ROCK INHIBITORS AND USES THEREOF

Disclosed herein are compounds of Formula (I), and pharmaceutically acceptable salts thereof, that are useful as ROCK inhibitors. Also disclosed are pharmaceutical compositions comprising a compound of Formula (I), and methods of using such compounds or compositions to treat ROCK-related disorder (e.g., glaucoma).

FIELD OF THE DISCLOSURE

The present disclosure generally relates to novel compounds inhibiting Rho-related protein kinase (“ROCK”), and pharmaceutically acceptable salts thereof. The present disclosure also relates to pharmaceutical compositions comprising the compound as an active ingredient and use of the compounds in the treatment of ROCK-related disorder, including glaucoma.

BACKGROUND

Rho-related protein kinase (ROCK) is a member of the serine-threonine protein kinase family. ROCK exists in two isoforms, ROCK1 and ROCK2. Both isoforms are activated by GTP-bound forms of Rho GTPase. ROCK plays important roles in numerous cellular processes including smooth muscle cell contraction, cell proliferation, adhesion and migration. Inhibition of ROCK activity has demonstrated potential therapeutic applicability in a wide range of pathological conditions.

Currently available ROCK inhibitor drugs include Eril (for the treatment of cerebral vasospasm) from Asahi Kasei, Glanatec (for the treatment of ocular hypertension and glaucoma) from Kowa, and Rhopressa (for the reduction of elevated intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension) from Aerie.

Therefore, novel compounds that inhibiting ROCK are needed as pharmacological tools and are of considerable interest as drugs for treating ROCK related disorders such as glaucoma.

SUMMARY OF THE DISCLOSURE

Disclosed herein are novel compounds that are capable of inhibiting ROCK. As a result, the compounds of the present disclosure are useful in the treatment of ROCK-related diseases such as glaucoma.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the compound of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a further aspect, the present disclosure provides a method for inhibiting ROCK activity in a subject in need thereof, comprising administering an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present disclosure to the subject.

In a further aspect, the present disclosure provides a method for treating a ROCK related disorder comprising administering an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present disclosure to a subject in need thereof.

In another aspect, the present disclosure provides use of the compound of the present disclosure or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating ROCK-related disorders.

In another aspect, the present disclosure provides a compound of present disclosure or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present disclosure, for use in the treatment of ROCK-related disorder.

DETAILED DESCRIPTION OF THE DISCLOSURE

Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. While the present disclosure will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present disclosure as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. In the event that one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, the present disclosure controls. All references, patents, patent applications cited in the present disclosure are hereby incorporated by reference in their entireties.

It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural forms of the same unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of compounds.

Definition

At various places in the present disclosure, linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl”, then it is understood that the “alkyl” represents a linking alkylene group.

When any variable (e.g., Ri) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 Ri moieties, then the group may optionally be substituted with up to two Ri moieties and Ri at each occurrence is selected independently from the definition of Ri. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

As used herein, a dash “—” at the front or end of a chemical group is used, a matter of convenience, to indicate a point of attachment for a substituent. For example, —OH is attached through the oxygen atom; chemical groups may be depicted with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or named. As used herein, a solid line coming out of the center of a ring indicates that the point of attachment for a substituent on the ring can be at any ring atom. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. A range used herein, unless otherwise specified, includes the two limits of the range. For example, the expressions “n is an integer between 1 and 6” and “n being an integer of 1 to 6” both mean “n being 1, 2, 3, 4, 5, or 6”.

As used herein, the term “compounds provided herein”, or “compounds disclosed herein” or “compounds of the present disclosure” refers to the compounds of Formula (I) as well as the specific compounds disclosed herein.

As used herein, the term “Ci-j” indicates a range of the carbon atoms numbers, wherein i and j are integers and the range of the carbon atoms numbers includes the endpoints (i.e. i and j) and each integer point in between, and wherein j is greater than i. For examples, C1-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms. In some embodiments, the term “C1-12” indicates 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms.

As used herein, the term “alkyl”, whether as part of another term or used independently, refers to a saturated linear or branched-chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. The term “Ci-j alkyl” refers to an alkyl having i to j carbon atoms. In some embodiments, alkyl groups contain 1 to 10 carbon atoms. In some embodiments, alkyl groups contain 1 to 9 carbon atoms. In some embodiments, alkyl groups contain 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of “C1-10 alkyl” include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Examples of “C1-6 alkyl” are methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

As used herein, the term “alkenyl”, whether as part of another term or used independently, refers to linear or branched-chain hydrocarbon radical having at least one carbon-carbon double bond, which may be optionally substituted independently with one or more substituents described herein, and includes radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkenyl groups contain 2 carbon atoms. Examples of alkenyl group include, but are not limited to, ethylenyl (or vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2 buten-1-yl, 5-hexenyl, and the like.

As used herein, the term “alkynyl”, whether as part of another term or used independently, refers to a linear or branched hydrocarbon radical having at least one carbon-carbon triple bond, which may be optionally substituted independently with one or more substituents described herein. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkynyl groups contain 2 carbon atoms. Examples of alkynyl group include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

As used herein, the term “amino” refers to —NH2 group. Amino groups may also be substituted with one or more groups such as alkyl, aryl, carbonyl or other amino groups.

As used herein, the term “aryl”, whether as part of another term or used independently, refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 12 ring members. Examples of “aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic ring system, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline). The second ring can also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. Aryl groups can be substituted at one or more ring positions with substituents as described above.

As used herein, the term “cyano” refers to —CN.

As used herein, the term “cycloalkyl”, whether as part of another term or used independently, refer to a monovalent non-aromatic, saturated or partially unsaturated monocyclic and polycyclic ring system, in which all the ring atoms are carbon and which contains at least three ring forming carbon atoms. In some embodiments, the cycloalkyl may contain 3 to 12 ring forming carbon atoms, 3 to 10 ring forming carbon atoms, 3 to 9 ring forming carbon atoms, 3 to 8 ring forming carbon atoms, 3 to 7 ring forming carbon atoms, 3 to 6 ring forming carbon atoms, 3 to 5 ring forming carbon atoms, 4 to 12 ring forming carbon atoms, 4 to 10 ring forming carbon atoms, 4 to 9 ring forming carbon atoms, 4 to 8 ring forming carbon atoms, 4 to 7 ring forming carbon atoms, 4 to 6 ring forming carbon atoms, 4 to 5 ring forming carbon atoms. Cycloalkyl groups may be saturated or partially unsaturated. Cycloalkyl groups may be substituted. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group that contains at least one double bond or triple bond in its ring system. In some embodiments, the cycloalkyl group may be monocyclic or polycyclic. The fused, spiro and bridged ring systems are also included within the scope of this definition. Examples of monocyclic cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl. Examples of polycyclic cycloalkyl group include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiro-pentadienyl, spiro[3.6]-decanyl, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, and the like.

As used herein, the term “halogen” refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo) and iodine (or iodo).

As used herein, the term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen (including N-oxides).

As used herein, the term “heteroalkyl” refers to an alkyl, at least one of the carbon atoms of which is replaced with a heteroatom selected from N, O, or S. The heteroalkyl may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical), and may be optionally substituted independently with one or more substituents described herein. The term “heteroalkyl” encompasses alkoxy and heteroalkoxy radicals.

As used herein, the term “heteroalkenyl” refers to an alkenyl, at least one of the carbon atoms of which is replaced with a heteroatom selected from N, O, or S. The heteroalkenyl may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical), and may be optionally substituted independently with one or more substituents described herein.

As used herein, the term “heteroalkynyl” refers to an alkynyl, at least one of the carbon atoms of which is replaced with a heteroatom selected from N, O, or S. The heteroalkynyl may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical), and may be optionally substituted independently with one or more substituents described herein.

As used herein, the term “heteroaryl”, whether as part of another term or used independently, refers to an aryl group having, in addition to carbon atoms, one or more heteroatoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. The heteroaryl group also includes polycyclic groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of polycyclic heteroaryl include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

As used herein, the term “heterocyclyl” refers to a saturated or partially unsaturated carbocyclyl group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, and the like, the remaining ring atoms being carbon, wherein one or more ring atoms may be optionally substituted independently with one or more substituents. In some embodiments, the heterocyclyl is a saturated heterocyclyl. In some embodiments, the heterocyclyl is a partially unsaturated heterocyclyl having one or more double bonds in its ring system. In some embodiments, the heterocyclyl may contains any oxidized form of carbon, nitrogen or sulfur, and any quaternized form of a basic nitrogen. “Heterocyclyl” also includes radicals wherein the heterocyclyl radicals are fused with a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. The heterocyclyl radical may be carbon linked or nitrogen linked where such is possible. In some embodiments, the heterocycle is carbon linked. In some embodiments, the heterocycle is nitrogen linked. For example, a group derived from pyrrole may be pyrrol-1-yl (nitrogen linked) or pyrrol-3-yl (carbon linked). Further, a group derived from imidazole may be imidazol-1-yl (nitrogen linked) or imidazol-3-yl (carbon linked).

As used herein, the term “hydroxyl” or “hydroxy” refers to —OH.

As used herein, the term “partially unsaturated” refers to a radical that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

As used herein, the term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the said event or circumstance occurs and instances in which it does not. As used herein, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents may include, but not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halo, haloalkyl, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thione, or combinations thereof. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

As used herein, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

The symbols “R” and “S” represent the configuration of substituents around a chiral carbon atom(s). The isomeric descriptors “R” and “S” are used as described herein for indicating atom configuration(s) relative to a core molecule and are intended to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry, 68:2193-2222 (1996)).

Compound

In one aspect, the present disclosure provides compounds of Formula (I):

In some embodiments, each of R1 and R2 is independently hydrogen, halogen, hydrogen, cyano, amino, or alkyl optionally substituted with one or more halogens.

In some embodiments, R1 is hydrogen or alkyl, and R2 is hydrogen, halogen or alkyl. In certain embodiments, R1 is hydrogen or C1-6 alkyl, and R2 is hydrogen, halogen or C1-6 alkyl.

In some embodiments, R1 and R2 are both hydrogen.

In some embodiments, R1 is hydrogen, and R2 is halogen. In certain embodiments, R1 is hydrogen, and R2 is fluoro.

In some embodiments, X, W, U and Z are C(R3). In certain embodiments, W, U and Z are C(R3), and R3 is hydrogen.

In some embodiments, X and U are C(R3), and W and Z are N. In certain embodiments, X and U are C(R3) where R3 is hydrogen, and W and Z are N.

In some embodiments, E is —N(R4)C(═O)— or —C(═O)N(R4)—. In certain embodiments, E is —N(R4)C(═O)— or —C(═O)N(R4)—, and R4 is hydrogen.

In some embodiments, Y1 is null.

In some embodiments, Y1 is —C(R5)2—.

In certain embodiments, Y1 is —C(R5)2—, one R5 is hydrogen, the other R5 is selected from —N(Ra)2, alkyl, cycloalkyl, heterocyclyl and -alkyl-heterocyclyl, wherein the alkyl, cycloalkyl, heterocyclyl and -alkyl-heterocyclyl are optionally substituted with one or more R6.

In certain embodiments, Y1 is —C(R5)2—, one R5 is alkyl, the other R5 is selected from —N(Ra)2, alkyl, cycloalkyl, or heterocyclyl, wherein the alkyl, cycloalkyl, and heterocyclyl are optionally substituted with one or more R6.

In certain embodiments, Y1 is —C(R5)2—, one R5 is C1-6 alkyl, the other R5 is selected from —N(Ra)2, C1-6 alkyl, C3-12 cycloalkyl, or 5- to 12-membered heterocyclyl, wherein the C1-6 alkyl, C3-12 cycloalkyl, or 5- to 12-membered heterocyclyl are optionally substituted with one or more R6.

In some embodiments, R6 is selected from amino, alkyl or -alkyl-N(Ra)2. In certain embodiments, R6 is selected from amino, C1-6 alkyl or —(C1-6 alkyl)-N(Ra)2.

In some embodiments, Y1 is selected from the group consisting of:

In some embodiments, Y2 is —(CH2)p-cycloalkyl-*.

In certain embodiments, Y2 is —(CH2)p—(C3-10 cycloalkyl)-*, and p is 0 or 1 In certain embodiments, Y2 is cyclopentyl or cyclohexyl.

In certain embodiments, Y2 is —(CH2)p-heterocyclyl-*.

In certain embodiments, Y2 is —(CH2)p-(5- to 12-membered heterocyclyl)-*, and p is 0 or 1.

In some embodiments, Y2 is —(CH2)p-aryl-*.

In certain embodiments, Y2 is —(CH2)p—(C5-12 aryl)-*, and p is 0 or 1.

In certain embodiments, Y2 is phenyl or —CH2-phenyl-*.

In some embodiments, Y2 is —(CH2)p-heteroaryl-*.

In certain embodiments, Y2 is —(CH2)p-(5- to 12-membered heteroaryl)-*, and p is 0 or 1.

In some embodiments, Y31 is null, Y32 is selected from null, —O—#, —C(═O)O—#, —P(═O)(Rb)—#, —N(Rb)C(═O)—#, or —C(═O)N(Rb)—#, and Y33 is selected from —N(Rc)2, alkyl, or aryl, wherein the alkyl and aryl are optionally substituted with one or more R7.

In some embodiments, Y31 is null, Y32 is selected from null, —O—#, —C(═O)O—#, —P(═O)(Rb)—#, —N(Rb)C(═O)—#, or —C(═O)N(Rb)—#, and Y33 is selected from —N(Rc)2, C1-6 alkyl, or C5-12 aryl, wherein the C1-6 alkyl and C5-12 aryl are optionally substituted with one or more R7.

In certain embodiments, Y31 is null, Y32 is selected from null, —O—#, —C(═O)O—#, —P(═O)(Rb)—#, —N(Rb)C(═O)—#, or —C(═O)N(Rb)—#, and Y33 is selected from —N(Rc)2, C1-6 alkyl, or C5-12 aryl, wherein the C1-6 alkyl and C5-12 aryl are optionally substituted with one or more R7 independently selected from halogen, hydroxy, amino, cyano, nitrooxy, or alkyl.

In some embodiments, Y31 is alkyl, and Y32 is selected from null, —O—#, —OC(═O)—#, —OC(═O)N(Rb)—#, or —N(Rb)C(═O)—#.

In some embodiments, Y31 is C1-6 alkyl, C1-5 alkyl, C1-4 alkyl, C1-3 alkyl or C1-2 alkyl, Y32 is selected from null, —O—#, —OC(═O)—#, —OC(═O)N(Rb)—#, or —N(Rb)C(═O)—#, and Y33 is selected from —N(Rc)2, alkyl, or aryl, wherein the alkyl and aryl are optionally substituted with one or more R7.

In some embodiments, Y31 is C1-6 alkyl, C1-5 alkyl, C1-4 alkyl, C1-3 alkyl or C1-2 alkyl, Y32 is selected from null, —O—#, —OC(═O)—#, —OC(—O)N(Rb)—#, or —N(Rb)C(═O)—#, and Y33 is selected from —NH2, —N(CH3)2, methyl, dimethylphenyl, or nitrooxypentyl, wherein the alkyl and aryl are optionally substituted with one or more R7.

In some embodiments, n is 1 or 2.

Exemplary compounds of Formula (I) are set forth in Table 1 below.

Exemplary Compounds of Formula (I)

Compounds provided herein are described with reference to both generic formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a number of different forms or derivatives, including but not limited to prodrugs, active metabolic derivatives (active metabolites), solvates, pharmaceutically acceptable salts or isotope derivatives, all of which are within the scope of the present disclosure.

As used herein, the term “prodrugs” refers to compounds or pharmaceutically acceptable salts thereof which, when metabolized under physiological conditions or when converted by solvolysis, yield the desired active compound. Prodrugs include, without limitation, esters, amides, carbamates, carbonates, ureides, solvates, or hydrates of the active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide one or more advantageous handling, administration, and/or metabolic properties. For example, some prodrugs are esters of the active compound; during metabolysis, the ester group is cleaved to yield the active drug. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. Prodrugs may proceed from prodrug form to active form in a single step or may have one or more intermediate forms which may themselves have activity or may be inactive. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems”, Vol. 14 of the A.C.S. Symposium Series, in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987; in Prodrugs: Challenges and Rewards, ed. V. Stella, R. Borchardt, M. Hageman, R. Oliyai, H. Maag, J. Tilley, Springer-Verlag New York, 2007, all of which are hereby incorporated by reference in their entirety.

As used herein, the term “metabolite”, e.g., active metabolite overlaps with prodrug as described above. Thus, such metabolites are pharmacologically active compounds or compounds that further metabolize to pharmacologically active compounds that are derivatives resulting from metabolic process in the body of a subject. For example, such metabolites may result from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound or salt or prodrug. Of these, active metabolites are such pharmacologically active derivative compounds. For prodrugs, the prodrug compound is generally inactive or of lower activity than the metabolic product. For active metabolites, the parent compound may be either an active compound or may be an inactive prodrug.

Prodrugs and active metabolites may be identified using routine techniques know in the art. See, e.g., Bertolini et al, 1997, J Med Chem 40:2011-2016; Shan et al., J Pharm Sci 86:756-757; Bagshawe, 1995, DrugDev Res 34:220-230; Wermuth, supra.

As used herein, the term “pharmaceutically acceptable” indicates that the substance or composition is compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the subjects being treated therewith.

As used herein, the term “pharmaceutically acceptable salt”, unless otherwise indicated, includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable. Contemplated pharmaceutically acceptable salt forms include, but are not limited to, mono, bis, tris, tetrakis, and so on. Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

Similarly, if the particular compound is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include organic salts derived from amino acids, such as L-glycine, L-lysine, and L-arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as hydroxyethylpyrrolidine, piperidine, morpholine or piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

It is also to be understood that the compounds of present disclosure can exist in unsolvated forms, solvated forms (e.g., hydrated forms), and solid forms (e.g., crystal or polymorphic forms), and the present disclosure is intended to encompass all such forms.

As used herein, the term “solvate” or “solvated form” refers to solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The present disclosure is also intended to include all isotopes of atoms in the compounds. Isotopes of an atom include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, chlorine, bromide or iodine in the compounds of present disclosure are meant to also include their isotopes, such as but not limited to 1H, 2H, 3H, 11C, 12C, 13C, 14C, 14N, 15N, 16O, 17O, 18O, 31P, 32P, 32S, 33S, 34S, 36S, 17F, 18F, 19F, 35Cl, 37Cl, 79Br, 81Br, 124I, 127I and 131I. In some embodiments, hydrogen includes protium, deuterium and tritium. In some embodiments, carbon includes 12C and 13C.

Compounds provided herein or pharmaceutically acceptable salts thereof may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids, or in terms of relative configuration, as rel-(R)- or rel-(S)-. The present disclosure includes all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved by conventional techniques, such as, chromatography and fractional crystallization. Traditional techniques for the preparation, isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Wherever compounds are represented in their chiral form, it is understood that the embodiment includes, but is not limited to, the specific diastereomerically or enantiomerically enriched form. In situations that the chirality is not specified but is present, it is understood that the embodiment is intended to include either the specific diastereomerically or enantiomerically enriched form; or a racemic or scalemic mixture of such compound(s).

The term “enantiomers” represent a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. A mixture of enantiomers at a ratio other than 1:1 is a “scalemic” mixture.

The term “diastereoisomers” represent stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.

The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. By way of examples, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol, amide-imidic acid, lactam-lactim, imine-enamine isomerizations and annular forms where a proton can occupy two or more positions of a heterocyclic system. Valence tautomers include interconversions by reorganization of some of the bonding electrons. Tautomers can be in equilibrium or sterically locked into one form by appropriate substitution. Compounds of the present disclosure identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

When the compounds provided herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, these compounds include both E and Z geometric isomers.

Synthetic Method

The compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes

Preparation of compounds of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), in P. Kocienski, Protecting Groups, Georg Thieme Verlag, 2003, and in Peter G. M. Wuts, Greene's Protective Groups in Organic Synthesis, 5th Edition, Wiley, 2014, all of which are incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g. 1H or 13C), infrared spectroscopy, spectrophotometry (e.g. UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC). Compounds can be purified by one skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6), 874-883, which is incorporated herein by reference in its entirety), and normal phase silica chromatography.

Pharmaceutical Composition

In a further aspect, there is provided pharmaceutical compositions comprising one or more compounds of the present disclosure, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions of the present disclosure comprise a first compound of Formula (I) or a pharmaceutically acceptable salt thereof and one or more additional compounds of the same formula but said first compound and additional compounds are not the same molecules.

In another aspect, there is provided pharmaceutical composition comprising one or more compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient.

In some embodiments, the pharmaceutical compositions of the present disclosure comprises a therapeutically effective amount of one or more compounds of the present disclosure or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical compositions of the present disclosure comprises a therapeutically effective amount of one or more compounds of the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient.

As used herein, the term “therapeutically effective amount” refers to an amount of a molecule, compound, or composition comprising the molecule or compound to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; the rate of administration; the therapeutic or combination of therapeutics selected for administration; and the discretion of the prescribing physician. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

As used herein, the term “pharmaceutical composition” refers to a formulation containing the molecules or compounds of the present disclosure in a form suitable for administration to a subject. The pharmaceutical compositions include compositions suitable adapted for oral administration, rectal administration, topical administration, parenteral (including subcutaneous, intramuscular, and intravenous) administration, sublingual administration, ocular administration, transdermal administration or nasal administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used herein includes both one and more than one such excipient. The term “pharmaceutically acceptable excipient” also encompasses “pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent”.

The particular excipient used will depend upon the means and purpose for which the compounds of the present disclosure are being applied. Solvents are generally selected based on solvents recognized by persons skilled in the art as safe to be administered to a mammal including humans. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof.

In some embodiments, suitable excipients may include one or more stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present disclosure or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament). The active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the compounds disclosed herein and, optionally, a chemotherapeutic agent) to a mammal including humans. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject, including, but not limited to a human, and formulated to be compatible with an intended route of administration.

A variety of routes are contemplated for the pharmaceutical compositions provided herein, and accordingly the pharmaceutical composition provided herein may be supplied in bulk or in unit dosage form depending on the intended administration route. For example, for oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets may be acceptable as solid dosage forms, and emulsions, syrups, elixirs, suspensions, and solutions may be acceptable as liquid dosage forms. For injection administration, emulsions and suspensions may be acceptable as liquid dosage forms, and a powder suitable for reconstitution with an appropriate solution as solid dosage forms. For inhalation administration, solutions, sprays, dry powders, and aerosols may be acceptable dosage form. For topical (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches may be acceptable dosage form. For vaginal administration, pessaries, tampons, creams, gels, pastes, foams and spray may be acceptable dosage form.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for oral administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of tablet formulations. Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case using conventional coating agents and procedures well known in the art.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in a form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of aqueous suspensions, which generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene 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 (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), coloring agents, flavoring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of oily suspensions, which generally contain suspended active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

In certain embodiments, the pharmaceutical compositions provided herein may be in the form of syrups and elixirs, which may contain sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, a demulcent, a preservative, a flavoring and/or coloring agent.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for injection administration.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for inhalation administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of aqueous and nonaqueous (e.g., in a fluorocarbon propellant) aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for topical or transdermal administration. In certain embodiments, the pharmaceutical compositions provided herein may be in the form of creams, ointments, gels and aqueous or oily solutions or suspensions, which may generally be obtained by formulating an active ingredient with a conventional, topically acceptable excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

In certain embodiments, the pharmaceutical compositions provided herein may be formulated for administration ocularly. In certain embodiments, the pharmaceutical compostions provided herein may be in the form of ophthalmic formulation, such as eye ointments, powders, solutions and the like. In certain embodiments, ophthalmic formulations are prepared at a comfortable pH with an appropriate buffer system.

Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present disclosure. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), in “Remington: The Science and Practice of Pharmacy”, Ed. University of the Sciences in Philadelphia, 21st Edition, LWW (2005), which are incorporated herein by reference.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated so that a dosage of between 0.001-1000 mg/kg body weight/day, for example, 0.01-800 mg/kg body weight/day, 0.01-700 mg/kg body weight/day, 0.01-600 mg/kg body weight/day, 0.01-500 mg/kg body weight/day, 0.01-400 mg/kg body weight/day, 0.01-300 mg/kg body weight/day, 0.1-200 mg/kg body weight/day, 0.1-150 mg/kg body weight/day, 0.1-100 mg/kg body weight/day, 0.5-100 mg/kg body weight/day, 0.5-80 mg/kg body weight/day, 0.5-60 mg/kg body weight/day, 0.5-50 mg/kg body weight/day, 1-50 mg/kg body weight/day, 1-45 mg/kg body weight/day, 1-40 mg/kg body weight/day, 1-35 mg/kg body weight/day, 1-30 mg/kg body weight/day, 1-25 mg/kg body weight/day of the compounds provided herein, or a pharmaceutically acceptable salt thereof, can be administered. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day. For further information on routes of administration and dosage regimes, see Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990, which is specifically incorporated herein by reference.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as a single dosage form. The amount of the compounds provided herein in the single dosage form will vary depending on the subject treated and particular mode of administration.

In some embodiments, dosage forms suitable for administration may contain from about 1 mg to about 1000 mg of active ingredient per dosage unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.1-95% by weight based on the total weight of the composition.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as short-acting, fast-releasing, long-acting, and sustained-releasing. Accordingly, the pharmaceutical formulations of the present disclosure may also be formulated for controlled release or for slow release.

In some embodiments, a dose of the compounds provided herein or the pharmaceutical compositions provided herein is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, a dose of the compounds provided herein or the pharmaceutical compositions provided herein is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, 1 month, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

In a further aspect, there is also provided veterinary compositions comprising one or more molecules or compounds of the present disclosure or pharmaceutically acceptable salts thereof and a veterinary carrier. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

The pharmaceutical compositions or veterinary compositions may be packaged in a variety of ways depending upon the method used for administering the drug. For example, an article for distribution can include a container having deposited therein the compositions in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings. The compositions may also be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.

In some embodiments, the pharmaceutical composition of the present disclosure comprising one or more compounds provided herein or pharmaceutically acceptable salts thereof further comprises one or more additional therapeutically active agents.

The additional therapeutically active agents have complementary activities to the compound provided herein such that they do not adversely affect each other. Such agents are suitably present in combination in amounts that are effective for the purpose intended.

In certain embodiments, the additional therapeutic agent is selected from beta blockers, alpha-agonists, carbonic anhydrase inhibitors, prostaglandin-like compounds, miotic or cholinergic agents, or epinephrine compounds.

Beta blockers reduce the production of aqueous humor. Examples include levobunolol (BETAGAN®), timolol (BETIMOL®, TIMOPTIC®), betaxolol (BETOPTIC®) and metipranolol (OPTIPRANOLOL®).

Alpha-agonists reduce the production of aqueous humor and increase drainage. Examples include apraclonidine (IOPIDINE®) and brimonidine (ALPHAGAN®).

Carbonic anhydrase inhibitors reduce the production of aqueous humor. Examples include dorzolamide (TRUSOPT®) and brinzolamide (AZOPT®).

Prostaglandins and prostaglandin-like compounds increase the outflow of aqueous humor. Examples include latanoprost (XALATAN®), bimatoprost (LUMIGAN®), and travoprost (TRAVATAN™).

Miotic or cholinergic agents increase the outflow of aqueous humor. Examples include pilocarpine (ISOPTO CARPINE®, PILOPINE®) and carbachol (ISOPTO CARBACHOL®).

Epinephrine compounds, such as dipivefrin (PROPINE®), also increase the outflow of aqueous humor.

The additional therapeutic agent or agents may be administered simultaneously or sequentially with the compounds provided herein. Sequential administration includes administration before or after the compounds provided herein. In some embodiments, the additional therapeutic agent or agents may be administered in the same composition as the compounds provided herein. In other embodiments, there may be an interval of time between administration of the additional therapeutic agent and the compounds provided herein.

In some embodiments, the administration of an additional therapeutic agent with a compound provided herein may enable lower doses of the other therapeutic agents and/or administration at less frequent intervals.

Method for Treatment

Compounds of the present disclosure and pharmaceutical composition comprising the same are capable of inhibiting ROCK, and thus can be useful for inhibiting ROCK activity in a subject in need thereof, and for preventing or treating ROCK-related disorders.

In a further aspect, the present disclosure provides a method of treating ROCK-related disorders, comprising administering an effective amount of the compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition provided herein to a subject in need thereof.

As used herein, the term “treating”, “treatment” or “therapy” is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology, thereby achieving beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treating” can also mean prolonging survival as compared to expected survival if not receiving it. Those in need of therapy include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

As used herein, the term “preventing”, “prevention” or “prophylaxis” is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease and secondary prophylaxis whereby the disease has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the disease.

In some embodiments, the compounds or pharmaceutically acceptable salts thereof and the compositions provided herein may be used for the treatment of a wide variety of ROCK-related disorders including cancer, cardiovascular diseases, autoimmune diseases, ocular diseases, metabolic syndrome, respiratory distress syndrome, kidney disease, overactive bladder, epilepsy, migraine, diabetes, high altitude pulmonary edema, psychiatric disorders, etc.

In certain embodiments, the eye disease that can be treated with the compounds or pharmaceutically acceptable salts thereof and the compositions provided herein is glaucoma.

The concentration and route of administration to the subject will vary depending on the ROCK-related disorders to be treated. In certain embodiments, the administering is conducted via a route selected from the group consisting of parenteral, intraperitoneal, intradermal, intracardiac, intraventricular, intracranial, intracerebrospinal, intrasynovial, intrathecal administration, intramuscular injection, intravitreous injection, intravenous injection, intra-arterial injection, oral, buccal, sublingual, transdermal, topical, intratracheal, intrarectal, subcutaneous, and ocular administration.

EXAMPLES

The followings further explain the general methods of the present disclosure. The compounds of the present disclosure may be prepared by the methods known in the art. The following illustrates the detailed preparation methods of the preferred compounds of the present disclosure. However, they are by no means limiting the preparation methods of the compounds of the present disclosure.

Synthetic Examples

For the purpose of illustration, the following examples are included. The Examples provided herein describe the synthesis of compounds disclosed herein as well as intermediates used to prepare the compounds. However, it is to be understood that these examples do not limit the present disclosure and are only meant to suggest a method of practicing the present disclosure. Persons skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other compounds of the present disclosure, and alternative methods for preparing the compounds of the present disclosure are deemed to be within the scope of the present disclosure. For example, the synthesis of non-exemplified compounds according to the present disclosure may be successfully performed by modifications apparent to those skilled in the art, e.g., by appropriately protecting interfering groups, by utilizing other suitable reagents and building blocks known in the art other than those described, and/or by making routine modifications of reaction conditions. Besides, persons skilled in the art will also understand that individual steps described herein or in the separate batches of a compound may be combined. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure. The following description is, therefore, not intended to limit the scope of the present disclosure, but rather is specified by the claims appended hereto.

Abbreviations for chemicals used in the synthesis of the compounds provided herein are listed below:

1H NMR
Proton Nuclear Magnetic Resonance Spectroscopy

aq
aqueous

CH3CN or MeCN or ACN
Acetonitrile

ee
Enantiomeric excess

EtOAc or EA
Ethyl acetate

EtOH
Ethanol

HCl
hydrochloric acid

LCMS
Liquid chromatography mass spectrometry

NaCl
Sodium chloride

PE
Petroleum ether

Pd/C
palladium on carbon

Prep-TLC
Preparatory thin layer chromatography

SFC
Supercritical fluid chromatography

Silica
Silica

TEA or Et3N
Triethylamine

tR
Retention time

Synthesis of Intermediates

Synthesis of Intermediate I-1

Step 1: A solution of p-cyanomethyl benzoic acid I-1a (20 g, 124.10 mmol, 1 equiv) and CDI (22.14 g, 136.51 mmol, 1.1 equiv) in THF (50 mL) was stirred for 3 h at room temperature under nitrogen atmosphere. Into the mixture was added NaBH4 (14.08 g, 372.30 mmol, 3 equiv) in H2O (45 mL) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (3×500 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with CH2Cl2/EtOAc (8:1, v/v) to afford 2-[4-(hydroxymethyl)phenyl]acetonitrile I-1b (10.9 g, 59.67%, crude) as a light yellow liquid. 1H NMR (CD3OD, 400 MHZ) δ 3.87-3.91 (2H, m), 4.62 (2H, s), 7.37 (4H, q).

Step 3: To a stirred solution of nitrile I-1c (14.0 g, 53.55 mmol, 1 equiv) in THF (20 mL) was added sodium hydride (1.93 g, 80.33 mmol, 1.5 equiv) in portions. The resulting mixture was stirred for 1 h at 0° C. under nitrogen atmosphere. Dimethyl carbonate (19.29 g, 214.20 mmol, 4 equiv) was added to the above mixture in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. The reaction was quenched with water (200 ml) at room temperature. The resulting mixture was extracted with ethyl acetate (3×250 mL), the combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography (PE/EA=6:1, v/v) to afford methyl 2-(4-{[(tert-butyldimethylsilyl)oxy]methyl}phenyl)-2-cyanoacetate I-1d (12.6 g, 73.65%) as a light yellow liquid. 1H NMR (CD3OD, 400 MHZ) δ 0.13 (6H, s), 0.97 (9H, s), 3.78 (3H, s), 4.78 (2H, s), 7.37-7.47 (4H, m).

Synthesis of Intermediate I-2

Step 1: To a solution of 2-(3-methoxyphenyl) acetonitrile I-2a (3 g, 20.38 mmol, 1 equiv) in THF (50 mL) was added sodium hydride (0.54 g, 22.42 mmol, 1.1 equiv) at 0° C. The mixture was stirred for 50 min. Dimethyl carbonate (14.69 g, 163.07 mmol, 8 equiv) was added and the mixture and the resulting mixture was allowed to warm to RT and stirred for 2 h. The reaction mixture was quenched by water (50 mL) and extracted with DCM (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (12:1, v/v) to afford methyl 2-cyano-2-(3-methoxyphenyl)acetate I-2b (2.84 g, 67.89%) as a yellow oil. 1H NMR (400 MHZ, DMSO-d6) δ 3.76 (d, 6H), 5.63 (s, 1H), 6.81-7.11 (m, 3H), 7.39 (t, 1H).

Step 3: Into a 50 mL round-bottom flask were added ester I-2c (760 mg, 2.46 mmol, 1 equiv) and LiOH (117.67 mg, 4.91 mmol, 2.0 equiv) in THF (3 mL) and water (3 mL) at room temperature. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was concentrated under reduced pressure. The mixture was acidified to pH 5 with HCl (1M). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, water in methanol, 0% to 100% gradient in 50 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum, to afford 3-[(tert-butoxycarbonyl)amino]-2-(3-methoxyphenyl)propanoic acid I-2 (200 mg, 27.57%) as a brown yellow solid. LCMS: m/z (ESI), [M-tBu]+=240.25.

Synthesis of Intermediate I-3

Step 2: To a stirred mixture of ester I-3b (5.5 g, 21.65 mmol, 1 equiv), CoCl2·6H2O (15.45 g, 64.94 mmol, 3 equiv) and Boc2O (14.17 g, 64.94 mmol, 3 equiv) in methanol (100 mL) was added NaBH4 (4.91 g, 129.88 mmol, 6 equiv) dropwise at 0° C. under air atmosphere, the mixture was allowed to warm to RT and stirred for 2 h. The resulting mixture was quenched with water at 0° C. (200 mL). The resulting mixture was filtered. The filtered cake was washed with DCM (4×100 mL). The filtrate was extracted with CH2Cl2 (2×300 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (10:1, v/v) to afford methyl 2-(3-bromophenyl)-3-[(tert-butoxycarbonyl)amino]propanoate I-3c (4 g, 30.95%) as a yellow oil. LCMS: m/z (ESI), [M+H−tBu]+=303.85.

Synthesis of Intermediate I-4

Step 1: To a stirred mixture of 2-(4-bromophenyl)-3-[(tert-butoxycarbonyl)amino]propanoic acid (2.0 g, 5.81 mmol, 1 equiv) and DMAP (141.97 mg, 1.16 mmol, 0.2 equiv) in DCM (10 mL) and methanol (1.0 mL, 24.70 mmol, 4.25 equiv) was added EDCI (2227.74 mg, 11.620 mmol, 2 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. After reaction, the resulting mixture was diluted with water (40 mL). The resulting mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with water (3×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (4:1, v/v) to afford methyl 2-(4-bromophenyl)-3-[(tert-butoxycarbonyl)amino]propanoate I-4a (1.58 g, 75.91%) as a white solid. LCMS: m/z (ESI), [M-t-Bu]+=301.80.

Step 3: To a stirred mixture of nitrile I-4b (300 mg, 0.99 mmol, 1 equiv) and (Boc)2O (430.26 mg, 1.97 mmol, 2 equiv), CoCl2·6H2O (469.05 mg, 1.97 mmol, 2 equiv) in methanol (10 mL) was added NaBH4 (298.32 mg, 7.89 mmol, 8 equiv) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 3 h at 25° C. under nitrogen atmosphere. The reaction was quenched with ice water (30 mL) at 0° C. The resulting mixture was filtered and the filtered cake was washed with methanol (3×20 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with PE/EA (5:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)propanoate I-4c (170 mg, 42.22%) as a yellow oil. LCMS: m/z (ESI), [M+Na]+=431.10.

Step 4: To a stirred mixture of ester I-4c (160 mg, 0.39 mmol, 1 equiv) in methanol (4.0 mL) was added LiOH·H2O (65.74 mg, 1.57 mmol, 4.0 equiv) in water (2.0 mL) dropwise at 25° C. under nitrogen atmosphere. The resulting mixture was stirred for 3 h at 60° C. under nitrogen atmosphere. After cooling to room temperature, the mixture was acidified to pH 4 with HCl (1 M). The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 20 min at 30%. After evaporated the solvent afforded 3-[(tert-butoxycarbonyl)amino]-2-(4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)propanoic acid I-4 (90 mg, 58.25%) as a white solid. LCMS: m/z (ESI), [M−H]=393.15.

Synthesis of Intermediate I-5

Synthesis of Intermediate I-6

Synthesis of Intermediate I-7

Step 1: Into a 250 mL round-bottom flask were added 2-(pyridin-3-yl) acetonitrile I-7a (6 g, 50.79 mmol, 1 equiv) and THF (40 mL, 493.71 mmol), sodium hydride (2.23 g, 55.87 mmol, 1.1 equiv, 60%) at 0° C. The resulting mixture was stirred for 1 h at room temperature, to the above mixture was add dimethyl carbonate (36.60 g, 406.30 mmol, 8 equiv). The resulting mixture was stirred for 4 h at room temperature. The resulting mixture was extracted with CH2Cl2/methanol=20:1 (3×30 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (1:1, v/v) to afford methyl 2-cyano-2-(pyridin-3-yl)acetate I-7b (7.4 g, 54.83%) as a brown yellow solid. LCMS: m/z (ESI), [M+H]+=177.25.

Synthesis of Intermediate I-8

Step 2: A solution of ester I-8b (250 mg, 1.50 mmol, 1 equiv) and 1-methylpiperazine (568.42 mg, 5.68 mmol, 4 equiv) in DMF (3 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. The residue was dissolved in PE (20 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure afford ethyl 3-(4-methylpiperazin-1-yl)-2-phenylpropanoate I-8c (330 mg, 84.16%) as an off-white solid. LCMS: m/z (ESI), [M+H]+=277.25.

Step 3: To a stirred solution of piperazin I-8c (500 mg, 1.81 mmol, 1 equiv) in methanol (10 mL) and H2O (5 mL) was added LiOH (173.31 mg, 7.24 mmol, 4 equiv) dropwise at room temperature under air atmosphere. The resulting mixture was stirred for 3 h at room temperature under air atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 20% to 30% gradient in 10 min; detector, UV 254 nm to afford 3-(4-methylpiperazin-1-yl)-2-phenylpropanoic acid I-8 (220 mg, 48.97%) as a white solid. LCMS: m/z (ESI), [M+H]+=249.15.

Synthesis of Intermediate I-9

Step 2: To a stirred solution of tert-butyl 4-(2-ethoxy-2-oxo-1-phenylethyl) piperidine-1-carboxylate (500 mg, 1.44 mmol, 1 equiv) in DCM (6 mL) was added TFA (2 mL). The resulting mixture was stirred for 2 h at room temperature under air atmosphere. The resulting mixture was concentrated under vacuum to afford ethyl 2-phenyl-2-(piperidin-4-yl)acetate I-9c (340 mg, 95.52%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=248.15.

Step 3: To a stirred mixture of ester I-9c (350 mg, 1.42 mmol, 1 equiv) and formaldehyde (424.89 mg, 14.15 mmol, 10 equiv) in DCM (10 mL) was added DIEA (914.47 mg, 7.08 mmol, 5 equiv). The resulting mixture was stirred for 0.5 h at room temperature under air atmosphere. To the above mixture was added NaBH(OAc)3 (899.73 mg, 4.25 mmol, 3 equiv). The resulting mixture was stirred for additional 2 h at room temperature. The reaction was quenched by the addition of water (4 mL) at 0° C. The resulting mixture was extracted with ethyl acetate (3×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in ethyl 2-(1-methylpiperidin-4-yl)-2-phenylacetate (350 mg, 94.63%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=262.10.

Synthesis of Intermediate I-10

Step 2: Into a 50 mL round-bottom flask were added ester I-10b (2.7 g, 10.87 mmol, 1 equiv) and LiOH·H2O (0.91 g, 21.75 mmol, 2.0 equiv) in methanol (16 mL) and water (8 mL) at room temperature. The resulting mixture was stirred for 5 h at room temperature under nitrogen atmosphere. The mixture was acidified to pH 6 with HCl (1 M). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 0% to 5% gradient in 30 min; detector, UV220 nm to afford (4-methylpiperazin-1-yl) (phenyl) acetic acid I-10 (1.6 g, 62.81%) as a white solid. LCMS: m/z (ESI), [M+H]+=235.15.

Synthesis of Intermediate I-11

Step 1: To a stirred solution of methyl 3-(cyanomethyl)benzoate I-11a (15 g, 85.62 mmol, 1 equiv) in methanol (250 mL) were added NaBH4 (16.20 g, 428.12 mmol, 5 equiv) in portions at 0° C. under air atmosphere. The resulting mixture was stirred for overnight at 70° C. under air atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The reaction was quenched by the addition of water (100 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (1:10, v/v) to afford 2-[3-(hydroxymethyl)phenyl]acetonitrile (11.14 g, 88.40%) as a yellow oil. 1H NMR (DMSO-d6, 400 MHz) δ 4.10 (2H, s), 4.52 (2H, d), 5.28 (1H, t), 7.21 (1H, d), 7.27 (1H, d), 7.32-7.39 (2H, m).

Step 3: To a stirred solution of nitrile I-11b (19.5 g, 74.59 mmol, 1 equiv) in THF (100 mL) were added sodium hydride (3.58 g, 149.17 mmol, 2 equiv) dropwise at 0° C. under air atmosphere. The resulting mixture was stirred for 1 h at room temperature under air atmosphere. To the above mixture was added dimethyl carbonate (53.75 g, 596.70 mmol, 8 equiv) dropwise at room temperature. The resulting mixture was stirred for additional 3 h at room temperature. The reaction was quenched by the addition of water/ice (100 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (1:1, v/v) to afford methyl 2-(3-{[(tert-butyldimethylsilyl)oxy]methyl}phenyl)-2-cyanoacetate (10.1 g, 42.39%) as a yellow oil. 1H NMR (CD3OD, 400 MHZ) δ 0.13 (6H, d), 0.97 (9H, s), 3.78 (3H, s), 4.79 (2H, d), 7.22-7.46 (3H, m), 7.50 (1H, dq).

Step 6: To a stirred solution of methyl 3-[(tert-butoxycarbonyl)amino]-2-[3-(hydroxymethyl)phenyl]propanoate (300 mg, 0.97 mmol, 1 equiv) in DCM (6 mL, 94.38 mmol, 97.33 equiv) was added N-methylcarbamoyl chloride (108.82 mg, 1.16 mmol, 1.2 equiv) and Et3N (294.39 mg, 2.91 mmol, 3 equiv) dropwise at room temperature under air atmosphere. The resulting mixture was stirred for overnight at room temperature under air atmosphere. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with CH2Cl2 (3×10 mL). The combined organic layers dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 100% to 100% gradient in 10 min; detector, UV 254 nm to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(3-{[(methylcarbamoyl)oxy]methyl}-phenyl)propanoate I-11d (90 mg, 25.33%) as a colorless oil. LCMS: m/z (ESI), [M+H]+=367.15.

Step 7: To a stirred solution of ester I-11d (270 mg, 0.74 mmol, 1 equiv) in methanol (8 mL) and H2O (2 mL) were added LiOH (70.59 mg, 2.95 mmol, 4 equiv) at room temperature under air atmosphere. The resulting mixture was stirred for 2 h at room temperature under air atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 50% to 60% gradient in 10 min; detector, UV 254 nm to afford 3-[(tert-butoxycarbonyl)amino]-2-(3-{[(methylcarbamoyl)oxy]methyl}phenyl)propanoic acid I-11 (90 mg, 34.66%) as a white solid. LCMS: m/z (ESI), [M+H]+=353.20.

Synthesis of Intermediate I-12

Step 1: To a stirred mixture of ethyl 2-cyano-2-(4-nitrophenyl)acetate I-12a (2.0 g, 8.539 mmol, 1 equiv) and zinc (4.47 g, 68.31 mmol, 8 equiv) in THF (40 mL) was added NH4Cl (4.57 g, 85.39 mmol, 10 equiv) in water (10 mL) dropwise at 25° C. under nitrogen atmosphere. The resulting mixture was stirred for 48 h at room temperature under nitrogen atmosphere. After reaction, the resulting mixture was filtered, the filter cake was washed with methanol (3×20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with CH2Cl2/methanol (60:1, v/v) to afford ethyl 2-(4-aminophenyl)-2-cyanoacetate I-12b (1.2 g, 68.81%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=205.15.

Step 3: To a stirred mixture of ester I-12c (1.1 g, 4.47 mmol, 1 equiv) and CoCl2·6H2O (2.13 g, 8.93 mmol, 2 equiv) in methanol (40 mL) was added (Boc) 20 (1.95 g, 8.93 mmol, 2 equiv) and NaBH4 (0.68 g, 17.87 mmol, 4 equiv) in portions at 0° C. under air atmosphere. The resulting mixture was stirred for overnight at room temperature under air atmosphere. After reaction, the resulting mixture was filtered, the filter cake was washed with water (3×30 mL). The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with ethyl acetate (3×40 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (5:1, v/v) to afford ethyl 3-[(tert-butoxycarbonyl)amino]-2-(4-acetamidophenyl)propanoate I-12d (900 mg, 57.50%) as a yellow oil. LCMS: m/z (ESI), [M-Boc]+=251.15.

Step 4. To a stirred mixture of ester I-12d (500 mg, 2.03 mmol, 1 equiv) in methanol (4.0 mL) was added LiOH (194.51 mg, 8.12 mmol, 4 equiv) in water (1.0 mL) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for overnight at 60° C. under nitrogen atmosphere. After cooling to room temperature, the resulting mixture was concentrated under vacuum. The resulting mixture was diluted with methanol (3.0 mL). The mixture was acidified to pH 5 with HCl (1 M). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 30 min; detector, UV 254 nm, and hold for 10 min at 16%. After lyophilization to afford 2-(4-acetamidophenyl)-3-((tert-butoxycarbonyl)amino) propanoic acid I-12 (260 mg, 36.54%) as a white solid. LCMS: m/z (ESI), [M-t-Bu]+=267.05.

Synthesis of Intermediate I-13

Synthesis of Intermediate I-14

Step 1: To a stirred solution of ester I-26c (2.08 g, 5.81 mmol, 1 equiv) and Pd(PPh3)4 (67.10 mg, 0.058 mmol, 0.01 equiv) in DMF (10 mL) was added Zn(CN)2 (688.59 mg, 5.86 mmol, 1.01 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2.0 h at 80° C. under nitrogen atmosphere. After cooling to room temperature, the resulting mixture was diluted with water (60 mL). The resulting mixture was extracted with ethyl acetate (3×40 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC with PE/EA (2:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(4-cyanophenyl)propanoate I-14c (1.3 g, 73.57%) as a yellow solid.

Step 2: To a stirred solution of nitrile I-14c (600 mg, 1.97 mmol, 1 equiv) in methanol (8.0 mL) and LiOH·H2O (330.89 mg, 7.88 mmol, 4.0 equiv) in water (2.0 mL) was added dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2.0 h at 70° C. under nitrogen atmosphere. After cooling to room temperature, the resulting mixture was concentrated under vacuum. After evaporated, the residue was dissolved in methanol (2.0 mL) and basified to pH 9 with NH3 aq. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 20 min at 18.2% to afford 3-[(tert-butoxycarbonyl)amino]-2-(4-cyanophenyl)propanoic acid I-14 (400 mg, 69.89%) as a white solid. LCMS: m/z (ESI), [M-tBu]+=235.05.

Synthesis of Intermediate I-15

Step 3: Into a 50 mL round-bottom flask were added nitrile I-15b (2.24 g, 6.99 mmol, 1 equiv), CoCl2·6H2O (2.49 g, 10.49 mmol, 1.5 equiv), Boc2O (3.05 g, 13.98 mmol, 2.0 equiv) and methanol (20 mL) at room temperature was added NaBH4 (1.06 g, 27.96 mmol, 4.0 equiv) in portions at 0° C. The resulting mixture was stirred for overnight at room temperature. The reaction was quenched with water (100 mL) at room temperature. The resulting mixture was filtered and the filtered cake was washed with CH2Cl2 (3×30 mL). The filtrate was extracted by DCM (2×100 mL), the combined organil layer was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol 17:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(6-{[(tert-butyldimethylsilyl)oxy]methyl}pyridin-3-yl)propanoate (625 mg, 14.74%) as a brown yellow solid. LCMS: m/z (ESI), [M+H]+=425.30.

Synthesis of Intermediate I-16

Step 2: To a stirred mixture of nitrile I-16b (800 mg, 4.21 mmol, 1 equiv) and Boc2O (3671.87 mg, 16.82 mmol, 4 equiv), CoCl2·6H2O (2601.83 mg, 10.94 mmol, 2.6 equiv) in methanol (50 mL) was added NaBH4 (1272.92 mg, 33.65 mmol, 8 equiv) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. The reaction was quenched with water at room temperature. The resulting mixture was extracted with CH2Cl2 (2×100 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (32:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(5-methylpyridin-3-yl)propanoate I-16c (310 mg, 43.66%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=295.15.

Step 3: A solution of ester I-16c (300 mg, 1.02 mmol, 1 equiv) in methanol (3 mL) and LiOH·H2O (85.53 mg, 2.04 mmol, 2 equiv) in H2O (1.5 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. The residue was acidified to pH 5 with HCl (1M). The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 10% to 23% gradient in 30 min; detector, UV 220 nm to afford 3-[(tert-butoxycarbonyl)amino]-2-(5-methylpyridin-3-yl)propanoic acid I-16 (140 mg, 49.00%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=281.10.

Synthesis of Intermediate I-17

Synthesis of Intermediate I-18

Step 2: To a stirred mixture of enolate I-18b (757 mg, 4.64 mmol, 1 equiv) in DMF (10 mL) was added 1-methylpiperazine (2323.41 mg, 23.20 mmol, 5 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum. This resulted in methyl 3-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl)propanoate I-18c (884 mg, 72.36%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=264.30.

Step 3: To a stirred mixture of piperazin I-18c (150 mg, 0.57 mmol, 1 equiv) in THF (8 mL) and H2O (2 mL) was added LiOH (68.2 mg, 2.85 mmol, 5 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 3-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl)propanoic acid I-18 (120 mg, 84.5%) as a white solid. LCMS: m/z (ESI), [M+H]+=250.35.

Synthesis of Intermediate I-19

Step 2: Into a 100 mL round-bottom flask were added nitrile I-19b (3.7 g, 13.44 mmol, 1 equiv) and CoCl2·6H2O (2.62 g, 20.16 mmol, 1.5 equiv), Boc2O (5.87 g, 26.88 mmol, 2 equiv) in methanol (20 mL) at room temperature. To the above mixture was added NaBH4 (1.53 g, 40.32 mmol, 3 equiv) in portions over 30 min at 0° C. The resulting mixture was stirred for additional 15 h at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was extracted with DCM (3×40 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (5:1, v/v) to afford tert-butyl 3-{3-[(tert-butoxycarbonyl)amino]-1-methoxy-1-oxopropan-2-yl}benzoate I-19c (2.07 g, 40.59%) as a white oil. LCMS: m/z (ESI), [M+H-Boc-tBu]+=223.95.

Step 3: Into a 100 mL round-bottom flask were added ester I-19c (2 g, 5.27 mmol, 1 equiv) and LiOH (252.47 mg, 10.54 mmol, 2 equiv) in water (15 mL) and methanol (15 mL) at room temperature. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The mixture was acidified to pH 5 with HCl (1M). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 12% to 22% gradient in 25 min; detector, UV 254 nm. This resulted in 3-[(tert-butoxycarbonyl)amino]-2-[3-(tert-butoxycarbonyl)phenyl]propanoic acid I-19 (650 mg, 33.75%) as an off-white solid. LCMS: m/z (ESI), [M+H]+=366.00.

Synthesis of Intermediate I-20

Step 1. Into a 40 mL vial were added methyl 2-bromo-2-phenylacetate I-20a (1 g, 4.37 mmol, 1 equiv) and tert-butyl N-[2-(piperazin-1-yl)ethyl]carbamate (1.20 g, 5.24 mmol, 1.2 equiv), triethylamine (1.33 g, 13.10 mmol, 3 equiv) in THF (20 mL) at room temperature. The resulting mixture was stirred for 15 h at 70° C. under nitrogen atmosphere. The reaction was quenched by the addition of water (60 mL) at room temperature. The resulting mixture was extracted with EA (3×50 mL). The combined organic layers were washed with saturated brine (1×100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (4 mL). The residue was purified by prep-TLC with PE/EA (1:1, v/v) to afford methyl 2-(4-{2-[(tert-butoxycarbonyl)amino]ethyl}piperazin-1-yl)-2-phenylacetate I-20b (1.54 g, 93.45%) as a light yellow oil. LCMS: m/z (ESI), [M+H]+=378.10.

Synthesis of Intermediate I-21

Step 3. To a stirred mixture of nitrile I-21b (2.1 g, 8.03 mmol, 1 equiv) in THF (25 mL) was added sodium hydride (289.14 mg, 12.05 mmol, 1.5 equiv) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. Next added dimethyl carbonate (2.89 g, 32.13 mmol, 4 equiv) in portions at 0° C. under nitrogen atmosphere. The final reaction mixture was stirred for overnight at room temperature. The reaction was monitored by TLC with PE/EA (6:1, v/v). The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (3×150 mL), the combined organic layer was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with PE/EA (6:1, v/v) to afford methyl 2-(3-{[(tert-butyldimethylsilyl)oxy]methyl}phenyl)-2-cyanoacetate (1.9 g, 74.04%) as a light yellow liquid. 1H NMR (CD3OD, 400 MHZ) δ 0.13 (6H, s), 0.97 (9H, s), 3.78 (3H, s), 4.80 (2H, q), 7.32-7.51 (4H, m).

Synthesis of Intermediate I-22

Step 2: A solution of nitrile I-22b (1 g, 4.54 mmol, 1 equiv) in methanol was treated with Boc2O (3.96 g, 18.17 mmol, 4 equiv) and CoCl2·6H2O (2.81 g, 11.81 mmol, 2.6 equiv) for 5 min at 0° C. under nitrogen atmosphere followed by the addition of NaBH4 (1.37 g, 36.34 mmol, 8 equiv) in portions at 0° C. The resulting mixture was stirred for 2 h at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was filtered and the filtered cake was washed with methanol (3×20 mL). The aqueous layer was extracted with CH2Cl2 (3×100 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (2:1, v/v) to afford ethyl 3-((tert-butoxycarbonyl)amino)-2-(4-((tert-butoxycarbonyl)amino)phenyl)propanoate I-22c (500 mg, 27.91%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=409.25.

Step 3: Into a 100 mL round-bottom flask were added ester I-22c (500 mg, 1.27 mmol, 1 equiv) and LiOH·H2O (106.37 mg, 2.54 mmol, 2 equiv) in THF (5 mL) and H2O (5 mL) at room temperature. The resulting mixture was stirred for 15 h at room temperature under air atmosphere. The mixture was acidified to pH 5 with HCl (1M). The resulting mixture was concentrated under reduced pressure. The resulting oil was dried by lyophilization. This resulted in 3-[(tert-butoxycarbonyl)amino]-2-{4-[(tert-butoxycarbonyl)amino]phenyl}propanoic acid I-22 (420 mg, 68.74%) as a yellow solid. LCMS: m/z (ESI), [M−H]−=379.15.

Synthesis of Intermediate I-23

Synthesis of Intermediate I-24

Step 1: A solution of 4-fluorobenzeneacetonitrile I-24a (3 g, 22.20 mmol, 1 equiv) in THF was treated with sodium hydride (1.1 g, 45.84 mmol, 2.06 equiv) for 1 h at 0° C. under nitrogen atmosphere followed by the addition of dimethyl carbonate (4.40 g, 48.84 mmol, 2.2 equiv) in portions at room temperature. The resulting mixture was stirred for 12 h at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (3×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with PE/EA (3:1, v/v) to afford methyl 2-cyano-2-(4-fluorophenyl)acetate (1.1 g, 15.01%) as a yellow oil. 1H NMR (DMSO-d6, 400 MHZ) δ 3.74 (3H, s), 5.73 (1H, s), 7.25-7.35 (2H, m), 7.46-7.55 (2H, m).

Synthesis of Intermediate I-25

Synthesis of Intermediate I-26

Step 3: To a stirred mixture of ester I-26c (4.0 g, 11.17 mmol, 1 equiv) in methanol (16 mL) was added LiOH·H2O (1874.09 mg, 44.66 mmol, 4 equiv) in water (4 mL) The resulting mixture was stirred at 60° C. for 8 hour under nitrogen atmosphere. After cooled to 25° C., the resulting mixture was concentrated under reduced pressure. The reaction mixture was diluted with water (20 mL), and adjust pH to 2 with HCl (1 M, 30 mL). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, phase A, water. Phase B ACN, from 0% to 50% gradient in 40 min; detector, UV 254 nm, and hold for 20 min at 30%. After evaporated the solvent, 2-(4-bromophenyl)-3-[(tert-butoxycarbonyl)amino]propanoic acid I-26 (3.2 g, 83.26%) was obtained as a yellow solid. LCMS: m/z (ESI), [M-Bu]+=289.95.

Synthesis of Intermediate I-27

Step 3: To a stirred mixture of ester I-27b (200 mg, 0.57 mmol, 1 equiv) in THF (40 mL) and H2O (10 mL) was added LiOH (40.66 mg, 1.70 mmol, 3 equiv) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for overnight at 60° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water (0.1% FA), 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 3-[(tert-butoxycarbonyl)amino]-2-[4-(2-methoxyethoxy)phenyl]propanoic acid I-27 (90 mg, 46.86%) as a white solid. LCMS: m/z (ESI), [M+H-boc]+=240.

Synthesis of Intermediate I-28

Step 1: To a solution of 2-(4-methoxyphenyl) acetonitrile I-28a (6 g, 40.77 mmol, 1 equiv) in THF (100 mL) was added sodium hydride (1.47 g, 61.15 mmol, 1.5 equiv) 60% in oil at 0° C. The mixture was stirred for 80 min. dimethyl carbonate (29.38 g, 326.14 mmol, 8 equiv) was added at 0° C. and the mixture was allowed to warm to RT and stirred for 4 h. The reaction mixture was quenched by water (100 mL) and extracted with DCM (3×100 mL). The residue was purified by silica column chromatography, eluted with PE/EA (8:1, v/v) to afford methyl 2-cyano-2-(4-methoxyphenyl)acetate (5.96 g, 70.03%) as a yellow oil.

Step 3: Into a 50 mL round-bottom flask were added ester I-28b (2 g, 6.47 mmol, 1 equiv) and LiOH·H2O (0.54 g, 12.93 mmol, 2 equiv) and H2O (4 mL) and methanol (8 mL) at room temperature. The resulting mixture was stirred for 2 h at room temperature under air atmosphere. The residue was acidified to pH=5 with HCl (1M). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 0% to 15% gradient in 30 min; detector, UV 220 nm. The resulting liquid was dried by lyophilization. This resulted in 3-[(tert-butoxycarbonyl)amino]-2-(4-methoxyphenyl)propanoic acid I-28 (1.38 g, 70.83%) as a white solid. LCMS: m/z (ESI), [M+H−tBu]+=240.25.

Synthesis of Intermediate I-29

Step 1: To a stirred solution of 2-(6-methylpyridin-2-yl) acetonitrile I-29a (500 mg, 3.78 mmol, 1 equiv) in THF (20 mL) was added sodium hydride (181.57 mg, 7.57 mmol, 2 equiv) in portion at 0° C. under air atmosphere. The resulting mixture was stirred for 1 h at room temperature under air atmosphere. To the above mixture was added dimethylcarbonate (436.34 mg, 4.54 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for additional 2 h at room temperature. The reaction was quenched by the addition of water/ice (20 mL) at room temperature. The resulting mixture was extracted with CH2Cl2 (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford methyl 2-cyano-2-(6-methylpyridin-2-yl)acetate (330 mg, 45.86%) as orange solid. LCMS: m/z (ESI), [M+H]+=191.20. 1H NMR (CDCl3, 400 MHz) δ 2.49 (3H, s), 3.82 (3H, s), 6.49 (1H, d), 7.18 (1H, d), 7.54 (1H, dd).

Synthesis of Intermediate I-30

Synthesis of Intermediate I-31

Synthesis of Intermediate I-32

Step 3: To a stirred mixture of ester I-32b (310 mg, 0.87 mmol, 1 equiv) in methanol (4 mL) was added LiOH·H2O (146.41 mg, 3.49 mmol, 4 equiv) in water (1 mL) dropwise at 25° C. under nitrogen atmosphere. The resulting mixture was stirred for overnight at 60° C. under nitrogen atmosphere. After cooled to 25° C., the resulting mixture was concentrated under reduced pressure and diluted with methanol (5 mL). The solution was acidified with HCl (1M, 10 mL). To the above solution was added DCM (10 mL). After extraction, the reaction mixture was evaporated under reduced pressure. The residue was diluted with DMF (2 mL) and purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, Phase B MeCN, Phase A water, from 10% to 50% gradient in 10 min; detector, UV 254 nm and hold for 20 min at 16.5%. After lyophilization, 3-[(tert-butoxycarbonyl)amino]-2-[4-(dimethylphosphoryl)phenyl]propanoic acid I-32 (200 mg, 67.17%) was obtained as a white solid. 1H NMR (DMSO-d6, 400 MHZ) δ 1.33 (9H, s), 1.60 (6H, d), 3.27 (2H, m), 7.37 (2H, dd), 7.60 (2H, m).

Synthesis of Intermediate I-33

Synthesis of Intermediate I-34

Synthesis of Intermediate I-35

Step 2: To a stirred mixture of methyl 2-cyano-2-(5-methoxypyridin-3-yl)acetate (700 mg, 3.40 mmol, 1 equiv) and CoCl2·6H2O (1.21 g, 5.09 mmol, 1.5 equiv) in methanol (15 mL) was added NaBH4 (385.27 mg, 10.19 mmol, 3 equiv) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. The reaction was quenched with water at room temperature. The resulting mixture was filtered and the filter cake was washed with methanol (1×10 mL). The resulting mixture was extracted with DCM (3×100 mL). The combined organic layers were washed with DCM (2×3 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (35:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(5-methoxypyridin-3-yl)propanoate I-35b (250 mg, 23.73%) as a light yellow solid. LCMS: m/z (ESI), [M+H]+=311.15.

Step 3: Into a 50 mL round-bottom flask were added ester I-35b (80 mg, 0.26 mmol, 1 equiv) and LiOH (12.35 mg, 0.52 mmol, 2 equiv) in water (3 mL) and methanol (3 mL) at room temperature. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The mixture was acidified to pH 5 with conc. HCl. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 28% to 32% gradient in 15 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum. This resulted in 3-[(tert-butoxycarbonyl)amino]-2-(5-methoxypyridin-3-yl)propanoic acid I-35 (40 mg, 52.37%) as an off-white solid. LCMS: m/z (ESI), [M+H]+=297.05.

Synthesis of Intermediate I-36

Step 2: A solution of methyl 2-cyano-2-(1-methyl-2-oxopyridin-4-yl)acetate (1.1 g, 5.34 mmol, 1 equiv) in methanol (15 mL) was treated with Boc2O (4.66 g, 21.34 mmol, 4 equiv) and CoCl2·6H2O (3.30 g, 13.87 mmol, 2.6 equiv) for 5 min at 0° C. under air atmosphere followed by the addition of NaBH4 (1.61 g, 42.68 mmol, 8 equiv) in portions at 0° C. The resulting mixture was stirred for 15 h at 70° C. under air atmosphere. The reaction was quenched by the addition of water (80 mL) at room temperature. The resulting mixture was filtered and the filter cake was washed with DCM (3×10 mL). The aqueous layer was extracted with CH2Cl2 (3×80 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (18:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-(1-methyl-2-oxopyridin-4-yl)propanoate I-36b (445 mg, 26.88%) as a white oil. LCMS: m/z (ESI), [M+H]+=311.15.

Synthesis of Intermediate I-37

Step 4: Into a 20 mL vial were added methyl 3-[(tert-butoxycarbonyl)amino]-2-(4-cyanophenyl)propanoate (900 mg, 2.96 mmol, 1 equiv), methanol (3.75 mL) and LiOH·H2O (248.16 mg, 5.91 mmol, 2 equiv) in water (2.5 mL) at room temperature. The resulting mixture was stirred for 1.5 h at 70° C. The mixture was acidified to pH 5 with HCl (aq.). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 0% to 100% gradient in 30 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum, to afford 3-[(tert-butoxycarbonyl)amino]-2-(4-carbamoylphenyl)propanoic acid I-37 (800 mg, 87.74%) as a white solid. LCMS: m/z (ESI), [M+H−tBu]+=253.00.

Synthesis of Intermediate I-38

Step 2: A mixture of methyl 2-[(tert-butoxycarbonyl)amino]-2-[4-(hydroxymethyl)phenyl]acetate (390 mg, 1.30 mmol, 1 equiv) and LiOH·H2O (109 mg, 2.60 mmol, 2 equiv) in THF (4 mL) and H2O (1 mL) for 5 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC with the following conditions (The mobile phase consisted of a mixture of solvent 0.1% NH4HCO3 in water and 0.1% NH4OH in acetonitrile. A constant gradient from 95% aqueous/5% organic to 5% aqueous/95% organic mobile phase over the course of 25 minutes was utilized. The flow rate was constant at 40 mL/min.) to give 312 mg of [(tert-butoxycarbonyl)amino][4-(hydroxymethyl)phenyl]acetic acid I-38 (84%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=282.13.

Synthesis of Intermediate I-39

Step 2: A mixture of methyl 2-(3-bromophenyl)prop-2-enoate (800 mg, 3.32 mmol, 1 equiv) in DMF (5 mL) was added 1-methylpiperazine (1661.86 mg, 16.59 mmol, 5 equiv). The resulting mixture was stirred for 2 h at 25° C. under nitrogen atmosphere. The reaction mixture was diluted with EA (20 mL). The residue was washed with saturated NaHCO3 (2×10 mL), then water (2×20 mL), and saturated NaCl (3×20 mL). The resulting organic layer was dried with Na2SO4. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 30 min at 65%. After evaporated the water, methyl 2-(3-bromophenyl)-3-(4-methylpiperazin-1-yl)propanoate I-39b (740 mg, 65.35%) was obtained as a yellow oil. LCMS: m/z (ESI), [M+H]+=341.00, 343.00.

Step 3: To a stirred mixture of methylpiperazine I-39b (740 mg, 1.32 mmol, 1 equiv) and Zn(CN)2 (154.84 mg, 1.32 mmol, 1.0 equiv) in DMF (3.0 mL) was added Pd(PPh3)4 (152.39 mg, 0.13 mmol, 0.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2.0 h at 80° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 30 min at 28%, after evaporated the solvent, methyl 2-(3-cyanophenyl)-3-(4-methylpiperazin-1-yl)propanoate (400 mg, 52.50%) was obtained as a yellow oil. LCMS: m/z (ESI), [M+H]+=288.05.

Step 4: To a stirred mixture of methyl 2-(3-cyanophenyl)-3-(4-methylpiperazin-1-yl)propanoate (400 mg, 1.39 mmol, 1 equiv) in THF (8.0 mL) and LiOH·H2O (175.22 mg, 4.18 mmol, 3.0 equiv) in water (2.0 mL) was added dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 4.0 h at 25° C. under nitrogen atmosphere. After reaction, the resulting mixture was concentrated under vacuum. The resulting mixture was diluted with methanol (4.0 mL). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 0% to 50% gradient in 10 min; detector, UV 254 nm, and hold for 20 min at 9.8% to afford 2-(3-cyanophenyl)-3-(4-methylpiperazin-1-yl)propanoic acid I-39 (240 mg, 63.08%) as a white solid. LCMS: m/z (ESI), [M+H]+=274.10.

Synthesis of Intermediate I-40

Step 2: Into a 50 mL round-bottom flask were added ester I-40b (2 g, 9.56 mmol, 1 equiv), hydroxylamine hydrochloride (0.86 g, 12.43 mmol, 1.3 equiv), pyridine (0.83 g, 10.52 mmol, 1.1 equiv) and methanol (10 mL) at 0° C. The resulting mixture was stirred for 6 h at room temperature. The resulting mixture was diluted with CH2Cl2 (40 mL). The resulting mixture was washed with 1×40 mL of HCl (0.5M), 1×40 ml of water, and 1×40 mL of saturated NaCl (aq). The organic layer was concentrated under vacuum. The precipitated solids in water phase were collected by filtration and washed with water (3×10 mL). This resulted in ethyl (2E)-2-(N-hydroxyimino)-2-(5-methoxypyridin-3-yl)acetate (1.78 g, 83.04%) as a white solid. LCMS: m/z (ESI), [M+H]+=225.15.

Synthesis of Intermediate I-41

Synthesis of Intermediate I-42

Step 2: To a stirred mixture of tert-butyl N-[2-oxo-2-(pyridin-3-yl)ethyl]carbamate (640 mg, 2.71 mmol, 1 equiv) and hydroxylamine hydrochloride (564.70 mg, 8.13 mmol, 3 equiv) in methanol (10 mL) was added pyridine (642.79 mg, 8.13 mmol, 3 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for overnight room temperature. After reaction, the resulting mixture was quenched by water (20 mL). The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford tert-butyl N-[2-(hydroxyimino)-2-(pyridin-3-yl)ethyl]carbamate I-42b (400 mg, 58.77%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=252.00.

Synthesis of Intermediate I-43

Step 3: Into a 40 mL vial were added amine I-43c (1.5 g, 5.70 mmol, 1 equiv) and BH3·C4H8O (20 mL, 208.98 mmol, 36.69 equiv) at room temperature. The resulting mixture was stirred for 24 h at 70° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The reaction was quenched by the addition of methanol (20 mL) at 0° C. And The resulting mixture was stirred for 2 h, concentrated under reduced pressure and The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 0% to 50% gradient in 40 min; detector, UV 220 nm to afford 1-(3-methoxyphenyl)-2-(4-methylpiperazin-1-yl) ethanamine I-43 (500 mg, 35.20%) as a colorless oil. LCMS: m/z (ESI), [M+H−tBu]+=250.05.

Synthesis of Intermediate I-44

Step 1: A mixture of tribromane-tetrabutylamine (6.42 g, 13.32 mmol, 1 equiv) and 1-[4-(hydroxymethyl)phenyl]ethanone I-44a (2 g, 13.32 mmol, 1 equiv) in ACN (100 mL) and acetone (10 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with MTBE (2×20 mL). The combined organic layers were washed with water (7×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 2-bromo-1-[4-(hydroxymethyl)phenyl]ethanone I-44b (2 g, 65.56%) as a yellow oil. The resulting mixture was used in the next step directly.

Synthesis of Intermediate I-45

Synthesis of Intermediate I-46

Synthesis of Intermediate I-47

Step 2: To a stirred mixture of methylpiperidine I-47b (880 mg, 3.77 mmol, 1 equiv) in methanol (10 mL) was added NaBH3CN (948.08 mg, 15.09 mmol, 4 equiv) and CH3COONH4 (3488.89 mg, 45.26 mmol, 12 equiv) in portions at 0° C. under air atmosphere. The resulting mixture was stirred for overnight at 70° C. under air atmosphere. After cooling to room temperature, the reaction was quenched by the addition of sat. NH4Cl (aq.) (30 mL) at 0° C. The resulting mixture was extracted with CH2Cl2 (3×40 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm hold for 25 min at 40% to afford 1-(3-methoxyphenyl)-1-(1-methylpiperidin-4-yl) methanamine I-47 (340 mg, 38.47%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=235.15.

Synthesis of Intermediate I-48

Synthesis of Intermediate I-49

Step 2: Into a 100 mL round-bottom flask were added nitro-indole I-49b (2.5 g, 7.61 mmol, 1 equiv) and Zn (3.98 g, 60.91 mmol, 8 equiv) at 0° C. A solution of NH4Cl (1.79 g, 33.51 mmol, 5 equiv) in water (10 mL) was added to the above mixture at 0° C. The resulting mixture was stirred for 1 h at 0° C. under nitrogen atmosphere. The resulting mixture was filtered and the filtered cake was washed with ethyl acetate (2×20 mL). The filtrate was diluted with water (50 mL) and the residue was extracted with EA (2×50 mL), the combined organic layer was dried over anhydrous Na2SO4, filtered and evaporated to afford a crude solid. The residue was purified by prep-TLC with CH2Cl2/methanol (25:1, v/v) to afford tert-butyl 4-(7-amino-1H-indol-3-yl) pyrazole-1-carboxylate I-49 (1 g, 44.02%) as a brown solid. LCMS: m/z (ESI), [M−tBu]+=243.10.

Synthesis of Intermediate I-50

Step 2: To a mixture of nitro-indole I-50b (200 mg, 557.93 μmol) and ammonia hydrochloride (298.45 mg, 5.58 mmol) in ethanol (10 mL) and water (10 mL) was added Iron powder (155.79 mg, 2.79 mmol). Then the reaction mixture was stirred and heated at 80° C. for 3 h. The reaction mixture was filtered through celite and washed with ethanol (3×10 mL), and the filtrate was concentrated under reduced pressure. The residue was dissolved 50 mL of ethyl acetate, filtered and the filtration was concentrated under reduced pressure to afford 3-[1-(2-trimethylsilylethoxymethyl) pyrazol-4-yl]-1H-indol-7-amine I-50 (160 mg, 87% yield) as brown solid. LCMS: m/z (ESI), [M+H]+=329.3.

Synthesis of Intermediate I-51

Synthesis of Intermediate I-52

Step 3: A mixture of ester I-52c (990 mg, 3.41 mmol, 1 equiv) in THF (4.0 mL) was added LiOH·H2O (572.34 mg, 13.64 mmol, 4 equiv) in water (1.0 mL). The resulting mixture was stirred for 6 h at room temperature under nitrogen atmosphere. After reaction, the resulting mixture was concentrated under vacuum and diluted with DMF (2 mL). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 25 min at 25%, after evaporated the water to afford [(tert-butoxycarbonyl)amino](3-cyanophenyl) acetic acid I-52 (540 mg, 57.31% yield) as a white solid. LCMS: m/z (ESI), [M+H]+=277.00.

Synthesis of Intermediate I-53

Synthesis of Intermediate I-54

Step 2: To a solution of ester I-54a (180 mg, 0.44 mmol, 1 equiv) in THF (10 mL) and H2O (5 mL) was added LiOH·H2O (36.98 mg, 0.88 mmol, 2 equiv) at room temperature under nitrogen atmosphere. The mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, ACN in water, 10% to 50% gradient in 10 min; detector, UV 220 nm. This resulted in 3-[(tert-butoxycarbonyl)amino]-2-(3-{[(tert-butoxycarbonyl)amino]methyl}phenyl)-propanoic acid I-54 (159 mg, 91.27% yield) as a white solid. LCMS: m/z (ESI), [M+H]+=395.10.

Synthesis of Intermediate I-55

Step 2: To a stirred mixture of nitrile I-55b (500 mg, 1.78 mmol, 1 equiv) and CoCl2·6H2O (1268.63 mg, 5.33 mmol, 3 equiv) in MeOH (2 mL) were added Boc2O (1163.74 mg, 5.33 mmol, 3 equiv) and NaBH4 (168.10 mg, 4.44 mmol, 2.5 equiv) in portions at 0° C. under air atmosphere. The resulting mixture was stirred for 1 h at room temperature under air atmosphere. The reaction was quenched with Water at room temperature. The resulting mixture was filtered and the filter cake was washed with MeOH (2×4 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography, eluted with PE/EA (3:1, v/v) to afford methyl 2-[4-(benzyloxy)phenyl]-3-[(tert-butoxycarbonyl)amino]propanoate I-55c (371 mg, 54.15% yield) as a white solid. LCMS: m/z (ESI), [M−tBu]+=330.15.

Synthesis of Intermediate I-56

Step 1: Into a 50 mL round-bottom flask were added cyclohexaneacetic acid I-56a (2500 mg, 17.58 mmol, 1.0 equiv) and SOCl2 (1.5 mL, 20.68 mmol) at room temperature. The resulting mixture was stirred for 1 h at 80° C. under nitrogen atmosphere. The reaction mixture was cooled down to room temperature. To the above mixture was added phosphorus tribromide (4.26 mL, 44.83 mmol, 2.55 equiv), bromine (3512.00 mg, 21.98 mmol, 1.25 equiv) at room temperature. The resulting mixture was stirred for additional 2 h at 80° C. The mixture was allowed to cool down to room temperature. Then MeOH (2.5 mL) was added to the above solution. The resulting mixture was stirred for 2 h at 70° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The reaction was quenched with sat. NaS2O3 (aq) (50 mL) at room temperature. The resulting mixture was extracted with DCM (2×50 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and evaporated to afford a crude solid. The crude solid was purified by silica column chromatography, eluted with PE/EA (5:1, v/v) to afford methyl 2-bromo-2-cyclohexylacetate I-56b (2000 mg, 40.25% yield) as an off-white oil. 1H NMR: (CDCl3, 400 MHz) δ 0.90-1.21 (4H, m), 1.27 (2H, qt), 1.60-1.81 (5H, m), 1.88 (1H, tdt), 2.06 (1H, dtd), 3.77 (3H, s), 4.02 (1H, d).

Step 3: A solution of methylpiperazine I-56c (590 mg, 2.32 mmol, 1 equiv) and LiOH·H2O (194.66 mg, 4.64 mmol, 2 equiv) in MeOH (3.0 mL) and H2O (3.0 mL) was stirred for 2 h at 70° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The mixture was acidified to pH 5 with 1M HCl (aq). The resulting mixture was concentrated under vacuum. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeOH in water, 10% to 50% gradient in 30 min; detector, UV 220 nm. This resulted in cyclohexyl (4-methylpiperazin-1-yl) acetic acid I-56 (240 mg, 35.8% yield) as an off-white solid. LCMS: m/z (ESI), [M+H]+=241.15.

Synthesis of Intermediate I-57

Step 1: To a stirred mixture of methyl 3-[(tert-butoxycarbonyl)amino]-2-(4-cyanophenyl)propanoate I-14c (740 mg, 2.43 mmol, 1 equiv) and CoCl2·6H2O (1156.98 mg, 4.86 mmol, 2 equiv) in MeOH (10 mL) was added NaBH4 (919.81 mg, 24.31 mmol, 10 equiv) in portions at 0° C. under air atmosphere. The resulting mixture was stirred for 5 h at room temperature under air atmosphere. After reaction, the reaction was quenched with water (30 mL) at 0° C. The resulting mixture was filtered and the filter cake was washed with MeOH (3×10 mL). The filtrate was extracted with EtOAc (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the crude product. The crude was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 20 min at 15%, after evaporated the solvent under reduced pressure to afford methyl 2-[4-(aminomethyl)phenyl]-3-[(tert-butoxycarbonyl)amino]propanoate I-57b (300 mg, 40.01% yield) as a white solid. LCMS: m/z (ESI), [2M+H]+=617.40.

Step 2: To a stirred mixture of amine I-57b (200 mg, 0.649 mmol, 1 equiv) and acetyl chloride (51.93 mg, 0.66 mmol, 1.02 equiv) in DCM (3.0 mL) was added Et3N (196.89 mg, 1.95 mmol, 3 equiv) in portions at 0° C. under air atmosphere. The resulting mixture was stirred for 3 h at room temperature under air atmosphere. After reaction, the resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with CH2Cl2 (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/MeOH (30:1, v/v) to afford methyl 3-[(tert-butoxycarbonyl)amino]-2-[4-(acetamidomethyl)phenyl]propanoate I-57c (100 mg, 44.00% yield) as a white solid. LCMS: m/z (ESI), [M-Boc]+=251.15.

Step 3: To a stirred mixture of ester I-57c (150 mg, 0.43 mmol, 1 equiv) in MeOH (4.0 mL) was added LiOH·H2O (71.85 mg, 1.71 mmol, 4 equiv) in water (1.0 mL) dropwise at room temperature under air atmosphere. The resulting mixture was stirred for overnight at 60° C. under air atmosphere. After cooling to room temperature, the resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with MeOH (2.0 mL). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in Water, 10% to 50% gradient in 10 min; detector, UV 254 nm, hold for 30 min at 26%. After evaporated solvent under reduced pressure to afford 3-[(tert-butoxycarbonyl)amino]-2-[4-(acetamidomethyl)phenyl]propanoic acid I-57 (92 mg, 63.89% yield) as a white solid. LCMS: m/z (ESI), [M+Na]+=359.00.

Synthesis of Intermediate I-58

Step 3: Into a 100 mL round-bottom flask were added ester I-58b (570 mg, 1.56 mmol, 1.00 equiv) and LiOH·H2O (163.18 mg, 3.89 mmol, 2.5 equiv) and MeOH (4 mL) and water (2 mL) at room temperature. The resulting mixture was stirred for 16 h at room temperature under air atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeOH in water (0.1% FA), 0% to 25% gradient in 20 min; detector, UV 254 nm. The resulting mixture was concentrated under reduced pressure to afford 3-[(tert-butoxycarbonyl)amino]-2-(4-{[(methylcarbamoyl)-oxy]methyl}phenyl)propanoic acid I-58 (520 mg, 94.86% yield) as an off-white solid. LCMS: m/z (ESI), [M+H-Boc]+=253.15.

Synthesis of Intermediate I-59

Synthesis of Intermediate I-60

Synthesis of Compounds

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(4-(hydroxymethyl)phenyl)propanamide

Following the procedure above starting with amide C1-b, Example 2 (enantiomer 1) can be obtained.

Preparation of 4-(1-((3-(1H-pyrazol-4-yl)-1H-indol-7-yl)amino)-3-amino-1-oxopropan-2-yl)benzyl 3,5-dimethylbenzoate

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-amino-2-phenylacetamide (racemic)

Step 1: To a mixture of nitro-indole I-7a (5 g, 11.30 mmol) and ammonia hydrochloride (6.05 g, 113.05 mmol) in EtOH (30 mL) and water (30 mL) was added iron powder (3.16 g, 56.52 mmol, 401.63 μL). Then the reaction mixture was stirred and heated at 80 CC for 3 h. The reaction mixture was filtered through celite and washed with ethanol (50 mL×3), and the filtrate was concentrated. The residue was dissolved into 200 ml of ethyl acetate, filtered and the filtrate was concentrated under reduced pressure to afford amine I-7b (3.10 g, 67% yield) as brown solid. LCMS: tR=2.07 min in 3 min chromatography (3 min-5-95% MeCN in water (0.02% TFA), Waters Acquity UPLC BEH C18 1.7 μm, 2.1*50 mm, 40° C.), MS (ESI) m/z=413.2 [M+H]+.

Preparation of (2R)-2-amino-2-phenyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of (2S)-2-amino-2-phenyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of [4-[(1S)-1-(aminomethyl)-2-oxo-2-[[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]amino]ethyl]phenyl]methyl 6-nitrooxyhexanoate

Step 2: To a mixture of 5-(nitrooxy) pentanoic acid I-11b (120 mg, 0.74 mmol, 5 equiv) in DCM (10 mL) were added EDCI (57 mg, 0.30 mmol, 2 equiv) and DMAP (2 mg, 0.016 mmol, 0.1 equiv) for 30 min at 0° C. under nitrogen atmosphere followed by the addition of alcohol C-1c (85 mg, 0.15 mmol, 1 equiv) dropwise at 0° C. The reaction was stirred for 16 h after the mixture warmed to room temperature. The reaction was quenched with water (20 mL) at room temperature and extracted with CH2Cl2 (3×20 mL). The combined organic layers were washed with sat. NaCl (3×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC eluted with dichloromethane/methanol (30:1, v/v) to give 60 mg of enantiomer C-11 as an orange solid (40%). LCMS: m/z (ESI), [M+H]+=721.31.

Step 2: A mixture of bromoester C-12b (120 mg, 0.17 mmol, 1 equiv) and AgNO3 (56 mg, 0.34 mmol, 2 equiv) in MeCN (15 mL) was stirred for 2 h at 70° C. under nitrogen atmosphere. The reaction was light sensitive and the light should be avoided. The mixture was cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with CH2Cl2 (3×50 mL) and concentrated under reduced pressure to afford nitrooxy ester C-12c as an orange solid (25 mg, 26% yield). LCMS: m/z (ESI), [M+H]+=707.2.

Preparation of (R)-N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-phenylpropanamide

Step 2: To a stirred mixture of amine I-14b (85 mg, 135.09 μmol), (R)-3-((tert-butoxycarbonyl)amino)-2-phenylpropanoic acid (39.42 mg, 148.60 μmol) and DIPEA (26.19 mg, 202.64 μmol, 35.29 μL) in NMP (2.00 mL) was cooled and stirred at 0° C. Then HATU (56.80 mg, 149.39 μmol) was added in one portion at this temperature. The resulting mixture was stirred at room temperature for 17 h. Then detected the reaction by LCMS, the reaction was incomplete. More (R)-3-((tert-butoxycarbonyl)amino)-2-phenylpropanoic acid (107.52 mg, 405.27 μmol) was added into the reaction. The reaction mixture was cooled and stirred at 0° C. Then more DIPEA (69.84 mg, 540.36 μmol, 94.12 μL) was added in one portion at this temperature. The resulting mixture was stirred at room temperature for 31 h. The solution was purified by C18 flash chromatography column, elution gradient from 0 to 80% MeCN in water (6 mmol/L NH4HCO3). Pure fractions were lyophilized to dryness to afford amide (R)-enantiomer C-14c (15 mg, 16% yield) as white solid. LCMS: tR=2.18 min in 3 min chromatography (3 min-5-95% MeCN in water (0.02% TFA), Waters Acquity UPLC BEH C18 1.7 μm, 2.1*50 mm, 40° C.), MS (ESI) m/z=700.5 [M+H]+.

Step 2: To a mixture of pyrazole I-15b (200 mg, 557.93 μmol) and ammonia hydrochloride (298.45 mg, 5.58 mmol) in ethanol (10 mL) and water (10 mL) was added iron powder (155.79 mg, 2.79 mmol). Then the reaction mixture was stirred and heated at 80° C. for 3 h. The reaction mixture was filtered through celite and washed with ethanol (10 mL×3), and the filtrate was concentrated. The residue was dissolved 50 mL of ethyl acetate, filtered and the filtration was concentrated under reduced pressure to afford amine I-15c (160 mg, 87% yield) as brown solid. LCMS: tR=1.56 min in 3 min chromatography (3 min-5-95% MeCN in water (0.02% TFA), Waters Acquity UPLC BEH C18 1.7 μm, 2.1*50 mm, 40° C.), MS (ESI) m/z=329.3 [M+H]+.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(3-methoxyphenyl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(3-cyanophenyl)propanamide

Step 4: A mixture of TFA (2 mL) and racemic amide C-20a (170 mg, 0.30 mmol, 1 equiv) in DCM (6 mL) was stirred for 1 h at room temperature under air atmosphere. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (20 mL). The mixture was basified to pH 8 with saturated NaHCO3 (aq.).

Preparation of 3-amino-2-cyclohexyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide (racemic)

Step 2: A solution of racemic amide C-22a (40 mg, 0.069 mmol, 1 equiv) in TBAF (7M) in THF (2 mL) was stirred for overnight at room temperature under nitrogen atmosphere. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with DCM (3×20 mL). The combined organic layers dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with DCM/methanol (15:1, v/v) to afford pyrazole C-22b (15 mg, 48.32%) as a white solid. LCMS: m/z (ESI), [M+H]+=452.30.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-cyclohexylpropanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(tetrahydro-2H-pyran-4-yl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(3-methoxyphenyl)-2-(1-methylpiperidin-4-yl) acetamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(pyridin-3-yl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(3-methoxyphenyl)-3-(4-methylpiperazin-1-yl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-(4-methylpiperazin-1-yl)-2-phenylpropanamide

Preparation of (2S)-2-amino-2-(3-methoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of (2R)-2-amino-2-(3-methoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(1-methylpiperidin-4-yl)-2-phenylacetamide

Preparation of 2-(4-methylpiperazin-1-yl)-2-phenyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(4-methylpiperazin-1-yl)-2-phenylacetamide

Preparation of 3-(1-((3-(1H-pyrazol-4-yl)-1H-indol-7-yl)amino)-3-amino-1-oxopropan-2-yl)benzyl methylcarbamate

Preparation of 3-amino-2-benzyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propenamide

Preparation of (2R)-3-amino-2-benzyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of (2S)-3-amino-2-benzyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of (2S)-3-amino-N-[3-(3,5-dimethyl-1H-pyrazol-4-yl)-1H-indol-7-yl]-2-phenyl-propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(4-cyanophenyl)propanamide

Step 1: To a stirred mixture of acid I-14 (400 mg, 1.38 mmol, 1 equiv) in CH3CN (8.0 mL) was added NMI (339.38 mg, 4.13 mmol, 3.0 equiv), amine I-49 (419.28 mg, 1.41 mmol, 1.02 equiv) and TCFH (773.16 mg, 2.76 mmol, 2.0 equiv) in portion at 25° C. under air atmosphere. The resulting mixture was stirred for overnight at 25° C. under air atmosphere. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford racemic amide C-58a (450 mg, 57.23%) as a yellow oil. LCMS: m/z (ESI), [M-Boc-tBu]+=471.25.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(6-(hydroxymethyl)pyridin-3-yl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(5-methylpyridin-3-yl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(3-hydroxyphenyl)propanamide

Step 2: Into a 50 mL round-bottom flask were added tert-butyl 4-(7-{2-[3-(benzyloxy)phenyl]-3-[(tert-butoxycarbonyl)amino]propanamido}-1H-indol-3-yl) pyrazole-1-carboxylate (330 mg, 0.51 mmol, 1 equiv) and Pd/C (20 mg, 0.19 mmol, 0.37 equiv) in methanol (5 mL) at room temperature. The resulting mixture was stirred for overnight at room temperature under hydrogen atmosphere. The resulting mixture was filtered and the filtered cake was washed with methanol (4×20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 0% to 100% gradient in 30 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to afford racemic amide C-64a (80 mg, 28.13%) as a white solid. LCMS: m/z (ESI), [M+H]+=562.20.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl)propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-amino-2-(3-cyanophenyl) acetamide

Step 2: A mixture of racemic amide C-68 (320 mg, 0.58 mmol, 1 equiv) in TFA (1.0 mL) and DCM (4.0 mL) was stirred for 1.0 h at room temperature under nitrogen atmosphere. After reaction, the resulting mixture was concentrated under vacuum and diluted with methanol (2.0 mL), the residue was basified to pH 9 with NH3 aq. The crude product was purified by prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 30*150 mm, 5 um; Mobile Phase A: water (10 mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 15% B to 40% B in 8 min; wavelength: 254 nm; tR=7.05 min) to afford Example 100, racemic (200 mg, 97.62%) as a white solid.

Preparation of 3-(2-amino-1-{[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]carbamoyl}ethyl)benzoic acid

Preparation of 4-(2-amino-1-{[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]carbamoyl}ethyl)benzoic acid

Step 1: To a stirred solution of acid I-53 (50 mg, 0.14 mmol, 1 equiv) and amine I-49 (48.99 mg, 0.16 mmol, 1.2 equiv) in DMF (4 mL) were added HATU (104.05 mg, 0.27 mmol, 2 equiv) and DIEA (53.05 mg, 0.411 mmol, 3 equiv) at room temperature under air atmosphere. The resulting mixture was stirred for 3 h at room temperature under air atmosphere. The reaction was quenched by the addition of water (5 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by pPrep-TLC with CH2Cl2/methanol (10:1, v/v) to afford racemic amide C-71 (50 mg, 56.59%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=646.40.

Preparation of 4-(2-amino-1-{[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]carbamoyl}ethyl)-N,N-dimethylbenzamide

Step 1: To a stirred solution of racemic amide C-71 (350 mg, 0.54 mmol, 1 equiv) in methanol (20 mL), H2O (5 mL) were added LiOH (51.92 mg, 2.17 mmol, 4 equiv) at room temperature. The mixture was stirred for 2 h at room temperature under air atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 50% to 60% gradient in 10 min; detector, UV 254 nm to afford racemic acid C-72a (300 mg, 113.07%) as a pink solid. LCMS: m/z (ESI), [M+H]+=490.53.

Step 2: To a stirred solution of racemic acid C-72a (120 mg, 0.25 mmol, 1 equiv) and dimethylamine (13.26 mg, 0.29 mmol, 1.2 equiv) in DMF (8 mL, 103.37 mmol) were added HATU (186.42 mg, 0.49 mmol, 2 equiv) and DIEA (95.05 mg, 0.74 mmol, 3 equiv) at room temperature under air atmosphere. The resulting mixture was stirred for 3 h at room temperature under air atmosphere. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. The residue was purified by prep-TLC with PE/EA (1:1, v/v) to afford racemic amide C-72b (90 mg, 71.07%) as a white solid. LCMS: m/z (ESI), [M+H]+=517.60.

Preparation of 4-(2-amino-1-{[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]carbamoyl}ethyl)-N-methylbenzamide

Step 1: To a stirred solution of racemic acid C-72a (120 mg, 0.25 mmol, 1 equiv) and methylamine (9.14 mg, 0.29 mmol, 1.2 equiv) in DMF (8 mL, 103.37 mmol) were added HATU (186.42 mg, 0.49 mmol, 2 equiv) and DIEA (95.05 mg, 0.74 mmol, 3 equiv) at room temperature under air atmosphere. The resulting mixture was stirred for 2 h at room temperature under air atmosphere. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (10:1, v/v) to afford racemic amide C-73 (50 mg, 40.59%) as a white solid. LCMS: m/z (ESI), [M+H]+=503.45.

Preparation of 4-(1-((3-(1H-pyrazol-4-yl)-1H-indol-7-yl)amino)-3-amino-1-oxopropan-2-yl)benzamide

Preparation of 3-amino-2-[3-(hydroxymethyl)phenyl]-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-[3-(aminomethyl)phenyl]-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-[4-(aminomethyl)phenyl]-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 1: To a stirred mixture of acid I-4 (80 mg, 0.20 mmol, 1 equiv) and DIEA (78.64 mg, 0.61 mmol, 3 equiv) in DMF (1.0 mL) was added amine I-49 (61.72 mg, 0.21 mmol, 1.02 equiv), and HATU (154.23 mg, 0.41 mmol, 2 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2.0 h at room temperature under nitrogen atmosphere. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with CH2Cl2 (3×10 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford racemic amide C-77 (130 mg, 94.99%) as a yellow oil. LCMS: m/z (ESI), [M−+H]+=675.40.

Preparation of 3-amino-2-{3-[(dimethylamino)methyl]phenyl}-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(4-hydroxyphenyl)-propanamide

Preparation of 3-amino-2-(4-aminophenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-(3-aminophenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 2-amino-2-(3,4-dimethoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of 2-amino-2-(3,5-dimethoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of 3-amino-2-(2-methoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 1: To a stirred mixture of acid I-23 (210 mg, 0.71 mmol, 1 equiv) and NMI (175.15 mg, 2.13 mmol, 3 equiv) in DMF were added amine I-50 (256.93 mg, 0.782 mmol, 1.1 equiv) and TCFH (997.54 mg, 3.56 mmol, 5 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The resulting mixture was diluted with water (30 mL). The aqueous layer was extracted with CH2Cl2 (3×20 mL). The resulting mixture was concentrated under reduced pressure. The crude product was purified by reverse phase flash with to afford racemic amide C-84 (230 mg, 53.39%) as a white solid. LCMS: m/z (ESI), [M+H]+=606.81.

Preparation of 3-amino-2-(4-fluorophenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-(4-cyanophenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 2: To a stirred solution of tert-butyl 4-{7-[2-(4-bromophenyl)-3-[(tert-butoxycarbonyl)amino]propanamido]-1H-indol-3-yl}pyrazole-1-carboxylate (290 mg, 0.46 mmol, 1 equiv) and Zn(CN)2 (30.36 mg, 0.46 mmol, 1 equiv) in DMF (3.0 mL) was added Pd(PPh3)4 (53.66 mg, 0.046 mmol, 0.1 equiv) at 25° C. under nitrogen atmosphere. The resulting mixture was stirred for 16 h at 80° C. under nitrogen atmosphere. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with ethyl acetate (3×40 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford racemic amide C-86 (200 mg, 75.4 8%) as a yellow solid. LCMS m/z (ESI), [M+H]+=471.20.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(m-tolyl)propanamide

Preparation of 3-amino-2-[4-(2-methoxyethoxy)phenyl]-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 1: A mixture of acid I-27 (90 mg, 0.27 mmol, 1 equiv) and amine I-49 (79.12 mg, 0.27 mmol, 1 equiv) and NMI (123.67 mg, 1.51 mmol, 5.68 equiv) and TCFH (148.81 mg, 0.53 mmol, 2 equiv) in CH3CN (10 mL) was stirred for overnight at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford racemic amide C-88 (122 mg, 74.24%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=620.

Preparation of 3-amino-2-(3-chlorophenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-(4-methoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-(oxan-3-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 1: To a stirred mixture of acid I-12 (130 mg, 0.40 mmol, 1 equiv) and NMI (99.33 mg, 1.21 mmol, 3 equiv) in CH3CN (3.0 mL) was added TCFH (226.30 mg, 0.81 mmol, 2 equiv) and amine I-49 (122.72 mg, 0.41 mmol, 1.02 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2.0 h at room temperature under nitrogen atmosphere. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford racemic amide C-92 (190 mg, 93.75%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=603.40.

Preparation of 3-amino-2-(6-methylpyridin-2-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-cyclopentyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 1: To a stirred mixture of acid I-30 (200 mg, 0.78 mmol, 1 equiv) and amine I-50 (306.37 mg, 0.93 mmol, 1.2 equiv) in CH3CN (10 mL) were added NMI (63.81 mg, 0.78 mmol, 1 equiv) and TCFH (261.68 mg, 0.93 mmol, 1.2 equiv) dropwise at room temperature under air atmosphere. The resulting mixture was stirred for overnight at room temperature under air atmosphere. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with CH2Cl2 (3×20 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with DCM/methanol (20:1, v/v) to afford racemic amide C-95 (385 mg, 87.24%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=568.80.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-3-amino-2-(tetrahydrofuran-3-yl)propanamide

Step 2: To a stirred solution of racemic amide C-94a (100 mg, 0.18 mmol, 1 equiv) in DCM (2 mL) was added HCl (gas) in 1,4-dioxane (2 mL) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The residue was basified to pH 8 with saturated NH3 (aq) at 0° C.

Preparation of 3-amino-2-[4-(dimethylphosphoryl)phenyl]-N-[3-(1H-pyrazol-4-yl)-1H-Indol-7-yl]propanamide

Preparation of 3-amino-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]-2-(pyridin-2-yl)propanamide

Step 1: To a stirred mixture of acid I-33 (80 mg, 0.30 mmol, 1 equiv) in DMF (1.2 mL) was added HATU (228.46 mg, 0.60 mmol, 2 equiv), DIEA (155.31 mg, 1.20 mmol, 4 equiv) and amine I-49 (90.52 mg, 0.30 mmol, 1.01 equiv) was added at 25° C. The resulting mixture was stirred at 25° C. for 2 hour under air atmosphere. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with water (3×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure and diluted with DCM (2 mL). The residue was purified by prep-TLC with CH2Cl2/methanol (20:1, v/v) to afford racemic amide C-98 (80 mg, 48.72%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=577.30.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(4-(acetamidomethyl)phenyl)-3-aminopropanamide

Step 1: To a stirred mixture of acid I-57 (82 mg, 0.24 mmol, 1 equiv) and NMI (60.04 mg, 0.73 mmol, 3 equiv) in MeCN (3.0 mL) was added amine I-49 (87.27 mg, 0.29 mmol, 1.2 equiv) and TCFH (136.79 mg, 0.49 mmol, 2 equiv) in portions at room temperature under air atmosphere. The resulting mixture was stirred for 2 h at room temperature under air atmosphere. After reaction, the resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with EtOAc (3×10 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/MeOH (20:1, v/v) to afford racemic amide C-99 (42 mg, 27.94% yield) as a yellow oil. LCMS: m/z (ESI), [M+Na]+=639.35.

Preparation of 3-amino-2-[4-(hydroxymethyl)-3-methoxyphenyl]-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-(5-methoxypyridin-3-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of 3-amino-2-(1-methyl-2-oxopyridin-4-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Step 1: Into a 20 mL vial were added acid I-36 (158.92 mg, 0.54 mmol, 1 equiv) and amine I-49 (160 mg, 0.54 mmol, 1.00 equiv) and HATU (305.87 mg, 0.80 mmol, 1.5 equiv) and triethylamine (162.81 mg, 1.61 mmol, 3 equiv) and DMF (5 mL) at room temperature. The resulting mixture was stirred for 1.5 h at room temperature under nitrogen atmosphere. The resulting mixture was diluted with ethyl acetate (100 mL). The resulting mixture was washed with 1×60 mL of saturated NaHCO3 (aq) and 2×60 mL of water and 1×40 mL of saturated NaCl (aq). The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (17:1, v/v) to afford racemic amide C-103 (100 mg, 32.34%) as a yellow solid. LCMS: m/z (ESI), [M+H]+=577.40.

Preparation of 2-amino-2-(4-methoxyphenyl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of 2-[4-(2-aminoethyl) piperazin-1-yl]-2-phenyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of 4-amino-2-phenyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]butanamide

Preparation of 2-amino-2-phenyl-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of (2S)-2-amino-3-(1H-imidazol-4-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of (2S)-2-amino-3-(1H-indol-3-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(5-methoxypyridin-3-yl)-3-(4-methylpiperazin-1-yl)propanamide

Preparation of 4-(1-((3-(1H-pyrazol-4-yl)-1H-indol-7-yl)amino)-3-amino-1-oxopropan-2-yl)benzamide

Preparation of 4-(1-((3-(1H-pyrazol-4-yl)-1H-indol-7-yl)amino)-3-amino-1-oxopropan-2-yl)benzyl methylcarbamate

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-(3-cyanophenyl)-3-(4-methylpiperazin-1-yl)propanamide

Step 1: To a stirred mixture of acid I-39 (200 mg, 0.37 mmol, 1 equiv) in CH3CN (6.0 mL) was added NMI (141.86 mg, 1.10 mmol, 3 equiv), TCFH (278.22 mg, 0.73 mmol, 2 equiv) and amine I-49 (110.24 mg, 0.37 mmol, 1.01 equiv) in portions at room temperature under nitrogen atmosphere. After reaction, the resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water, 10% to 100% gradient in 10 min; detector, UV 254 nm and hold for 30 min at 90%. After evaporated we afford racemic amide C-116a (180 mg, 44.44%) as a yellow oil. LCMS m/z (ESI), [M+H]+=554.25.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-amino-2-(4-(hydroxymethyl)phenyl) acetamide

Preparation of (2R)-3-methoxy-2-(4-methylpiperazin-1-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]propanamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-amino-2-cyclohexylacetamide

Preparation of 3-(1-((3-(1H-pyrazol-4-yl)-1H-indol-7-yl)amino)-3-amino-1-oxopropan-2-yl)benzamide

Preparation of (R)-N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-amino-3-phenylpropanamide

Step 1: A solution of tert-butyl 4-[7-amino-1-(p-tolylsulfonyl) indol-3-yl]pyrazole-1-carboxylate I-14b (142 mg, 313.79 μmol, TFA), (2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoic acid (249.75 mg, 941.38 μmol) and DIPEA (243.33 mg, 1.88 mmol, 327.94 μL) in NMP (2.00 mL) was stirred and cooled at 0° C. by ice/water bath for 15 min. Then HATU (395.82 mg, 1.04 mmol) was added into the reaction at this temperature. The resulting suspension was stirred and warmed slowly to room temperature for 16 h. Then the reaction mixture was poured into water (50 mL), and extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with brine (60 mL×1), dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica flash chromatography column, elution gradient from 15% to 40% ethyl acetate in petroleum ether. Pure fractions were evaporated to dryness to afford racemic amide C-126a (25 mg, 11% yield) as white solid. LCMS: tR=2.23 min in 3 min chromatography (3 min-5-95% MeCN in water (0.02% TFA), Waters Acquity UPLC BEH C18 1.7 μm, 2.1*50 mm, 40° C.), MS (ESI) m/z=700.5 [M+H]+.

Preparation of Methyl 2-(4-bromophenyl)-3-[(tert-butoxycarbonyl)amino]propanoate

Preparation of (2S)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]pyrrolidine-2-carboxamide

Step 1: Into a 20 mL vial were added amine I-49 (150 mg, 0.50 mmol, 1 equiv) and (2S)-1-(tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid (129.87 mg, 0.60 mmol, 1.2 equiv) and TCFH (169.28 mg, 0.60 mmol, 1.2 equiv) and NMI (165.12 mg, 2.01 mmol, 4 equiv) and MeCN (5 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (40 mL). The aqueous layer was extracted with CH2Cl2 (3×40 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and evaporated to afford a crude solid. The residue was purified by prep-TLC with CH2Cl2/methanol (37:1, v/v) to afford tert-butyl 4-{7-[(2S)-1-(tert-butoxycarbonyl) pyrrolidine-2-amido]-1H-indol-3-yl}pyrazole-1-carboxylate C-128 (240 mg, 96.32%) as a yellow oil. LCMS: m/z (ESI), [M+H−tBu]+=440.20.

Preparation of 2-cyclohexyl-2-(4-methylpiperazin-1-yl)-N-[3-(1H-pyrazol-4-yl)-1H-indol-7-yl]acetamide

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-2-amino-2-(pyridin-3-yl) acetamide

Step 2: Into a 100 mL round-bottom flask were added racemic amide C131a (300 mg, 0.56 mmol, 1 equiv) and DCM (3 mL) and TFA (0.86 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature under air atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in DMF (2 mL). The mixture was basified to pH 9 with NH3 (aq). The resulting mixture was stirred for 30 min at 0° C. under air atmosphere. The crude product (250 mg) was purified by prep-HPLC with the following conditions (Column: XBridge Shield RP18 OBD 30*150 mm, 5 um; Mobile Phase A: water (10 mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 5% B to 30% B in 8 min; wavelength: 220 nm; tR=7.93 min) to afford racemic amine C-131b (150 mg, 80.21%) as a white solid. LCMS: m/z (ESI), [M+H]+=333.3.

Preparation of N-(3-(1H-pyrazol-4-yl)-1H-indol-7-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide

Preparation of N-(2-amino-1-phenylethyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-(2-amino-1-(3-methoxyphenyl)ethyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Step 1: To a stirred solution of acid I-51 (100 mg, 0.44 mmol, 1 equiv) and tert-butyl N-[2-amino-2-(3-methoxyphenyl)ethyl]carbamate (117.22 mg, 0.44 mmol, 1 equiv) in DMF (5 mL) were added HATU (251.01 mg, 0.66 mmol, 1.5 equiv) and DIEA (170.64 mg, 1.32 mmol, 3 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The crude product was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, MeCN in water, 0% to 100% gradient in 25 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum. This resulted in racemic amide C-136a (70 mg, 33.45%) as a white solid. LCMS: m/z (ESI), [M+H]+=476.10.

Preparation of N-(1-(3-methoxyphenyl)-2-(4-methylpiperazin-1-yl)ethyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-(3-amino-1-phenylpropyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-((3-methoxyphenyl) (1-methylpiperidin-4-yl)methyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-[cyclohexyl (1-methylpiperidin-4-yl)methyl]-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-(2-amino-1-(4-methoxyphenyl)ethyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-(2-amino-1-(4-(hydroxymethyl)phenyl)ethyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-(1-amino-3-phenylpropan-2-yl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of N-[(1S)-2-amino-1-phenyl-ethyl]-3-(1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-7-carboxamide

Preparation of N-[2-amino-1-(pyridin-3-yl)ethyl]-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Step 1: To a stirred mixture of acid I-51 (130 mg, 0.572 mmol, 1.00 equiv) and DIEA (221.84 mg, 1.716 mmol, 3.00 equiv), amine I-42 (149.34 mg, 0.629 mmol, 1.1 equiv) in DMF (2.0 mL) was added and HATU (337.19 mg, 0.887 mmol, 1.55 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 hour at room temperature. After reaction, 10 mL water was added to the mixture. The resulting mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (12:1, v/v) to afford racemic amide C-149 (130 mg, 50.89%) as a yellow oil. LCMS: m/z (ESI), [M+H]+=447.15.

Preparation of (rac)-N-{2-amino-1-[3-(dimethylphosphoryl)phenyl]ethyl}-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Preparation of 3-amino-2-(3-cyanophenyl)-N-[7-(1H-pyrazol-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl]propanamide

Step 1: Into a 40 mL vial were added 7-bromo-4-chloro-5H-pyrrolo[3,2-d]pyrimidine C-152a (1 g, 4.30 mmol, 1 equiv) and NH3 aq (20 mL) at room temperature. The resulting mixture was stirred for 72 h at 100° C. under air atmosphere. The product was precipitated by the addition of water. The precipitated solids were collected by filtration and washed with water (3×10 mL). The resulting solid was dried by lyophilization. This resulted in 7-bromo-5H-pyrrolo[3,2-d]pyrimidin-4-amine C-152b (0.84 g, 91.66%) as a brown solid. LCMS: m/z (ESI), [M+H]+=212.95. 1H NMR (CD3OD, 400 MHZ) δ 7.60 (1H, s), 8.19 (1H, s).

Step 2: Into a 40 mL vial were added amine C-152b (232.37 mg, 1.09 mmol, 1 equiv) and acid I-3 (380.00 mg, 1.31 mmol, 1.2 equiv) and TCFH (1.84 g, 6.545 mmol, 6 equiv) and NMI (716.46 mg, 8.73 mmol, 8 equiv) and MeCN (8 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. The resulting mixture was filtered and the filtered cake was washed with CH2Cl2 (3×10 mL). The filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC with CH2Cl2/methanol (30:1, v/v) to afford a crude solid. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica; mobile phase, methanol in water (0.1% FA), 50% to 70% gradient in 20 min; detector, UV 254 nm. The resulting mixture was concentrated under reduced pressure. This resulted in racemic amide C-152c (290 mg, 54.78%) as a brown yellow solid. LCMS: m/z (ESI), [M+H]+=487.10.

Preparation of N-(7-(1H-pyrazol-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-3-amino-2-(3-methoxyphenyl)propanamide

Preparation of (2S)-3-amino-2-phenyl-N-[7-(1H-pyrazol-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl]propanamide

Preparation of (R)-N-(2-amino-1-phenylethyl)-3-(1H-pyrazol-4-yl)-1H-indole-7-carboxamide

Biological Examples

Exemplary compounds disclosed herein have been characterized in one or more of the following biological assays.

Recombinant ROCK1 and ROCK2 was purchased from Thermo Fisher with catalogue numbers PV3691 and PR71168. The inhibition potency of compounds against these enzymes was assessed using Lance Ultra Kinase Assay. In brief, recombinant kinases were pre-incubated in the presence or absence of compound at room temperature for 30 minutes. The reaction was initiated by the addition of the ATP (Km) and substrate peptide which could be phosphorylated by kinases in the reaction. After 1 h incubation, the reaction was stopped by the addition of the detection reagent mix containing EDTA. The fluorescence was measured at 615 nm and 665 nm, respectively with excitation wavelength at 320 nm. The calculated signal ratio of 665 nm/615 nm is proportional to the kinase activity. The concentration of compound producing 50% inhibition of the respective kinase (IC50) was calculated using four-parameter logistic.

Biological Example 2: Cellular PD Assay

PASMC (Human Pulmonary Artery Smooth Muscle Cells) were grown in Smooth Muscle Cell Growth medium (Promocell #C-22162). The cells were resuspended at 3×105 cells per well in 6-well plate in Smooth Muscle Cell Growth medium (Promocell #C-22162) and incubated compound concentration range: (10000/1111/333/111/37/12.3/4.1/0.41/0 nM) for 30 min.

Cells were then placed on ice and washed with ice-cold PBS. Cell lysates were prepared with 2×SDS. Separate proteins by SDS-PAGE 2 hr, transfer of proteins form gel to membrane 90 min, Blocking the membrane with 5% milk 1 hr and incubation in primary antibody overnight at 4° C., Incubate the membrane in HRP-conjugated secondary antibody diluted in blocking buffer (1:2000) for 1 hour at room temperature. Detection using Amersham ECL plus western blotting detection system (LAS4000). The purified product was analysed by SDS PAGE and the amount of target protein calculated by gray scanning by Multi-Gauge V3.0 software.

The assay plates were incubated at 37° C. for 4 h to allow the cells to attach to wells, followed by addition of 30 μL of 1:250 LipidTOX Red dye (1:1000 final, then 120 nL Test compounds were added for the final concentration (50 uM start, 3-fold dilution, 8 doses, in duplicate). The 20 uM of Amiodarone and DMSO (0.1% final) were used for maximum and minimum control.

The assay plates were then incubated at 37° C. for 24 h, 120 μL of 8% paraformaldehyde (4% final) fixative solution containing 8 μg/ml Hoechst 33342 (4 ug/mL final) in DPBS was added to each well. After incubation at room temperature for 25 min, the assay plates were washed with DPBS. The assay plates were sealed and stored at 4° C. before imaging.

The fluorescence intensities (595 nm excitation, 615 nm emission for LipidTOX Red; 352 nm excitation, 461 nm emission for Hoechst 33342) were measured using an High Content Screening System (Operetta, PerkinElmer) with a 10×Fluor objective. Images were acquired for 4 fields in each well and analyzed with the software. Average intensities were subjected to Prism and EC50 were calculated. The present disclosure provided compounds that are potent ROCK1 and ROCK2 inhibitors without liability of inducing phospholipidosis.

The results for some exemplary compounds of the present disclosure are shown in Tables 2-4 below.

IC50 for ROCK1 and ROCK2 inhibition

Cellular Pharmacodynamic Assay Data

Example
in PASMC
in PASMC

The foregoing description is considered as illustrative only of the principles of the present disclosure. Further, since numerous modifications and changes will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the invention as defined by the claims that follow.