WO2025159870A1 - Alkylated inhibitors of plasma kallikrein - Google Patents

Abstract

The present disclosure provides compounds of Formula (I) and their pharmaceutical compositions: The compounds and compositions are useful for inhibiting the activity of plasma kallikrein, and they are useful in therapy and in methods of treating diseases and conditions, such as ocular disorders.

Description

ALKYLATED INHIBITORS OF PLASMA KALLIKREIN

[0001] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 63/624, 644 filed on January 24, 2023, the content of which application is incorporated as if fully set forth herein.

BACKGROUND

[0002] Plasma kallikrein is a serine protease that circulates in blood as prekallikrein, an inactive precursor, and participates in the surface-mediated defense system via activation of factor XII and high molecular weight kininogen (HK) involved signaling. Elements of the kallikrein-kinin system (KKS) are involved in activities such as surface-mediated defense reactions, regulation of blood flow, fibrin deposition, blood pressure, smooth muscle contractility, nociception, electrolyte transport, and mediator release. See Donald H. Miller, Hany S. Margolins, 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

[0003] The present disclosure provides small molecule inhibitors of plasma kallikrein and methods of using the inhibitors to treat disease. Thus, in various embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0004] In Formula (I), D¹ is N or CR¹; and D² is N or CR².

[0005] R ¹ and R² are independently selected from the group consisting of H and C₁-C₆-alkyl. At least one of R ^: and R² is C₁-C₆-alkyl.

[0006] R^a1, R^a2, and R^a3 are independently selected from group consisting of H, C₁-C₆-alkyl, and C₁-C₆-haloalkyl. [0007] Qi, Q2, and Q3 are independently selected from the group consisting of CR⁵, N, O and S.

[0008] R⁵ is selected from the group consisting

[0009] P is C or N.

[0010] L¹ is -SO2- or -Ci-C₈-alkylene-.

[0011] Ring ® is a bivalent monocyclic or bicyclic moiety selected from the heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof,

[0012] L² is selected from the group consisting of heteroaryl (wherein 1 -4 heteroaryl members are independently selected from N, O, and S), wherein each cycloalkyl, cycloalkenyl, aryl, and heteroaryl is independently monocyclic or bicyclic.

[0013] In some embodiments, L² is optionally substituted by 1 to 3 substituents selected from the group consisting of C₁-C₆-alkyl, C₁-C₆-haloalkyl, and CN.

[0014] Z is selected from the group consisting of -OR^C, -OC(O)R^C, -

[0015] Each instance of R^a, R^c, and R^d is independently selected from H, C₁-C₆- alkyl , Ci-Cs-haloalkyl, and C₆-C₁₀-aryl.

[0016] In additional embodiments, the present disclosure provides a pharmaceutical composition comprising a compound as described herein or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0017] In more embodiments, the present disclosure provides a method for treating a subject suffering from a disease or condition as described herein. The method comprises administering to the subject a compound or pharmaceutically acceptable salt thereof as described herein.

DETAILED DESCRIPTION

[0018] The present disclosure provides compounds, compositions, and methods for inhibiting plasma kallikrein (“PKal”). The compounds are useful, in exemplary embodiments, for the treatment of inflammatory and ocular disorders.

[0019] One advantage of the compounds described herein resides in their unexpectedly superior inhibition of PKal in comparison to known small molecule PKal inhibitors (see, e.g., WO 2009/097141 , US 2021/078999, and US 2017/253561). In this context, the observed maximum limit of binding affinities of small molecules for macromolecular targets is about -15 kcal/mol, which corresponds to picomolar dissociation constants. I D. Kuntz el al., Proc Natl Acad Sci U S A. 96(18) (1999) 9997-10002; R. D. Smith et al., JChemlnf Model. 52(8) (2012) 2098-2106; A. J. T. Smith etal., J Med Chem. 52(2) (2009) 225-233. Hence, at or near this limit, even small improvements in binding affinity are to be viewed as significant. The compounds of the present disclosure surprisingly exhibit inhibition constants (ICso) in the picomolar range, constituting about two-fold or even more than 100-fold improvements over known PKal inhibitors that already exhibit ICso’s in the nanomolar range.

Another advantage of the present disclosure is moreover premised upon the surprising discovery that structure of the PKal inhibitor compounds described herein, according to Formula (I), exerts profound effect upon compound potency as evidenced by the various examples and corresponding comparative examples described below. These advantages and others are apparent in the more detailed description that follows. [0020] Definitions

[0021] Unless defined otherwise, 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 pertains.

[0022] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the cell" includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth.

[0023] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range.

[0024] In the present disclosure, the number of atoms of a particular element in a substituent group is generally given as a range, e.g., an alkyl group containing from 1 to 4 carbon atoms or C 1-4 alkyl. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range. For example, an alkyl group from 1 to 4 carbon atoms includes each of C1, C2, C3, and C4. A C1.12 heteroalkyl, for example, includes from 1 to 12 carbon atoms in addition to one or more heteroatoms.

Other numbers of atoms and other types of atoms may be indicated in a similar manner.

[0025] The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, "consist of' or "consist essentially of" the described features. [0026] “Alkyl” refers to straight or branched chain hydrocarbyi including from 1 to about 20 carbon atoms. For instance, an alkyl can have from 1 to 10 carbon atoms or 1 to 6 carbon atoms. Exemplary alkyl includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like, and also includes branched chain isomers of straight chain alkyl groups, for example without limitation, -CH(CH₃)₂, -CH(CH₃)(CH₂CH₃), -CH(CH₂CH3)₂, -C(CH₃)₃, -C(CH₂ CH₃)₃, -CH₂CH(CH₃)₂, -CI-I₂CH(CH₃)(CH₂CH₃), -CH₂CH(CH₂CH₃)₂, -CH₂C(C H₃)₃, -CH₂C(CH₂CH₃)₃, -CH(CH₃)CH(CH₃)(CH₂CH₃), -CH₂CH₂CH(CH₃)₂, -CH ₂CH₂CH(CH₃)(CH₂CH₃), -CH₂CH₂CH(CH₂CH₃)₂, -CH₂CH₂C(CH₃)₃, -CH₂CH₂ C(CH₂CH₃)₃, -CH(CH₃)CH₂CH(CH₃)₂, -CH(CH₃)CH(CH₃)CH(CH₃)₂, and the like. Thus, alkyl groups include primary' alkyl groups, secondary alkyl groups, and tertiary' alkyl groups. An alkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein, such as halogen(s), for example.

[0027] Each of the terms “halogen,” “halide,” and “halo” refers to -F or fluoro, -Cl or chloro, -Br or bromo, or -I or iodo.

[0028] The term “alkenyl” refers to straight or branched chain hydrocarbyi groups including from 2 to about 20 carbon atoms having 1-3, 1-2, or at least one carbon to carbon double bond. An alkenyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0029] “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 a (C₂-C₈)alkynyl group 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. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0030] The term “cycloalkyl” refers to a saturated monocyclic, bicyclic, tricyclic, or polycyclic, 3- to 14-membered ring system, such as a C₃-C₈- cycloalkyl. The cycloalkyl may be attached via any atom. Representative examples of cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0031] Embodiments of inhibitors of plasma kallikrein include compounds comprising a C₁-C₈ alkyl linker. In embodiments, a "Cns alkyl” may be characterized by a branched or unbranched hydrocarbon group having from 1 to 8 carbon atoms. C₁-C₈alkyls include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tertbutyl, cyclobutyl, pentyl, and cyclopentyl.

[0032] “Aryl” (Ar) when used alone or as part of another term means a carbocyclic aromatic group whether or not fused having the number of carbon atoms designated or if no number is designated, up to 14 carbon atoms, such as a . In embodiments, an Ar may be characterized by an aromatic group having a ring system comprised of carbon atoms with conjugated π electrons (e.g., phenyl). The term includes aryl groups having from 6 to 12 carbon atoms. Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring has five or six members. Examples of aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang’s Handbook of Chemistry (Dean, J. A., ed) 13th ed. Table 7-2 [1985]). “Aryl” also contemplates an aryl ring that is part of a fused polycyclic system, such as aryl fused to cycloalkyl as defined herein. An exemplary aryl is phenyl. An aryl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0033] The term “heteroatom” refers to N, O, and S. Compounds of the present disclosure that contain N or S atoms can be optionally oxidized to the corresponding N -oxi de, sulfoxide, or sulfone compounds.

[0034] “Heteroaryl,” alone or in combination with any other moiety described herein, is a monocyclic aromatic ring structure containing 5 to 10, such as 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing one or more, such as 1-4, 1-3, or 1-2, 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 is produced. 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, oxathiadi azolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, and indolyl. A heteroaryl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0035] “Heterocycloalkyl” is a saturated or partially unsaturated non-aromatic monocyclic, bicyclic, tricyclic or polycyclic ring system that, has from 3 to 14, such as 3 to 6, atoms in which 1 to 3 carbon atoms in the ring are replaced by heteroatoms of O, S or N. The ring heteroatoms can also include oxidized S or N, such as sulfinyl, sulfonyl, and N-oxides of a tertiary ring nitrogen. A heterocycloalkyl can be fused to another ring system, such as with an aryl or heteroaryl of 5-6 ring members. The point of attachment of the heterocycloalkyl ring is at a carbon or heteroatom such that a stable ring is retained. Examples of heterocycloalkyl groups include without limitation morpholino, tetrahydrofuranyl, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydrobenzofuryl , and dihydroindolyl. A heterocycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0036] The term “nitrile” or “cyano” can be used interchangeably and refers to a -CN group.

[0037] Compounds described herein can exist in various isomeric forms, including configurational, geometric, and conformational isomers, including, for example, cis- or trans- conformations. The compounds may also exist in one or more tautomeric forms, including both single tautomers and mixtures of tautomers. The term “isomer” is intended to encompass all isomeric forms of a compound of this disclosure, including tautomeric forms of the compound. The compounds of the present disclosure may also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms may result from the loss of water. The specific composition of the open-chain and cyclized forms may be dependent on how the compound is isolated, stored or administered. For example, the compound may exist primarily in an open-chained form under acidic conditions but cyclize under neutral conditions. All forms are included in the disclosure.

[0038] Some compounds described herein can have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound as described herein can be in the form of an optical isomer or a diastereomer. Accordingly, the disclosure encompasses compounds and their uses as described herein in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology or via chemical separation of stereoisomers through the employment of optically active resolving agents.

[0039] 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 the compound. A stereomerically pure compound having two chiral centers wall be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomers of the compound. The stereoisomer as described above can be viewed as composition comprising two stereoisomers that are present in their respective weight percentages described herein.

[0040] If there is a discrepancy between a depicted structure and a. name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds are prepared as single enantiomers from the methods used to prepare them.

[0041] As used herein, and unless otherwise specified to the contrary, the term “compound” is inclusive in that it encompasses a compound or a pharmaceutically acceptable salt, stereoisomer, isotopologue, and/or tautomer thereof. Thus, for instance, a compound includes a pharmaceutically acceptable salt of a tautomer of the compound. Similarly, a compound of includes a pharmaceutically acceptable salt of an isotopologue of the compound.

[0042] In this 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, e.g, alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene- 2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, ni trate, N-rn ethylglucamine ammonium salt, 3 -hydroxy-2 -naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2- hydroxy-3 -naphthoate, einbonate), pantothenate, phosphate/ diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

[0043] The terms “treat’, “treating” and “treatment” refer to the amelioration or eradication of a disease or symptoms associated with a disease. In various embodiments, the terms refer to minimizing or slowing the spread, progression, or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient with such a disease.

[0044] The terms “prevent,” “preventing,” and “prevention” refer to the prevention of the onset, recurrence, or spread of the disease in a patient resulting from the administration of a compound described herein.

[0045] The term “effective amount” refers to an amount of a compound as described herein or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease. Further, a therapeutically effective amount with respect to a compound as described herein means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound as described herein, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or is synergistic with another therapeutic agent.

[0046] A “patient” or subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. In accordance with some embodiments, the animal is a mammal such as a non-primate and a primate (c.g, monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent or adult. In the present disclosure, the terms “patient” and “subject” are used interchangeably.

[0047] COMPOUNDS

[0048] In various embodiments, the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0049 ] In some embodiments, D¹ is N or CR¹. In other embodiments, D² is N or CR².

[0050] R¹ and R² are independently selected from the group consisting of H and At least one of R¹ and R² is Ci-Cg-alkyl.

[0051] R³¹, R^a2, and R³³ are independently selected from group consisting of H,

[0052] Ring members Q1, Q2, and Q3 are independently selected from the group consisting of CR⁵, N, O and S.

[0053] R⁵ is selected from the group consisting of H, C₁-C₆-alkyl, OR^a, -(C₁-C₆- alkyl)OR.^a, and C₈-Cio-cycloalkyl.

[0054] In some embodiments, ring member P is C. In other embodiments, P is

N.

[0055] L¹ is -SO2- or -Ci-C₈-alkylene-.

[0056] Ring ® is a bivalent monocyclic or bicyclic moiety selected from the group consisting y y y , heterocycloalkyl (wherein 1-4 ring members are independently selected from N, (), and S), 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof.

[0057] L² is selected from the group consisting of -Ci-C₈-alkylene, -C2-C8- alkenylene, -Ck-C₈-alkynylene, and a bivalent moiety selected from the group heteroaryl (wherein 1-4 heteroaryl members are independently selected from N,

O, and S). In L², each cycloalkyl, cycloalkenyl, aryl, and heteroaryl is independently monocyclic or bicyclic. In some embodiments, L² is optionally substituted by 1 to 3 substituents selected from the group consisting of C₁-C₆- alkyl, C₁-C₆-haloalkyl, and CN.

[0058] Z is selected from the group consisting of -OR^C, ~OC(O)R^C, - N (' (). A C S. and AR AR' R^d

[0059] Each instance of R^a, R^c, and R^d is independently selected from H, C₁-C₆- alkyl, C₁-C₆-haloalkyl, and Cs-Cio-aryl.

[0060] In some embodiments, only one of D¹ and D^z is N. In other embodiments, the ring containing D¹ and D² is:

[0061] In a particular embodiment, the ring containing D¹ and D² is

[0062] In additional embodiments, each of R^{ and R² is independently C₁-C₆- alkyl. In other embodiments, one of R¹ and R² is C₁-C₆-alkyl and the other is H.

[0063] In various embodiments optionally in combination with any other embodiment described herein, D¹ is CR¹, D² is CR², and R¹ and R² are independently selected from H, methyl, ethyl, n-propyl, iso-propyl, n-butyl, secbutyl, and tert-butyl. All combinations of R^l and R² are contemplated. In exemplary embodiments, R’ is H and R² is methyl or ethyl, or each of R¹ and R² is selected from methyl and ethyl.

[0064] In further embodiments, the present disclosure provides formula (I) compounds wherein the ring containing D¹ and D² is selected from the group consisting of:

[0065] In some embodiments, each of R^al, R^a2, and R^a3 is H.

[0066] In various embodiments:

Q¹ is CR⁵; each of Q² and Q³ is independently selected from the group consisting of

CR⁵, N, O and S; and P is selected from the group consisting of C and N.

[0067] In an embodiment, P is N. In additional embodiments, Q² is CR⁵ and O ’ is N, In other embodiments, Q² is N and Q³ is CR⁵.

[0068] In embodiments, In an illustrative embodiment, L¹ is methylene.

[0069] In further embodiments, ® is bivalent monocyclic Cs-Cio-aryl. For example, in one embodiment, ® is .

[0070] In various embodiments, L² is selected from -Ci-C₈-alkylene, Ce-Cio-aryl

[0071] In additional embodiments, Z is selected from the group consisting of - OR^C, CN, -C(O)OR^C, and -C(O)NR^cR^a. In an exemplary embodiment, Z is CN.

[0072] In various embodiments, the present disclosure provides formula (I) compounds wherein: the ring containing

[0073] In illustrative embodiments, the present disclosure provides the following specific compounds:

[0074] PHARMACEUTICAL COMPOSITION

[0075] The disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds as described herein, or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavor imparting agents. The pharmaceutical composition can be administered by any suitable means that results in a concentration of the compound in a subject that is effective at treating a disease or condition suitable for treatment with the compounds of the disclosure.

[0076] 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 a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof that is administered is governed by such considerations and is the minimum amount necessary' to elicit inhibition of PKal. Such amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure that is administered is in 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 the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In other embodiments, the compound is administered into a dosage form for administration into or around the eye, as described herein, for which the therapeutically effective amount of the compound can 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 can be administered once a day or twice per day.

[0077] While the attending physician ultimately will decide the appropriate amount and dosage regimen, in addition embodiments, the therapeutically effective amount of a compound described herein can be, for example, in the range of 0.0035 pg to 20 pg/kg body weight/day or 0.010 pg to 140 pg/kg body weight/week. In some embodiments, a therapeutically effective amount is in the range of 0.025 pg to 10 pg/kg, for example, 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 pg/kg body weight administered daily, every other day, or twice a week. In some embodiments, a therapeutically effective amount may be in the range of 0.05 pg to 20 pg/kg, for example, 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 pg/kg body weight administered weekly, every other week, or once a month. In some embodiments, a therapeutically effective amount of a compound may be, for example, in the range of 100 pg/m² to 100,000 pg/m² (of subject body surface area) administered daily, every other day, once weekly, or every other week. In some embodiments, the therapeutically effective amount is in the range of 1000 pg/m² to 20,000 pg/m², for example, at least 1000, 1500, 4000, or 14,000 pg/m² of the compound administered daily, every other day, twice weekly, weekly, or even,- other week.

[0078] In some embodiments, a compounds of the disclosure is administered at 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, 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) per day for an adult human.

[0079] In certain embodiments, the compound as described herein or a pharmaceutically acceptable salt or solvate thereof, is substantially pure, in that it contains 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 other organic small molecules, such as unreacted intermediates or synthesis by-products that are created, for example, in one or more of the steps of a synthesis method.

[0080] In various embodiments, the composition is provided in a dosage form that is suitable for oral, parenteral (e.g., intravenously, intramuscularly, subcutaneous, intraarterial), buccal, sublingual, rectal, cutaneous, nasal, vaginal, intranasal, inhalation, transdermal, ocular, intraosseous, otic, or intracranial administration route. Thus, in some embodiments, the composition dosage form is chosen from tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, patches, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, and aerosols. The 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. S warbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0081] Pharmaceutical compositions can be formulated to release the active compound immediately upon administration, at any predetermined time, or during any predetermined time period after administration (e.g., controlled release formulations). Examples of controlled release formulations include (i) formulations that create substantially constant concentrations of the agent(s) of the disclosure within the body over an extended period of time; (ii) formulations that after a predetermined lag time create substantially constant concentrations of the agents of the disclosure within the body over an extended period of time; (iii) formulations that sustain the agent(s) action during a predetermined time period by maintaining a relatively constant, effective level of the agent(s) in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the agent(s) (sawtooth kinetic pattern); (iv) formulations that localize action of agent(s), e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; (v) formulations that achieve convenience of dosing, e.g., administering the composition once per week or once every two weeks; and (vi) formulations that target the action of the agentts) bv using carriers or chemical derivatives to deliver the compound to a particular target cell type. Administration of the compound in the form of a controlled release formulation is desirable, in some embodiments, for compounds having a narrow absorption window in the gastrointestinal tract or a relatively short biological half-life.

[0082] In some embodiments, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. In some embodiments, the compound is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the compound in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes. [0083] A pharmaceutical composition comprising a compound as described herein can be administered parenterally by injection, infusion, or implantation (e.g., intraocular, subcutaneous, intravenous, intramuscular, intraperitoneal) via 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.

[0084] Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs.

[0085] In another embodiment, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise a compound of the disclosure or its pharmaceutically acceptable stereoisomer, salt, or tautomer and a pharmaceutically acceptable carrier.

[0086] The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the compounds of the present disclosure contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of a compound of the present disclosure.

[0087] For tablet compositions, a compound of the present disclosure in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. [0088] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0089] For aqueous suspensions, a compound of the present disclosure is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydroxpropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

[0090] Oral suspensions can also contain dispersing or wetting agents, such as naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example 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.

[0091] Oily suspensions may be formulated by suspending a compound of the present disclosure in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

[0092] Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0093] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide a compound of the present disclosure in admixture with a dispersing or wetting agent, 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 sweetening, flavoring and coloring agents, may also be present.

[0094] Pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents.

[0095] Syrups and elixirs may be formulated with sweetening agents, for example 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, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as 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 find use in the preparation of injectables.

[0096] A compound of the present disclosure can be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drag. Such materials are cocoa butter and polyethylene glycols.

[0097] Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the drug in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved drag. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions.

[0098] In some embodiments, the composition is especially adapted for administration into or around the eye. For example, a composition can be adapted to be used as eye drops, or injected into the eye, e.g., using peribulbar or intravitreal injection. Such compositions should be sterile and substantially endotoxin-free, and within an acceptable range of pH. In some embodiments a formulation without preservatives is used. Formulation of eye medications is known in the art., see, e.g., 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)).

[0099] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry' powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent(s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents. [00100] In some embodiments, the pharmaceutical compositions of the disclosure are in a form suitable for sterile injection. To prepare such a composition, 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 addition of 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. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). In cases where the compound has limited solubility in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol.

[00101] The pharmaceutical compositions can be administered to a subject in a single dose or in multiple doses. For example, a compound described herein can be administered once a week or for 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of a health care provider administering or supervising the administration of the compound. For example, the dosage of a compound can be increased if the lower 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, if the disease or condition is reduced or eliminated, or to reduce undesirable side-effects.

[00102] METHODS OF USE AND TREATMENT

[00103] As one advantage of the present disclosure, the compounds described herein are potent inhibitors of plasma kallikrein, that is, the compounds are particularly effective in reducing the activity of plasma kallikrein. In various embodiments, a compound described herein can be characterized by an inhibitory constant, IC50 (half maximal inhibitory' concentration), no higher than 500 nM (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 10, 1, 0.1 , 0.08, 0.05, or 0.01 nM). [00104] In an embodiment, the present disclosure provides a method for inhibiting plasma kallikrein are provided. In some embodiments, the method comprises contacting PKal with a compound of the present disclosure in an amount effective to inhibit the activity of PKal. In some embodiments, the compounds of the disclosure inhibit PKal acti vity with an IC50 value in the range of 0.01 to 500 11M (e.g., 0.01, 0.05, 0.08, 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, 1 1, 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). It will be understood that all ranges in the disclosure include one or both endpoints, all values in between the endpoints to one significant digit, and any subranges between the endpoints. In some embodiments, the compounds of the disclosure inhibit PKal activity with an IC50 less than or equal to 500 nM, such as less than or equal to 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, 0.1, 0.08, 0.05, or 0.01 nM.

[00105] In some embodiments, the method comprises administering to a subject an effective amount of a compound of the disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, the method inhibits PKal activity in vivo with an IC50 (half maximal inhibitory' concentration) value in the range of 0. 1 to 500 nM (e.g., 0.01, 0.05, 0.08, 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).

[00106] The present disclosure provides, in additional embodiments, a method for treating a subject suffering from a disease or condition in a subject. The method comprises administering an effective amount of compound or pharmaceutically acceptable salt thereof as disclosed herein, or a pharmaceutical composition thereof, to the subject. 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.

[00107] In various embodiments, the disease or condition is selected from the group consisting of ischemic stroke, hemorrhagic stroke, hypertension, retinopathy, diabetic retinopathy, nephropathy, cerebral edema, pulmonary hypertension, inflammation, acute myocardial infarction, deep vein thrombosis, complications from fibrinolytic treatment, stroke, angina, angioedema, sepsis, arthritis, complications of cardiopulmonary' bypass, capillary leak syndrome, inflammatory bowel disease, vascular complications from diabetes, diabetic macular edema, macular degeneration, neuropathy, age related macular degeneration, retinal vein occlusions, brain edema, ischemia reperfusion injury, angiogenesis, asthma, anaphylaxis, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, glioblastoma multiforme, complications of fibrinolysis treatment, increased albumin excretion, macroalbuminuria, pain, amyotrophic lateral sclerosis, Creutzfeld-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, enterocolitis, arteritis, glomerulonephritis, psoriasis, endometriosis, preeclampsia, malaria, arthritis, periodic and recurrent fever, Chagas disease, Reynaud’s disease, systemic sclerosis, granulomatosis with polyangiitis, small vessel vasculitis, medium vessel vasculitis, large vessel vasculitis, pan-vasculitis, systemic autoinflammatory diseases, renal insufficiency, cerebral malaria, Clarkson’s disease (systemic vascular leakage syndrome), Hantavirus infection, Hantavirus renal syndrome, Hantavirus pulmonary syndrome, viral associated inflammatory disorders, retinal vasculitis, uveitis, Eales' disease, Behcet's disease, sarcoidosis, whooping cough, acute cough, chronic cough, coronavirus infection, and non-infectious posterior uveitis.

[00108] In some embodiments, the 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 undergoing medical treatment by a health care provider.

[00109] Combination Therapy

[00110] The disclosure also provides combination pharmaceutical compositions comprising: (a) at least one compound or a pharmaceutically acceptable salt thereof as disclosed herein, and (b) at least one inhibitor of inflammation, pain, or edema. In some embodiments, orally delivered pharmaceutical compositions comprising the combination pharmaceutical compositions of the disclosure are provided. The disclosure further provides tablets, capsules, orally delivered particles, injectable suspensions and solutions, and compositions for pulmonary or nasal delivery comprising the combination pharmaceutical composition.

[00111] Kits

[00112] In another embodiment, the disclosure provides a kit comprising a compound or a pharmaceutically acceptable salt thereof as described herein.

The kit also comprises instructions to a health care provider for administering the compound to a patient.

[00113] EXAMPLES

[00114] The following examples provide further embodiments of the present disclosure. The examples are illustrative and non-limiting.

[00115] Synthesis of Compounds

[00116] As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following procedures. It will be appreciated that, although the described methods depict the synthesis of certain compounds of the present disclosure, these and other methods known to one of ordinary skill in the art can be adapted to achieve the synthesis of all compounds as described herein. Thus, additional compounds of the invention were prepared by methods substantially similar to those described herein as illustrated throughout the following Examples.

[00117] Preparation of Intermediates

[00118] Example 1A: Preparation of ethyl l-(4-(2-cyanopropan-2- yI)benzyi)-lH-pyrazoIe-4-carboxyIate (5_Int-5)

[00119] Step-1. Synthesis of 2-methyl~2~(p-toIyI)propanenitrile (5_Int~

2)

[00120] A stirred solution of 2-(p-tolyl)acetonitrile (5 Int-1) (200g, 1524mmol, l.0eq), in N-methyl-2-pyrrolidone (lOOOmL, 5V) and tetrahydrofuran (1000mL, 5 V) was prepared at 0°C to 5°C and sodium tert- butoxide (586.07g, 6098mmol, 4.0eq) was added portion wise under nitrogen atmosphere. The RM was stirred at 0°C to 5°C for 30 minutes and iodomethane (865.62g, 6098mmol, 4.0eq), was added drop wise. The RM was stirred at 0°C to RT for 2.5h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were washed by cold water to remove N-methyl-2 -pyrrolidone, evaporated and then purified by column chromatography (2-5% ethyl acetate in hexanes) to give compound (5 Int-2). ¹H NMR (400 MHz, DMSO-d₆) 5 1.65 Its, 6H), 2.298 (s, 3H), 7.226 d, J 8.0 Hz, 2H), 7.392 (d, J 6.8 Hz, 2H).

[00121] Step-2. Synthesis of 2-(4-(bromomethyl)phenyl)~2- methylpropasienitrile (S Int-3)

[00122] A 10,000mL 4N (four-neck) round bottom flask (RBF) attached to mechanical stirrer and condenser was charged with 2-methyl-2-(p- tolyl)propanenitrile (5_Int-2) (170g, 1067mmol, l.Oeq) and carbon tetrachloride (3400mL, 20V) at RT. AIBN (17.53g, 106mmol, 0. leq), was added to the reaction mixture (RM). N-bromosuccinimide (209.04g, 1174mmol, l .leq) was added portion wise to the RM at room temperature (RT), heated to 90°C, and stirred for 2h. The reaction mixture was cooled to RT, quenched into water and extracted using DCM. The combined organic fractions were dried over NazSCh, evaporated and purified by column chromatography (7% ethyl acetate in hexanes) to give compound (5 __Int-3). ¹H NMR (400 MHz, DMSO-dr,) 5 1 .654 (s, 6H), 4.714 (s, 2H), 7.507 (s, 4H).

[00123] Step-3. Synthesis of ethyl l”(4"(2-cyam>pr0pan~2~yi)benzyl)"

1 H-pyrazoIe-4-carboxylate (5_Int-5).

[00124] A 3000mL 4N RBF attached to mechanical stirrer and condenser was charged with ethyl lH-pyrazole-4-carboxylate (5 Int-4) (70g, 499mmol,

1. Oeq), 2-(4-(bromomethy l)pheny I )-2-m ethylpropan enitri I e (5_In t-3 ) ( 130.84g, 549mmol, l. leq) and acetone (700mL, 10V) at RT. CS2CO3 (390.8g,

1198mmol, 2.4eq) was added to the RM, heated to 60-65°C, and stirred for 6h. The RM was cooled to RT, filtered to remove CS2CO3, and washed with ethyl acetate. After discarding the solid residue, the filtrate was evaporated and purified by column chromatography (15% ethyl acetate in hexane) to give compound (5 Int-5). TI NMR (400 MHz, DMSO~d₆) 5 1.656 (s, 6H), 4.204 (q, J 6.8 Hz, 2H), 5.372 (s, 2H), 7.328 (d, J 8 0 Hz, 2H), 7.498 (d, J 8.0 Hz, 2H), 8.053 (s, 1H), 8.482 (s, 1H).

[00125] Example 2A: Preparation of ethyl l-(4-(cyanomethyI)benzyl)- lH-pyrazole-4-earboxyiate (7A_Int-4)

7AJnt-1 7AJnt-2 7AJnt-3 7AJnt-4

[00126] Step-1. Synthesis of ethyl l-(4-methylbenzyl)-lH-pyrazoIe-4- carboxylate (7A_Int-2).

[00127] A 10000mL 4N RBF attached to mechanical stirrer and condenser was charged with ethyl lH-pyrazole-4-carboxylate (5 Int-4) (250g, 1783mmol, l.Oeq), l-(bromomethyl)-4-m ethylbenzene (7A_Int-l) (363.1g, 1962mmol, 1 . 1 eq) and acetone (6250mL, 25V) at RT. CS2CO3 (390.8g,

1198mmol, 2.4eq) was added to the RM, heated to 60-65°C, and stirred for 16h. The RM was cooled to RT, filtered, and washed with ethyl acetate. After discarding the solid residue, the filtrate evaporated under reduced pressure to obtain crude. The crude was triturated in hexanes to give compound (7A Int-2). ' l l NMR (400 MHz, DMSO-d₆) 5 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, 1 H)

[00128] Step-2. Synthesis of ethyl l-(4-(bromomethyI)benzyl)-lH- pyrazoIe-4-carboxyIate (7A__Int-3).

[00129] A lOOOOmL 4N RBF attached to mechanical stirrer and condenser was charged with ethyl l-(4-methylbenzyl)-lH-pyrazole-4- carboxylate (7 A Int-2) (300g, 1228mmol, 1.0eq) and 1,2 dichloroethane (6000mL, 20V) at RT. Benzoyl peroxide (29.71g, 122.8mmol, 0.1 eq) was added to the RM followed by portion wise addition of N-bromosuccinimide (240.4g, 1350mmol, l. leq) at RT. Resulting RM was heated to 90°C and stirred for 4h. The RM was cooled to RT, quenched in a saturated Na₂SO4 solution, and extracted using DCM. The combined organic fractions were dried over NazSCfi, evaporated, and purified by column chromatography (17-25% ethyl acetate in Hexanes) to give compound (7A Int-3). ¹ H NMR (400 MHz, DMSO-de) 8 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=8.4 Hz, 2H), 7.868 (s, 1H), 8.478 (s, 1H).

[00130] Step-3. Synthesis of ethyl l-(4-(cyanomethyI)benzyl)-lH- pyrazoIe-4-carboxylate (7A_Int-4).

[00131] A 5000mL 4H RBF attached to mechanical stirrer and condenser was charged with ethyl l-(4-(bromomethyl)benzyl)-lH-pyrazole-4-carboxylate (7A_Int-3) (242g, 499748mmol, l.Oeq), McCN (2420ml.. I0V) and Cs₂CO₃ (488g, 1497mmo1, 2.0eq), followed by slow addition of trimethyl silyl cyanide (334g, 3369mniol, 4.5eq) at RT. The RM was heated to 80-85°C and stirred for 16h. The RM was then cooled to RT and filtered. After discarding the solid residue, the filtrate was evaporated and purified by column chromatography (12- 15% ethyl acetate in hexanes) to give compound (7A Int-4). ^fHNMR (400 MHz, DMSO-de) 6 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). [00132] Example 3A: Preparation of 4-(aminomethyi)-3- methylbenzimidamide dihydrochloride (20__Int-7)

[00133] Step-1. Synthesis of 4-(hydroxymethyI)-3-methyIbenzoiiitrile (20_Int-2)

[00134] A stirred solution of 4-cyano-2 -methyl benzoic acid (20_Int-l)

[00135] Step-2. Synthesis of 4-(bromomethyI)-3~methylbenzonitrile

(20__Int~3)

[00136] A stirred solution of 4-(hydroxymethyl)-3-methylbenzonitrile

(20 Int-2) (2g, 13.5mmol, l.Oeq) in DCM (40mL, 20V) was prepared and triphenylphosphine (3.9g, 14.9mmol, l . leq) and CBn (4.95g, 14.9mmol, l . leq) were added at RT. The RM was stirred at RT for 4h. The RM was quenched in water and extracted using DCM. The combined organic fractions w'ere dried over NaiSOi, evaporated, and purified by column chromatography (10-12% [00137] Step-3. Synthesis of tert-butyl (tert-butoxycarbonyi)(4-cyano- 2~methylbenzyl)carbamate (20___Int-4)

[00138] A stirred solution of 4-(bromomethyl)-3-methylbenzonitrile (20_Int-3) (2.5g, 11 ,9mmol, l .Oeq) in DMF (25mL, 10V) was prepared and di- tert-butyl-iminodicarboxylate (2.84g, 13.09mmol, 1.1 eq) and K2CO3 (3.28g, 13.80mmol, 1.16eq) were added at RT. The RM was stirred at 70°C for 3h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over N evaporated, and purified by column chromatography (15-18% ethyl acetate in hexanes) to give compound (20_ _Int-4). 1H NMR (400 MHz, DMSO-d₆) 5 1.399 (d, J 4.8 Hz 18H), 2.310 (s, 3H), 4.734 (s, 2H), 7.122 (d, 1=4.8 Hz, 1H), 7.688 (d, J=5.4 Hz 2H).

[00139] Step-4. Synthesis of tert-butyl (tert-butoxycarbonyI)(4-(N- hydroxycarbamimidoyl)~2~methyibenzyi)carbamate (20_lnt-5)

[00140] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-2- methylbenzyljcarbamate (20 Int-4) (3.6g, 10.3mmol, l.Oeq) in methanol (36mL, 10V) was prepared and hydroxyl am monium chloride (1.22g, 17.66mmol, 1 .7eq) and DIPEA (3.0mL, 17.66mmol, 1.7eq) were added at RT. The RM was stirred at 70°C for 16h. The RM was evaporated, and the residue was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried

[00141] Step-5. Synthesis of tert-bntyl (tert-butoxycarbonyl) (4- carbamimidoyl~2-methylbenzyl) carbamate (20_Int-6)

[00142] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-(N- hydroxycarbamimidoyl)-2-methylbenzyl)carbamate (20 Int-5) (3.8g, lO.Ommol, l.Oeq) in methanol (38mL, 10V) was prepared and ammonium formate (3.15g, 50.0mmol, 5.0eq) and 10% Pd/C (50% rvet base) (0.19g, 0.065% w/w) were added at RT. The RM was stirred at 70°C for 16h under 20kg H2 pressure in an autoclave. The RM was filtered through a celite bed, and the filtrate was evaporated to give compound (20_Int-6). MS (ES): 364.0 m/z [M+I]’, LCMS purity: 94.2%.

[00143] Step-6. Synthesis of 4-(ammomethyl)-3-methylbenzimidamide dihydrochloride (20_Int-7)

[00144] A stirred solution of tert-butyl (tert-butoxycarbonyl) (4- carbamimidoyl-2-methylbenzyl) carbamate (20 Int-6) (3.8g, 10.4mmol, l.Oeq) in water (41.8mL, 1 IV) was prepared and concentrated HC1 (12.5mL, 3.3V) was added at RT and stirred at RT for 16h. The RM was evaporated, and the residue triturated in methanol to give compound (20_Int-7). MS (ES): 164.2 m/z [M+l]⁺, LCMS purity: 90.2%, ’HNMR (400 MHz, DMSO-d₆) 8 2.498 (s, 3H), 4.101 (d, J=5.6 Hz, 2H), 7.251(s, 2H), 8.772 (s, 1H), 7.378 (s, 1H), 7.505 (s, 1H), 7.663 (d, J=8.0 Hz, 1H), 7.752 (d, J= 8.0 Hz, 1H), 8.780 (s, 2H), 9.326 (s, 1H), 9.502 (s, 1H).

[00145] Example 4A: Preparation of ethyl l-(4-(2-(m ethylamino)- 2- oxoethyl)benzyl)-lH-pyrazoIe-4-carboxylate (l_Int-3)

[00146] Step-1. Synthesis of 2-(4-(bromomethyl)phenyl)~N- methylacetamide (l_Int-2).

[00147] A stirred solution of 2-(4-(bromomethyl)phenyl)acetic acid (1 Int-1) (1.0g, 4.36mmol, l.Oeq) in toluene (10mL, 10V) was prepared and (0.54g, 4.53mmol, I.04eq) and DMF (0.05 Ig, 0.69mmol, 0.16eq) were added at R.T. The RM was heated to 85°C and stirred for 3h. The RM was then cooled to 0°C followed by the addition of methylamine (2M in THE) (2 mL, 2V). The RM was stirred at 0°C to RT for 6h. The RM was filtered, and the solids were washed with ethyl acetate and dried to give compound (l__Int-2). MS (ES): 242.1 m/z [M]⁺ 244.1 m/z [M+2]⁺, LCMS purity: 96.6%, *HNMR (400 MHz, DMSO-dJ 3 3.389 (s, 2H), 4.726 (s, 2H), 7.245 (d, J 8 0 Hz, 2H), 7.344 (d, J=8.0 Hz, 2H), 8.019 (s, 1H).

[00148] Step-2. Synthesis of ethyl l-(4-(2-(methyIamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylate (l_hit-3).

[00149] A stirred solution of ethyl lH-pyrazole-4-carboxylate (0.5g, 3.56mmol, l .Oeq) in acetone (10mL, 20V) was prepared and 2-(4- (bromomethyOphenyl)-N-methylacetamide (l_Int-2) (1.03g, 4.28mmol, 1.2eq) and CS2CO3 (2.78g, 8.56mmol, 2.4eq) were added at RT. The RM was heated to 65°C and stirred for 16h. The RM was cooled to RT, filtered to remove CS2CO3, and washed with ethyl acetate. The filtrate was evaporated and then purified by column chromatography (12-15% ethyl acetate in hexanes) to give compound (l_Int-3) MS (ES): 302.4 m/z [ M H ]⁺, LCMS purity: 86.5%, ¹H NMR (400 MHz, DMSO-d₆) 6 1.658 (s, 6H), 4.556 (s, 2H), 5.366 (s, 2H), 7.331 (d, J 6.0 Hz, 2H), 7.505 (d, J=8.0 Hz, 4H), 7.983-7.934 (m, 2H), 8.314 (s, IH), 8.595 (s, IH), 8.813 (s, IH).

[00150] Example SA: Preparation of ethyl l-(4~(2-(dimethylamino)~2~ oxoethyl)benzyl)-lH-pyrazoIe-4-carboxylate (3_Int-3)

[00151] Step-1. Synthesis of 2-(4-(bromomethyl)phenyl)-N- methylacetamide (3_Int-2)

[00152] A stirred solution of 2-(4-(bromomethyl)phenyl) acetic acid (3 Int-1 ) (1.0g, 4.36mmol, l .Oeq) in toluene (lOrnL, 10V) was prepared and SOCI2 (0.54g, 4.53mmol, 1.04eq) and DMF (0.051g, 0.69mmol, 0.16eq) were added at RT. The RM was stirred at 85°C for 3h. The RM was cooled to 0°C followed by the addition of dimethylamine (2M in THF) (2.0mL, 2V). The RM: then brought to RT and stirred for 6h. The RM was quenched in saturated NaHCCh solution and extracted using ethyl acetate. The combined organic fractions were dried over NazSO4 and evaporated to give compound (3_Int-2). MS (ES): 256.6 m/z [M]⁺ 258.1 m/z [Mv2]⁺, LCMS purity: 99.0%, l i XMR (400 MHz, DMSO-de) 5 2.826 (s, 3H), 2.997 (s, 3H), 3.694 (s, 2H), 4.706 (s, 3H), 7.210 (d, J=8.0 Hz, 2H), 7.385 (d, J=8.0 Hz, 2H).

[00153] Step-2. Synthesis of ethyl l-(4~(2-(dimethyIamino)-2~ oxoethyl)benzyl)-lH~pyrazole~4~carboxylate (3 _Int-3).

[00154] A stirred solution of ethyl 1 H-pyrazole-4-carboxylate (0.3g, 2.14mmol, l.Oeq) in acetone (6.0mL, 20V) was prepared and 2-(4- (bromomethyr)pheny])-N,N-dimethylacetamide (3_Int-2) (0.657g, 2.56mmol, 1.2eq) and CS2CO3 ( 1 .67g, 5.13mniol, 2.4eq) were added at RT. The RM was heated to 65°C and stirred for 16h. The RM was then cooled to RT, filtered to remove CS2CO3, and washed with ethyl acetate. The filtrate was evaporated and then purified by column chromatography (25-30% ethyl acetate in hexanes) to give compound (3_Int-3) MS (ES): 316.3 m/z [M+l]^;, LCMS purity: 100%, ^TH NMR (400 MHz, DMSO-d₆) 8 1.252 (t, J= 2.8 Hz, 3H), 2.802 (s, 3H), 2.974 (s, 311), 3.396-3.295 (m, 2H), 4.188 (s, 2H), 5.324 (s, 2H), 7.189 (s, 411). 7.852 (s, IH), 8.466 (s, IH).

[00155] Example 6A: Preparation of methyl l-(4-(2-cyanopropan-2~ yi)benzyI)-5-(methoxymethyl)-lH-pyrazoIe-4-carboxylate (22_Int-3)

[00156] Step-1. Synthesis of methyl 5-(methoxymethyl)-lH-pyrazoIe- 4-carboxyiate (22_Int-2)

[00157] A solution of methyl 4-m ethoxy-3 -oxobutanoate (22_Int-l)

(15.0g, 102mmol, 1.0 eq) and dimethylformamide dimethyl acetal (12.23g,

102mmol, l.Oeq) was prepared and stirred at 110°C for Ih. The RM was cooled to RT followed by addition of ethanol (150mL, 10V) and hydrazine hydrate (99%) (5.13g, 102mmol, l.Oeq). The RM was heated to 70°C and stirred for 2h. The RM was evaporated and then purified by column chromatography (25-30% ethyl acetate in hexanes) to give compound to give compound (22_Int-2). ^{H NMR (400 MHz, DMSO-d₆) 8 3.258 (s, 3H), 3.758 (s, 3H), 4.702(s, 2H), 7.837 (s, 1H), 8.281 (s, 1H).

[00158] Step-2. Synthesis of methyl l-(4-(2-cyanopropan-2-yI)benzyI)” 5-(methoxymethyI)-1.H-pyrazoIe-4-carboxylate (22_Int-3)

[00159] A stirred solution of methyl 5-(methoxymethyl)-lH-pyrazole-4- carboxylate (22 Int-2) (1.0g, 5.87mmol, l.Oeq) in DMF (lOmL, 10V) was prepared and NaHMDS (I ML in THF) (5.8mL, 5.87mmol, l.Oeq) was added at 0°C and stirred for 30 minutes. 2-(4-(bromomethyl)phenyl)-2- methylpropanenitrile (5 Int-3) (1.39g, 5.87mmol, l.Oeq) was added to the RM at 0°C. The RM was brought to RT and stirred for 16h. The RM was quenched in water and extracted using ethyl acetate. The RM was dried over NazSCfi, evaporated, and then purified by flash column chromatography (15% ethyl acetate in hexanes) (22 Int-3). Confirmed by 2D NMR (ROE analysis). MS (ES): 328.0 m/z [M+I]^;, LCMS purity: 99.1%, ’H NMR (400 MHz, DMSO-d₆) 8 1.654 (s, 6H), 3.258 (s, 3H), 3.758 (s, 3H), 4.797 (s, 2H), 5.394 (s, 2H), 7.258 (d, J 8.0 Hz, 2H), 7.478 (d, J 8.0 Hz, 2H), 7.903 (s,lH).

[00160] Example 7A: Preparation of methyl l-(4- (cyanomethyl)benzyI)-5-(methoxymethyl)-lH-pyrazok~4-carboxyIate (21__Int-3) [00161] Step-1. Synthesis of methyl l-(4-(cyanomethyl)benzyi)-5-

(methoxymethyI)-lH-pyrazoIe-4-carboxyiate (21_Int-3)

[00162] A stirred solution of methyl 5-(methoxymethyl)-lH-pyrazole-4- carboxylate (22_Int-2) (1.0g, 5.87mmol, I .Oeq) in DMF (l OmL, 10V) was prepared and NaHMDS (IM in THF) (5.8mL, 5.87mmol, I.Oeq) was added at 0°C and stirred for 30 minutes. 2-(4-(bromomethyl)phenyl)acetonitrile (1 ,4g, 7.05mmol, 1.2eq) was added to the RM at 0°C. The RM was brought to RT and stirred for 16h. The RM was quenched in water and extracted using ethyl acetate. The RM was dried over Na2SO/i, evaporated, and then purified by flash column chromatography (25-30% ethyl acetate in hexanes) to give compound (21 Int-3). Confirmed by 2D NMR (ROE analysis). MS (ES): 300.0 m/z 8 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).

[00163] Example SA: Preparation of 4-(aminomethyI)-2- methyibenzimidamide dihydrochloride (19_Int~8)

[00164] Step-1. Synthesis of ethyl 4-cyano-3-methyIbenzoate (19 Ini-

2)

[00165] A stirred solution of 4-cyano-3 -methylbenzoic acid (19_Int- 1) (7.0g, 43.43mmol, I .Oeq) in ethanol (70mL, 10V) was prepared and H?.SO4 (3.5mL, 36.16mmol, 1.5eq) was added at RT. The RM was heated to 80°C and stirred for 4h. The RM was evaporated, the residue was basified with saturated NaHCO3 solution, extracted using ethyl acetate, dried over Na?SO4, and evaporated to give compound (19 Int-2). ’H NMR (400 MHz, DMSO-de) 6 1.340 (t, .1 6 8 Hz, 3H), 2,509 (s, 3H), 4.356 (q, J 1 .2, 7.2 Hz, 2H), 7.904 (d, J=8 Hz, IH), 7.944 (d, J=8 Hz, 1H), 8.020 (s, 1H). [00166] Step-2. Synthesis of 4-(HydroxymethyI)"2-methyibenzonitrile

(19 Int-3)

[00167] A stirred solution of 4-cyano-3 -methylbenzoate (19 Int-2) (8.0g, 42.32mmol, 1 .Oeq) in THF (80mL, 10V) was prepared and LiBEU (4M solution in THF) (12.6mL, 50.79mmol, 1.2eq) was added at RT and stirred for 16h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over Na2SO4 and evaporated to give compound 8 2.499 (s, 3H), 4.815 (s, 2H), 5.411 (s, 1H), 7.303 (d, J =8.0 Hz, 1H), 7.391 (s, 1H), 7.709 (d, J 8.0 Hz, 1H).

[00168] Step-3. Synthesis of 4-(BromoethyI)”2-methylbenzonitriIe (19__Int-4)

[00169] A stirred solution of 4-(Hydroxymethyl)-2 -methylbenzonitrile (19 Int-3) (6.0g, 40.77mmol, l.Oeq) in DCM (60mL, 10V) was prepared and triphenylphosphine (11.6gm, 44.84mmol, l . leq) and CBiu (14.87g, 44.84mmol, 1.1 eq) were added at RT and stirred for 3h. The RM was quenched in water and extracted using DCM. The combined organic fractions were dried over NaiSCU evaporated and purified by column chromatography (8-10% ethyl acetate in hexanes) to give compound (19 Int-4). ¹H NMR (400 MHz, DMSO-dr,) 82.479 (s, 3H), 4.724 (s, 2H), 7.451 (d, J=8.0 Hz, 1H), 7.544 (s, 1H), 7.770 (d, 1=8.0 Hz 1H).

[00170] Step-4. Synthesis of tert-butyl (tert-butoxycarbonyl)(4-cyano- 3~methylbenzyl)carbamate (19___Int-5)

[00171] A stirred solution of 4-(bromomethyl)-2 -methylbenzonitrile (19_Int-4) (5.2g, 24.76mmol, l.Oeq) in DMF (52mL, 10V) and THF (52ml, 10V) was prepared and di-tert-butyl-iminodicarboxylate (8.07g, 37. 13mmol, 1 ,5eq) and K2CO3 (5.12g, 37. 13mmol, 1 ,5eq) were added at RT. The RM was heated to 90°C and stirred for 3h. The RM was quenched in cold water and extracted using ethyl acetate. The combined organic fractions were dried over NaiSCti, evaporated, and then purified by column chromatography (8-10% ethyl acetate in hexanes) to give compound (19 Int-5). MS (ES): 191.0 m/z [M-l 56] \ LCMS purity: 83.8%, ¹H NMR (400 MHz, DMSO-d₆) 6 1.406 (s, 18H), 2.504 (s. 3H), 4.728 (s, 2H). 7.186 (d, J 8 0 Hz, 1H), 7.283 (s, 1 H), 7.765 (d, J 8.0 Hz, 1H).

[00172] Step-5. Synthesis of tert-butyl (tert-butoxycarbonyI)(4-(N~ hydroxycarbamimidoyl)-3-methylbenzyl)carbamate (19_lnt-6)

[00173] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-3- m ethylbenzyl )carbamate (19 Int-5) (5.0g, 13.73mmol, l.Oeq) in methanol (50mL, 10V) was prepared and hydroxylamine hydrochloride (1.64g, 23.69mmol, 1.7eq) and DIPEA (4.2mL, 23.69mmol, 1.7eq) were added at RT. The RM was heated to 70°C and stirred for 16h. The RM was evaporated to remove methanol and the residue was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over NajSCh and evaporated to give compound (19_Int-6). MS (ES): 380.3 m/z [M+l]⁺, LCMS purity: 59.5%, H i NMR (400 MHz, DMSO-d₆) 8 1.431 (s, 18H), 2.338 (s, 31 1). 4.667 (s, 2H), 5.791 (s, 1H) 7.029-7.074 (m, 2H), 7.259 (d, J=8.0 Hz, 1H), 7.355 (d, 1 8 0 Hz, 1H), 9.319 (d, J 2.0 Hz, 1H).

[00174] Step-6. Synthesis of tert-butyl (tert-butoxycarbouyl)(4- carbamimidoyl-3-methyIbenzyl)carbamate (19_Int-7)

[00175] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-(N- hydroxycarbamimidoyl)-3-methylbenzyl)carbamate (19__Int~6) (4.5g, 11.87mmol, l.Oeq) in methanol (45mL, 10V) was prepared and ammonium formate (3.75g, 59.36mmol, 5.0eq) and 10% Pd/C (50% wet base) (0.22g, 0.05% w/w) were added at RT. The RM was stirred at 70°C for 16h under 20kg H2 pressure in autoclave. The RM was filtered through celite bed and washed with methanol. The filtrate was evaporated to give compound (19__Int-7). MS (ES): 364.3 m/z [M+1]⁺ LCMS purity: 66.1%.

[00176] Step-7. Synthesis of 4-(aiuinomethyI)-2-niethylbenzimidamide dihydrochloride

[00177] A stirred solution of tert-butyl (tert-butoxy carbonyl) (4- carbamimidoyl-3-methylbenzyl) carbamate (19_Int-7) (4.5g, 10.4mmol, l.Oeq) in water (50mL, 1 IV) was prepared and concentrated HC1 (14.83mL, 3.3V) was added at RT and stirred for 16h. The RM w'as evaporated, and residue w'ere triturated by methanol to give compound (19_Int-8). MS (ES): 164.2 m/z [M + 1 ] ^h, LCMS purity: 80.9%

[00178] Exampie 9A: Preparation of 4-(aminomethyI)~3,5~ dimethylbenzimidamide dihydrochloride (7 Int-8)

[00179] Step-1. Synthesis of 4-formyl-3,5-dimethyIbenzonitriIe (7_lnt-

2)

[00180] A stirred solution of 4-bromo-3,5-dimethylbenzonitrile (7_Int-l)

(15.0g, 74.40mmol, I .Oeq) in THF (75mL, 5V) was prepared and n-BuLi (2.5M in hexane) (31.4mL, 78.54mmol, 1.1 eq) was added dropwise at -65°C over 45 minutes under N2 environment. The RM was stirred at -65°C for 30 minutes. DMF (7.3g, 99.96mmol, 1.4eq) was added drop wise to the RM at -65°C and stirred for 16h. The RM was slowly quenched in cold IN HC1 solution and extracted using ethyl acetate. The combined organic fractions were dried over Na2SO4, evaporated, and purified by column chromatography (0.4-0.6% ethyl acetate in hexanes) to give compound (7_Int-l). ^]H NMR (400 MHz, DMSO- d₆) 8 2.552 (s, 6H), 7.668 (s, 2H), 10.551 (s, 1H).

[00181] Step-2. Synthesis of 4-(hydroxymethyl)-3,5~ dimethylbenzonitrile (7__Int-3)

[00182] A stirred solution of 4-formyl-3,5-dimethylbenzonitrile (7_Int-2) (6.5g, 40.8mmol, I.Oeq) in THF (195mL, 30V) was prepared and NaBHU (2.0g, 52.86mmol, 1.3eq) was added portion wise over 30 minutes. The RM was stirred at RT for Ih, The RM was then quenched in 2N HO solution and extracted using ethyl acetate. The combined organic fractions were dried over NasSCh and evaporated to give compound (7_Int-3). NMR (400 MHz, DMSO-de) 3

2.386 (s, 6H), 4.552 (s, 2H), 4.967 (s, 2H), 7.495 (s, 2H).

[00183] Step-3. Synthesis of 4-(bromomethyl)-3,5~ dimethylbenzonitrile (7_Int-4)

[00184] A stirred solution of 4-(hydroxymethyl)-3,5-dimethylbenzonitrile

(7 Int-3) (6.2g, 38.46mmo1, 1 ,0eq) in DCM (124mL, 20V) was prepared and CBt4 (12.76g, 38.46mmol, l.Oeq) and triphenylphosphine (10.09g, 38.46mmol, l.Oeq) were prepared at RT and stirred for 4h. The RM was quenched in water and extracted using DCM. The combined organic fractions were dried over Na2SO4, evaporated, and then purified by column chromatography (2% ethyl acetate in hexanes) to give compound (7_Int-4). ^SH NMR (400 MHz, DMSO-dc,) 6 2.464 (s, 6H), 4.525 (s, 2H), 7.285 (s, 1H), 7.355 (s, IH).

[00185] Step-4. Synthesis of tert-butyl (tert-butoxycarbonyl)(4-cyano- 2,6-dimethylbenzyl)carbamate (7_Int-5)

[00186] A stirred solution of 4-(bromomethyl)-3,5-dimethylbenzonitrile (7 Int-4) (2.8g, 12.5mmol, l.Oeq) in DMF (28mL, 10V) was prepared and di- tert-butyl-im in odi carboxyl ate (3.2g, 15.0mmol, 1.2eq) and K2CO3 (3.4mL, 25.0mmol, 2.0eq) were added at RT. The RM was heated to 50°C and stirred for 16h. The RM was cooled to RT, quenched in water, and extracted using ethyl acetate. The combined organic fractions were dried over Na2SC>4, evaporated, and purified by column chromatography (2% ethyl acetate in hexanes) to give compound (7 Int-5). MS (ES): 205. 1 m/z [M-156]⁺, 41 NMR (400 MHz, DMSO-d₆) 5 1.326 (s, 18H), 2.333 (s, 6H), 4.792 (s, 2H), 7.504 (s, 2H).

[00187] Step-5. Synthesis of tert-butyl (tert-butoxycarbonyl)(4-(N- hydroxycarbamimidoyI)-2,6-dimethylbenzyi)carbamate (7__Iut-6)

[00188] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-2,6- dimethylbenzyl)carbamate (7_Int-5) (3.0g, 8.3mmol, l.Oeq) in methanol (30mL, 10V) was prepared and hydroxylamine hydrochloride (0.984g, 14.1mmol, 1.7eq) and DIPEA (1 ,8g, 14.1 mmol, 1.7eq) w'ere added at RT. The RM w'as heated to 70°C and stirred for 16h. The RM was evaporated, the residue quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over Na2SO4, evaporated, and purified by column chromatography (8-10% ethyl acetate in hexanes) to give compound (7_Int-6). MS (ES): 394.3 m/z [M+l]⁺ LCMS purity: 72.5%. ^lH NMR (400 MHz, DMSO-d₆) 6 1.375 (s, 18H), 2.303 (s, 6H), 4.734 (s, 2H), 7.200 (s, 1H), 7.322 (s, 211). 9.847 (s, 1 H), 10.279 (s, 1 H).

[00189] Step-6. Synthesis of tert-butyl (tert-butoxycarbonyl)(4- carbamimidoyl-2,6~dimethylbenzyl)carbamate (7__Int-7)

[00190] A stirred solution of tert-butyl (tert-butoxy carbonyl )(4-(N- hydroxycarbamimidoyl )-2,6-dimethylbenzyl)carbamate (7_Int-6) (2.85g, 7.2mmol, l.Oeq) in methanol (28.5mL, 10V) was prepared and ammonium formate (2.26g, 36.0mmol, 5.0eq) and 10% Pd/C (50% wet basis) (0. 142g, 0.05w/w) were added at RT. The RM was heated to 70°C, put under 20kg H2 pressure, and stirred for 16h. The RM was cooled to RT and filtered through a celite bed. The filtrate was evaporated to give compound (7_Int-7). MS (ES): 378.5 m/z [M+l ]\ LCMS purity: 84.9%.

[00191] Step-7. Synthesis of 4-(aminomethyl)-3,5- dimethylbenzimidamide dihydrochloride (7_Jnt-8)

[00192] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4- carbamimidoyl-2,6-dimethylbenzyl)carbamate (7_Int-7) (2.4g, 6.3mmol, l.Oeq) in water (14.4niL, 6V) was prepared and concentrated HC1 (7.2mL, 3V) was added at RT and stirred for 16h. The RM was evaporated, and residue was triturated in ethyl acetate to give compound (7_Int-8). MS (ES): 178.0 m/z [M+If, LCMS purity: 80.15%, H l NMR (400 MHz, DMSO-de) 8 2.251 (s, 6H), 4.071 (d, J=4.4 Hz, 2H), 7.368 (t, J=5.2 Hz, 1H), 7.541 (s, 2H), 8.616 (s, 1H), 9.312 (s, 1H), 9.461 (s, 1 H).

[00193] Example 10A: Preparation of 4-(aminomethyl)-3- ethylbenzimidamide dihydroehloride (10___Int-7)

[00194] Step-1. Synthesis of 3-bromo-4-(bromomethyI)benzonitriIe

(10__Int-2)

[00195] A stirred solution of 3-bromo-4-methylbenzonitrile (l()__Int-I) (20.0g, 102.04mmol, I.Oeq) in carbon tetrachloride (200mL, 10V) was prepared and AIBN (1.64g, 10.2mmol, 0.1 eq) and N-bromosuccinimide (20g,

112.35mmol, l . leq) were added at RT. The RM was heated to 90°C and stirred for 2h. The RM was cooled to RT, quenched in water, and extracted using DCM. The combined organic fractions were dried over Na2SO4, evaporated, and purified by column chromatography (0.5% ethyl acetate in hexanes) to give compound (10Int-2). ¹H NAIR (400 MHz, l)MSO-d₆ ) 5 5.770 (s, 2H), 7.561 (d, J=8.0 Hz, 1H), 7.792 (dd, J=1.2, 8.0 Hz, 1H), 8.163 (d, J=1.2 Hz, 1H).

[00196] Step-2. Synthesis of tert-butyl (2-bromo-4-cyanobenzyl)(tert~ butoxycarbonyl)carbamate (10___Int~3)

[00197] A stirred solution of 3-brorno-4-(bromomethyl)benzonitrile (10_Int-2) (30.0g, 109.0mmol, I .Oeq) in DMF (30mL, 10V) and THF (30mL, 10V) was prepared and K2CO3 (22.63g, 163.6mmol, 1.5eq) and di -tert-butyl - iminodicarboxylate (35g, 161.29mmol, 1.5 eq) were added at RT. The RM was heated to 90°C and stirred for 16h. The RM was cooled to RT, quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over Na2SO4, evaporated, and purified by column chromatography (5% ethyl acetate in hexanes) to give compound (10 Int-3). MS (ES): 313.1 m/z. [M- 98]⁺, LCMS purity: 96.9%, 'HNMR (400 MHz, CDCI3-d) 5 1.522 (s, 18H), 4.921 (s, 2H), 7.290 (dd, J =1 .2, 8.0 Hz, 1H), 7.632 (d, 1 8 Hz, 1 H ), 7.884 (s, 1H).

[00198] Step-3. Synthesis of tert-butyl (4-cyano-2~ ethylbenzyljcarbamate (10_Int-4)

[00199] A stirred solution of tert-butyl (2-bromo-4-cyanobenzyl)(tert- butoxycarbonyl)carbamate (10Jmt-3) (1.0g, 2.43mmol, l.Oeq) in 1,4-dioxane (lOmL, 10V) was prepared and purged with argon for 5 minutes. Pd(dppf)Cb (0.178g, 0.24mmol, 0. leq) and ZnCh (0.331g, 2.43mmol, l.Oeq) were added to the RM at RT. The RM was cooled to 0-5°C followed by the addition of ethyl magnesium bromide (12.16mL, 12.16mmol, 5.0eq) and stirred for 15 minutes. The RM was heated to 100°C and stirred for 16h. The RM was then cooled to RT, quenched in water, and extracted using ethyl acetate. The combined organic fractions were dried over NazSCL, evaporated and purified by column chromatography (8-10% ethyl acetate in hexanes) to give compound ( 10_ _Int-4) MS (ES): 205.1 m/z [M-56J⁺, LCMS purity: 100% TI NMR (400 MHz, DMSO- d₆) 8 1.172 (t, J 7.2 Hz, 3H), 1.402 (s, 9H), 2.674 (q, J 7 2 Hz, 2H), 4.212 (d, J=6.0 Hz, 2H), 7.369 (d, 1=8.0 Hz, 1H), 7.472 (t, 1=4.8 Hz, 1H), 7.636 (d, J=8.0 Hz, 1 H), 7.663 (s, H i).

[00200] Step-4. Synthesis of tert-butyl (2-ethyI-4-(N- hydroxycarbamimidoyl)benzyl)carbamate (10 Int-5)

[00201] A stirred solution of tert-butyl (4-cyano-2-ethylbenzyl)carbamate (10 Int-4) (5.5g, 21.21mmol, l.Oeq) in methanol (55mL, 10V) was prepared and hydroxylammonium chloride (2.49g, 35.83mmol, 1.7eq) and DIPEA (6.23mL, 35.83mmol, 1.7eq) were added at RT. The RM was heated to 70°C and stirred for 3h. The RM was evaporated, and the residue was quenched in cold water and stirred for 15 minutes. The solids were collected by filtration and dried to give compound (10 Int-5). MS (ES): 294.2 m/z [M+l]⁺, LCMS purity: 82.6%

[00202] Step-5. Synthesis of tert-butyl (4-carbamimidoyI~2~ ethylbenzyl)carbamate (10_Int-6)

[00203] A stirred solution of tert-butyl (2-ethyl-4-(N- hydroxycarbamimidoyl)benzyl)carbamate ( 10_Int-5) (4.5g, 15.33mmol, l.Oeq) in methanol (45mL, 10V) was prepared and ammonium formate (4.83g, 76.30mmol, 5.0eq) and 10% Pd/C (50% wet base) (0.225g, 0.05% w/w) were added at RT. The RM was heated to 70°C, put under 20kg Eh pressure, and stirred for 16h in an autoclave. The RM: was filtered through celite bed and washed by methanol. The filtrate was evaporated and the residue was quenched in cold water and stirred for 15 minutes. The solid residue was dried to give compound (10_Int-6). MS (ES): 278.2 m/z [M+l]⁺ LCMS purity: 45.1%

[00204] Step-6. Synthesis of 4-(aminomethyI)-3-ethyIbenzimidamide dihydrochloride (1.0_Int-7)

[00205] A stirred solution of tert-butyl (4-carbamimidoyl-2- ethylbenzyl)carbamate (10 Int-6) (2.0g, 72.10mmol, l.Oeq) in water (22mL,

I I V) was prepared and concentrated HC1 (6.6mL, 3.3V) was added at RT and stirred for 16h. The RM was evaporated, and residue was triturated in ethyl acetate to give compound (10 Int-7). MS (ES): 178.1 m/z [M+l] ⁺, LCMS purity: 98.0%, ¹H NMR (400 MHz, DMSO-d₆) 5 1.237 (t, 1=7.2 Hz, 3H), 2.728- 2.785 (m, 2H), 4.131-4.172 (m, 2H), 7.664 (s, 1H), 7.330-7.773 (ra, 2H), 8.551 (br s, 2H), 9. 131 (s, 2H), 9.417 (s, 2H).

[00206] Example 11 A: Preparation of 4-(aminomethyI)-3- isopropyibenzimidamide dihydrochloride (ll_Int-4)

[00207] Step-1. Synthesis of tert-bntyl (tert-butoxycarbonyl)(4-cyano- 2-(prop-l-en-2-yI)benzyI)carbamate (11 Int-1)

[00208] A stirred solution of tert-butyl (2~bromo-4-cyanobenzyl)(tert- butoxycarbonyl)carbamate (10_Int-3) (9.5g, 23.09mmol, l. leq) in 1,4-dioxane (142.5mL, 15V) and water (19mL, 2V) was prepared and CS2CO3 (15.06g, 46.19mmol, 2.0eq) and 2-isopropenyl boronic acid pinacol ester (5.82g, 34.64mmol, 1.5 eq) were added at RT. The RM was purged by argon gas for 20 minutes followed by the addition of Pd(dppf)Ch (0.81g, 1.15mmoL 0.05eq) at RT. The RM was heated to 120°C and stirred for 16h. The RM was cooled to RT and quenched in water and extracted using ethyl acetate. The combined layers were passed through celite bed and separated. The combined organic fractions were dried overNa2SO4, evaporated and purified by column chromatography (3- 4% ethyl acetate in hexanes) to give compound (1 1 Int-1) NMR (400 MHz, DMSO-dr,) 5 1.403 (s, 18H), 2.048 (s, 3H), 4.782 (s, 2H), 4.895 (s, 2H), 7.220 (dd, J=4.0, 4.0 Hz IH), 7.658 (d, J=2.0 Hz, 1H), 7.996 (dd, J=1.6, 6.4 Hz, IH).

[00209] Step-2. Synthesis of tert-butyl (tert-butoxycarbonyI)(4-(N- hydroxycarbamimidoyI)-2-(prop-l-en-2-yI)benzyI)carbamate (llJtat-2)

[00210] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-2- (prop-I-en-2-yl)benzyl)carbamate (1 1 Int-1) (6.0g, 15.40mmol, I .0eq) in methanol (60mL, 10V) was prepared and hydroxylamine hydrochloride (3.2g, 46.21mmol, 3.0eq) and NazCCh (4.89g, 46.21mmol, 3.0eq) were added at RT. The RM was heated to 80°C and stirred for 16h. The RM was evaporated, quenched in water, and extracted using ethyl acetate. The combined organic fractions were dried over NazSCfi and evaporated to give compound (11 _Int-2). MS (ES): 406.4 m/z [M+l]⁺, LCMS purity: 72.7%

[00211] Step-3. Synthesis of tert-butyl (4-carbamimidoyI-2-(prop-l- en-2-yl)benzyl)carbamate acetate (ll_ltat-3)

[00212] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-(N- hydroxycarbamimidoyl)-2-(prop-l -en-2-yl)benzyl)carbamate (1 l_Int-2) (1.0g, 2.46mmol, l.Oeq) in acetic acid (5mL, 5V) was prepared and zinc dust (0.483g, 7.35mmol, 3.0eq) was added at RT. The RM was heated to 90°C and stirred for Ih followed by addition of excess zinc dust (0.483g, 7.35mniol, 3.0eq). The RM was stirred for an additional Ih at 90°C. The RM was cooled to RT, filtered through celite bed, and washed with methanol. The filtrate was evaporated to give compound (l l_Int-3). MS (ES): 290.2 m/z [M+l]⁺, LCMS purity: 69.0%. [00213] Step-4. Synthesis of 4-(aminomethyI)-3- isopropylbenzimidamide dihydrochloride (ll Int-4)

[00214] A stirred solution of tert-butyl (4-carbamimidoyl-2-(prop-l-en-2- yl)benzyl)carbamate acetate (1 l_Int-3) (4.0g, 13.73mmol, l.Oeq) in ethanol (80mL, 20V) was prepared and concentrated HC1 (0.8mL, 0.2V) and 10% Pd/C (50% wet base) (0.4g, 0.1 w/w) were added at RT. The RM was heated to 70°C and stirred for 16h. The RM was filtered through celite bed and washed with methanol. The filtrate was evaporated to give compound (1 1 Int-4). MS (ES): 192.2 m/z [M+l/p, LCMS purity: 65.0%

[00215] Example 12A: Preparation of l-(4-(2-(methyIamino)-2- oxoethyl)benzyl)-lH"pyrazok"4-carboxylic acid (l_Int-l)

[00216] Step-1. Synthesis of l-(4-(2-(methyiamino)-2- oxoethyl)benzyl)~l H-pyrazok-4-carboxylic acid (l__Int-4)

[00217] A stirred solution of ethyl l-(4-(2-(methylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylate (1 Int-3) (9.0g, 29.86mmol, l.Oeq) in THE (90mL, I0V) was prepared and IM aqueous NaOH (119mL,

119.46mmol, 4.0eq) was added at RT and stirred for 16h. The RM was evaporated to remove THF, and the remaining RM was acidified by concentration HO to a final pH ~4. Solid precipitate were collected by filtration and dried to give compound (1 Int-4) MS (ES): 274.3 m/z i M ■ H | , LCMS purity: 89.5%, ^fH NMR (400 MHz, DMSO-ds) 5 2.560 (s, 3H), 3.367 (s, 2H), 5.336 (s, 2H), 7.201-7.249 (m, 4H), 7.812 (s, 1H), 7.950 (br s, 1H), 8.380 (s, 1H), 12.356 (s, 1H). [00218] Example 13A: Preparation of l-(4-(2-(dimethylamino)-2- oxoethyl)benzyI)~lH”pyrazok”4”€arboxylk add (3 Im-4)

[00219] Step-1. Synthesis of l-(4-(2-(dimethyIamino)~2- oxoethyl)benzyl)~l H-pyrazole-4-carboxylic add (3_Int-4)

[00220] A stirred solution of ethyl I -(4-(2-(dimethylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylate (3 Int-3) (0.5g, 0. 158mmol, l.Oeq) in water:methanol:THF (1 : 1 : 1) (5mL, 10V) was prepared and LiOH (0.3g, 0.793mmol, 5.0eq) were added at RT and stirred for 16h. The RM was quenched in 4N HC1 (pH -~4) and extracted using 10% methanol in DCM. The combined organic fractions were dried over Na2SO4 and evaporated to give compound (3 Int-4) MS (ES): 288.4 m/z [M+H]⁴, I CMS purity: 97.5%, TI NMR (400 MHz, DMSO-d₆) 6 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).

[00221] Example 14A: Preparation of l-(4-(2-cyanopropan-2- yI)benzyI)-lH-pyrazoIe-4-carboxylic add (5___Int-6) [00222] Step-l. Synthesis of l-(4-(2-cyanopropan-2-yi)benzyI)-lH- pyrazoIe-4-carboxyIic acid (5 Int-6)

[00223] A stirred solution of ethyl l-(4-(2-cyanopropan-2-yl)benzyl)-lH- pyrazole-4-carboxylate (5_Int-5) (0,7g, 23.5mmol, l .Oeq) in water:niethanol:THF (1 : 1 : 1) (140mL, 20V) was prepared and LiOH (3.95g, 94.27mmol, 4.0eq) was added at RT and stirred for 16h. The RM was evaporated to remove organic solvents and extracted using ethyl acetate. The organic fraction was discarded, and the aqueous layer was acidified with concentrated HC1 to a final pH --4 and extracted using ethyl acetate. The combined organic fractions were dried over Na2SC>4 and evaporated to give compound (5_Int-6) MS (ES): 270.3 m/z [ M H j . LCMS purity: 77.2%, ¹H NMR (400 MHz, DMSO-d₆) 8 1.667 (s, 6H), 5.373 (s, 2H), 7.335 (d, J=8.0 Hz, 211), 7.510 (d, J 8.0 Hz, 21 h, 7.823 (s, 1 H), 8.396 (s, 1H), 12.298 (s, H I).

[00224] Example ISA: Preparation of l-(4-(2-methoxyethyI)benzyI)-

I H-pyrazoIe-4-carboxyKc acid (6 __Int~4)

[00225] Step-l. Synthesis of ethyl l-(4-(2-hydroxyethyi)benzyI)-lH- pyrazoIe-4-carboxyIate (6 Int-2)

[00226] A stirred solution ethyl lH-pyrazole-4-carboxylate (1g, 7.14mmol, 1 ,0eq) in acetone (10ml, 10V) was prepared and 2-(4- (broniomethyl)phenyl)ethan-l-ol (6 Int-1) (1.6g, 7.85mmol, l. leq) and CS2CO3 (5.58g, 17.14mmol, 2.4eq) was added at RT. The RM was heated to 65°C and stirred for 6h. The RM was cooled to RT, quenched in water, and extracted using ethyl acetate. The combined organic fractions were dried over NazSCE, concentrated, and purified by flash column chromatography (25% ethyl acetate in hexanes) to give compound (6 Int-2). MS (ES): 274.2 rn/z i M 1 1 .. LCMS purity : 97.0%, ' 1 1 NMR (400 MHz, DMSO-de) 8 1.22 (t, J === 7.20 Hz, 3 H ) 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, 1 H), 5.31 (s, 2H), 7.19 (s, 4H), 0.00 (s, 1H), 8.43 (s, 1 H).

[00227] Step-2. Synthesis of ethyl l-(4-(2-methoxyethyl)benzyI)-lH- pyrazole-4-carboxyiate (6_Int-3)

[00228] A stirred solution ethyl l-(4-(2 -hydroxy ethyl)benzyl)-lH- pyrazole-4-carboxylate (6_Int-2) (0.900g, 5.17mmol, l.Oeq) in DMF (10ml, 10V) was prepared and NaH (2.85g, 10.34mmol, 2eq) was added and stirred 15 minutes at 0°C. Mel (0.948g, 6.724mmol, I .3eq) was then added at 0°C and the RM was stirred for 3h. The RM was quenched in cold water and extracted using ethyl acetate. The combined organic fractions were dried over NajSCh, concentrated, and purified by flash column chromatography (12% ethyl acetate in hexanes) to give compound (6 Int-3). MS (ES): 289.3 m/z [M+l]⁴, ¹H NMR (400 MHz, DMSO-d₆) 6 400 MHz, DMSO-d₆: 8 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).

[00229] Step-3. Synthesis of l-(4-(2-methoxyethyl)benzyl)-lH- pyrazole~4~carboxyhc add (6_Int-4)

[00230] A stirred solution of ethyl 1 ~(4~(2 -methoxy ethyl)benzyl )- 1 H- pyrazole-4-carboxylate (6_Int-3) (0.5g, 1 ,73mmol, l.Oeq) in water : methanol :THF (1 : 1 : 1) (lOrnL, 20V) was prepared and LiOH (0.3g, 0.69mmol, 4.0eq) was added at RT and stirred for 16h. The RM was quenched in 2N HC1 (pH ~4) and extracted using ethyl acetate. The combined organic fractions were dried over Na2SCri and evaporated to give compound (6_Int-4) MS (ES): 261.4 m/z [M+H] , LCMS purity: DMSO-de) 8 2.686 (t, J=6.8 Hz, 3H), 3.211 (s, 3H), 3.544 (t, J=6.8 Hz, 2H), 5.303 (s, 2H), 7.172-7.217 (m, 4H), 7.796 (s, 1H), 8.348 (s, 1H), 12.301 (s, I I I} [00231] Example 16A: Preparation of l-(4-(cyanomethyl)benzyl)-lH- pyrazoIe-4-carboxyIic acid (7A__Int-5)

[00232] Step-1. Synthesis of l-(4-(cyanomethyl)benzyl)-lH-pyrazole- 4-carboxyIic acid (7A_Int-5)

[00233] A stirred solution of ethyl l-(4-(cyanomethyl)benzyl)-lH- pyrazole-4-carboxylate (7A-Int-4) (1.6g, 5.94mmol, l.Oeq) in water:methanol:THF (1:1: 1) (32mL, 20V) was prepared and LiOH (0.99g, 23.70mmol, 4.0eq) was added at R.T and stirred for 16h. The RM was evaporated to remove organic solvents. The remaining aqueous layer was acidified with concentrated HO to a final pH ~4. Solid precipitate was collected by filtration and dried to give compound (7A_Int-5) MS (ES): 242.0 m/z [M+H]⁺, LCMS purity: 39.7%, 'HNMR (400 MHz, DMSO-dg) 5 4.021 (s, 2H), 5.344 (s, 2H), 7.133-7.223 (m, 4H), 7.749 (s, 1H), 8.262 (s, 1H), 12.301 (s, 1H).

[00234] Synthesis of Final Compounds

[00235] Comparative Example 1: Preparation of N-(4- carbamimidoyl-3-methyIbenzyl)-l-(4-(2-(methyIamino)-2-oxoethyl)benzyl)- lH-pyrazoIe~4-carboxamide (1)

1 -!nt-4 [00236] Step-1. Synthesis of N-(4-carbamimidoyl-3-methylbenzyl)-l-

(4-(2-(methylamino)-2~oxoethyi)benzyl)~lH-pyrazoIe~4”Carboxamide (1)

[00237] A stirred solution of l-(4-(2-(methylamino)-2-oxoethyl)benzyl)- lH-pyraz.ole-4-carboxylic acid (1_Int-4) (0.098g, 0.36mmol, l .Oeq) in pyridine (0.98mL, 10V) was prepared and 4-(aminomethyl)-2-methylbenzimidamide tri fluoroacetate ( 19__Int~8) (0.168g, 0.51 mmol, 1.4eq) and EDC.HCi (0.34g, 1.79mmol, 5.0eq) were added at RT and stirred for 16h. The RM was evaporated and purified by reverse phase column chromatography. The pure fraction was evaporated, and the residue was dissolved in water and extracted using 20% IPA in chloroform. The combined organic fractions were dried over NasSO* and evaporated to give compound (1) MS (ES): 419.5 m/'z [M+H] \ LCMS purity: 95.1%, HPLC purity: 99.5%, ¹H NMR (400 MHz, DMSO-d₆) 52.363 (s, 3H), 2.560 (d, J 4 8 Hz, 3H), 3.370 (s, 2H), 4.405 (d, J 5.6 Hz, 211). 5.312 (s, 2H), 7.193-7.257 (m, 4H), 7.283 (d, J=4.8 Hz, 1H), 7.391 (d, 1=8.0 Hz, 1H), 7.899 (s, 1 H), 7.971 (d, >4.4 Hz, 1H), 8.256 (s, 1H), 8.444 (s, 1H), 8.744 (t, J 6.0 Hz, 1H).

[00238] Example 2: Preparation of N-(4-carbamimidoyI"2- niethyibenzyI)-l-(4-(2“(niethylamino)-2-oxoethyl)benzyl)-lH-pyrazoie-4- carboxamide (2)

[00239] Step-1. Synthesis of N-(4-carbamimidoyl-2-methylbenzyl)-l- (4-(2-cyanopropan-2-yI)benzyI)~5-(methoxymethyI)-lH~pyrazoIe-4- carboxamide (2)

[00240] A stirred solution of ethyl 1 -(4-(2-(methylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylate (1 Int-3) (0.15g, 0.49mmol, l.Oeq) in THE ( 1.5mL, 10V) was prepared and 4-(aminomethyl)-3- methylbenzimidamide dihydrochloride (20__Int-7) (0.175g, 0.74mmol, 1.5eq) and DIPEA (0.34mL, 1.99mmol, 4.0eq) was added at RT. The RM was cooled to 0°C followed by the addition of TMA (2M in toluene) (1 ,49mL, 2.98mmol, 6.0eq). The RM was heated to 85°C and stirred for 16h. The RM was quenched by water (Iv) and evaporated. The residue was washed with 20% methanol in di chi or om ethane and filtered. The solid residue was discarded and the filtrate was evaporated and purified by reverse phase column chromatography ((A) 0.1% TFA in water (B) 100% MeCN). Pure fractions were lyophilized to give compound (2). MS (ES): 419.4 m/z [M+H] LCMS purity: 100%, HPLC purity: 100%, ¹H NMR (400 MHz, DMSO-d₆) 5 2.382 (s, 3H), 2.544 (d, J 4.8 Hz, 3 H ) 3.356 (s, 2H), 4.430 (d, J=5.6 Hz, 2H), 5.304 (s, 2H), 7.187-7.234 (m, 4H), 7.380 (d, J 8 0 Hz, 1H), 7.569-7.608 (m, 2H), 7.913-7.959 (m, 2H), 8.268 (s, 1H), 8.656 (t, J=6.0 Hz, 1H), 8.928 (s, 2H), 9.216 (s, 2H).

[00241] Comparative Example 3: Preparation of N-(4~ carbamimidoyl-3-methyIbenzyl)-l-(4-(2-(dimethyIainino)-2- oxoethyl)benzyl)-lH-pyrazoIe-4-carboxamide (3)

[00242] Step-1. Synthesis of N-(4-carbamimidoyl-3-methylbenzyl)-l-

(4-(2~(methylamino)-2-oxoethyl)benzyl)-lH-pyrazoIe-4-carboxamide (1)

[00243] A stirred solution of l-(4-(2-(dimethylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylic acid (3_Int-4) (0.25g, 0.87mmol, l.Oeq) in pyridine (3.3mL, 15V) was prepared and 4-(aminomethyl)-2- methylbenzimidamide tri fluoroacetate (19_Int-8) (0.24g, 0.87mmol, l.Oeq) and EDC.HC1 (0.83g, 4.35mmoL 5.0eq) were added at RT and stirred for 16h. The RM was evaporated and purified by reverse phase column chromatography ((A) 1% TFA in water (B) 100% ACN). The pure fraction was lyophilized to give compound (3). MS (ES): 433.2 m/z [M+H]⁺, LCMS purity: 100%, HPLC purity: 99.3%, ’H NMR (400 MHz, DMSO-d₆) 52.353 (s, 3H), 2.817 (s, 3H), 2.993 (s, 3H), 3.665 (s, 2H), 4.411 (d, J=6.0 Hz, 2H), 5.320 (s, 2H), 7.203 (s, 411), 7.299 (m, 2H), 7.403 (d, J =7.6 Hz, 1 H), 7.903 (s, 1 H), 8.260 (s, 1 H), 8.735 (I, 1=6.0 Hz, 1 H), 9.083 (s, 2H), 9.207 (s, 2H).

[00244] Example 4: Preparation of N-(4-carbamimidoyI-2~ methylbenzyi)-l-(4-(2-(dimethyIamino)-2"Oxoethyi)benzyI)-lH-pyrazole-4- rarboxamide (4)

[00245] Step-1. Synthesis of N-(4-carbamimidoyI-2-methylbenzyI)-l- (4-(2-(dimethyIamino)-2-oxoethyi)benzyI)-lH-pyrazoIe-4~carboxamide (4)

[00246] Example 4 was prepared from ethyl l-(4-(2-(dimethylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylate (3 Int-3) and 4-(aminomethyl)-3- methylbenzimidamide di hydrochloride (20 Int-7) in a similar fashion to that described in Example 2. MS (ES): 433.7 m/z [M+H]⁺, LCMS purity: 100%, HPLC purity: 100%, ‘H NMR (400 MHz, DMSO-d₆) 8 2.388 (s, 3H), 2.807 (s, 3H), 2.984 (s, 3H), 3.658 (s, 2H), 4.439 (d, J=5.6 Hz, 2H), 5.318 (s, 2H), 7.178- 7.224 (m, 4H), 3.788 (d, J=8.0 Hz, 1H), 7.574-7.612 (m, 2H), 7.925 (s, 1H), 8.286 (s, 1H), 8.667 (t, J 6.0 Hz, 1H), 8.926 (s, 2H), 9.219 (s, 2H).

[00247] Example 5: Preparation of N-(4-carbamimidoyl-2- methyIbenzyI)~l~(4”(2-cyanopropan-2~yI)beiizyI)"lH-pyrazole-4- earboxamide (5)

[00248] Step-1. Synthesis of N-(4-carbamimidoyI-2-methylbenzyI)-l-

(4-(2-cyanopropan-2-yI)benzyI)”lH”pyrazoIe”4-carboxamide (5)

[00249] Example 5 was prepared from l-(4-(2-cyanopropan-2-yl)benzyl)- lH-pyrazole-4-carboxylic acid (5_Int-6) and 4-(aminomethyl)-3- methylbenzimidamide dihydrochloride (20 Int-7) in a similar fashion to that described in Comparative Example 1. MS (ES): 415.2 m/z [M+H]⁺, LCMS purity: 96.6%, HPLC purity: 96.7%, ^lH NMR (400 MHz, DMSO-de) 8 1.673 (s, 611), 2.395 (s. 3H), 4.449 (d, J 5.6 Hz, 2H), 5.372 (s, 2H), 7.333-7.402 (m, 3H), 7.519 (d, 1=8.0 Hz, 2H), 7.584-7.623 (m, 2H), 7.944 (s, 1H), 8.314 (s, 1H), 8.451 (s, 1 H), 8.653 (t, .1 6.0 Hz, 1 H), 9.607 (br s, 2H).

[00250] Example 6: Preparation of N-(4-carbamimidoyI-2- methyibenzyI)-l-(4-(2-methoxyethyI)benzyl)~lH~pyrazoie~4-carboxamide (6) [00251] Step-1. Synthesis of N-(4-carbamimidoyl-2-methylbenzyl)-l-

(4-(2-methoxyethyl)benzyI)-lH-pyrazoIe~4”Carboxamide (6)

[00252] Example 6 was prepared from l-(4-(2 -methoxy ethyl)benzyl)-lH- pyrazole-4-carboxylic acid (6_ Int-4) and 4-(aminomethyl)-3- methylbenzimidamide dihydrochloride (20 Int-7) in a similar fashion to that described in Comparative Example 3. MS (ES): 406,6 m/z. [M+H]⁺, LCMS purity: 100%, HPLC purity: 99.8%, ’HNMR (400 MHz, DMSO-d₆) 52.486 (s, 311), 2.789 (L J 5.2 Hz, 2H), 3.231 (s, 3H), 3.514 (d, J 5 2 Hz, 2H), 4.449 (d, J=5.6 Hz, 2H), 5.316 (s, 2H), 7.215-7.255 (m, 4H), 7.399 (d, J=8.0 Hz, 1H), 7.587 (d, J I .6 Hz, H I). 7.618 (d, 1 8 0 Hz, 1H), 7.933 (s, 1H), 8.286 (s, H I), 8.674 (t, J=6.0 Hz, 1H), 8.982 (s, 2H), 9.234 (s, 2H).

[00253] Example 7: Preparation of N-(4-carbamimidoyI-2,6- diuiethyIbenzyl)-l-(4-(cyanomethyI)benzyl)~lH~pyrazoie-4-carboxamide (7)

[00254] Step-1. Synthesis of N-(4-carbamimidoyI-2,6-dimethyIbenzyl)- l-(4-(cyanomethyI)benzyl)-lH-pyrazoIe-4-carboxamide (7)

[00255] Example 7 was prepared from l-(4-(cyanomethyl)benzyl)-lH- pyrazole-4-carboxylic acid (7A_Int-5) and 4-(aminomethyl)-3,5- dimethylbenzimidamide dihydrochloride (7 Int-8) in a similar fashion to that described in Comparative Example 3. MS (ES): 401.3 m/z [M+H]⁺, LCMS purity: 99.6%, HPLC purity: 97.3%, ^lHNMR (400 MHz, DMSO-d₆) 62.442 (s, 6H), 4.018 (s, 2H), 4.669 (d, J =5.6 Hz, 2H), 5.331 (s, 2H), 7.256 (d, J 8 0 Hz, 2H), 7.332 (d, J=8.0 Hz, 2H), 7.497 (s, 1H), 7.881 (s, 1H), 8.202 (t, J=6.0 Hz, H I), 8.254 (s, 1 H), 8.962 (s, 2H), 9.212 (s, 2H). [00256] Example 8: Preparation of N-(4-carbamimidoyl-2,6- dimethylbenzyl)-l”(4-(cyanomethyl)benzyl)-lH-pyrazoIe~4”Carboxamide (8)

[00257] Step-1. Synthesis of N-(4-carbamimidoyI-2,6-diinethyIbenzyI)- l-(4-(cyanomethyI)benzyl)~lH-pyrazoie-4-carboxamide (8)

[00258] Example 8 was prepared from l-(4-(2-cyanopropan-2-yl)benzyl)- lH-pyrazole-4-carboxylic acid (5 Int-6) and 4-(aminomethyl)-3,5- dimethylbenzimidamide dihydrochloride (7 Int-8) in a similar fashion to that described in Comparative Example 3. MS (ES): 429.4 m/z [M+H]⁺, LCMS purity: 100%, HPLC purity: 99.0%, H i NMR (400 MHz, DMSO-d₆) 8 1.662 (s, 6H), 2.443 (s, 6H), 4.671 (d, J=5.6 Hz, 2H), 5.340 (s, 2H), 7.309 (d, J=8.0 Hz, 2H), 7.490-7.511 (m, 4H), 7.884 (s, 1H), 8.208 (t, J=6.0 Hz, 1H), 8.272 (s, 1H), 8.947 (s, 2H), 9.211 (s, 2H).

[00259] Example 9: Preparation of N-(4-carbamimidoyI-2,6- dimethyIbenzyl)-l-(4-(2-(methyiamino)-2~oxoethyI)benzyi)-lH-pyrazoIe-4- carboxamide (9) [00260] Step-l. Synthesis of N-(4-carbamimidoyl-2,6-dimethylbenzyI)- l~(4-(2-(methyiamino)~2-oxoethyI)benzyl)-lH-pyrazoIe-4-carboxamide (9)

[00261] Exampie 9 was prepared from l-(4-(2-(m ethyl amino)-2- oxoethyl)benzyl)-1H-pyrazole-4-carboxylic acid (l_Int-4) and 4-(aminomethyl)- 3,5-dimethylbenzimidamide dihydrochloride (7 Int-8) in a similar fashion to that described in Comparative Example 3. MS (ES): 433.4 m/z. [M+H]⁺, I . CMS purity: 100%, HPLC purity: 100%, ’HNMR (400 MHz, DMSO-d₆) 5 2.338 (s, 6H), 2.559 (s, 2H), 3.350 (s, 2H), 4.465 (d, J 5 6 Hz, 2H), 5.318 (s, 2H), 7.169- 7.231 (m, 4H), 7.493 (s, 2H), 7.864 (s, 1H), 7.933 (br s, 1H), 8.194 (t, 1=6.0 Hz, I H ). 8.232 (s, 2H), 8.930 (s, 2H), 9.211 (s, 2H).

[00262] Example 10: Preparation of N-(4-carbamimidoyl-2- ethyIbenzyl)-l-(4-(2-(methylamino)-2-oxoethyl)benzyl)-lH-pyrazoIe-4- carboxamide (10)

[00263] Step-1. Synthesis of N-(4-carbamimidoyI-2~ethyIbenzyl)-l~(4- (2-(methy8amino)-2-oxoethyl)benzyl)-l H-pyrazok-4-carhoxamsde (10)

[00264] Example 10 was prepared from l-(4-(2-(methylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylic acid (1 Int-4) and 4-(aminomethyl)- 3-ethylbenzimidamide dihydrochloride ( 10_Int-7) in a similar fashion to that described in Comparative Example 3. MS (ES): 433.4 m/z [M+H]⁺, LCMS purity: 98.2%, HPLC purity: 99.5%, ¹H NMR (400 MHz, DMSO-de) 6 1.232 (t, J=8.0 Hz, 3H), 2.752 (d, J=7.2 Hz, 2H), 3.352 (s, 2H), 4.480 (d, J=5.6 Hz, 2H), 5.310 (s, 2H), 7.191-7.242 (m, 4H), 7.416 (d, J 8.0 Hz, I H ) 7.586-7.624 (m, 2H), 7.914 (s, 1H), 7.949 (br s, 1H), 8.675 (t, J=6.0 Hz, 1H), 8.982 (s, 2H), 9.246 (s, 2H). [00265] Example 11: Preparation of N-(4-carbamimidoyl-2- isopropyIbenzyI)-l-(4-(2-(methylamino)-2-oxoethyI)benzyl)-lH-pyrazole-4~ carboxamide (11)

[00266] Step-1. Synthesis of N-(4-carbamimidoyl-2-isopropylbenzyI)- l-(4-(2"(methyiamino)-2-oxoethyI)benzyl)-l H-pyrazoIe-4-earboxamide (11)

[00267] .A stirred solution l-(4-(2-(methylamino)-2-oxoethyl)benzyl)-lH- pyrazole-4-carboxylic acid (1 Int-4) (0.25g, 0.91mmol, l.Oeq) in pyridine (2.5mL, 10V) was prepared and 4-(aminomethyl)-3-isopropylbenzimidamide dihydrochloride (11 Int-4) (0.358g, 1.37mmol, 1.5eq) and EDC.HC1 (0.876g, 4.57mmol, 5.0eq) were added at RT and stirred for 16h. The RM was evaporated and purified by reverse phase column chromatography ((A) 1% TFA in water (B) 100% ACN). The pure fractions were lyophilized to give compound (11). MS (ES): 447.6 ni/z [M+H]⁺, LCMS purity: 99.4%, HPLC purity: 99.2%, ¹H NMR (400 MHz, DMSO-d₆) 6 1.236 (d, .1 6 8 Hz, 6H), 2.557 (d, J 4.4 Hz, 3 H ) 3.296 (s, 1H), 3.366 (s, 2H), 4.516 (d, 1=6.0 Hz, 2H), 5.310 (s, 2H), 7.189-7.244 (m, 4H), 7.411 (d, J 8.0 Hz, 1H), 7.572-7.597 (m, 1 H), 7.120 (d, J 2.0 Hz, 1 H), 7.911 (s, 1H), 7.955 (d, J=4.4 Hz, 1H), 8.265 (s, 1H), 8.478 (s, 1H), 8.635 (t, J 6 0 Hz, 1H), 9.342 (s, 1H), 10.714 (s, 1H).

[00268] Example 12: Preparation of N-(4-carbamimidoyl-2- ethyIbenzyl)-l~(4-(2-cyanopropan-2-yl)benzyl)~lH~pyrazoIe~4”Carboxamide (12)

[00269] Step-1. Synthesis of N-(4-carbamimidoyI-2-ethyIbenzyl)-l-(4- (2-cyanopropan~2-yl)benzyi)-lH-pyrazoIe-4-carboxamide (12)

[00270] Example 12 was prepared from l-(4-(2-cyanopropan-2- yl)benzyl)-lH-pyrazole-4-carboxylic acid (5 Int-6) and 4-(aminomethyl)-3- ethylbenzimidamide di hydrochloride (10 Int-7) in a similar fashion to that described in Comparative Example 3. MS (ES): 429.4 m/z | Xi • I H , LCMS purity: 100%, HPLC purity: 95.8%, NM¹HR (400 MHz, DMSO-d₆) 5 1.241 (t, J=8.0 Hz, 3H), 1.672 (s, 6H), 2.760 (q, 2H), 4.491 (d, J=5.6 Hz, 2H), 5.373 (s, 211), 7.340 (d, J 8.0 Hz, 2H), 7.424 (d, J 8.0 Hz, 1 H), 7.517 (d, J 8.0 Hz, 2H), 7.620 (d, 1 8 0 Hz, 2H), 7.938 (s, 1 H), 8.304 (s, 1 H), 8.677 (t, J 6.0 Hz, H I). 8.908 (s, 2H), 9.235 (s, 2H).

[00271] Example 13: Preparation of N-(4-carbamimidoyl-2- isopropyIbenzyI)-l-(4~(2-cyanopropaH”2-yI)benzyI)~lH~pyrazoIe~4- carboxamide (13) [00272] Step-1. Synthesis of N-(4-carbamimidoyl-2-isopropylbenzyi)- l~(4-(2-cyanopropan-2~yl)benzyI)-lH-pyrazole-4-carboxamide (13)

[00273] Exampie 13 was prepared from l-(4-(2-cy anopropan -2- yl)benzyl)-1H-pyrazole-4-carboxylic acid (5_Int-6) and 4-(aminomethyl)-3- isopropylbenzimidamide dihydrochloride (11 Int-4) in a similar fashion to that described in Example 11. MS (ES): 443.6 m/z [M+H]⁺, LCMS purity: 99.9%, HPLC purity: 99.2%, hi W (400 MHz, DMSO-d₆) 8 1.256 (d, J=8.8 Hz, 6H), 1.682 (s, 6H), 3.298-2.342 (m, IH), 4.530 (d, J=5.6 Hz, 2H), 5.379 (s, 2H), 7.344 (d, 1=8.0 Hz, 2H), 7.428 (d, J=8.0 Hz, 1H), 7.526 (d, 1=8.0 Hz, 2H), 7.601 (d, 1 8 0 Hz, 2H), 7.737 (s, IH), 7.942 (s, 1 H), 8.310 (s, IH), 8.487 (s, IH), 8.668 (t, 1=6.0 Hz, IH), 9.779 (br s, IH).

[00274] Comparative Example 14: Preparation of N-(4- carbauiiuiidoyI-2-(triniioromethyl)benzyI)-1~(4~(2-cyanopropan-2- yI)benzyi)-lH-pyrazole-4-carboxamide (14) [00275] Step-l. Synthesis of 4-(bromomethyI)-3-

(trifluoromethyl)benzonitrile (14 Int-2)

[00276] A stirred solution of 4-methyl-3-(trifluoromethyl)benzonitrile

(14__Int-l) (2.5g, 13.50mmol, l.Oeq) in carbon tetrachloride (25mL, 10V) was prepared and AIBN (0.22g, 1.35mmol, O. leq) and N-bromosuccinimide (2.6g, 14.85mmol, l . leq) were added at RT. The RM was heated to 90°C and stirred for 4h. The RM was cooled to RT, quenched in water, and extracted using DCM. The combined organic fractions were dried over NazSCU, evaporated, and purified by column chromatography (0.4% ethyl acetate in hexanes) to give compound ( 14 lm-2) (400 MHz, DMSO-d₆) 8 4.835 (s, 2H ), 7.979 (d, J=8.0 Hz, 1H), 8.208 (dd, J=1.2, 8.0 Hz, 1H), 8.312 (s, 1H).

[00277] Step-2. Synthesis of tert-butyl (tert-butoxycarbonyl)(4-cyano- 2-(trifluoromethyI)benzyl)carbamate (14_Int-3)

[00278] A stirred solution of 4-(bromomethyl)-3-

(tritluoromethyl)benzonitrile (14 Int-2) (0.227g, 0.85mmol, l.Oeq) in DMF (2.27rnL, 10V) was prepared and K2CO3 (0.237g, 1.72mmol, 2.0eq) and di-tert- butyl -iminodi carboxylate (0.224g, 1.03mmol, I .2eq) were added at RT. The RM was heated to 60°C and stirred for 2h. The RM was cooled to RT quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over NazSCti, evaporated, and purified by column chromatography (5% ethyl acetate in hexanes) to give compound (14_Int-3). MS (ES): 301.2 m/z [M- 100]⁺, I .CMS purity: 98.4%, ^-INMR. (400 MHz, DMSO-d₆) 6 1.393 (s, 18H), 4.950 (s, 2H), 7.415 (d, 1=8.0 Hz, 1H), 7.209 (d, J=8.0 Hz, 1H), 8.318 (s, 1H).

[00279] Step-3. Synthesis of tert-bntyl (tert-butoxycarbonyI)(4-(N- hydroxycarbamimidoyl)-2-(trifluoromethyl)benz;yl)carbamate (14__Int~4)

[00280] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-2- (trifluoromethyl)benzyl)carbamate (14 Int-3) (0.355g, 0.88mmol, l.Oeq) in methanol (3.5mL, 10V) was prepared and hydroxylammonium chloride (0. 104g, 1.50mmol, 1.7eq) and DIPEA (0.194mL, 1.50mmol, 1.7eq) were added at RT. The RM was heated to 70°C and stirred for 16h. The RM was evaporated, and the residue was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over NazSCh and evaporated to give compound (14_Int-4). MS (ES): 434.3 m/z [M+l]⁴, LCMS purity: 97.0%, ¹H NMR (400 MHz, DMSO-d₆) 8 1.271 (s, 18H), 4.018 (t, J=5.2 Hz, 1H), 4.906 (s, 211), 6.003 (s, 2H), 7.221 (d, J 8.0 Hz, 1 H), 7.972 (d, J 8.0 Hz, 1H), 8.027 (s, 1H), 9.838 (s, 1H).

[00281] Step-4. Synthesis of tert-butyl (tert-butoxycarbonyl)(4- carbamimidoyl-2-(trifluoromethyI)benzyl)carbamate (14_Int-5)

[00282] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-(N- hydroxycarbamimidoyl)-2-(trifluoromethyl)benzyl)carbamate (14 Int-4) (0.25g, 0.57mmol, 1 .Oeq) in methanol (2.5mL, 10V) was prepared and ammonium formate (0.18g, 2.88mmol, 5. Oeq) and 10% Pd/C (50% wet base) (0.012g, 0.05% w/w) were added at RT. The R]M was heated to 70°C, put under 20kg Ifc pressure in an autoclave, and stirred for 16h. The RM was filtered through celite bed and washed with methanol. The filtrate was evaporated, and the residue was quenched in cold water and stirred for 15 minutes. Solid precipitate was collected by filtration and dried to give compound (14 Int-5). MS (ES): 418.3 m/z [M+l ]⁺, LCMS purity: 74.6%

[00283] Step-5. Synthesis of 4-(aminomethyI)-3- (trifluoromethyl)benzimidamide dihydrochloride (14__Int~6)

[00284] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4- carbamimidoyl-2-(trifIuoromethyl)benzyl)carbamate ( 14_Int-5) (0.158g, 3.78mmol, l .Oeq) in water (1.73mL, l l V) was prepared and concentrated HQ (0 5 L 3 3V) dd d t RT d ti d f 16h Th RM t d

[00285] Step-6. Synthesis of N-(4-carbamimidoyI-2- (trifluoromethyl)benzyI)-l~(4-(2-cyanopropan-2-yI)benzyl)-lH-pyrazoIe-4- carbnxamide (14)

[00286] Comparative example 14 was prepared from l-(4-(2- cyanopropan-2-yl)benzyl)-lH-pyrazole-4-carboxylic acid (5_Int-6) and 4- (aminomethyl)-3-(trifluoromethyl)benzimidamide dihydrochloride (14__Int-6) in a similar fashion to that described in Comparative Example 3. MS (ES): 469.3 m/z [M+H]'^h, LCMS purity: 100%, HPLC purity: 99.3%, ‘H NMR (400 MHz, DMSO-df,) 5 1.677 (s, 6H), 4.646 (d, J=5.6 Hz, 2H), 5.392 (s, 2H), 7.360 (d, 1 =8.0 Hz, 211 ). 7.519 (d, .1 2.0 Hz, 1H), 7.535 (d, 1=2.0 Hz, 1 H), 7.698 (d, .1 8.0 Hz, 1 H), 7.970 (s, 1H), 8.033 (d, J 8.0 Hz, 1 H), 8.137 (s, 1H), 8.346 (s, 1 H), 8.876 (t, 1=5.6 Hz, 1H), 9.128 (s, 211 ). 9.444 (s, 211 ).

[00287] Comparative Example 15: Preparation of N-(4- carbamimidoyI-3~(trinuoromethyl)benzyI)-1.~(4~(2-cyanopropan-2~ yI)benzyl)-lH-pyrazole-4-carboxamide (15)

[00288] Step-1. Synthesis of 4-(bromomethyi)-2-

(trifluoromethyl)benzonitrile (15_Int-2)

[00289] A stirred solution of 4-methyl-2-(trifluoromethyl)benzonitrile

(15 Int-1) (2.5g, 13,50mmol, I .Oeq) in carbon tetrachloride (25mL, 10V) was prepared and N-bromosuccinimide (2.6g, 14.80mmol, l. leq) and AIBN (0.220g, 1.35mmol, 0. leq) were added at RT. The RM was heated to 90°C and stirred for 6h. The RM was cooled to RT, quenched in water, and extracted using DCM. The combined organic fractions were dried over Na2SOi, evaporated, and then purified by column chromatography (3-5% ethyl acetate in hexanes) to give compound (15 _Int-2). MS (ES): ¹H NMR (400 MHz, DMSO-d₆) 8 4.585 (s, 2H), 7.731 (dd, J=1.2, 8.0 Hz, 1H), 7.842-7.911 (m, 2H).

[00290] Step-2. Synthesis of tert-butyl (tert-butoxycarbonyl)(4-cyano~ 3-(trifliioromethyI)benzyl) carbamate (15_Int-3)

[00291] A stirred solution of 4-(bromomethyl)-2-

(trifluoromethyl)benzonitrile (15 Int-2) (1.6g, 6.00mmol, l.Oeq) in DMF

( 16mL, 10V) was prepared and K2CO3 (1.65g, 11 ,93mmol, 2.0eq) and di-tert- butyl-iminodicarboxylate (1.5g, 6.91mmol, 1.2eq) were added at RT. The RM was heated to 60°C and stirred for 3h. The RM: was cooled to RT, quenched in water, and extracted using ethyl acetate. The combined organic fractions were dried over Na?SO4, evaporated, and then purified by column chromatography (7- 8 % ethyl acetate in hexanes) to give compound (15_Int-3). ^jH NMR (400 MHz, DMSO-d₆) 5 1.397 (s, 18H), 4.859 (s, 2H), 7.699 (d, J-8.0 Hz, 1H), 7.834 (s, 1H), 8. 185 (d, J 8.0 Hz. H i).

[00292] Step-3. Synthesis of tert-bntyl (tert-butoxycarbonyI)(4-(N- hydroxycarbamimidoyl)-3-(trifluoromethyl)bei»yl)carbamate (15__Int~4)

[00293] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-3- (trifluoromethyl)benzyl) carbamate (15 Int-3) (1.95g, 4.8mmol, l.Oeq) in methanol (19.5mL, 10V) was prepared and hydroxyl am monium chloride (0.575g, 8.2mmol, 1.7eq) and DIPEA (1.05g, 8.2mniol, I .7eq) were added at RT. The RM was heated to 80°C and stirred for 16h. The RM was evaporated, and the residue was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over Na?SO4 to give compound (15__Int- 4). MS (ES): 434.4 m/z [M+l]⁺, LCMS purity: 44.6%

[00294] Step-4. Synthesis of tert-bntyl (tert-butoxycarbonyl)(4- carbamimidoyl-3-(trifluoromethyI)benzyl)carbamate (15_Int-5)

[00295] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-(N- hydroxycarbamimidoyl)-3 -(trifluoromethyl)benzyl)carbamate ( 15 Int-4) (2.1g, 4.8mmol, 1 .Oeq) in methanol (2 ImL, 10V) was prepared and ammonium formate (1.51g, 24.0mmol, 5. Oeq) and 10% Pd/C (50% wet base) (0.105g, 0.05% w7w) were added at RT. The RM: was heated to 80°C, put under 20kg IE pressure in an autoclave, and stirred for 16h. The RM was filtered through celite bed and the filtrate was evaporated to give compound (15_Int-5). MS (ES): 318.2 m/z [M-100]⁺, LCMS purity: 34.7%

[00296] Step-5. Synthesis of 4-(aminomethyl)-2- (trifluoromethyl)benzimidamide dihydrochloride (15_Int~6)

[00297] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4- carbamimidoyl-3-(trifluoromethyl)benzyl)carbamate (15 Int-5) (1.53g, 3.7mmol, l.Oeq) in water (9.18mL, 6.0V) was prepared and concentrated HC1 (4.5mL, 3.0V) was added at RT and stirred for 16h. The RM was evaporated, and the residue was triturated in ethyl acetate and purified by reverse phase column chromatography (12-15 % water in MeCN) to give compound (15 Int- 6). MS (ES): 218.2 m/z | M • l | . LCMS purity: 90.0%, NMR¹ (H400 MHz, DMSO-dg) 6 4.208 (s, 2H), 7.832-8.833 (m, 3H), 9.628 (s, 2H).

[00298] Step-6. Synthesis of N-(4-carbamimidoyI-3- (trifluoromethyl)benzyl)-l-(4-(2-eyanopropan"2-yI)benzy8)-lH-pyrazoIe-4- carboxamide (15)

[00299] Comparative Example 15 was prepared from l-(4-(2- cyanopropan-2-yl)benzyl)-lH-pyrazole-4-carboxylic acid (5_Int-6) and 4- (aminomethyl)-2-(trifluorom ethyl )benzimidamide dihydrochloride ( 15 Int-6) in a similar fashion to that described in Comparative Example 3. MS (ES): 469.3 m/z [M+H]⁺, LCMS purity: 100%, HPLC purity: 95.7%, 'HNMR (400 MHz, DMSO-de) 5 1.678 (s, 6H), 4.541 (d, 1 5 6 Hz, 2H), 5.380 (s, 2H), 7.339 (d, J=8.0 Hz, 2H), 7.523 (d, J=8.0 Hz, 2H), 7.729 (d, J=7.6 Hz, 1H), 7.775 (d, J=7.6 Hz, I H), 7.870 (s, 1 H), 7.925 (s, 1 H), 8.307 (s, 1H), 8.873 (t, J 5 0 Hz, 1 H), 9.295 (s, 21 1), 9.495 (s, 2H).

[00300] Comparative Example 16: Preparation of N-(4- carbamimidoyl-3-methyIbenzyl)-l-(4~(2-cyanopropan-2-yI)beiizyI)-lH- pyrazole-4-carboxamide (16)

[00301] Step-1. Synthesis of N-(4-carbamimidoyI-3~methylbenzyI)~l- (4-(2-cyanopropan-2-yi)benzyI)-lH-pyrazole-4-carboxamide (16)

[00302] Comparative Example 16 was prepared from l-(4-(2- cyanopropan-2-yl)benzyi)-lH-pyrazole-4-carboxylic acid (5 Int-6) and 4- (aminomethyl)-2-methylbenzimidamide tri fluoroacetate (19_Int-8) in a similar fashion to that described in Comparative Example 3. MS (ES): 415.3 m/z I M H | . LCMS purity: 99.8%, HPLC purity: 97.0%, ¹H NMR (400 MHz, DMSO-d₆) 8 1.680 (s, 6H), 2.365 (s, 3H), 4.425 (d, 1=6.0 Hz, 2H), 5.358 (s, 2H), 7.283-7.323 (m, 4H), 7.384 (d, J=8.0 Hz, 1H), 7.504 (d, .>8.0 Hz, 2H), 7.892 (s, 1H), 8.222 (s, 1H), 8.520 (br s, 1H), 8.930 (s, 1H), 9.102 (s, 2H).

[00303] Comparative Example 17: Preparation of N-(4- carbamimidoyl-3-methyIbenzyl)-l-(4-(2-methoxyethyl)benzyl)-lH-pyrazoIe- 4~carboxamide (17) [00304] Step-1. Synthesis of N-(4-carbamimidoyl-3-methylbenzyl)-l-

(4-(2-methoxyethyi)benzyl)-lH-pyrazoIe-4-carboxamide (17)

[00305] Comparative Example 17 was prepared from l-(4-(2- methoxyethyl)benzyl)-lH-pyrazole-4-carboxylic acid (6_Int-4) and 4- (aminomethyl)-2-methylbenzimidamide trifluoroacetate (19 Int-8) in a similar

[00306] Comparative Example 18: Preparation of N-(4- carbamimidoyI-3-isopropyIbenzyI)-l-(4-(2-(methylamino)-2- oxoethyi)beazyl)~lH~pyrazoie"4~earboxamide (18)

[00307] Step-1. Synthesis of 2-bromo-4-(bromomethyI)benzonitriIe (18_Int-2)

[00308] A stirred solution of 2-bromo-4-m ethylbenzonitrile (18_Int-l ) (5.0g, 25.50mmol, l.Oeq) in carbon tetrachloride (50mL, 10V) was prepared and AIBN (0.418g, 2.55mmol, O. leq) and N-bromosuccinimide (4.99g, 28.05mmol, 1.1 eq) were added at RT. The RM was heated to 90°C and stirred for 6h. The RM was cooled to RT, quenched in water, and extracted using DCM. The combined organic fractions were dried over Na^SCL, evaporated, and then purified by column chromatography (5-7% ethyl acetate in hexanes) to give compound (18_Int-2). ¹H NMR (400 MHz, CDC13-d) 8 4.457 (s, 2H), 7.472 (d, J 8.0 Hz, H i}. 7,671 (d. J 8 0 Hz, 1 H), 7.757 (s, 1H).

[00309] Step-2. Synthesis of tert-butyl (3-bromo-4-cyanobenzyI)(tert- butoxycarbonyl)carbamate (18 Int-3)

[00310] A stirred solution of 2-bromo-4-(bromomethyl)benzonitrile (18_Int-2) (2.0g, 7.20mmol, l .Oeq) in DMF (20mL, 10V) and THF (20mL, I0V) were prepared and K2CO3 (1.99g, 14.0mniol, 2.0eq) and di-tert-butyl- iminodicarboxylate (2.3g, 10.90mmol, 1.5eq) were added at RT. The RM was heated to 90°C and stirred for 5h. The RM was cooled to RT, quenched in water, and extracted using ethyl acetate. The combined organic fractions were dried over Na2SO4, evaporated, and then purified by column chromatography (5-7% ethyl acetate in hexanes) to give compound (18 Int-3). MS (ES): 313.1 m/z [M- 98]⁺, LCMS purity: 100%, ⁵H NMR (400 MHz, DMSO-d₆) 8 1.423 (s, I8H), 4.778 (s, 2H), 7.401 (d, J=7.2 Hz, 1H), 7.720 (s, 1H), 7.966 (d, 1=8.0 Hz, 1H).

[00311] Step-3. Synthesis of tert-butyl (tert-butoxycarbonyl)(4-cyano~ 3-(prop-l-eii"2-yI)benzyl)carbamate (18_Int-4)

[00312] A stirred solution of tert-butyl (3-bromo-4-cyanobenzyl)(tert- butoxycarbonyl)carbamate (18 Int-3) (0.45g, l .Olmmol, l.Oeq) in 1,4-dioxane (6.7niL, 15V) and water (0.9mL, 2V) was prepared and CS2CO3 (0.65g, 2.00mmol, 2.0eq) and 2-isopropenyl boronic acid pinacol ester (0.275g, 1.60mmol, 1.5eq) were added at RT. The RM was purged by argon gas for 20 minutes followed by the addition of Pd(dppf)Ch (0.035g, O.OSmmol, 0.05eq) at RT. The RAI was heated to 120°C and stirred for 2h. The RM was cooled to RT and quenched in water and extracted using ethyl acetate. The combined layers were passed through celite bed and separated. The combined organic fractions were dried overNa2SO4, evaporated and purified by column chromatography (3- 4% ethyl acetate in hexanes) to give compound ( 18_Int-4) MS (ES): 217.3 m/z [M-156]⁺, LCMS purity: 95.1%, ¹H NMR (400 MHz, DMSO-de) 8 1.411 (s, 18H), 2.138 (s, 3H), 4.775 (s, 2H), 5.213 (s, 1H), 5.437 (s, 1H), 7.292-7.335 (m, 2H), 7.850 (d, J=8.0 Hz, IH). [00313] Step-4. Synthesis of tert-butyl (4-(N-hydroxycar bam imidoyl)- 3-(prop-l-en-2-yI)benzyI)carbamate (18 Im-5)

[00314] A stirred solution of tert-butyl (tert-butoxycarbonyl)(4-cyano-3- (prop-1-en-2-yl)benzyl)carbamate ( 18_ Jnt-4) (0.7g, 1.80mmol, 1 .Oeq) in methanol (7mL, 10V) was prepared and hydroxylamine hydrochloride (0.26g, 3.60mmol, 2. Oeq) and DIPEA (0.6mL, 3.2mmol, 1.8eq) were added at RT. The RM was heated to 80°C and stirred for 36h. The RAI was evaporated, and the residue was quenched in water and extracted using ethyl acetate. The combined

[00315] Step-5. Sy nthesis of tert-butyl (4-carbamimidoyl-3- isopropylbenzyl)carbamate (18__Int-6)

[00316] A stirred solution of tert-butyl (4-(N-hydroxycarbamimidoyl)-3- (prop-l -en-2-yl)benzyl)carbamate ( 18_ Int-5 ) ( 1.0g, 2.46mmol, l.Oeq) in methanol (lOmL, 10V) was prepared and ammonium formate (0.77g, 12.30mmol, 5. Oeq) and 10% Pd/C (50% wet base) (0. 1g, 0. Iw/w) were added at RT. The RM was heated to 80°C, put under 20kg H2 pressure in an autoclave, and stirred for 24h. The RM was cooled to RT, filtered through celite bed and washed with methanol. The filtrate was evaporated to give compound (18_Int-6). MS (ES): 237.1 m/z [M-56]⁺, LCMS purity: 33.3%.

[00317] Step-6. Synthesis of 4-(ammomethyI)-2- isopropylbenzimidamide dihydrochloride (18__Int-7)

[00318] A stirred solution of tert-butyl (4-carbamimidoyl-3- isopropylbenzyl)carbamate (18_Int-6) (0.33g, 11.34mmol, l.Oeq) in water (2.6mL, 8V) was prepared and concentrated HC1 (ImL, 3.3V) was added at RT and stirred for 16h. The RM was evaporated to give compound (18_Int-7). MS (ES): 192.3 m/z [M+l], LCMS purity: 54.1%.

[00319] Step-7. Synthesis of N-(4-carbamimidoyI-3-isopropylbenzyI)~ l-(4-(2-(methyIamino)-2-oxoethyI)benzyl)-lH-pyrazole-4-carboxamide (18)

[00320] Comparative Example 18 was prepared from l-(4-(2- (methylamino)-2~oxoethyl)benzyl)-lH-pyrazole-4~carboxylic acid (1 Int-4) and 4-(antinomethyl)-2-isopropylbenzimidamide di hydrochloride ( 18_Int-7) in a similar fashion to that described in Example 11 . MS (ES): 447.6 m/z [M+H] 4 LCMS purity: 99.5%, HPLC purity: 99.3%, ’H NMR (400 MHz, DMSO-cU) 8 1.227 (d, 1 6 8 Hz, 6H), 2.518 (s, 3H), 2.970-3.004 (m, 1 H), 3.336 (s, 2H), 4.429 (d, 1=6.0 Hz, 2H), 5.311 (s, 2H), 7.183-7.274 (m, 5H), 7.331 (d, 1=8.0 Hz, 1H), 7.449 (s, 1H), 7.923 (s, 1H), 7.949 (d, J 4.4 Hz, 1 H), 8.250 (s, H i}. 8.437 (s, 1H), 8.738 (t, J=6.0 Hz, 1H), 9.221 (br s, 1H), 10.080 (br s, 1H).

[00321] Comparative Example 19: Preparation of N-(4- carbamimidoyI-3-methyibenzyI)-1.-(4-(cyanomethyi)benzyI)-lH~pyrazole-4- carboxamide (19)

[00322] Step-1. Synthesis of N-(4~carbamimidoyI-3-methyIbenzyI)-l~

(4-(cyanomethyI)benzyI)“lH-pyrazoIe-4-carboxamide (19)

[00323] Comparative Example 19 was prepared from l-(4- (cyanomet.hyl)benzyl)-lH-pyrazole-4-carboxylic acid (7A Jnt-5) and 4- (aminomethyl)-2-methylbenzimidamide trifluoroacetate (19 Int-8) in a similar fashion to that described in (Example 3) (19) MS (ES): 387.5 m/z. [M+H]⁺, LCMS purity: 93.1%, HPLC purity: 99.1%, ^JH NMR (400 MHz, DMSO-de) 8 2.359 (s, 31 1). 4.037 (s, 2H), 4.419 (d, 1=5.2 Hz, 2H), 5.364 (s, 211), 7.252-7.363 (m, 6H), 7.411 (d, 1=7.2 Hz, 1H), 7.918 (s, 1H), 8.277 (s, 1H), 8.757 (t, 1=6.0 Hz, H I), 9.087 (s, 2H), 9.216 (s, 2H). [00324] Example 20: Preparation of N-(4-carbamimidoyl-2- methylbenzyl)~l~(4”(cyanomethyl) benzyI)-lH-pyrazole-4-carboxamide (20)

[00325] Step-1. Synthesis of N-(4-carbamimidoyI-2-methylbenzyI)-l-

(4-(cyanomethyI)benzyl)-lH-pyrazole-4-carboxamide (20)

[00326] Example 20 was prepared from ethyl l-(4-(cyanomethyl)benzyl)- lH-pyrazole-4-carboxylate (7A_Int-4) and 4-(aminomethyl)-3- methylbenzimidamide di hydrochloride (20 Int-7) in a similar fashion to that described in Example 2. MS (ES): 387.4 m/z [M+H]⁺, LCMS purity: 100%, HPLC purity: 100%, ’H NMR (400 MHz, DMSO-d₆) 5 2.396 (s, 3H), 4.032 (s, 2H), 4.446 (d, >6.0 Hz, 2H), 5.363 (s, 2H), 7.297-7.405 (m, 4H), 7.586-7.623 (m, 2H), 7.940 (s, 1H), 8.299 (s, 1H), 8.673 (t, >6.0 Hz, 1H), 8.940 (s, 2H), 9.226 (s, 2H).

[00327] Example 21: N-(4-carbamimidoyl-2-methyIbenzyl)-l-(4-

(cyanomethyl) benzyl)-5-(methoxymethyI)”lH-pyrazoIe~4-carboxamide (21) [00328] Step-l. Synthesis of N-(4-carbamimidoyL2-methylbenzyl)-l-

(4-(cyanomethyI)benzyI)-5-(methoxymethyl)-lH-pyrazoIe-4-carboxamide

(21)

[00329] Example 21 was prepared from methyl l-(4- (cyanomethyl)benzyl)-5-(niethoxymethyl)-lH-pyrazole-4-carboxylate (21 Int-3) and 4-(aminomethyl)-3-methylbenzimidamide dihydrochloride (20_Int-7) in a similar fashion to that described in Example 2. MS (ES): 431.4 m/z [M+H]⁺, I .CMS purity: 100%, HPLC purity: 100%, (400 MHz, DMSO-d₆) 6 2.409 (s, 3H), 3.221 (s, 3H), 4.023 (s, 2H), 4.464 (d, J=6.0 Hz, 2H), 4.820 (s, 2H), 5.369 (s, 2H), 7.231 (d, J 8.0 Hz, 2H), 7.321 (d, 1 8 0 Hz, 2H), 7.420 (d, J=8.0 Hz, 1H), 7.595-7.633 (m, 2H), 8.063 (s, 1H), 8.738 (t, J=6.0 Hz, 1H), 8.954 (s, 2H), 9.233 (s, 2H).

[00330] Example 22: Preparation of N-(4-carbaminiidoyl-2- methyIbenzyI)-l-(4-(2-cyanopropan -2-yI)benzyl)-5-(methoxymethyl)-lH- pyrazok-4-carboxamide (22)

[00331] Step-1. Synthesis of N-(4-carbamimidoyI”2-methylbenzyI)-l-

(4-(2-cyanopropaii"2-yI)benzyl)-5-(methoxymethyI)-lH-pyrazole-4- carboxamide (22)

[00332] Example 22 was prepared from methyl l-(4-(2-cyanopropan-2- yl)benzyl)-5-(methoxymethyl)-1H-pyrazole-4-carboxylate (22_Int-3) and 4- (aminomethyl)-3-methylbenzimidamide dihydrochloride (20 Int-7) in a similar fashion to that described in Example 2. MS (ES): 459.4 m/z [M+H]⁺, LCMS purity: 100%, HPLC purity: 100%, H M1R (400 MHz, DMSO-de) 5 1.666 (s, 6H), 2.407 (s, 3H), 3.233 (s, 3H), 4.463 (d, J 4.8 Hz, 2H), 4.830 (s, 2H), 5.372 (s, 2H), 7.272 (d, J=8.0 Hz, 2H), 4.416 (d, J=8.0 Hz, 1 H), '4.488 (d, J=8.0 Hz, 2H), 7.592-7.633 (m, 2H), 8.060 (s, IH), 8.735 (t, J 6.0 Hz, IH), 8.975 (s, 2H),

9.232 (s, 2H).

[00333] Exampie 23: Preparation of N-(4-carbamimidoyi-2,6- diethyIbenzyI)-l-(4-(2-(methylamino)-2-oxoethyl)benzyl)-lH-pyrazoIe-4- carboxamide (23)

[00334] Step-1 . Synthesis of 4-bromo-2,6-diethylbenzaldehyde (23_Int-2) [00335] A stirred solution of 5-bromo-l,3-diethyS-2-iodobenzene (23_Int- I) (5.0g, 14.83mmol, l .Oeq) in diethylether (70mL, 14V) was prepared and n- Buli (2.5M in hexane) (2.37mL, 37.09mmol, 2.5eq) was added dropwise at - 78°C under nitrogen atmosphere. The RM was stirred at -78°C for Ih followed by dropwise addition of DMF (4.87g, 66.76mmol, 4.5eq). The RM was stirred for an additional 3h at brought to RT. The RM was slowly quenched in cold water and extracted using ethyl acetate. The combined organic fractions were dried over and evaporated to give compound (23_Int-2). IH NMR (400 MHz, DMSO-d6) 5 1.132-1.240 (m, 6H), 2.880-2.967 (m, 4H), 7.443 (s, 2H), 10.462 (s, IH).

[00336] Step-2, Synthesis of 3, 5-diethyl-4-formylbenzonitrile (23 Int-3)

[00337] A stirred solution of 4-bromo-2,6-diethylbenzaldehyde (23_Int~2) (2.0g, 0.83mmol, l.Oeq) in DMF (20mL, 10V) was prepared and (3,5g, 0.83mmol, l.Oeq) and ) were added at RT. Argon gas was purged into the RM for 15 min followed by the addition of palladium acetate (0.186g, 0.08mmol, O. leq). The RM was heated to 130°C and stirred for 4h. The RM: was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over NaiSCU evaporated, and purified by column chromatography (3-4% ethyl acetate in hexanes) to give compound (23__Int-3). 1H NMR (400 MHz, DMSO-d6) 5 1.138-1.232 (m, 6H), 2.865-3.020 (m, 4H)„ 7.578 (s, IH), 7.697 (s, IH), 10.544 (s, IH), CMR (400 MHz, DMS0-d6) 3 16.502, 25.701, 1 15. 126, 118.673, 127.400, 131.813, 136.428, 147.195, 195.315.

[00338] Step-3. Synthesis of (E)-3,5-diethyl-N-hydroxy-4-

((hydroxyimi no)methyl)benzimi damide (23_Int-4)

[00339] A stirred solution of 3,5-diethyl-4-formylbenzonitrile (23_Int-3) (0.434g, 2.32mmol, 1 .Oeq) in Ethanol .'water (4: 1) (5mL, 1 IV) was prepared and sodium carbonate (2.5g, 23.20mmol, 10. Oeq) and hydroxylamine hydrochloride (1.62g, 23.20mmol, 10. Oeq) were added at RT. The RM was stirred at 90°C for 16h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over Na2SO4, evaporated, and purified by reverse phase column chromatography to give compound (23_Int-4). MS (ES): 236.3ni/z [M+l]+, LCMS purity: 89.8%.

[00340] Step-4. Synthesis of 4-(aminomethyl)-3,5-diethylbenzimidamide (23_Int-5)

[00341] An autoclave was charged with a solution of (E)-3,5-diethyl-N- hydroxy-4-((hydroxyimino)methyl)benzimidamide (23 Int-4) (0.166g, 0.706mmol, l .Oeq) in methanol (4.15mL, 25V ). Ammonium formate (0.222g, 3.532mmol, 5. Oeq) and 10% palladium on activated carbon, (50% wet with water) (0.108g, 0.65w/w) were then added at RT and stirred at 60°C for 6h under 20kg H2 pressure. After completion of reaction, the RM was cooled to RT, filtered, and evaporated to give compound (23_Int-5). MS (ES): 206.0m/z purity: 54.2%.

[00342] Step-5. Synthesis of N-(4-carbamimidoyl-2,6-diethylbenzyl)-l-

(4”(2-(methylamino)-2-oxoethyl)benzyl)-lH”pyrazole-4-carboxamide (23)

[00343] A stirred solution of l-(4-(2-(methylamino)-2-oxoethyl)benzyl)- lH-pyrazole-4-carboxylic acid (l int-4) (0.15g, 0.54mmol, l.Oeq) in DCM (3.0mL, 20V) was prepared and 1 -propanephosphonic acid anhydride (T3P) 50% solution in ethyl acetate (0.44g, 1.37mmol, 2.5eq), trimethylamine (0.28g, 2.74mmol, 5.0eq) and 4-(aminomethyl)-3,5-diethylbenzimidamide (23__Int-5) (0. 11g, 0.54mmol, 1 ,0eq) were added at RT and stirred for 16h. The RM was evaporated and purified by PREP HPLC ((A) 0.1% TEA in water (B) 100% ACN). The product fractions were evaporated and further purified by PREP HPLC ((A) 10 mM ammonium acetate + 0, 1% formic acid in water (B) 100% MeCN). The pure fraction were lyophilized to give compound (23) AIS (ES): 461.3m/z [M+H]+, LCMS purity: 97.1%, HPLC purity: 98.6%, 1H NMR (4OO MHz, DMSO-d6) 6 1.182 (q, 1=7.6 Hz, 6H), 2.542 (d, J 4.4 Hz, 3H), 2.769 (q, 1 7 6 Hz, 4H), 3.349 (s, 2H), 4.475 (s, 2H), 5.265 (s, 2H), 7. 154-7. 199 (ra, 4H), 7.510 (d, 1=2.8 Hz, 2H), 7.860 (d, J 2.4 Hz, 2H), 8.187 (s, 1H), 8.229 (d. 1=2.8 Hz, 1H), 8.970 (s, 2H), 9.242 (s, 2H). [00344] Example 24: Preparation of N-(4-carbamimidoyl-2,6- diisopropylbenzyl)-l-(4-(2-(methyIamino)-2-oxoethyI)benzyI)-lH~pyrazoIe~ 4-carboxamide (24)

[00345] Step-1. Synthesis of 5-bromo-2-iodo-l,3-diisopropylbenzene

(24_Int-2)

[00346] A stirred solution of 4-bromo-2,6-diisopropylaniline (24___Int-l) (0.1g, 0.35mmol, 1 .Oeq) in acetic acid (ImL, 10V) was prepared and sulfuric acid (0.3mL, 3 V) was added dropwise at 0°C. The RM was allowed to come to RT and stirred for 30 min. The RM was again cooled to 0°C followed by drop wise addition of isoamyl nitrite (0.09g, 0.78mmol, 2. Oeq) and stirred for 20 min. A solution of potassium iodide (0.08g, 5.50mmol, 1.3eq) in water (2mL, 20V) was added to the RM at 0°C and stirred at RT for 12h. The RM was slowly quenched in sodium bisulfite solution and extracted by hexanes. The combined organic fractions were washed with a sodium bicarbonate solution, dried over Na2SO4, and evaporated to give compound (24_Int-2). 1H NMR (400 MHz, CDC1.0 8 1.219 (d, J 0.4 Hz, 121 1), 3.344-3.395 (m, 2H), 7.195 (s, 2H).

[00347] Step-2. Synthesis of 4-bromo-2,6-diisopropylbenzaldehyde (24 Int-3)

[00348] A stirred solution of 5-bromo-2-iodo-l ,3-diisopropylbenzene (24_Int-2) (1.0g, 2.72mmol, l.Oeq) in toluene ( lOmL, 10V) was prepared and n- Buli (2.5M in hexane) (2.7mL, 6.8mmol, 2.5eq) was added at -78°C under nitrogen atmosphere and stirred for 30min. DMF (3.0mL, 3V) was added dropwise to the RM at -78°C and the RM was brought to RT and stirred for 3h. The RM was slowly quenched in cold water and extracted using ethyl acetate. The combined organic fractions were dried over Na?SO4 and evaporated to give compound (24_Int-3). 1H NMR (400 MHz, CDCh) 6 1.272 (d, J=6.4 Hz, 12H), 3.491-3.559 (m, 2H), 7.403 (s, 2H), 10.643 (s, IH).

[00349] Step-3. Synthesis of 4-formyl-3,5-diisopropylbenzonitrile

(24 Int-4)

[00350] A stirred solution of 4-bromo-2,6-diisopropylbenzaldehyde

(24_Int-3) (0.5g, I .85mmol, l.Oeq) in DMF (5mL, 10V) was prepared. lUFeCNe (1.17g, 2.78mmol, 1.5eq) and were added at RT. Argon was purged into the RM for 15 min followed by the addition of palladium acetate (0.041g, 0.18mmol, O. leq). The RM was heated to 130°C and stirred for 12h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over Na2SOa, evaporated, and purified by column chromatography (3-4% ethyl acetate in hexanes) to give compound (24_Int-4) IH NMR (400 MHz, CD('h) 5 1 .265 (d, 1 6.4 Hz, 12H), 3.365-3.433 (m, 2H), 7.540 (s, 2H), 10.696 (s, IH).

[00351] Step-4. Synthesis of (E)-N-hydroxy-4-((hydroxyimino)methyl)- 3,5-diisopropylbenzimidamide (24_Int-5)

[00352] A stirred solution of 3,5-diethyl-4-formylbenzonitrile (24__Int-4) (0.05g, 0.23mmol, l .Oeq) in ethanol: water (4: 1) (5rnL, 1 IV) was prepared.

Sodium carbonate (0.25g, 2.32mmol, lO.Oeq) and hydroxylamine hydrochloride (0.16g, 2.32mmol, lO.Oeq) were added at RT. The RM was stirred at 90°C for 16h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over and evaporated to give compound (24__Int-5). MS (ES): 264.2m/z [M+l]+, LCMS purity: 84.2%.

[00353] Step-5. Synthesis of 4-(aminomethyl)-3,5- diisopropylbenzimidamide (24 Int-6)

[00354] An autoclave was charged with a solution of (E)-N-hydroxy-4- ((hydroxyimino)methyl)-3,5-diisopropylbenzimidamide (24___ In t-5) (0.5g, I .90mmol, l.Oeq) in methanol (5mL, 10V). ammonium formate (0.6g, 9.50mmol, 5.0eq) and 10% palladium on activated carbon, (50% wet with water) (0.32g, 0.65w/w) were added at RT. The RM was stirred at 60°C for 12h under 20kg H2 pressure. The RM was cooled to RT, filtered, and the filtrate evaporated to give compound (24_Int-6). MS (ES): 234.2m/z [M+l]+, LCMS purity: 70.4%.

[00355] Step-6. Synthesis ofN-(4-carbamimidoyl-2,6- diisopropylbenzyl)-1-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4- carb oxami de (24)

[00356] A stirred solution of l-(4-(2-(methylamino)-2-oxoethyl)benzyl)- lH-pyrazole-4-carboxylic acid (l__Im-4) (0.1g, 0.36mmol, l.Oeq) in DCM (2.0mL, 20V) was prepared and 1-propanephosphonic acid anhydride (T3P) 50% solution in ethylacetate (0.58mL, 0.9mmol, 2.5eq), trimethylamine (0.38g, 2.9mmol, 8.0eq) and 4-(aminomethyl)-3,5-diisopropylbenzimidamide (24_Int-6) (0.10g, 0.44mmol, l.Oeq) were added at RT and stirred for 16h. The RM was evaporated and purified by PREP HPLC ((A) 0.05% TFA in water (B) 0.05% TFA in ACN) product fractions were lyophilized to give compound (24) MS (ES): 489.3m/z [ \1 + H ]■ + , LCMS purity: 98.5%, HPLC purity: 99.2%, 1 H NMR (400 MHz, DMSO-d6) 5 1.218 (d, 1=6.4 Hz, 12H), 2.550 (d, J 4.8 Hz, 3H), 3.296-3.357 (m, 4H), 4.519 (d, 1 4.4 Hz, 2H), 5.283 (s, 2H), 7.160-7.228 (m, 4H), 7.615 (s, 2H), 7.878 (s, 1H), 7934 (d, J=4.4 Hz, 1H), 8.202 (t, J=4.4Hz, 1H), 8.246 (s, 1H), 8.994 (s, 2H), 9.292 (s, 2H). [00357] Example 25: Preparation of of N-(4-carbamimidoyI-2,3- dimethylbenzyl)-l”(4-(2~(methyIamino)~2-oxoethyI)benzyI)-lH-pyrazole-4- earboxamide (25)

[00358] Step-1. Synthesis of (4-bromo-2,3-dimethylphenyl)methanol (25 Int-2)

[00359] A stirred solution of 4-bromo-2,3-dimethylbenzoic acid (25_Int- 1) (5.0g, 21.82mmol, l.Oeq) in tetrahydrofuran (50mL, 10V) was prepared and lithium aluminium hydride (1.0M in tetrahydrofuran) (33.0mL, 32,74rnmol, 1.5eq) was added dropwise at 0°C under nitrogen atmosphere. The RM was brought to RT and stirred for 2h. The RM was then slowly quenched in cold water and extracted using ethyl acetate. The combined organic fractions were dried over Nio.SOs, evaporated, and purified by column chromatography (12- 15% ethyl acetate in hexanes) to give compound (25_Int-2). 1H NMR (400 MHz, DMSO-d6) 5 20203 (s, 3H), 2.288 (s, 3H), 4.453 (d, J 5.2 Hz, 2H), 5.144 (t, J 5.2 Hz, 1 H), 7.135 (d, J 8.0 Hz, 1H), 7.398 (d, 1 8.0 Hz, 1H).

[00360] Step-2. Synthesis of 4-bromo-2,3-dimethylbenzaldehyde (25_Int- 3)

[00361] A stirred solution of (4-bromo-2,3-dimethylphenyl )methanol (25__Int-2) (1.7g, 7.90mmol, l.Oeq) in dichl or om ethane (5 I ml ., 30V) was prepared and Dess-Martin periodinane (5.0g, 11.85mmol, 1.5eq) was added at 0°C. The RM was brought to RT and stirred for 3h. The RM was quenched in a saturated sodium bicarbonate solution and extracted using ethyl acetate. The combined organic fractions were dried over Na2SC>4, evaporated, and purified by column chromatography (5-7% ethyl acetate in hexanes) to give compound (25_Int-3). 1H NMR (400 MHz, DMSO-d6) 5 2.414 (s, 3H), 2.621 (s, 3H), 7.555 (d, J=8.0 Hz, 1H), 7.675 (d, J=8.0 Hz, 1H), 10.280 (s, 1H).

[00362] Step-3. Synthesis of 4-formyl-2,3-dimethylbenzonitrile (25 Int-4)

[00363] A stirred solution of 4-bromo-2,3-dimethylbenzaldehyde (25__Int- 3) (1.0g, 4.69mmol, l .Oeq) in dimethylformamide (lOmL, I0V) was prepared and sodium carbonate (0.74g, 7.04mmol, 1.5eq), K^FeCNe (1.98g, 4.69mmol, l .Oeq) and tetramethylethylenediamine (0.54g, 4.69mmol, l.Oeq) were added at RT. The RM was purged with argon for 15 min followed by the addition of Pd(OAc)2 (0.11g, 0.46mmol, 0.1 eq). The RM was heated to 130°C and stirred for 4h. The RM was quenched in water and extracted using ethyl acetate. The organic fraction was dried over NazSCk evaporated, and purified by column chromatography (5-7% ethyl acetate in hexanes) to give compound (25_Int-4).

1 H NMR (400 MHz, DMSO-d6) 5 2.410 (s, 3H), 2.621 (s, 3H), 7.761 (d, J 8.0 Hz, 1H), 7.827 (d, J=8.0 Hz, 1H), 10.380 (s, 1H).

[00364] Step-4. Synthesis of (E)-N-hydroxy~4-((hydroxyimino)methyl)- 2,3-dimethylbenzimidamide (25 Int-5)

[00365] A stirred solution of hydroxylamine hydrochloride (4.36g, 62.81nimol, lO.Oeq) in water (lOmL, 10V) was prepared and sodium carbonate (6.78g, 62.81mmol, lO.Oeq) was added at RT and stirred for 10 min. A solution of 4-formyl-2,3-dimethylbenzonitrile (25_Int-4) (1.0g, 6.25mmol, l.Oeq) in ethanol (40mL, 40V) was then added at RT, heated to 90°C, and stirred for 16h. The RM was quenched in water and extracted using ethyl acetate. The combined organic fractions were dried over NazSCfo evaporated, and purified by reverse phase column chromatography ((A) 0.1% formic acid in water (B) 100% ACN) to give compound (25_Int-5). MS (ES): 208.26m/z [M+1 J+, LCMS purity: 86.8%.

[00366] Step-5. Synthesis of 4-(aminomethyl)-2,3- dimethylbenzimidamide (25 Int-6)

[00367] A solution of (E)-N-hydroxy-4-((hydroxyimino)methyl)-2,3- dimethylbenzimidamide (25___Int-5) (0.1g, 0.48mmol, l.Oeq) in acetic acid (2.0mL, 20V) was prepared and zinc metal powder (0.12g, 1.93mmol, 4.0eq) was added at RT. The RM was heated to 90°C and stirred for 16h. The RM was evaporated to give compound (25_Int-5). MS (ES): 178.2m/z [M+l]+, LCMS purity: 18.8%.

[00368] Step-6. Preparation of N-(4-carbamimidoyl-2,3-dimethylbenzyl)- l-(4-(2-(methylamino)-2-oxoethyl)benzyl)-1H-pyrazole-4-carboxamide (25)

25Jnt-6

[00369 ] Example 25 was prepared from l-(4-(2-(methylamino)-2- oxoethyl)benzyl)-lH-pyrazole-4-carboxylic acid (l_Int-4) and 4- (aminomethyl)-2,3-dimethylbenzimidamide (25_Int-6) in a similar fashion to that, described in (Example 24) (25). MS (ES): 433.4 m/z [M+H]+, LCMS purity: 99.6%, HPLC purity: 99.1%, 1HNMR (400 MHz, DMSO-d6) 5 2.282 (s, 3H), 2.554 (s, 311). 3.329 (s, 2H), 4.446 (d. J-5.6 Hz, 2H), 5.311 (s, 2H), 7.196-7.247 (m, 5H), 7.909 (s, 1H), 7.946 (d, .1 4.4 Hz, 1H), 8.258 (s, 1H), 8.620 (t, J=6.0 Hz, 1H), 9.051 (s, 2H), 9.214 (s, 2H).

[00370] Example 26: Preparation of N-(4-carbamimidoyl-2,6- diethyIbenzyI)"l-(4-(2-cyanopropan-2”yI)benzyI)-lH-pyrazoIe-4~ carboxamide (26) SJnt-6 [00371] Example 26 was prepared from l-(4-(2-cyanopropan-2- yl)benzyl)-lH-pyrazole-4-carboxylic acid (5_Int-6) and 4-(aminomethyl)-3,5- diethyibenzimidamide (23_Int~6) in a similar fashion to that described in (Example 24) (26). MS (ES): 457.3m/z [M+H]+, LCMS purity: 97.5%, HPLC purity: 95.1%, 1H NMR (400 MHz, DMSO-d6) 5 1.203 (t, >7.6 Hz, 6H), 1.661 (s, 6H), 2.785 (q, J=7.6 Hz, 4H), 4.498 (d, J=4.8 Hz, 2H), 5.339 (s, 2H), 7.304 (d, >8.0 Hz. 2H), 7.499 (d, >8.0 Hz, 2H), 7.526 (s, 2H), 7.894 (s, 1 H), 8.215 (t, >6.0 Hz, H i ). 8.279 (s, H i). 8.969 (s, 2H), 9.254 (s, 2H).

[00372] Example 27: Preparation of N-(4-carbamimidoyI-2,6- diisopropyIbenzyl)-l-(4-(2~cyanopropan~2~yl)benzyi)-lH-pyrazok-4- carboxamide (27)

[00373] Example 27 was prepared from l-(4-(2-cyanopropan-2- yl)benzyl)-lH-pyrazole-4-carboxylic acid (5_Int-6) and 4-(aminomethyl)-3,5- diisopropylbenzimidamide (24_Int-6) in a similar fashion to that, described in (Example 24) (27). MS (ES): 485.2m/z [M+H]+, LCMS purity: 98.9%, HPLC purity: 99.3%, 1H NMR (400 MHz, DMSO-d6) 3 1.228 (d, >6.8 Hz, 12H), 1.661 (s, 6H), 3.315-3.349 (m, 2H), 4.521 (d, >4.4 Hz, 2H), 5.340 (s, 2H), 7.301 (d, >8.0 Hz, 2H), 7.499 (d, >8.0 Hz, 2H), 7.619 (s, 2H), 7.897 (s, 1H), 8.210 (t, >6.0 Hz, 1H), 8.282 (s, 1H), 8.986 (s, 2H), 9.292 (s, 2H). [00374] Example 28: Preparation of N-(4-carbamimidoyl-2,3- dimethylbenzyl)~l-(4-(2-cyanopropaii-2~yl)benzyI)-lH-pyrazoIe-4- earboxamide (28)

[00375] Example 28 was prepared from l-(4-(2-cyanopropan-2- yl)benzyl)-lH-pyrazole-4-carboxylic acid (5_Int-6) and 4-(aminomethyl)-2,3- dimethylbenzimidamide (25_Int-6) in a similar fashion to that described in (Example 24) (28). MS (ES): 429.4m/z [M+H]+, LCMS purity: 82.6%, HPLC purity: 97.2%, 1H NMR (400 MHz, DMSO-d6) 5 1.622 (s, 6H), 2.235 (s, 3H), 2.267 (s, 3H), 4.446 (d, J=4.4 Hz, 2H), 5.368 (s, 2H), 7.235 (d, 1=8.0 Hz, 2H), 7337 (d, J 8.0 Hz, 2H), 7.517 (d, J 8.0 Hz, 2H), 7.932 (s, 1H), 8.374 (s, 1H), 8.641 (t, J=6.0 Hz, 1H), 9.093 (s, 2H), 9.213 (s, 2H).

[00376] Biological Examples

[00377] Plasma kadikrein protease inhibition assay. Two methods for determining the ICso of a test compound against Plasma Kallikrein are provided.

[00378] In the first method was used a reaction buffer composed 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 Cat# 2497-SE;

Recombinant Human Plasma Kallikrein, expressed in Mouse myeloma cell line, NSO-derived Gly20-Ala638, with a C -terminal 60-His tag. MW=70 kDa).

[00379] The enzyme was activated by dilution to 200 pg/mL in activation buffer (100 mM Tris, 10 mM CaC12, 150 mM NaCl, pH 7.5 (TCN), and then combined with an equal volume of 20 ug/mL thermolysin to form a reaction buffer. Each test compound was then dissolved in DMSO and delivered into the reaction buffer. The reaction was initiated by delivering a substrate solution containing 10 pM Z-FR-AMC (Enzo Cat# P-139, AMC: 7-Amino-4- methyl coumarin) into the reaction well after a 20-minute pre-incubation period.

[00380] Measurement was conducted with EnVision (PE) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. The reaction was stopped with EDTA. The enzyme activities were monitored even,- 5 minutes as a time-course measurement of the increase in signal from fluorescently labeled peptide substrate for 120 minutes at room temperature.

[00381] Data was analyzed by taking slope* (signal/time) of the linear portion of measurements. Slope was calculated using Excel, and curve fits were performed using Prism software.

[00382] Second, 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 delivered to the reaction mixture along with Z-FR-AMC substrate and pooled human plasma. The multiwell reaction plate was incubated for five minutes at room temperature. To initiate the reaction, the 10% Actin FS solution was added to each well, and kinetic measurements were taken at Ex, 'Em at 355/460nm. The fluorescence signal was recorded even,- 30 seconds for a total of 10 minutes.

[00383] Permeability Assay. A permeability study was conducted using Caco-2 cells (ECACC Cat. no. 09042001) seeded on cell inserts plate (Millicell, Cat#PSHT010R5) at a density of 80,000 cells per well, and the cells were maintained for 18-21 days in culture medium (lx DMEM with 10% FBS, 0. Img/mL Penicillin/Streptomycin). The culture medium was changed every alternate day. Prior to the experiments, the integrity of the cellular monolayer was evaluated by measuring the TEER value using a volt ohm meter and STX100C96 electrode. Only monolayers with TEER values greater than 800 ohm. cm² in buffer after the first wash were used.

[00384] An assay buffer (HBSS with Ca+2 and Mgt-2 buffered with 10 mM HEPES and 25 niM D- Glucose, pH -7.4) was used on apical side as well as on basolateral side. [00385] An intermediate stock solution of a test compound was prepared in DMSO at a concentration of 1 mM in DMSO. This stock solution was spiked in the assay buffer to achieve a target test compound concentration of 10 pM, The organic content of final drug preparation was 1.0% v/v. The bidirectional permeability experiment was done in singlet and the sample analysis was done in duplicate.

[00386] The cultured cell monolayer was washed twice with assay buffer (0.4 mL and 0.8 mL was added to the apical and basolateral sides, respectively, of the culture plate), and then buffers from both compartments were discarded.

[00387] For the apical to basal (AP>BL) experiment, aliquots of 0.4 mL donor solution (assay buffer, pH 7.4 containing test compound) and 0.8 mL of receiver solutions (assay buffer, pH 7.4, only) were added to the apical and basolateral compartments, respectively. For the basal to apical (BL>AP) experiment, aliquots of 0.8 mL donor solution (assay buffer pH - 7.4 containing test compound) and 0.4 mL of receiver solutions (assay buffer, pH -7.4) were added to the basolateral and apical compartments, respectively. The plate was then kept in an incubator at 37°C for 120 minutes.

[00388] Control experiments with Propranolol (High Permeability), Atenolol (Low⁷ Permeability), Digoxin (High Efflux - Pgp substrate), and Digoxin+ Verapamil (Pgp Inhibitor) in both directions (AP>BL and BL>AP) were performed in separate webs on the same experiment day.

[00389] After the completion of the transport experiment, the integrity of the cell monolayer was evaluated by measuring the Lucifer Yellow (LY) rejection. To do this, 400 uL of 10 pM LY was added to each well of the filter plate and incubated for 1 hour at 37 °C. Subsequently, the samples were collected from the basolateral compartments, and the 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 fluorescence in the receiver plate (basolateral compartment) compared to theoretical equilibrium standard.

[00390] The study samples (collected from apical and basolateral compartments after 120 min incubation) were analyzed by LCMS/MS, following which the Papp of the compound(s) was calculated in both apical to basal and basal to apical directions. Papp = ([DBL] x VBL)/(A x t * [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.

[00391] Results from the assays described above for representative compounds of the present disclosure are presented in Table 1 below. Scores for selected compounds in each assay are presented as follows:

[00392] Table 1. Plasma KaHikrem Inhibition and Cell Permeation of

Representative Compounds

[00393] The examples and embodiments described herein are for illustrative purposes only. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WE CLAIM:

I . A compound of formula (I) or a pharmaceutically acceptable salt thereof: wherein y ;

® is a bivalent monocyclic or bicyclic moiety selected from the group consisting of heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and fused combinations thereof; and (Ci-Cg-alkyl)5- to 10-membered heteroaryl (wherein 1 -4 heteroaryl members are independently selected from N, O, and S), wherein each cycloalkyl, cycloalkenyl, aryl, and heteroaryl is independently monocyclic or bicyclic, wherein L² is optionally substituted by 1 to 3 substituents selected from the group consisting of C₁-C₆-alkyl, C₁-C₆-haloalkyl, and CN;

Z is selected from the group consisting of -OR^C, -OC(O)R^C, -OC(O)NR^cR^a, - N=C=O, -N=C=S, and -NR^c-NR^cR^d; and each instance of R^a, R^c, and R^d is independently selected from H, Ci-Cg- alkyl, Cj-Ce-haloalkyl, and Ce-Cw-aryl.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein only one of D¹ and D² is N.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the ring containing D¹ and D² is:

4. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 3, wherein the ring containing

5. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 4, wherein each of R^l and R² is independently C₁-C₆-alkyl.

6. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 4, wherein one of R¹ and R² is C₁-C₆-alkyl and the other is H.

7. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 5, wherein the ring containing D¹ and D² is selected from the group consisting of:

8. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 7, wherein each of R^al, R^az, and R^a3 is H

9. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 8, wherein

Q¹ is CR³; each of Q² and Q³ is independently selected from the group consisting of CR⁵, N, O and S, and P is selected from the group consisting of C and N.

10. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 9, wherein P is N.

11 . The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 10, wherein Q² is CR⁵ and 0 ’ is N.

12. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 10, wherein Q² is N and Q^J is CR⁵.

13. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 12, wherein I? is -Ci-Cg-alkylene-.

14. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 13, wherein I? is -Ci-Cs-alkylene-.

15. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 14, wherein L¹ is methylene.

16. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 15, wherein ® is bivalent monocyclic Ce-Cio-aryl.

17. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 16, wherein

18. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 17, wherein L² is selected from -Ci-C₈-alkylene, Cs-Cio-aryl

19. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 18, wherein

20. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 19 wherein L.² is Ci-Cs-alkylene.

21. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 18, wherein

22. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 21, wherein L² is phenyl.

23. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 22, wherein Z is selected from the group consisting of -OR^C, CN, -C(O)OR^C, and -C(O)NR^cR^d

24. The compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 23, wherein Z is CN.

25. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein: the ring containing

L² is Ci-Cs-alkylene; and

Z is selected from the group consisting of -OR^C, CN, -C(O)OR^C, and - C(O)NR^cR^d.

26. The compound or pharmaceutically acceptable salt, thereof according to claim 1, wherein the compound is selected from the following table:

27. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 26 and a pharmaceutically acceptable carrier.

28. A method for treating a subject suffering from a disease or condition, comprising administering to the subject a compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 26, wherein the disease or condition is selected from the group consisting of ischemic stroke, hemorrhagic stroke, hypertension, retinopathy, diabetic retinopathy, nephropathy, cerebral edema, pulmonary hypertension, inflammation, acute myocardial infarction, deep vein thrombosis, complications from fibrinolytic treatment, stroke, angina, angi oedema, sepsis, arthritis, complications of cardiopulmonary bypass, capillary leak syndrome, inflammatory bowel disease, vascular complications from diabetes, diabetic macular edema, macular degeneration, neuropathy, age related macular degeneration, retinal vein occlusions, brain edema, ischemia reperfusion injury, angiogenesis, asthma, anaphylaxis, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, glioblastoma multiforme, complications of fibrinolysis treatment, increased albumin excretion, macroalbuminuria, pain, amyotrophic lateral sclerosis, Creutzfeld-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, enterocolitis, arteritis, glomerulonephritis, psoriasis, endometriosis, preeclampsia, malaria, arthritis, periodic and recurrent fever, Chagas disease, Reynaud’s disease, systemic sclerosis, granulomatosis with polyangiitis, small vessel vasculitis, medium vessel vasculitis, large vessel vasculitis, pan-vasculitis, systemic autoinflammatory diseases, renal insufficiency, cerebral malaria, Clarkson’s disease (systemic vascular leakage syndrome), Hantavirus infection, Hantavirus renal syndrome, Hantavirus pulmonary syndrome, viral associated inflammatory disorders, retinal vasculitis, uveitis, Eales' disease, Behcet's disease, sarcoidosis, whooping cough, acute cough, chronic cough, coronavirus infection, and non-infectious posterior uveitis.

29. The method according to claim 28, wherein the disease or condition is selected from the group consisting of diabetic macular edema, diabetic retinopathy, and uveitis.