3 (7.2 g, 0.20 mmol, 2.4 eq) was added at room temperature. The RM was heated to 65 °C and stirred for 8 h. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (30% ethyl acetate in hexane) to give compound (28_Int-2). MS (ES): 357.27 m / z [M+1]+, LCMS purity: 82%.

[0225] Step-2 Synthesis of ethyl 1-(4-(3-cyano-1H-pyrrol-1-yl)benzyl)-1H-pyrazole-4-carboxylate (25_Int-3) A stirred solution of ethyl 1-(4-iodobenzyl)-1H-pyrazole-4-carboxylate (28_Int-2) (0.800 g, 2.20 mmol, 1 eq) and 1H-pyrrole-3-carbonitrile (0.426 g, 8.95 mmol, 4 eq) in DMF (8 mL, 10 V) was prepared, and KCO (0.530 g, 6.78 mmol, 3 eq) and CuI (0.426 g, 2.23 mmol, 1 eq) were added at room temperature. The RM was heated to 100 °C and stirred for 8 h. After completion of the reaction, the RM was cooled at room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over NaSO, concentrated, and then purified by flash column chromatography (30% ethyl acetate in hexane) to give compound (25_Int-3). MS (ES): 319.04 m / z [M+1]+, LCMS purity: 70%.

[0226] Step-3 N-(4-carbamimidoylbenzyl)-1-(4-(3-cyano-1H-pyrrol-1-yl)benzyl)-1H-pyrazole-4-carboxamide (25) was synthesized from ethyl 1-(4-(3-cyano-1H-pyrrol-1-yl)benzyl)-1H-pyrazole-4-carboxylate (25_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 424.15 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR (400 MHz, DMSO-δ 4.49 (d, J = 5.20 Hz, 2H), 5.41 (s, 2H), 6.73 (s, 1H), 7.02 (s, 1H), 7.14 (s, 2H), 7.26 (s, 1H), 7.42 (d, J = 8.00 Hz, 1H), 0.00 (t, J = 10.8 Hz, 1H), 7.65 (d, J = 8.00 Hz, 1H), 7.76 (d, J = 7.60 Hz, 1H), 7.94 (s, 1H), 8.23 (s, 1H), 8.32 (s, 1H), 8.79 (s, 1H), 9.10 (s, 2H), 9.25 (s, 2H).19 F NMR 400 MHz, DMSO-d6: δ -73.478(1F).

[0227] Example 20: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(4-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (14) TIFF2025526464000050.tif125165 Step-1 Synthesis of 4-(4-(hydroxymethyl)benzyl)benzonitrile (14_Int-2) A stirred solution of (4-cyanophenyl)boronic acid (1.5 g, 7.46 mmol, 1.0 eq) (14_Int-1) in 1,4 dioxane (15 ml, 10 V) was prepared, and (4-(bromomethyl)phenyl)methanol (1.1 g, 8.20 mmol, 1.1 eq), Cs2CO3 (7.27 g, 22.38 mmol, 3 eq), PdCl2(dppf).DCM (3.04 g, 3.73 mol, 0.5 eq), and water were added at room temperature. The RM was heated to 100 °C and stirred for 8 h. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (20% ethyl acetate in hexane) to give compound (14_Int-2). 1 H NMR (400 MHz, DMSO-d6) δ 4.02 (s, 2H), 4.44 (s, 2H), 5.10 (t, J = 5.56 Hz, 1H), 7.22 (d, J = 8Hz, 4H), 7.44 (d, J = 8.40 Hz, 2H), 7.74 (d, J =8.40 Hz, 2H).

[0228] Step-2 Synthesis of 4-(4-(chloromethyl)benzyl)benzonitrile (14_Int-3) A stirred solution of ethyl 1H-pyrazole-4-carboxylate (0.300 g, 2.14 mmol, 1 eq) in DCM (10 ml, 10 V) was prepared, and DIPEA (0.115 g, 8.96 mmol, 2 eq) and MsCl (0.107 g, 9.41 mmol, 2.1 eq) were added at 0 °C. The RM was stirred at room temperature for 6 h. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (4% ethyl acetate in hexane) to give compound (14_Int-3). 1 H NMR (400 MHz, DMSO-d6) δ 400 MHz, DMSO-d6: δ 4.05 (s, 2H), 4.72 (s, 2H), 7.22 (d, J = 8 Hz, 4H), 7.44 (d, J = 8.40 Hz, 2H), 7.74 (d, J = 8.40Hz, 2H).

[0229] Step-3 Synthesis of ethyl 1-(4-(4-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (25_Int-4) A stirred solution of 1H-ethyl pyrazole-4-carboxylate (0.300 g, 2.14 mmol, 1 eq) and 4-(4-(chloromethyl)benzyl)benzonitrile (14_Int-3) (0.568 g, 2.35 mmol, 1.1 eq) in acetone (3 mL, 10 V) was prepared, and CsCO (1.67 g, 5.14 mmol, 2.4 eq) was added at room temperature. The RM was heated to 65 °C and stirred for 5 h. After completion of the reaction, the RM was cooled at room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over NaSO, concentrated, and then purified by flash column chromatography (16% ethyl acetate in hexane) to give compound (28_Int-2). MS (ES): 346.3 m / z [M+1], LCMS purity: 88%.

[0230] Step-4 N-(4-Carbamimidoylbenzyl)-1-(4-(4-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (14) was synthesized from ethyl 1-(4-(4-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (25_Int-4) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 448 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR (400 MHz, DMSO-d6)δ 4.02 (s, 2H), 4.47 (d, J = 6.00 Hz, 2H), 5.30 (s, 2H), 7.22 (s, 4H), 7.42 (d, J = 8.00 Hz, 2H), 7.49 (d, J = 8.40 Hz, 2H), 7.74 (d, J = 8.4Hz, 3H), 0.00 (s, 1H), 8.25 (s, 1H), 8.76 (s, 1H), 8.92 (s, 2H), 8.95 (s, 2H).

[0231] Example 21: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxamide (15) TIFF2025526464000051.tif109165 Step-1 Synthesis of ethyl 1-(4-(2-hydroxyethyl)benzyl)-1H-pyrazole-4-carboxylate (15_Int-2) A stirred solution of 1H-ethyl pyrazole-4-carboxylate (1 g, 7.14 mmol, 1.0 eq) (14_Int-1) in acetone (10 ml, 10 V) was prepared, and 2-(4-(bromomethyl)phenyl)ethan-1-ol (1.6 g, 7.85 mmol, 1.1 eq) and CsCO (5.58 g, 17.14 mmol, 2.4 eq) were added at room temperature. The RM was heated to 65 °C and stirred for 6 h. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The combined organic fractions were dried over NaSO, concentrated, and then purified by flash column chromatography (25% ethyl acetate in hexane) to give compound (14_Int-2). MS (ES): 274.2 m / z [M+1], LCMS purity: 97%, HPLC purity: % 1 H NMR (400 MHz, DMSO-d6) δδ 1.22 (t, J = 7.20 Hz, 3H), 2.69 (t, J = 6.80 Hz, 2H), 3.57 (t, J = 6.80 Hz, 2H), 4.21 (t, J = 7.20 Hz, 2H), 4.63 (t, J = 5.20 Hz, 1H), 5.31 (s, 2H), 7.19 (s, 4H), 0.00 (s, 1H), 8.43 (s, 1H).

[0232] Step-2 Synthesis of ethyl 1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxylate (15_Int-3) A stirred solution of ethyl 1-(4-(2-hydroxyethyl)benzyl)-1H-pyrazole-4-carboxylate (15_Int-2) (0.900 g, 5.17 mmol, 1.0 eq) in DMF (10 ml, 10 V) was prepared, and NaH (2.85 g, 10.34 mmol, 2 eq) was added and stirred at 0 °C for 15 min. Then, MeI (0.948 g, 6.724 mmol, 1.3 eq) was added at 0 °C, and the RM was stirred for 3 h. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (12% ethyl acetate in hexane) to give compound (14_Int-3). MS (ES): 289.26 m / z [M+1]+, 1 H NMR (400 MHz, DMSO-d6) δ 400 MHz, DMSO-d6: δ 1.26 (t, J = 6.80 Hz, 3H), 2.78 (t, J = 6.80 Hz, 2H), 3.27 (s, 3H), 3.51 (t, J = 6.80 Hz, 2H), 4.20 (q, J = 6.80 Hz, 2H), 5.31 (s, 2H), 7.20 (s, 2H), 0.00 (s, 2H), 8.43 (s, 2H).

[0233] Step-3 N-(4-carbamimidoylbenzyl)-1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxamide (15) was synthesized from ethyl 1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxylate (15_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in Example 2. MS (ES): 392.7 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1H NMR (400 MHz, DMSO-d6) δ 2.52 (t, J = 181.60 Hz, 2H), 3.21 (s, 3H), 3.50 (t, J = 6.8 Hz, 2H), 4.48 (d, J = 5.60 Hz, 2H), 5.30 (s, 2H), 7.20 (t, J = 8.40 Hz, 4H), 7.49 (d, J = 8.40 Hz, 2H), 0.00 (d, J = 8.00 Hz, 2H), 7.90 (s, 1H), 8.26 (s, 1H), 8.77 (s, 1H), 8.97 (s, 2H), 9.25 (s, 2H).

[0234] Example 22: Preparation of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (49) TIFF2025526464000052.tif68165 Step 1 N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (49) was prepared from ethyl 1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-4) and 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) in a manner similar to that described in Example 2. MS (ES): 467.14 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR 400 MHz, DMSO-d6: δ 3.99 (s, 2H), 4.46 (d, J = 6.00 Hz, 2H), 5.34 (s, 2H), 7.34-7.20 (m, 6H), 7.48 (t, J = 7.60 Hz, 1H), 7.71-7.56 (m, 3H), 7.89 (s, 1H), 8.25 (s, 1H), 8.76 (t, J = 6 Hz, 1H), 9.18 (s, 2H), 9.36 (s, 2H), 19F NMR 400 MHz, DMSO-d6: δ -73.492 (1F), -113.893 (0.35F).

[0235] Example 23: N-(4-carbamimidoyl-2-fluorobenzyl)-1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (50) N-(4-carbamimidoyl-2-fluorobenzyl)-1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxamide (50) was prepared from ethyl 1-(4-(3-cyanobenzyl)benzyl)-1H-pyrazole-4-carboxylate (28_Int-4) and 4-(aminomethyl)-3-fluorobenzimidamide dihydrochloride (45_Int5) in a manner similar to that described in Example 2. MS (ES): 466.52 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1 H NMR 400 MHz, DMSO-d6: δ 3.99 (s, 2H), 3.32 (s, 1H), 4.49 (d, J = 5.60 Hz, 1H), 5.30 (s, 1H), 7.23 (q, J = 8.00 Hz, 5H), 7.71-7.48 (m, 8H), 7.90 (s, 1H), 8.25 (s, 1H), 8.73 (s, 1H), 9.11 (s, 1H), 9.32 (s, 1H), 19 F NMR 400 MHz, DMSO-d6: δ -73.536 (1F), -117.182(0.68F).

[0236] Example 24: Preparation of N-(4-carbamimidoylbenzyl)-1-(3-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxamide (69) TIFF2025526464000054.tif125165 Step 1 Synthesis of 1-bromo-3-(2-methoxyethyl)benzene (69_Int-2) A stirred solution of 2-(3-bromophenyl)ethan-1-ol (69_Int-1) (3 g, 14.92 mmol, 1.0 eq) in DMF (30 mL, 10 V) was prepared, and NaH (60% in mineral oil) (0.72 g, 17.91 mmol, 1.2 eq) was slowly added at 0 °C and stirred for 1 h. Iodomethane (2.54 g, 17.91 mmol, 1.2 eq) was added to the RM at 0 °C, then brought to room temperature and stirred for 16 h. After completion of the reaction, the RM was slowly quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (4-5% ethyl acetate in hexane) to give compound (69_Int-2). MS (ES): 162.23 m / z [M-53]+, LCMS purity: 99.25%, 1H NMR (400 MHz, DMSO-d6) δ 2.808 (t, J=6.4 Hz, 2H), 3.235 (s, 3H), 3.533 (t, J=6.4 Hz, 2H), 7.250 (d, J=5.2 Hz, 2H), 7.383-7.410 (m, 1H), 7.458 (s, 1H).

[0237] Step 2 Synthesis of 3-(2-methoxyethyl)benzaldehyde (69_Int-3) A stirred solution of 1-bromo-3-(2-methoxyethyl)benzene (69_Int-2) (1.5 g, 7.0 mmol, 1.0 eq) in diethyl ether (15 mL, 10 V) was prepared, and tetramethylethylenediamine (1.75 g, 1.5 mmol, 2.16 eq) was added at room temperature. The RM was cooled to -75 °C, followed by the dropwise addition of n-BuLi (2.5 M in hexanes) (5.6 mL, 14.0 mmol, 2.0 eq) under N2. The RM was stirred at -75 °C for 1 h. The RM was then warmed to -20 °C and stirred for an additional 20 min. The RM was again cooled to -75 °C, followed by the dropwise addition of dry DMF (7.5 mL, 5 V) and stirring for 16 h. After completion of the reaction, the RM was slowly quenched with 1 N HCl and extracted with ethyl acetate. The combined organic fractions were dried over NaSO, concentrated, and then purified by flash column chromatography (10-12% ethyl acetate in hexanes) to give compound (69_Int-3). MS (ES): 132.8 m / z [M-32], LCMS purity: 100%, H NMR (400 MHz, DMSO-d) δ 2.902 (t, J=6.4 Hz, 2H), 3.325 (s, 3H), 3.581 (t, J=6.4 Hz, 2H), 7.518 (t, J=7.6 Hz, 1H), 7.588 (d, J=8.0 Hz, 1H), 7.742-7.767 (m, 2H), 9.983 (s, 1H).

[0238] Step 3 Synthesis of (3-(2-methoxyethyl)phenyl)methanol (69_Int-4) A stirred solution of 3-(2-methoxyethyl)benzaldehyde (69_Int-3) (0.23 g, 1.40 mmol, 1.0 eq) in methanol (4.6 mL, 20 V) was prepared, and sodium borohydride (0.106 g, 2.8 mmol, 2.0 eq) was slowly added at room temperature and stirred for 4 h. After completion of the reaction, the RM was evaporated. The residue was quenched with water, adjusted to pH ∼5 with 2 N HCl, and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (12–15% ethyl acetate in hexane) to give compound (69_Int-4). MS (ES): 149.1 m / z [M-18]+, LCMS purity: 97.53%, 1H NMR (400 MHz, DMSO-d6) δ 2.794 (t, J=6.8 Hz, 2H), 3.240 (s, 3H), 3.527 (t, J=6.8 Hz, 2H), 4.467 (d, J=5.6 Hz, 2H), 5.150 (t, J=6.0 Hz, 1H), 7.092 (d, J=7.6 Hz, 1H), 7.151 (t, J=7.6 Hz, 2H), 7.231 (t, J=7.6 Hz, 1H).

[0239] Step 4 Synthesis of 1-(bromomethyl)-3-(2-methoxyethyl)benzene (69_Int-5) A stirred solution of (3-(2-methoxyethyl)phenyl)methanol (69_Int-4) (0.2 g, 1.20 mmol, 1.0 eq) in DCM (4 mL, 20 V) was prepared. Then, triphenylphosphine (0.34 g, 1.32 mmol, 1.1 eq) and carbon tetrabromide (0.44 g, 1.32 mmol, 1.1 eq) were added at room temperature and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with DCM. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (8-10% ethyl acetate in hexanes) to give compound (69_Int-5). MS (ES): 248.2 m / z [M+18]+, LCMS purity: 100%, 1H NMR (400 MHz, DMSO-d6) δ 2.816 (t, J=6.8 Hz, 2H), 3.343 (s, 3H), 3.538 (t, J=6.8 Hz, 2H), 4.681 (s, 2H), 7.187 (s, 1H), 7.259-7.309 (m, 2H), 7.332-7.387 (m, 1H).

[0240] Step 5 Synthesis of ethyl 1-(3-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxylate (69_Int-6) A stirred solution of 1-(bromomethyl)-3-(2-methoxyethyl)benzene (69_Int-5) (0.176 g, 0.76 mmol, 1.0 eq) in acetone (3.52 mL, 20 V) was prepared. Then, 1H-ethyl pyrazole-4-carboxylate (0.4 g, 0.76 mmol, 1.0 eq) and cesium carbonate (0.6 g, 1.84 mmol, 2.4 eq) were added at room temperature. The RM was brought to 65 °C and stirred for 16 h. After completion of the reaction, the RM was filtered, and the filtrate was concentrated and then purified by flash column chromatography (20-25% ethyl acetate in hexanes) to give compound (69_Int-6). MS (ES): 289.4 m / z [M+H]+, LCMS purity: 100%, 1H NMR (400 MHz, DMSO-d6) δ 1.25 (t, J=8.0 Hz, 3H), 2.77 (t, J=8.0 Hz, 2H), 3.22 (s, 3H), 3.50 (d, 4.12 (q, J=8.0 Hz, 2H), 5.33 (s, 2H), 7.08 (d, J=8.0 Hz, 1H), 7.15-7.18 (m, 2H), 7.25 (t, J=8.0 Hz, 1H), 7.87 (s, 1H), 8.45 (s, 1H).

[0241] Step 6 Synthesis of N-(4-carbamimidoylbenzyl)-1-(3-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxamide (69) A stirred solution of ethyl 1-(3-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxylate (69_Int-6) (0.1 g, 0.34 mmol, 1.0 eq) in THF (1 mL, 10 V) was prepared. 4-(Aminomethyl)benzimidamide dihydrochloride (0.09 g, 0.41 mmol, 1.2 eq) and DIPEA (0.18 g, 1.38 mmol, 4.0 eq) were added at room temperature. The RM was then cooled to 0 °C, followed by the addition of TMA (2 M in toluene) (1.0 mL, 2.08 mmol, 6.0 eq). The RM was brought to 85 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with 1 V of water and evaporated. The residue was washed with 20% methanol in dichloromethane and filtered. The filtrate was concentrated and then purified by preparative HPLC ((A) 0.1% TFA in water (B) 100% MeCN). Finally, the pure fractions were lyophilized to give compound (69). MS (ES): 392.6 m / z [M+H]+, LCMS purity: 97.92%, HPLC purity: 100% 1H NMR (400 MHz, DMSO-d6) δ 2.790 (t, J=6.8 Hz, 2H), 3.226 (s, 3H), 3.522 (d, J=6.8 Hz, 2H), 4.492 (d, J=6.4 Hz, 2H), 5.326 (s, 2H), 7.088 (d, J=8.0 Hz, 1H), 7.077-7.131 (m, 2H), 7.181 (t, J=4.4 Hz, 1H), 7.403 (d, J=4.4 Hz, 2H), 7.761 (d, J=8.4 Hz, 2H), 7.924 (s, 1H), 8.274 (s, 1H), 8.776 (t, J=6.0 Hz, 1H), 9.043 (s, 2H), 9.256 (s, 2H).

[0242] Example 25: Preparation of N-(4-carbamimidoyl-3,5-difluorobenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (44) TIFF2025526464000055.tif53165 Step-1 Synthesis of N-(4-carbamimidoyl-3,5-difluorobenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (44) A stirred solution of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (7_Int-5) (0.1 g, 0.3367 mmol, 1.0 eq) in toluene (2 mL, 10 V) was prepared. 4-(Aminomethyl)-2,6-difluorobenzimidamide dihydrochloride (44_Int-8) (0.095 g, 0.505 mmol, 1.5 eq) and DIPEA (0.6 mL, 0.8417 mmol, 2.5 eq) were added to the RM at room temperature. The RM was cooled to 0 °C, and TMA (2.0 M in toluene, 0.6 mL, 1.0101 mmol, 3.0 eq) was added. The RM was then heated to 100 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with 1 V of water and evaporated. The residue was washed with 20% methanol in dichloromethane and filtered. The solid residue was discarded, and the filtrate was concentrated and then purified by preparative HPLC ((A) 0.1% TFA in water (B) 100% MeCN). Finally, the pure fractions were lyophilized to give compound 44. MS (ES): 437.21 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 94.56% 1H 400 MHz, DMSO-d6: δ 1.664 (s, 6H), 4.462 (d, J = 6.40 Hz, 2H), 5.369 (s, 2H), 7.252 (d, J=8.8 Hz, 2H), 7.331 (d, J = 8.0 Hz, 2H), 7.511 (d, J = 8.00 Hz, 2H), 7.921 (s, 1H), 8.310 (s, 1H), 8.840 (t, J=6.4 Hz, 1H), 9.513 (s, 2H), 9.669 (s, 2H).

[0243] Example 26: N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxamide (83) TIFF2025526464000056.tif109165 Step-1 Synthesis of tert-butyl 2-(4-(2-cyanopropan-2-yl)benzyl)hydrazine-1-carboxylate (83_Int-1) A stirred solution of 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (7_Int-3) (0.5 g, 2.1 mmol, 1.0 eq) in dimethylacetamide (5 mL, 10 V) was prepared. DIPEA (0.54 g, 4.2 mmol, 2.0 eq) and t-butyl carbazate (0.55 g, 4.2 mmol, 2.0 eq) were added at room temperature and stirred at 70 °C for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were washed with brine solution, dried over Na2SO4, concentrated, and purified by flash column chromatography (10-12% ethyl acetate in hexane) to give compound (83_Int-1). 1H 400 MHz, DMSO-d6: δ 1.637 (s, 6H), 1.760 (s, 9H), 4.206 (s, 2H), 7.514 (s, 4H), 7.744 (d, br, J = 8.00 Hz, 1H), 7.886-8.004 (d, br, J=47.2, 1H).

[0244] Step-2 Synthesis of 2-(4-(hydrazinylmethyl)phenyl)-2-methylpropanenitrile (83_Int-2) A stirred solution of tert-butyl 2-(4-(2-cyanopropan-2-yl)benzyl)hydrazine-1-carboxylate (83_Int-1) (2.0 g, 6.9 mmol, 1.0 eq) in methanol (10 mL, 5 V) and water (22 mL, 11 V) was prepared, and concentrated HCl (6.6 mL, 3.3 V) was added at room temperature. The RM was heated to 80 °C and stirred for 16 h. After completion of the reaction, the RM was evaporated and triturated in ethyl acetate to give compound (83_Int-2). MS (ES): 191.4 m / z [M+H]+, LCMS purity: 83.16%, 1H 400 MHz, DMSO-d6: δ 1.693 (s, 6H), 4.076 (s, 2H), 7.096 (s, br, 2H), 7.517 (d, J = 12.8 Hz, 4H).

[0245] Step-3 Synthesis of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxylate (7_Int-3) A solution of ethyl 3-cyclopropyl-3-oxopropanoate (0.22 g, 1.41 mmol, 1.0 eq) and dimethylformamide dimethyl acetal (0.185 g, 1.55 mmol, 1.1 eq) was prepared and stirred at 75 °C for 90 min. The RM was cooled to room temperature, followed by the addition of ethanol (4.4 mL, 20V), TEA (0.57 g, 5.63 mmol, 4.0 eq), and 2-(4-(hydrazinylmethyl)phenyl)-2-methylpropanenitrile (83_Int-2) (0.365 g, 1.41 mmol, 1.0 eq). The RM was heated to 80 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were washed with brine solution, dried over Na2SO4, concentrated, and purified by flash column chromatography (12-15% ethyl acetate in hexanes) to give compound (83_Int-3). MS (ES): 180.88 m / z [M+H]+, LCMS purity: 89%, 1H 400 MHz, DMSO-d6: δ 1.637 (s, 6H), 4.174-4.225 (m, 2H), 5.484 (s, 2H), 7.222 (d, J = 8.0 Hz, 2H), 7.505 (d, J = 8.0 Hz, 2H), 7.830 (s, 1H).

[0246] Step-4 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxamide (83) The final compound was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxylate (7_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (83). MS (ES): 441.4 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ 0.734-0.757 (m, 2H), 0.919-0.967 (m, 2H), 1.658 (s, 6H), 1.753-1.796 (m, 1H), 4.472 (d, J=6.0 Hz, 2H), 5.438 (s, 2H), 7.227 (d, J = 8 Hz, 2H), 7.501 (t, J=8.8 Hz, 4H), 7.762 (d, J=8.0 Hz, 2H), 7.801 (s, 1H), 8.534 (t, J=6.0 Hz, 1H), 8.977 (s, 2H), 9.258 (s, 2H).

[0247] Example 27: N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-propyl-1H-imidazole-4-carboxamide (84) TIFF2025526464000057.tif120165 Step-1 Synthesis of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-propyl-1H-imidazole-4-carboxylate (84_Int-1) A stirred solution of ethyl 5-propyl-1H-imidazole-4-carboxylate (0.45 g, 2.47 mmol, 1.0 eq) in DMF (4.5 mL, 10 V) was prepared, and NaHMDS (1 M in THF, 2.5 mL, 2.47 mmol, 1.0 eq) was added at 0 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (7_Int-3) (0.5 g, 3.56 mmol, 1.0 eq) was added to the RM at 0 °C, brought to room temperature, and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (20-25% ethyl acetate in hexane) to give 84_Int-1. 2D NMR (ROE analysis) confirmed the product. MS (ES): 339.9 m / z [M+H]+, LCMS purity: 84.72%, 1H 400 MHz, DMSO-d6: δ 0.807 (t, J = 7.2 Hz, 3H), 1.251 (q, J = 8.0 Hz, 3H), 1.297 (s, 2H), 1.654 (s, 6H), 2.734 (q, J = 6.4 Hz, 2H), 4.186 (q, J=7.2 Hz, 2H), 5.264 (s, 2H), 7.194 (d, J=8.0 Hz, 2H), 7.512 (d, J=8.0 Hz, 2H), 7.788 (s, 1H).

[0248] Step-2 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-propyl-1H-imidazole-4-carboxamide (84) The final compound was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-propyl-1H-imidazole-4-carboxylate (84_Int-1) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (84). MS (ES): 443.35 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 99.63% 1H 400 MHz, DMSO-d6: δ 0.782 (t, J = 7.2 Hz, 3H), 1.262-1302 (m, 2H), 1.656 (d, J = 6.00 Hz, 2H), 2.773 (t, J = 8 Hz, 2H), 4.473 (d, J=6.4 Hz, 2H), 5.260 (s, 2H), 7.226 (d, J = 8.4 Hz, 2H), 7.487-7.526 (m, 4H), 7.745 (d, J=8.4 Hz, 2H), 7.872 (s, 1H), 8.634 (t, J=6.0 Hz, 1H), 9.069 (s, 2H), 9.248 (s, 2H).

[0249] Example 28: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-methyl-1H-pyrazole-4-carboxamide (81) TIFF2025526464000058.tif94165 Step-1 Synthesis of ethyl 5-methyl-1H-pyrazole-4-carboxylate (81_Int-2) A solution of ethyl 3-oxobutanoate (81_Int-1) (0.5, 3.84 mmol, 1.0 eq) and dimethylformamide dimethyl acetal (0.46 g, 3.84 mmol, 1.0 eq) was prepared and stirred at 110 °C for 1 h. The RM was cooled to room temperature, followed by the addition of ethanol (5.0 mL, 10 V) and hydrazine hydrate (99%) (0.2 g, 3.84 mmol, 1.0 eq). The RM was heated to 70 °C and stirred for 2 h. After completion of the reaction, the reaction mixture was evaporated to give compound (81_Int-2). MS (ES): 154.96 m / z [M+H]+, LCMS purity: 100%, 1H NMR (400 MHz, DMSO-d6) δ 1.241 (t, J=8.00 Hz, 3H), 2.446 (s, 3H), 4.178 (q, J=4.2 Hz, 2H), 7.794 (s, 1H), 13.102 (s, 1H).

[0250] Step-2 Synthesis of ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-methyl-1H-pyrazole-4-carboxylate (81_Int-3) A stirred solution of ethyl 5-methyl-1H-pyrazole-4-carboxylate (81_Int-2) (0.25 g, 1.62 mmol, 1.0 eq) in DMF (2.5 mL, 10 V) was prepared, and NaHMDS (1 M in THF) (1.62 mL, 1.62 mmol, 1.0 eq) was added at 0 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)-2-methylpropanenitrile (7_Int-3) (0.4 g, 1.62 mmol, 1.0 eq) was added to the RM at 0 °C. The RM was allowed to reach room temperature and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (20-25% ethyl acetate in hexane) to give compound (81_Int-3). MS (ES): 312.2 m / z [M+H]+, LCMS purity: 100%, 1H 400 MHz, DMSO-d6: δ 1.239 (t, J = 8.00 Hz, 3H), 1.656 (s, 6H), 2.299 (s, 3H), 4.171 (t, J = 8.00 Hz, 2H), 5.273 (s, 2H), 7.190 (d, J=8.00 Hz, 2H), 7.322 (d, J=8.00 Hz, 2H), 7.829 (s, 1H).

[0251] Step-3 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-propyl-1H-imidazole-4-carboxamide (81) The final compound was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-propyl-1H-imidazole-4-carboxylate (81_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 25) (81). MS (ES): 415.35 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 99.47% 1H 400 MHz, DMSO-d6: δ 1.655 (s, 6H), 2.457 (s, 3H), 4.777 (d, J = 6.0 Hz, 2H), 5.341 (s, 2H), 7.191 (d, J=8 Hz, 2H), 7.515-7.477 (m, 4H), 7.750 (d, J = 8 Hz, 2H), 7.985 (s, 1H), 8.676(t, J=6 Hz, 1H), 8.886 (s, 2H), 9.247 (s, 2H).

[0252] Example 29: N-(4-Carbamimidoyl-3-fluoro-5-methoxybenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (48) TIFF2025526464000059.tif120165 Step-1 Synthesis of tert-butyl (4-cyano-3-fluoro-5-methoxybenzyl)carbamate (48_Int-1) A stirred solution of tert-butyl (4-cyano-3,5-difluorobenzyl)carbamate (44_Int-7) (5 g, 18.36 mmol, 1.0 eq) was prepared in THF (50 mL, 10 V) and MeOH (50 mL, 10 V). The RM was cooled to 0 °C, and sodium methoxide (4.02 g, 74.6 mmol, 4.0 eq) was added slowly. The RM was allowed to reach room temperature and stirred for 5 h. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (48_Int-1). 1H 400 MHz, DMSO-d6: δ 1.631 (s, 9H), 3.924 (s, 3H), 4.169 (d, J = 6.0 Hz, 2H), 6.871 (d, J=10 Hz, 1H), 6.982 (s, 1H), 7.544 (t, J=6.0 Hz, 1H).

[0253] Step-2 Synthesis of tert-butyl (3-fluoro-4-(N-hydroxycarbamimidoyl)-5-methoxybenzyl)carbamate (48_Int-2) A stirred solution of tert-butyl (4-cyano-3-fluoro-5-methoxybenzyl)carbamate (48_Int-1) (4.7 g, 16.78 mmol, 1.0 eq) in MeOH (50 mL, 10 V) was prepared. Hydroxylamine hydrochloride (1.96 gm, 28.536 mmol, 1.7 eq) and DIPEA (5.0 ml, 28.535 mmol, 1.7 eq) were then added at room temperature. The RM was heated to 70 °C and stirred for 16 hours. After completion of the reaction, the RM was cooled to room temperature, quenched with water, and extracted with DCM. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (48_Int-2). MS (ES): 314.29 m / z [M+1]+, LCMS purity: 80%.

[0254] Step-3 Synthesis of tert-butyl (4-carbamimidoyl-3-fluoro-5-methoxybenzyl)carbamate (48_Int-3) A stirred solution of tert-butyl (3-fluoro-4-(N-hydroxycarbamimidoyl)-5-methoxybenzyl)carbamate (48_Int-2) (2 g, 6.3397 mmol, 1.0 eq) in MeOH (20 mL, 10 V) was prepared. Ammonium chloride (1.7 g, 31.9485 mmol, 5.0 eq) and iron (1.7 g, 31.9485 mmol, 5.0 eq) were then added at room temperature. The RM was cooled to 0 °C, followed by the dropwise addition of acetic acid (20 mL, 5 V). The RM was heated to 70 °C and stirred for 16 h. After completion of the reaction, the RM was cooled to room temperature. The RM was concentrated, quenched with cold water, and basified to a target pH of 10 by slow addition of saturated NaOH solution. The solid was filtered, and the filtrate was extracted with DCM. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (48_Int-3). MS (ES): 298.23 m / z [M+1]+, LCMS purity: 73.2%.

[0255] Step-4 Synthesis of 4-(aminomethyl)-2-fluoro-6-methoxybenzimidamide (48_Int-4) A stirred solution of tert-butyl (4-carbamimidoyl-3-fluoro-5-methoxybenzyl)carbamate (48_Int-3) (0.8 g, 25.473 mmol, 1.0 eq) in water (8 mL, 10 V) was prepared, and concentrated HCl (2.6 mL, 3.3 V) was added at room temperature and stirred for 3 h. After completion of the reaction, the RM was concentrated and triturated in methanol to give compound (43_Int-4). MS (ES): 198.2 m / z [M+1]+, LCMS purity: 36%.

[0256] Step-5 Synthesis of N-(4-carbamimidoyl-3-fluoro-5-methoxybenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxamide (48) The final compound was prepared from ethyl 1-(4-(2-cyanopropan-2-yl)benzyl)-1H-pyrazole-4-carboxylate (7_Int-5) and 4-(aminomethyl)-2-fluoro-6-methoxybenzimidamide dihydrochloride (48_Int-4) in a manner similar to that described in (Example 25) (48). MS (ES): 449.22 m / z [M+1]+, LCMS purity: 93.36%, HPLC purity: 93.46% 1H 400 MHz, DMSO-d6: δ 1.660 (s, 6H), 3.843 (s, 3H), 4.436 (d, J = 5.60 Hz, 2H), 5.364 (s, 2H), 6.878 (d, J=10 Hz, 2H), 6.966 (s, 1H), 7.324 (d, J = 8 Hz, 2H), 7.507 (d, J=8.0 Hz, 2H), 7.919 (s, 1H), 8.299 (s, 1H), 8.778 (t, J= 6.4 Hz, 1H), 9.139 (s, 2H), 9.383 (s, 2H).

[0257] Example 30: N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxamide (72) TIFF2025526464000060.tif73165 Step-1 Synthesis of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxamide (72) The final compound was prepared from ethyl 1-(4-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (1_Int-4) and 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) in a manner similar to that described in (Example 25) (72). MS (ES): 391.22 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ 4.024 (s, 2H), 4.460 (s, 2H), 5.357 (s, 2H), 7.304-3.349 (m, 6H), 7.622 (s, 1H), 7.915 (s, 1H), 8.280 (s, 1H), 8.800 (t, J=6.4 Hz, 1H), 9.132 (s, 2H), 9.377 (s, 2H).

[0258] Example 31: N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxamide (73) TIFF2025526464000061.tif68165 Step-1 Synthesis of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxamide (73) The final compound was prepared from ethyl 1-(4-(2-methoxyethyl)benzyl)-1H-pyrazole-4-carboxylate (15_Int-3) and 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) in a manner similar to that described in (Example 25) (73). MS (ES): 410.4 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 99.67% 1H 400 MHz, DMSO-d6: δ 2.774 (t, J =6.80 Hz, 2H), 3.212 (s, 3H), 3.500 (t, J =6.80 Hz, 2H), 7.460 (d, J=6.0 Hz, 2H), 5.303 (s, 2H), 7.175-7.224 (m, 4H), 7.319 (t, J = 7.2 Hz, 2H), 7.899 (s, 1H), 8.260 (s, 1H), 8.797 (t, J=6.0 Hz, 1H), 9.182 (s, 2H), 9.377 (s, 2H).

[0259] Example 32: N-(4-Carbamimidoyl-3-fluorobenzyl)-1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (75) TIFF2025526464000062.tif63165 Step-1 Synthesis of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (75) The final compound was prepared from methyl 1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (85_Int-2) and 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) in a manner similar to that described in (Example 24) (75). MS (ES): 435.4 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ 3.232 (s, 3H), 4.019 (s, 2H), 4.85 (d, J = 6.0 Hz, 2H), 4.812 (s, 2H), 5.365 (s, 2H), 7.242 (d, J = 7.6 Hz, 2H), 7.307-7.374 (M, 4H), 7.640 (t, = Hz, 1H), 8.039 (s, 1H), 8.871 (t, J=6.0 Hz, 1H), 9.197 (s, 2H), 9.401 (s, 2H).

[0260] Example 33: N-(4-carbamimidoylbenzyl)-1-(4-(methoxymethyl)benzyl)-1H-pyrazole-4-carboxamide (71) TIFF2025526464000063.tif53165 Step-1 Synthesis of ethyl 1-(4-(methoxymethyl)benzyl)-1H-pyrazole-4-carboxylate (71_Int-1) A stirred solution of ethyl 1-(4-(hydroxymethyl)benzyl)-1H-pyrazole-4-carboxylate (36_Int-2) (2.5 g, 9.6153 mmol, 1.0 eq) in DMF (25 ml, 10 V) was prepared, and NaH (60% in mineral oil, 0.6 g, 12.4998 mmol, 2 eq) was added at 0 °C and stirred for 15 min. MeI (1.18 mL, 19.2306 mmol, 1.3 eq) was added dropwise to the RM at 0 °C. The RM was allowed to reach room temperature and stirred for 5 h. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (15% ethyl acetate in hexane) to give compound (71_Int-1). MS (ES): 274 m / z [M+1]+, 1H NMR (400 MHz, DMSO-d6) δ 1.267(t, J = 7.2 Hz, 3H), 3.268 (s, 3H), 4.235-4.182(m, 2H), 4.382 (s, 2H), 5.536(s, 2H), 7.248 (t, J=8Hz 4H), 7.877 (s, 1H), 8.468 (s, 1H).

[0261] Step-2 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(methoxymethyl)benzyl)-1H-pyrazole-4-carboxamide (71) The final compound was prepared from ethyl 1-(4-(methoxymethyl)benzyl)-1H-pyrazole-4-carboxylate (71_Int-1) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (71). MS (ES): 378.28 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 98.18% 1H 400 MHz, DMSO-d6: δ 3.275 (s,3H), 4.390 (s, 2H), 4.487 (d, J = 6.0 Hz, 2H), 5.353 (s, 2H), 7.248-7.318 (m, 4H), 7.50 (d, J =8.0 Hz, 2H), 7.758 (d, J =8.0 Hz, 2H), 7.924 (s, 1H), 8.281 (s, 1H), 8.791 (t, J=6.0 Hz, 1H), 9.074 (s, 2H), 9.264 (s, 2H).

[0262] Example 34: N-(4-Carbamimidoylbenzyl)-1-(4-(cyanomethyl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxamide (86) TIFF2025526464000064.tif99165 Step-1 Synthesis of tert-butyl 2-(4-(cyanomethyl)benzyl)hydrazine-1-carboxylate (86_Int-2) A stirred solution of 2-(4-(bromomethyl)phenyl)acetonitrile (86_Int-1) (3 g, 14.2857 mmol, 1.0 eq) in dimethylacetamide (30 mL, 10 V) was prepared at room temperature. DIPEA (3.68 g, 28.5714 mmol, 2.0 eq) and t-butyl carbazate (3.77 g, 28.5714 mmol, 2.0 eq) were then added at room temperature. The RM was then heated to 70 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with DM water and extracted with ethyl acetate. The combined organic fractions were washed with brine solution, dried over Na2SO4, concentrated, and then purified by flash column chromatography (10-14% ethyl acetate in hexane) to give compound (86_Int-2). MS (ES): 261 m / z [M+1]+, LCMS purity: 70%.

[0263] Step-2 Synthesis of 2-(4-(hydrazinylmethyl)phenyl)acetonitrile (86_Int-3) A stirred solution of tert-butyl 2-(4-(cyanomethyl)benzyl)hydrazine-1-carboxylate (86_Int-2) (1.0 g, 7.6628 mmol, 1.0 eq) in methanol (10 mL, 5 V) and water (22 mL, 11 V) was prepared. Concentrated HCl (6.6 mL, 3.3 V) was then added to the RM at room temperature. The RM was heated to 80° C. and stirred for 16 hours. After completion of the reaction, the RM was evaporated and triturated in ethyl acetate to give compound (86_Int-3). MS (ES): 162.3 m / z [M+1]+, LCMS purity: 64%.

[0264] Step-3 Synthesis of ethyl 1-(4-(cyanomethyl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxylate (86_Int-4) A solution of ethyl 3-cyclopropyl-3-oxopropanoate (1.0 g, 4.61531 mmol, 1.0 eq) and dimethylformamide dimethyl acetal (0.6 g, 4.2307 mmol, 1.1 eq) was prepared and stirred at 75 °C for 90 min. The RM was cooled to room temperature, followed by the addition of ethanol (12 mL, 20V), TEA (2.0 mL, 15.3844 mmol, 4.0 eq), and 2-(4-(hydrazinylmethyl)phenyl)acetonitrile (86_Int-3) (0.600 g, 3.8461 mmol, 1.0 eq). The RM was heated to 80 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were washed with brine solution, dried over Na2SO4, concentrated, and then purified by flash column chromatography (12-15% ethyl acetate in hexanes) to give compound (86_Int-4). MS (ES): 310 m / z [M+1]+, LCMS purity: 64%. 1H 400 MHz, DMSO-d6: δ 0.754 (d, J = 6.40 Hz, 2H), 0.963(q, J = 6.4 Hz, 2H), 1.790-1.756 (m, 1H), 4.106 (s, 2H), 4.205-4.152 (m, 2H), 5.459 (s, 2H), 7.166 (d, J= 8Hz, 2H), 7.240 (d, J= 8Hz 2H), 7.814 (s, 1H).

[0265] Step-4 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(cyanomethyl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxamide (86) The final compound was prepared from ethyl 1-(4-(cyanomethyl)benzyl)-5-cyclopropyl-1H-pyrazole-4-carboxylate (86_Int-4) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (86). MS (ES): 412.5 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ 0.754 (d, J = 6.40 Hz, 2H), 0.963(q, J = 6.4 Hz, 2H), 1.790-1.756 (m, 1H), 4.025 (s, 2H), 4.487 (d, J=6.4Hz, 2H), 5.440 (s, 2H),7.196 (d, J = 8Hz, 2H), 7.33 (d, J=8Hz, 2H), 7.522 (d, J= 8Hz 2H), 8.538 (d, J=6.4Hz, 2H), 8.964(s, 2H),9.266(s, 2h).

[0266] Example 35: N-(4-Carbamimidoylbenzyl)-1-(4-(2-methoxyethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (87) TIFF2025526464000065.tif104165 Step-1 Synthesis of methyl 1-(4-(2-hydroxyethyl)benzyl)-3-(methoxymethyl)-1H-pyrazole-4-carboxylate (87_Int-2) A stirred solution of methyl 5-(methoxymethyl)-1H-pyrazole-4-carboxylate (3.2 g, 1.88235 mmol, 1.0 eq) in DMF (32 mL, 10 V) was prepared, and NaHMDS (1 M in THF) (18.88 mL, 1.88235 mmol, 1.0 eq) was added at 0 °C and stirred for 30 min. 2-(4-(bromomethyl)phenyl)ethan-1-ol (87_Int-1) (4.0 g, 1.88235 mmol, 1.0 eq) was added to the RM at 0 °C. The RM was allowed to reach room temperature and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (5% ethyl acetate in hexane) (87_Int-2). The identity was confirmed by 2D NMR (ROE analysis). MS (ES): 304.9 m / z [M+1]+, LCMS purity: 99.72%, 1H 400 MHz, DMSO-d6: δ 2.679 (t, J=4 Hz, 3H), 3.242 (s, 3H), 3.555 (t, J=4 Hz, 2H), 3.724 (s, 3H), 4.490 (s, 2H), 5.276 (s, 2H), 7.203-7.184 (m, 4H), 8.416 (s, 1H).

[0267] Step-2 Synthesis of methyl 1-(4-(2-methoxyethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (87_Int-3) A stirred solution of methyl 1-(4-(2-hydroxyethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (87_Int-2) (0.4 g, 1.3157 mmol, 1.0 eq) in DMF (4 ml, 10 V) was prepared. NaH (60% in mineral oil) (0.126 g, 2.6314 mmol, 2 eq) was slowly added, and the RM was stirred at 0 °C for 15 min, followed by the dropwise addition of MeI (0.106 mL, 1.7106 mmol, 1.3 eq). The RM was allowed to reach room temperature and stirred for 5 h. After completion of the reaction, the RM was quenched with cold water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and then purified by flash column chromatography (15% ethyl acetate in hexane) to give compound (87_Int-3). MS (ES): 319 m / z [M+1]+, LCMS purity: 79%, 1H NMR (400 MHz, DMSO-d6) δ 400 MHz, DMSO-d6: δ 2.489 (t, J = 6.40 Hz, 2H), 3.211 (s, 3H), 3.225 (s, 3H), 3.518 (t, J=6.4Hz, 2H), 4.794 (s, 2H), 5.358 (s, 2H), 7.111 (d, J=8Hz, 2H), 7.200 (d, J=8Hz, 2H), 7.905 (s, 2H).

[0268] Step-3 N-(4-Carbamimidoylbenzyl)-1-(4-(2-methoxyethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (87) The final compound was prepared from methyl 1-(4-(2-methoxyethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (87_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (87). MS (ES): 435.53 m / z [M+1]+, LCMS purity: 100%, HPLC purity: 100% 1H 400 MHz, DMSO-d6: δ2.489 (t, J = 6.40 Hz, 2H), 3.211 (s, 3H), 3.225 (s, 3H),3.503(t, J=6.4Hz, 2H), 4.492 (d, J=6Hz, 2H), 4.802(d, 2H), 5.310 (d, 2H), 7.111 (d, J=8Hz, 2H), 7.183 (d, J=8Hz, 2H), 7.508 (d, J=8Hz, 2H), 7.752(d, J=8Hz, 2H).8.012(s, 1H), 8.817(t, J=6 Hz, 1H), 8.911(s, 2H), 9.252(s, 2H).

[0269] Example 36: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (33) TIFF2025526464000066.tif58165 Step-1 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (33) The final compound was prepared from ethyl 1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (33_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (33). MS (ES): 419.4 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 95.56% 1H NMR (400 MHz, DMSO-d6) δ 2.807 (s, 3H), 2.978 (s, 3H), 3.656 (s, 2H), 4.778 (d, J=6.0 Hz, 2H), 7.183-7.197 (m, 5H), 7.483-7.504 (m, 2H), 7.734-7.755 (m, 1H), 7.837 (s, 1H), 7.907 (s, 1H), 8.269 (s, 1H), 8.767 (t, J=6.0 Hz, 1H), 8.946 (s, 2H), 9.245 (s, 2H).

[0270] Example 37: Preparation of N-(4-carbamimidoylbenzyl)-1-(2-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxamide (70) TIFF2025526464000067.tif48165 Step-1 Synthesis of ethyl 1-(2-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (70_Int-2) A stirred solution of ethyl 1H-pyrazole-4-carboxylate (0.1 g, 0.71 mmol, 1.0 eq) in DMF (2 mL, 20 V) was prepared, and 2-(2-(bromomethyl)phenyl)acetonitrile (70_Int-1) (0.149 g, 0.71 mmol, 1.0 eq) was added at room temperature. The RM was then heated to 80 °C and stirred for 16 h. After completion of the reaction, the RM was quenched with water and extracted with ethyl acetate. The combined organic fractions were dried over Na2SO4, concentrated, and purified by flash column chromatography (25% ethyl acetate in hexane) to give the compound. MS (ES): 270.m / z [M+1]+, LCMS purity: 98.57%, 1H 400 MHz, DMSO-d6: δ 1.244 (t, J = 6.4 Hz, 3H), 4.225-4.172 (m, 4H), 5.448 (s, 2H), 7.119 (d, J = 7.20 Hz, 1H), 7.454-7.332 (m, 3H), 7.892 (s, 1H), 8.453 (s, 1H).

[0271] Step-2 Synthesis of N-(4-carbamimidoylbenzyl)-1-(2-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxamide (70) The final compound was prepared from ethyl 1-(2-(cyanomethyl)benzyl)-1H-pyrazole-4-carboxylate (70_Int-2) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (70). MS (ES): 373.45 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 98.64. 1H 400 MHz, DMSO-d6: δ 4.185 (s, 2H), 4.483 (d, J =6.0 Hz, 2H), 5.444 (s, 2H), 7.119 (s, 1H), 7.345-7.396 (m, 2H), 7.448-7.504 (m, 3H), 7.746 (d, J=8.0 Hz, 2H), 7.943 (s, 1H), 8.259 (s, 1H), 8.784 (t, J=6.0 Hz, 1H), 8.952 (s, 2H), 9.247 (s, 2H).

[0272] Example 38: Preparation of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (74) TIFF2025526464000068.tif58165 Step-1 Synthesis of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (74) The final compound was prepared from ethyl 1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (19_Int-3) and 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-6) in a manner similar to that described in (Example 24) (74). MS (ES): 423.4 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 98.51% 1H NMR (400 MHz, DMSO-d6) δ 2.678 (s, 3H), 3.367 (s, 2H), 4.467 (d, J=6 Hz, 2H), 5.317 (s, 2H), 7.196-7.243 (m, 4H), 7.328 (t, J=8.4 Hz, 2H), 7.262 (t, J=8.4 Hz, 1H), 7.906 (s, 1H), 7.961 (s, 1H), 8.268 (s, 1H), 8.807 (t, J=6.0 Hz, 1H), 9.186 (s, 2H), 9.387 (s, 2H).

[0273] Example 39: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (82) TIFF2025526464000069.tif53165 Step-1 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (82) The final compound was prepared from methyl 1-(4-(2-cyanopropan-2-yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (82_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (70). MS (ES): 445.4 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1H NMR (400 MHz, DMSO-d6) δ 1.675 (s, 6H), 3.246 (s, 3H), 4.514 (d, J=6 Hz, 2H), 4.480 (s, 2H), 5.380 (s, 2H), 7.274 (d, J=8.4 Hz, 2H), 7.486-7.537 (m, 4H), 7.777 (d, J=8.4 Hz, 2H), 8.046 (s, 1H), 8.843 (t, J=6 Hz, 1H), 9.022 (s, 2H), 9.274 (s, 2H).

[0274] Example 40: Preparation of N-(4-carbamimidoylbenzyl)-1-(4-(2-cyanopropan-2-yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (85) TIFF2025526464000070.tif58165 Step-1 Synthesis of N-(4-carbamimidoylbenzyl)-1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxamide (85) The final compound was prepared from methyl 1-(4-(cyanomethyl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (85_Int-3) and 4-(aminomethyl)benzimidamide dihydrochloride in a manner similar to that described in (Example 24) (70). MS (ES): 417.4 m / z [M+H]+, LCMS purity: 100%, HPLC purity: 100% 1H NMR (400 MHz, DMSO-d6) δ 3.233 (s, 3H), 4.029 (s, 2H), 4.514 (d, J=6 Hz, 2H), 4.829 (s, 2H), 5.375 (s, 2H), 7.232 (d, J=8 Hz, 2H), 7.327 (d, J=8 Hz, 2H), 7.529 (d, J=8.0 Hz, 2H), 7.774 (d, J= 8.0 Hz, 2H), 8.046 (s, 1H), 8.839 (t, J=6 Hz, 1H), 8.963 (s, 2H), 9.271 (s, 2H).

[0275] Example 41: Preparation of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (78) TIFF2025526464000071.tif53165 Step-1 Synthesis of 1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylic acid (78_Int-1) A stirred solution of ethyl 1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylate (33_Int-3) (0.5 g, 0.158 mmol, 1.0 eq) in water:methanol:THF (1:1:1) (5 mL, 10 V) was prepared, and LiOH (0.3 g, 0.793 mmol, 5 eq) was added at room temperature and stirred for 16 h. After completion of the reaction, the RM was quenched with 2 N HCl (pH approx. 4) and extracted with 10% methanol in DCM. The combined organic fractions were dried over Na2SO4 and concentrated to give compound (78_Int-1). MS (ES): 288.4.m / z [M+H]+, LCMS purity: 97.47%, 1H NMR (400 MHz, DMSO-d6) δ 2.804 (s, 3H), 2.975 (s, 3H), 5.315 (s, 2H), 7.191-7.219 (m, 4H), 7.795 (s, 1H), 8.357 (s, 1H), 12.301 (s, 1H).

[0276] Step-2 Synthesis of N-(4-carbamimidoyl-3-fluorobenzyl)-1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (78) A stirred solution of 1-(4-(2-(dimethylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxylic acid (78_Int-1) (0.2 g, 0.069 mmol, 1.0 eq) in pyridine (2 mL, 10 V) was prepared. 4-(aminomethyl)-2-fluorobenzimidamide dihydrochloride (43_Int-5) (0.2 g, 0.083 mmol, 1.2 eq) and EDC HCl (0.67 g, 0.348 mmol, 5.0 eq) were added to the RM at room temperature and stirred for 16 h. After completion of the reaction, the RM was concentrated and purified by preparative HPLC ((A) 0.1% TFA in water (B) 100% MeCN). The pure fractions were lyophilized to give compound (78). MS (ES): 437.36.m / z [M+H]+, LCMS purity: 98.37%, HPLC purity: 99.70% 1H NMR (400 MHz, DMSO-d6) δ 2.804 (s, 3H), 2.981 (s, 3H), 3.653 (s, 2H), 4.459 (d, J=6.4 Hz, 2H), 5.314 (s, 2H), 7.194 (s, 4H), 7.318 (t, J=11.6 Hz, 2H), 7.615 (t, J=11.6 Hz, 1H), 7.900 (s, 1H), 8.266 (s, 1H), 8.790 (t, J=6 Hz, 1H), 9.170 (s, 2H), 9.370 (s, 2H).

[0277] Biological Examples Plasma kallikrein protease inhibition assay IC of test compound against plasma kallikrein 50 We provide two methods for determining

[0278] The first method used a reaction buffer consisting of 25 mM Tris-HCl (pH 8.0), 100 mM NaCl (pH 8.5), 0.01% Brij35, and 1% DMSO (final). The enzyme used was plasma kallikrein (R&D Systems catalog #2497-SE, recombinant human plasma kallikrein expressed in a mouse myeloma cell line, NS0-derived Gly20-Ala638, containing a C-terminal 60-His tag, MW = 70 kDa).

[0279] The enzyme was activated by diluting it to 200 μg / mL with activation buffer (100 mM Tris, 10 mM CaCl, 150 mM NaCl, pH 7.5 (TCN)) and then combined with an equal volume of 20 μg / mL thermolysin to form the reaction buffer. Each test compound was then dissolved in DMSO and delivered to the reaction buffer. After a 20-minute preincubation period, the reaction was initiated by delivering a substrate solution containing 10 μM Z-FR-AMC (Enzo catalog #P-139, AMC: 7-amino-4-methylcoumarin) to the reaction well.

[0280] Measurements were performed using an EnVision (PE) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. The reaction was stopped with EDTA. Enzyme activity was monitored every 5 minutes for 120 minutes at room temperature as a time course measurement of the increase in signal from a fluorescently labeled peptide substrate.

[0281] Data were analyzed by taking the slope*(signal / time) of the linear portion of the measurements. The slope was calculated using Excel and curve fitting was performed using Prism software.

[0282] In the second method, plasma kallikrein activity was also measured in pooled human plasma. First, a 10% actin FS solution was prepared in assay buffer. Each test compound was dissolved in DMSO and added to the reaction mixture along with Z-FR-AMC substrate and pooled human plasma. The multiwell reaction plate was incubated at room temperature for 5 minutes. The 10% actin FS solution was added to each well to initiate the reaction, and kinetic measurements were taken at excitation / emission wavelengths of 355 / 460 nm. Fluorescence signals were recorded every 30 seconds for a total of 10 minutes.

[0283] Permeability assay Permeability studies were performed using Caco-2 cells (ECACC catalog number 09042001) seeded at a density of 80,000 cells per well on cell insert plates (Millicell, catalog #PSHT010R5). Cells were maintained in culture medium (1x DMEM containing 10% FBS, 0.1 mg / mL penicillin / streptomycin) for 18-21 days. The medium was changed every other day. Prior to the experiment, the integrity of the cell monolayer was assessed by measuring the TEER value using a volt-ohm meter and an STX100C96 electrode. After the first wash, the TEER value was measured at 800 ohms.cm in buffer. 2 Only monolayers with TEER values above 0.1 were used.

[0284] Assay buffer (HBSS containing Ca +2 and Mg +2 , pH 7.4, buffered with 10 mM HEPES and 25 mM D-glucose) was used on the basolateral as well as the apical side.

[0285] An intermediate stock solution of test compound was prepared in DMSO at a concentration of 1 mM in DMSO. This stock solution was added to assay buffer to achieve a target test compound concentration of 10 μM. The organic content of the final drug preparation was 1.0% (v / v). Bidirectional permeability experiments were performed in series, and sample analysis was performed in duplicate.

[0286] The cultured cell monolayers were washed twice with assay buffer (0.4 mL and 0.8 mL were added to the apical and basolateral sides of the culture plate, respectively), and then the buffer in both compartments was discarded.

[0287] In apical-to-basolateral (AP>BL) experiments, 0.4 mL of donor solution (pH 7.4 assay buffer containing test compound) and 0.8 mL of receiver solution (pH 7.4 assay buffer only) were added to the apical and basolateral compartments, respectively. In basolateral-to-apical (BL>AP) experiments, 0.8 mL of donor solution (pH 7.4 assay buffer containing test compound) and 0.4 mL of receiver solution (pH 7.4 assay buffer) were added to the basolateral and apical compartments, respectively. The plate was then placed in a 37°C incubator for 120 minutes.

[0288] On the same experimental day, bidirectional (AP>BL and BL>AP) control experiments were performed in separate wells using propranolol (high permeability), atenolol (low permeability), digoxin (high efflux - Pgp substrate), and digoxin + verapamil (Pgp inhibitor).

[0289] After the transport experiments were completed, the integrity of the cell monolayer was assessed by measuring the rejection of Lucifer Yellow (LY). To do this, 400 μL of 10 μM LY was added to each well of the filter plate and incubated at 37°C for 1 hour. Samples were then collected from the basal compartment, and LY fluorescence was measured using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The percent LY rejection across the cell monolayer was calculated by measuring the fluorescence in the receiver plate (basal compartment) relative to a theoretical equilibrium standard.

[0290] Test samples (collected from the apical and basolateral compartments after 120 min of incubation) were analyzed by LCMS / MS, after which the Papp of the compound(s) was calculated in both the apical-to-basolateral and basolateral directions: Papp = ([DBL] x VBL) / (A x t x [DAP]), where [DBL] = final drug concentration on the basolateral side, VBL = volume of the basolateral compartment, A = surface area of the cell culture, t = total incubation time, and [DAP] = initial drug concentration on the apical side.

[0291] Results from the above assays for representative compounds of the present disclosure are presented below in Table 1. Scores for selected compounds in each assay are presented below. TIFF2025526464000072.tif35128

[0292] Table 1. Plasma kallikrein inhibition and cell permeability of representative compounds TIFF2025526464000073.tif194145TIFF2025526464000074.tif247145TIFF2025526464000075.t if247145TIFF2025526464000076.tif247145TIFF2025526464000077.tif247145TIFF20255264640 00078.tif194145TIFF2025526464000079.tif200145TIFF2025526464000080.tif247145TIFF202 5526464000081.tif247145TIFF2025526464000082.tif247145TIFF2025526464000083.tif247145 TIFF2025526464000084.tif195145TIFF2025526464000085.tif247145TIFF2025526464000086.t if247145TIFF2025526464000087.tif247145TIFF2025526464000088.tif247145TIFF20255264640 00089.tif247145TIFF2025526464000090.tif247145TIFF2025526464000091.tif247145TIFF202 5526464000092.tif247145TIFF2025526464000093.tif247145TIFF2025526464000094.tif141145

[0293] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will occur to those skilled in the art and will fall within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

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