Amino acid derivatives and method of making the same

Abstract

The present disclosure relates to an unnatural amino acid and method of making the same.

Description

WSGR Docket No.65728-703.601 AMINO ACID DERIVATIVES AND METHOD OF MAKING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 551,273, filed February 8, 2024, the entire content of which is incorporated herein by reference. BACKGROUND

[0002] Antibody conjugates can be used in many areas of medicine and biological research, andantibody-drug conjugates (ADCs) can be used in new therapeutics for cancer treatment or other treatment. Conjugation to the antibody may produce random conjugations to multiple amino acid side chains, resulting in heterogeneous mixtures of conjugated antibodies. In contrast, site- specific conjugation to the antibody may produce a homogeneous population of antibody conjugates with consistent and / or improved pharmacologic properties over randomly coupled ADC’s. Therefore, there remains a need to develop new methods to make site-specific antibody conjugates. SUMMARY

[0003] The present disclosure provides non-natural amino acid analogues of Formula (I), or anenantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, as described herein. There is also provided a mutant protein comprising as non-natural amino acid(s) one or more compounds according to Formula (I) as described herein.

[0004] In an aspect, provided herein is a compound of Formula (I):or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, wherein: R1is H, C1-C6 alkyl, C3-C10 cycloalkyl, or benzyl, wherein each C1-C6 alkyl, C3-C10 cycloalkyl, and benzyl is optionally substituted with 1 or 2 R9; is a 4-, 5-, 6-, or 7-membered ring consisting of C4-C7 cycloalkyl and aconsisting of 4-6 carbon atoms and one or two heteroatoms or groupsWSGR Docket No.65728-703.601 selected from –NH–, –NR2–, –O–, and –S–, wherein the 4-, 5-, 6-, or 7-membered ring is optionally substituted with 1 or 2 R9; R2is C1-C6alkyl, C1-C6haloalkyl, –C1-C6alkoxy, Linker is #–(L3)o–(L2)p–(L1)q–; # denotes a ; each of L1, L2, and L3a bond, –O–, –NH–, C1-C6alkylene, C1-C6haloalkylene, C1-C6heteroalkylene, C2-C6alkenylene, C2-C6alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C10aryl, or 5- to 10- membered heteroarylene, wherein each C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 alkoxy, C1-C6heteroalkylene, C2-C6alkenylene, C2-C6alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10 cycloalkylene, 3- to 10- membered heterocycloalkylene, C6-C10aryl, and 5- to 10-membered heteroarylene is optionally substituted with 1, 2, or 3 R9; each o and p is independently an integer of 0-6; q is an integer of 1-6; and R9is independently halogen, C1-C4alkyl, C2-C4alkenyl, C2-C4alkynyl, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C10cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10aryl, 5- to 10-membered heteroaryl, –C(O)(C1-C4alkyl), –C(O)O(C1-C4alkyl), –C(O)NH2, –C(O)NH(C1-C4alkyl), –C(O)N(C1-C4alkyl)2, –NH2, –NH(C1-C4alkyl), –N(C1-C4alkyl)2, –NH(C2-C4alkylene)-OH, –NH(C2-C4alkylene)-O-(C1-C4alkyl), –OH, –O(C1-C4alkyl), –O(C1-C4haloalkyl), –O(C2-C4alkylene)-NH2, –O(C2-C4alkylene)-NH-(C1-C4alkyl), –O(C2-C4alkylene)-N-(C1-C4alkyl)2, –O(C1-C4alkylene)-C(O)OH, –O(C1-C4alkylene)-C(O)O- (C1-C4 alkyl), –O(C2-C4 alkenyl), –O(C1-C4 alkylene)-(C6-C10 aryl), –O(C1-C4 alkylene)- (5- to 10-membered heteroaryl),–O(C6-C10aryl),–SH, S(O)2OH, –S(O)2(C1-C4alkyl), – S(O)2NH2, –S(O)2NH(C1-C4 alkyl), or –S(O)2N(C1-C4 alkyl)2; or two R9, together with atoms to which they are attached, form a C3-C10cycloalkyl or a 3- to 10-membered heterocycloalkyl ring.

[0005] In another aspect, provided herein is a mutant protein comprising one or more non-natural amino acids, wherein each of the one or more non-natural amino acids is independently a compound disclosed herein.WSGR Docket No.65728-703.601

[0006] In still another aspect, provided herein is a use of a compound disclosed herein in themanufacture of a mutant protein comprising one or more non-natural amino acids, wherein at least one of the one or more non-natural amino acids is the compound.

[0007] Additional aspects and advantages of the present disclosure will become readily apparentto those skilled in this art from the following detailed description, wherein only illustrative instances of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different instances, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. DETAILED DESCRIPTION

[0009] While various embodiments of the present disclosure have been shown and describedherein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0010] Compounds are generally described herein using standard nomenclature. For compoundshaving asymmetric centers, it should be understood that (unless otherwise specified) all of the optical isomers and mixtures thereof are encompassed. In addition, compounds with carbon- carbon double bonds may occur in Z- and E- forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. Definitions

[0011] As used herein, the singular forms “a,” “an,” and “the” include the plural referenceunless the context clearly dictates otherwise.WSGR Docket No.65728-703.601

[0012] When a range of values is provided, it is to be understood that each intervening valuebetween the upper and lower limit of that range, and any other stated or intervening value in that stated range is encompassed within the scope of the present disclosure. Where the stated range includes upper or lower limits, ranges excluding either of those included limits are also included in the present disclosure. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.

[0013] As used herein, the term “about” or “nearly” when referring to a number or a numericalrange means that the number or numerical range generally referred to is within + / - 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the stated number or numerical range.

[0014] As used herein, the term “comprising” (and related terms such as “comprise” or“comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein.

[0015] As used in the specification and appended claims, unless specified to the contrary, thefollowing terms have the meaning indicated below.

[0016] As used herein, the term “C1-C6 alkyl” generally refers to a straight or branchedhydrocarbon chain having from 1 to 6 carbon atoms, and the straight or branched hydrocarbon chain is attached to the rest of the molecule by a single bond. Likewise, an alkyl group comprising up to 3 carbon atoms is a C1-C3alkyl group, and an alkyl group comprising up to 4 carbon atoms is a C1-C4alkyl group. Examples of a C1-C6alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3- methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1- dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl. In some instances, a substituent of an alkyl group is specifically indicated. For example, “cyanoalkyl” refers to an alkyl group substituted with at least one cyano substituent.

[0017] The C1-C6 alkyl group may be optionally substituted with a C1-C3 alkoxy group.Examples include, but are not limited to, methoxyethyl, methoxypropyl, methoxyisopropyl, ethoxyethyl, ethoxypropyl, ethoxyisopropyl, propoxyethyl, propoxypropyl, and propoxyisopropyl.

[0018] The C1-C6 alkyl group may be optionally substituted with a C3-C6 cycloalkyl group.Examples include, but are not limited to, 1-methylcyclopropyl, 1-methylcyclobutyl, and 1- methylcyclohexyl.WSGR Docket No.65728-703.601

[0019] As used herein, the term “C1-C6 alkoxy” generally refers to a radical of the formula –ORwherein R is a C1-C6alkyl group as defined. Likewise, an alkoxy group comprising up to 3 carbon atoms is a C1-C3alkoxy group. Examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, n-pentoxy, isopentoxy, 2-methylbutoxy, neopentoxy, 1-ethylpropoxy, n-hexyloxy, isohexyloxy, 4- methylpentoxy, 3-methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3.3-dimethylbutoxy, 2,2- dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1.3-dimethylbutoxy, 2,3- dimethylbutoxy, and 2-ethylbutoxy.

[0020] The C1-C3 alkoxy group may be optionally substituted with a C1-C3 alkoxy group.Examples include, but are not limited to, methoxymethoxy, methoxyethoxy, methoxypropoxy, methoxyisopropoxy, ethoxymethoxy, ethoxyethoxy, ethoxypropoxy, ethoxyisopropoxy, propoxymethoxy, propoxyethoxy, propoxypropoxy, and propoxyisopropoxy.

[0021] As used herein, the term “C3-C6 cycloalkylamino” is, for example, azacyclobutyl,pyrrolidino, piperidino, or hexamethylenimino.

[0022] As used herein, the term “C3-C6 cycloalkyl” generally refers to a monocyclic non-aromatic radical having from 3 to 6 ring atoms, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “cycloalkenyl” as used herein generally refers to a group that comprises one or more unsaturated rings in which all ring members are carbon. Polycyclic cycloalkyls or carbocycles include, for example, adamantyl, norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless stated otherwise specifically in the specification, a cycloalkyl is optionally substituted.

[0023] As used herein, the term “alkenyl” generally refers to straight or branched chain alkenegroups, which comprise at least one unsaturated carbon-carbon double bond. Alkenyl groups include C2-8 alkenyl, C2-6 alkenyl and C2-4 alkenyl groups, which have from 2 to 8, 2 to 6, or 2 to 4 carbon atoms, respectively, including, for example, ethenyl, allyl or isopropenyl. The term “alkynyl” as used herein generally refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond. Alkynyl groups include C2-8alkynyl, C2-6alkynyl and C2-4alkynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively.

[0024] As used herein, the term “halogen” or “halide” generally refers to fluorine, chlorine,bromine, and iodine. The term “haloalkyl” as used herein generally refers to an alkyl group that is substituted with one or more independently chosen halogens (e.g., “C1-C6haloalkyl” groupsWSGR Docket No.65728-703.601 have from 1 to 6 carbon atoms and at least one halogen). Examples of haloalkyl groups include, but are not limited to, mono-, di- or tri-fluoromethyl; mono-, di- or tri-chloromethyl; mono-, di-, tri-, tetra- or penta-fluoroethyl; mono-, di-, tri-, tetra- or penta-chloroethyl; 2,2,2-trifluoroethyl; 1,2-difluoroethyl; 3-bromo-2-fluoropropyl; 1,2-dibromoethyl; and 1,2,2,2-tetrafluoro-l- trifluoromethyl-ethyl.

[0025] As used herein, the term “heteroalkyl” generally refers to an alkyl group in which one ormore skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or combinations thereof. In some instances, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In one aspect, a heteroalkyl is a C1-C6heteroalkyl wherein the heteroalkyl is comprised of 1 to 6 carbon atoms and one or more atoms other than carbon, e.g., oxygen, nitrogen, sulfur, or combinations thereof. In some instances, a carbon atom or heteroatom is optionally oxidized (e.g., - C(O)OCH2-, -CH2OCH2-, -CH2S(O)2NHCH2-, -NHC(O)NHCH2-, -CH2NHC(O)CH2-). Further examples of such heteroalkyl are, for example, -CH2OCH3, -CH2CH2OCH3, - CH2CH2OCH2CH2OCH3, or -CH(CH3)OCH3. Unless stated otherwise specifically in the specification, a heteroalkyl is optionally substituted for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, - CF3, -OH, or -OMe.

[0026] As used herein, the term “heteroaryl” generally refers to a monocyclic aryl group thatincludes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. Examples include, but are not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazyl.

[0027] The terms “heterocyclic” or “heterocycle” or “heterocyclyl” or “cycloheteroalkyl” or“heterocycloalkyl” as used herein generally refer to a ring structure (monocycle or polycycle) containing 3-12 ring atoms (3-12 membered heterocycle), 3-8 ring atoms (3-8 membered heterocycle or 3-8 membered cycloheteroalkyl), 3-6 ring atoms (3-6 membered heterocycle or 3- 6 membered cycloheteroalkyl), or 5-6 ring atoms (5-6 membered heterocycle or 5-6 membered cycloheteroalkyl), in which at least one ring atom is carbon, and at least one ring atom is a heteroatom selected from N, O, and S, or a heteroatom group selected from C(=O), S(=O), and S(=O)2. A heterocyclic group may be aromatic or non-aromatic. Piperidine and oxetane are non- limiting examples of non-aromatic heterocycles. Thiazole and pyridine are non-limitingWSGR Docket No.65728-703.601 examples of aromatic heterocycles. Other examples of heterocycle include: aziridinyl, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, morpholinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxothiomorpholinyl, butyrolactam, valerolactam, caprolactam, butyrolactone, valerolactone and caprolactone. Similarly, the term “cycloheteroalkenyl” refers to a monocycle or polycycle ring structure comprising carbon atom(s) and heteroatom(s) / heteroatom group(s), wherein the cycloheteroalkenyl comprises at least one C=C double bond, at least one ring atom that is carbon, and at least one ring atom that is a heteroatom selected from N, O, and S or a heteroatom group selected from C(=O), S(=O), and S(=O)2. Unless stated otherwise specifically in the specification, a heterocycle or heterocycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, or - OMe.

[0028] As used herein, the term “aryl” generally refers to an all-carbon monocyclic or fused-ring polycyclic groups of 6 to 12 (C6-12aryl) or 6 to 10 carbon atoms (C6-10aryl) having a completely conjugated pi-electron system. Examples include, but are not limited to, phenyl, naphthalenyl, tetrahydronaphthyl, indanyl, biphenyl, and anthracenyl. The aryl group may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NRXRY, wherein RXand RYare independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and, combined, a five- or six-membered heteroalicyclic ring. Illustrative substituted alkyl group include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, aminomethyl, aminoethyl, hydoxymethyl, methoxymethyl, 2-fluoroethyl, and 2- methoxyethyl, etc.

[0029] The term “heteroaryl” as used herein generally refers to an aromatic group in which atleast one aromatic ring comprises at least one heteroatom selected from N, O and S. Heteroaryls include, for example, 5-12 membered heteroaryls, 5-10 membered heteroaryls, 5-7 membered monocyclic structures or 7-12 membered bicyclic structures. The number of heteroatoms in a heteroaryl can be 1, 2, 3, 4, or more. Examples include, but are not limited to, thienyl, pyridyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridine-2(1H)-keto, pyridine-4(1H)-keto,WSGR Docket No.65728-703.601 pyrrolyl, pyrazolyl, thiazolyl, 1,2 ,3-triazolyl, 1,2,4-triazolyl, 1,2,5-oxadiazolyl, imidazolyl, furanyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, naphthyl, benzothienyl, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolinyl, isoquinolinyl, and quinazolinyl. The heteroaryl group may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NRXRY, with RXand RYas defined above.

[0030] 2),).

[0031] As used herein, the term “alkylamino” generally refers to a secondary or tertiary aminethat has the general structure -NH-R1or -N(R1)(R2), respectively, wherein R1and R2are selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Such alkylamino groups include, but are not limited to, mono- and di-(C1-6alkyl)amino groups, in which each C1-6alkyl may be the same or different. In this case, the definition of “alkyl” as used in the term “alkylamino” differs from the definition of “alkyl” used for all other alkyl-containing groups, in the inclusion of cycloalkyl and (cycloalkyl)alkyl groups.

[0032] The term “alkylthio” as used herein generally refers to an alkyl-substituted thio group,wherein the term alkyl is as defined above.

[0033] The terms “substituent” and “substituted,” as used herein, generally denote that amolecular moiety is covalently bonded to an atom within a molecule of interest. For example, a ring substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member. Substituents of aromatic groups are generally covalently bonded to a ring carbon atom. A straight chain substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a member of a straight chain.

[0034] The term “cycloalkylamine” as used herein generally refers to either a ring structure withan amino group attached to a carbon atom in the ring or a ring structure with a nitrogen atom as member of the ring.

[0035] As used herein, the term “C1-C3 alkylsulfonyl” generally refers to a sulfonyl radical thatis substituted by a C1-C3 alkyl radical as defined above. Examples include, but are not limited to, methanesulfonyl, ethanesulfonyl, n-propanesulfonyl, and isopropanesulfonyl.WSGR Docket No.65728-703.601

[0036] As used herein, the term “C1-C4 alkoxycarbonyl” generally refers to a carbonyl radicalthat is substituted by C1-C4alkoxy radical, as defined above. Examples thereof include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, and tert-butoxycarbonyl.

[0037] The term “pharmaceutically acceptable” as used herein generally refers to a form of thecompound that is safe for administration to a subject. For example, a free base, a salt form, a solvate, a hydrate, a prodrug or derivative form of a compound described herein, which has been approved for mammalian use, via oral ingestion or any other route of administration, by a governing authority or regulatory agency, such as the Food and Drug Administration (FDA) of the United States, is pharmaceutically acceptable.

[0038] Included in the compounds of Formulas (I), (II), (III), and (IV) are the pharmaceuticallyacceptable salt forms of the free-base compounds. The term “pharmaceutically-acceptable salts” as used herein generally refers to salts, commonly used to form alkali metal salts and to form addition salts of free acids or free bases, which have been approved by a regulatory agency. Salts are formed from ionic associations, charge-charge interactions, covalent bonding, complexation, coordination, etc. The nature of the salt is not critical, provided that it is pharmaceutically acceptable.

[0039] As used herein, the term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. For example, Berge et al. describes pharmaceutically acceptable salts in detail in Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Inorganic acids from which salts can be derived include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, but are not limited to, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate,WSGR Docket No.65728-703.601 camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like.

[0040] Pharmaceutically acceptable salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and other amine salt. Inorganic bases from which salts can be derived include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, but are not limited to, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, examples include, but are not limited to, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is ammonium, potassium, sodium, calcium, or magnesium salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts. Bis salts (i.e. , two counterions) and higher salts (e.g. , three or more counterions) are encompassed within the meaning of pharmaceutically acceptable salts.

[0041] The term “isomers” as used herein generally refers to different compounds that have thesame molecular formula, including any and all geometric isomers and stereoisomers. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. ForWSGR Docket No.65728-703.601 example, “isomers” include geometric double bond cis- and trans-isomers, also termed E- and Z- isomers; R- and S-enantiomers; diastereomers, (d)-isomers and (l)-isomers, racemic mixtures thereof; and other mixtures thereof, as falling within the scope of this disclosure, unless specified otherwise. As used herein, the term “tautomer” is a type of isomer that includes two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa).

[0042] In some embodiments, the compound(s) of Formulas (I), (II), (III), and (IV) is used totreat a subject by administering the compound(s) as a pharmaceutical composition. To this end, the compound(s), in one embodiment, is combined with one or more pharmaceutically acceptable excipients, including carriers, diluents or adjuvants, to form a suitable composition, which is described in more detail herein.

[0043] The term “excipient” as used herein generally refers to any pharmaceutically acceptableadditive, carrier, adjuvant, or other suitable ingredient, other than the active pharmaceutical ingredient (API), which is typically included for formulation and / or administration purposes.

[0044] The term “diluent” as used herein generally refers to an agent used as filler in order toachieve the desired composition volume or weight. The diluent may be present in the pharmaceutical composition within granules in the form of a single compound or in the form of a mixture of compounds. Non-limiting examples of diluent include lactose, starch, pregelatinized starch, microcrystalline cellulose, silicified microcrystalline cellulose, cellulose acetate, dextrose, mannitol, sodium phosphate, potassium phosphate, calcium phosphate, fructose, maltose, sorbitol, or sucrose.

[0045] The term “adjuvant,” as used herein generally refers to any substance or mixture ofsubstances that increases the efficacy or potency of a compound disclosed herein on a target where the adjuvant is used together with the compound disclosed herein. However, when the adjuvant is used alone, no pharmacological effect is observed on the same target. Unnatural Amino Acids

[0046] As used herein, the term “unnatural amino acid” or “non-natural amino acid” generallyrefers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term “unnatural amino acid” include “non-naturally encoded amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof.

[0047] Genetically incorporated unnatural amino acids (uAAs) may allow unique orthogonalcoupling strategies leading to antibody conjugates when compared to those antibodies made from the 20 naturally occurring amino acids. Thus, uAAs may provide a novel paradigm for theWSGR Docket No.65728-703.601 production of the next generation ADCs. Incorporation of uAAs may allow unique orthogonal biological recognition of the modified antibody by other proteins (e.g., enzymes). Such recognition may lead to selective biological reactions targeting the antibodies comprising specific uAAs.

[0048] Engineered orthogonal aminoacyl-tRNA synthetase (aaRS) and its respective cognatetransfer RNA (tRNA) pair(s) can be used to conduct site-specific incorporation of the uAAs disclosed herein by expanding the genetic code to encode these uAAs. For example, the engineered orthogonal aaRS / tRNA pair can suppress a repurposed nonsense codon to directly incorporate these uAAs at a specific site of a desired protein. The novel uAAs disclosed herein having a defined chemistry handle (i.e., a carbonyl group) to conjugate with other molecules are desirable for mutant proteins. Using these defined chemistry handle, the mutant protein comprising these uAAs can conjugate with another protein, a cytotoxic agent, a polymer, a nucleic acid, etc. For example, an amber stop codon (UAG) can be used for the incorporation of these uAAs. Unique aaRS / tRNA pairs as the machinery to introduce these uAAs can be developed in a range of hosts, include Escherichia coli, yeast, inset cells, and mammalian cells. Numbered Embodiments

[0049] The following embodiments recite non-limiting permutations of combinations of featuresdisclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed.

[0050] Embodiment 1. A compound of Formula (I):Formula (I) or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, wherein: R1is H, C1-C6 alkyl, C3-C10 cycloalkyl, or benzyl, wherein each C1-C6 alkyl, C3-C10 cycloalkyl, and benzyl is optionally substituted with 1 or 2 R9; is a 4-, 5-, 6-, or 7-membered ring consisting of C4-C7cycloalkyl and a heteroalkyl consisting of 4-6 carbon atoms and one or two heteroatoms or groups selected from –NH–, –NR2–, –O–, and –S–, wherein the 4-, 5-, 6-, or 7-membered ring is optionally substituted with 1 or 2 R9; R2is C1-C6alkyl, C1-C6haloalkyl, –C1-C6alkoxy,WSGR Docket No.65728-703.601 Linker is #–(L3)o–(L2)p–(L1)q–; # denotes a ; each of L1, L2, and L3a bond, –O–, –NH–, C1-C6alkylene, C1-C6haloalkylene, C1-C6 heteroalkylene, C2-C6alkenylene, C2-C6alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C10aryl, or 5- to 10- membered heteroarylene, wherein each C1-C6alkylene, C1-C6haloalkylene, C1-C6alkoxy, C1-C6heteroalkylene, C2-C6alkenylene, C2-C6alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10cycloalkylene, 3- to 10- membered heterocycloalkylene, C6-C10 aryl, and 5- to 10-membered heteroarylene is optionally substituted with 1, 2, or 3 R9; each o and p is independently an integer of 0-6; q is an integer of 1-6; and R9is independently halogen, C1-C4alkyl, C2-C4alkenyl, C2-C4alkynyl, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C10cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10aryl, 5- to 10-membered heteroaryl, –C(O)(C1-C4alkyl), –C(O)O(C1-C4alkyl), –C(O)NH2, –C(O)NH(C1-C4alkyl), –C(O)N(C1-C4alkyl)2, –NH2, –NH(C1-C4alkyl), –N(C1-C4alkyl)2, –NH(C2-C4alkylene)-OH, –NH(C2-C4alkylene)-O-(C1-C4alkyl), –OH, –O(C1-C4alkyl), –O(C1-C4haloalkyl), –O(C2-C4alkylene)-NH2, –O(C2-C4alkylene)-NH-(C1-C4alkyl), –O(C2-C4alkylene)-N-(C1-C4alkyl)2, –O(C1-C4alkylene)-C(O)OH, –O(C1-C4alkylene)-C(O)O- (C1-C4alkyl), –O(C2-C4alkenyl), –O(C1-C4alkylene)-(C6-C10aryl), –O(C1-C4alkylene)- (5- to 10-membered heteroaryl),–O(C6-C10aryl),–SH, S(O)2OH, –S(O)2(C1-C4alkyl), – S(O)2NH2, –S(O)2NH(C1-C4alkyl), or –S(O)2N(C1-C4alkyl)2; or two R9, together with atoms to which they are attached, form a C3-C10 cycloalkyl or a 3- to 10-membered heterocycloalkyl ring.

[0051] Embodiment 2. The compound of Embodiment 1, wherein the compound is Formula (II):or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof.

[0052] Embodiment 3. The compound of Embodiment 1, wherein the compound is Formula(III):WSGR Docket No.65728-703.601 or an enantiomer, asalt thereof.

[0053] Embodiment 4. The compound of Embodiment 1, wherein the compound is Formula(IV):or an enantiomer, a salt thereof.

[0054] Embodiment 5. The compound of any one of Embodiments 1-4, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: R1is H.

[0055] Embodiment 6. The compound of any one of Embodiments 1-4, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: R1is C1-C6 alkyl.

[0056] Embodiment 7. The compound of any one of Embodiments 1-6, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: is C4-C7cycloalkyl wherein the C4-C7cycloalkyl is optionally substituted2 R9.

[0057] Embodiment 8. The compound of any one of Embodiments 1-6, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: is a heteroalkyl consisting of 3-6 carbon atoms and one or two heteroatoms orfrom –NH–, –NR2–, –O–, and –S–, wherein the heteroalkyl is optionally substituted with 1 or 2 R9.

[0058] Embodiment 9. The compound of any one of Embodiments 1-6, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, is:WSGR Docket No.65728-703.601 ,

[0059] Embodiment 10. The compound of any one of Embodiments 1-6, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, is:,

[0060] Embodiment 11. The compound of any one of Embodiments 1-6, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, is:,WSGR Docket No.65728-703.601

[0061] Embodiment 12. The compound of any one of Embodiments 1-11, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: Linker is #–(L3)o–(L2)p–(L1)q–; # denotes a ; each of L1, L2, and L3a bond, –O–, –NH–, C1-C6alkylene, C1-C6haloalkylene, C1-C6 heteroalkylene, alkoxycarbonyl, C3-C10cycloalkylene, or 3- to 10-membered heterocycloalkylene, wherein each C1-C6alkylene, C1-C6haloalkylene, C1-C6alkoxy, C1-C6heteroalkylene, alkoxycarbonyl, C3-C10cycloalkylene, 3- to 10-membered heterocycloalkylene is optionally substituted with 1, 2, or 3 R9; each o and p is independently an integer of 0-6; and q is an integer of 1-6.

[0062] Embodiment 13. The compound of any one of Embodiments 1-11, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: Linker is methylene, –CF2–, –CH(CH3)–, ethylene, #–CH(CH3)–CH2–, #–CH2– CH(CH3)–, n-propylene, –CH2–CH(CH3)–CH2–, #–CH(CH3)–CH2–CH2–, #–CH2–CH2– CH(CH3)–, n-butylene, n-pentylene, –CH2–O–CH2–, #–CH2–O–CH2–CH2–, #–CH2– CH2–O–CH2–, –CH2–O–CH2–O–CH2–, or an enantiomer or a diastereomer thereof; and # denotes a .

[0063] Embodiment 14. Theone of Embodiments 1-11, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: Linker is methylene,–CH(CH3)–, ethylene, #–CH(CH3)–CH2–, #–CH2–CH(CH3)–, n- propylene, –CH2–CH(CH3)–CH2–, #–CH(CH3)–CH2–CH2–, #–CH2–CH2–CH(CH3)–, n- butylene, n-pentylene, –CH2–O–CH2–, #–CH2–O–CH2–CH2–, #–CH2–CH2–O–CH2–, or an enantiomer or a diastereomer thereof; and # denotes a .

[0064] Embodiment 15. Theany one of Embodiments 1-11, or an enantiomer, adiastereomer, or a pharmaceutically acceptable salt thereof, wherein: Linker is n-butylene, #–CH2–O–CH2–CH2–, #–CH2–CH2–O–CH2–, or an enantiomer or a diastereomer thereof; andWSGR Docket No.65728-703.601 # denotes a .

[0065] Embodiment 16. The Embodiment 1, or an enantiomer, a diastereomer, ora pharmaceutically acceptablewherein the compound is: ,WSGR Docket No.65728-703.601 ,saltthereof, wherein the compound is: ,WSGR Docket No.65728-703.601 ,,thereof, wherein the compound is: ,WSGR Docket No.65728-703.601 ,,thereof, wherein the compound is:WSGR Docket No.65728-703.601 ,thereof, wherein the compound is: ,WSGR Docket No.65728-703.601 orof,or a pharmaceutically acceptable salt thereof, wherein the compound is: orsalt thereof, wherein the compound is: orsalt thereof, wherein the compound is: .WSGR Docket No.65728-703.601

[0076] Embodiment 27. A mutant protein comprising one or more non-natural amino acids,wherein each of the one or more non-natural amino acids is independently a compound of any one of Embodiments 1-26.

[0077] Embodiment 28. The mutant protein of Embodiment 27, wherein the mutant proteincomprises one compound of any one of Embodiments 1-26.

[0078] Embodiment 29. The mutant protein of Embodiment 27, wherein the mutant proteincomprises two or more non-natural amino acids, and wherein each of the two or more non- natural amino acids is the same compound of any one of Embodiments 1-26.

[0079] Embodiment 30. Use of a compound of any one of Embodiments 1-26 in themanufacture of a mutant protein comprising one or more non-natural amino acids, wherein at least one of the one or more non-natural amino acids is the compound of any one of Embodiments 1-26.

[0080] Embodiment 31. The use of Embodiment 30, wherein at least two of the one or morenon-natural amino acids is the compound of any one of Embodiments 1-26.

[0081] Embodiment 32. A mutant protein comprising as non-natural amino acid(s) one or morecompounds of any one of Embodiments 1-26.

[0082] Embodiment 33. The mutant protein of Embodiment 32, further comprising one or moremoieties selected from the group consisting of a protein, an antibody, a cytotoxic agent, a drug, and a polymer, wherein the one or more moieties are conjugated to the non-natural amino acid(s).

[0083] Embodiment 34. The mutant protein of Embodiments 33, wherein the polymer comprisesa polyethylene glycol moiety.

[0084] Embodiment 35. An antibody comprising as non-natural amino acid one or morecompounds of any one of Embodiments 1-26 in each heavy chain and / or light chain.

[0085] Embodiment 36. The antibody of Embodiment 35, wherein an Fc region comprises theone or more compounds. EXAMPLES Chemical Synthesis

[0086] Methods of the present invention may include the use of at least one compound ofFormulas (I), (II), (III), and (IV), which inhibits programmed necrosis in the regulation of repair and / or functional performance of a wide range of cells, tissues and organs, and have therapeutic and cosmetic applications ranging from regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primitive gut, regulation of hematopoietic function,WSGR Docket No.65728-703.601 regulation of skin and hair growth, etc. Accordingly, the methods and compositions of the present invention include the use of the subject inhibitors for all such uses as inhibitors of programmed necrosis may be implicated. Moreover, the subject methods can be performed on cells which are provided in culture (in vitro), or on cells in a whole animal (in vivo).

[0087] The examples and preparations provided below illustrated and exemplify the compoundsdescribed herein and methods of preparing such compounds. In general, the compounds described herein may be prepared by processes known in the general chemical arts.

[0088] The compounds of the present invention can be prepared using various synthetic routes,including those described below, starting from commercially available materials. Starting materials of the invention, are either known, commercially available, or can be synthesized in analogy to or according to methods that are known in the art. Many starting materials may be prepared according to known processes and, in particular, can be prepared using processes described in the examples. In synthesizing starting materials, functional groups in some cases are protected with suitable protecting groups when necessary. Functional groups may be removed according to known procedures in the art.

[0089] The protection of functional groups by protecting groups, the protecting groupsthemselves, and their removal reactions (commonly referred to as “deprotection”) are described, for example, in standard reference works, such as J.F.W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, London and New York (1973), in T.W. Greene, Protective Groups in Organic Synthesis, Wiley, New York (1981), in The Peptides, Volume 3, E. Gross and J. Meienhofer editors, Academic Press, London and New York (1981).

[0090] All synthetic procedures described herein can be carried out under known reactionconditions, advantageously under those described herein, either in the absence or in the presence (usually) of solvents or diluents.

[0091] The invention further encompasses “intermediate” compounds, including structuresproduced from the synthetic procedures described, whether isolated or not, prior to obtaining the finally desired compound. Structures resulting from carrying out steps from a transient starting material, structures resulting from divergence from the described method(s) at any stage, and structures forming starting materials under the reaction conditions are all “intermediates” included in the invention. Further, structures produced by using starting materials in the form of a reactive derivative or salt, or produced by a compound obtainable by means of the process according to the invention and structures resulting from processing the compounds of the invention in situ are also within the scope of the invention.WSGR Docket No.65728-703.601

[0092] New starting materials and / or intermediates, as well as processes for the preparationthereof, are likewise the subject of this invention. In select embodiments, such starting materials are used and reaction conditions so selected as to obtain the desired compound(s).

[0093] Starting materials of the invention, are either known, commercially available, or can besynthesized in analogy to or according to methods that are known in the art. Many starting materials may be prepared according to known processes and, in particular, can be prepared using processes described in the examples. In synthesizing starting materials, functional groups in some cases are protected with suitable protecting groups when necessary. Protecting groups, their introduction and removal are described above.

[0094] All reagents and solvents are obtained commercially unless stated otherwise. Allcommercial reagents and solvent are used without purification unless stated otherwise. When required, some reagents and solvents are purified by standard techniques. For example, tetrahydrofuran may be purified by distillation from sodium.

[0095] Reaction progress is monitored by reverse-phase HPLC and / or thin-layerchromatography (TLC). Liquid chromatography-mass spectrometry is performed using either Waters or Shimadzu 2010EV LCMS instruments using water and acetonitrile or methanol doped with 0.1% formic acid. TLC is performed using silica gel 60 F254 pre-coated plates (0.25 mm). Flash chromatography is performed using silica gel (32-63 m particle size) or aluminum oxide (activated, basic, ~150 mesh size). Automated chromatographic purification is carried out using pre-packed silica or C18 cartridges (from RediSep and Luknova) and is eluted using an ISCO Companion system. Reverse phase purifications are conducted using water and acetonitrile or methanol doped with 0.1% formic acid. All final product compounds are purified using one of these two chromatographic methods. Purity and characterization of compounds is established by a combination of TLC, liquid chromatography-mass spectroscopy (LC-MS) and Nuclear Magnetic Resonance (NMR) analytical techniques.1H and13C NMR spectra are obtained on a (ppm) and are internally referenced to deuterated solvent signals.

[0096] The size and scale of the synthetic methods will vary depending on the desired amount ofend product. It is understood that while specific reactants and amounts are provided in the Examples, one of skill in the art knows other alternative and equally feasible sets of reactants that will also yield the same compounds. Thus, where general oxidizers, reducers, solvents of various nature (aprotic, apolar, polar, etc.) are utilized, equivalents will be known in the art and are herein contemplated for use in the present methods.

[0097] Many of the steps below indicate various work-ups following termination of the reaction.A work-up involves generally quenching of a reaction to terminate any remaining catalyticWSGR Docket No.65728-703.601 activity and starting reagents. This is generally followed by addition of an organic solvent and separation of the aqueous layer from the organic layer. The product is typically obtained from the organic layer and unused reactants and other spurious side products and unwanted chemicals are generally trapped in the aqueous layer and discarded. The work-up in standard organic synthetic procedures found throughout the literature is generally followed by drying the product by exposure to a drying agent, such as anhydrous Na2SO4, to remove any excess water or aqueous byproducts remaining partially dissolved in the organic layer and concentration of the remaining organic layer. Concentration of product dissolved in solvent may be achieved by any known means, such as evaporation under pressure, evaporation under increased temperature and pressure, and the like. Such concentrating may be achieved by use of standard laboratory equipment such as rotary-evaporator distillation, and the like. This is optionally followed by one or more purification steps which may include, but is not limited to, flash column chromatography, filtration through various media and / or other preparative methods known in the art and / or crystallization / recrystallization. (See, for instance, Addison Ault, “Techniques and Experiments for Organic Chemistry,” 6th Ed., University Science Books, Sausalito, Calif., 1998, Ann B. McGuire, Ed., pp.45-59). General Synthetic Routes

[0098] There are many routes available to synthesize compounds of Formulas (I), (II), (III), and(IV). Below are synthetic routes leading to specific compounds of Formulas (I), (II), (III), and (IV). The synthetic routes shown are not limiting but are examples from many available routes.

[0099] In one example leading to compounds of Formula (I), as shown in Scheme 1, the ketonegroup of Compound A-1 is protected as a ketal under acidic catalysis to afford an ester Compound A-2 without affecting the stereochemistry at the alpha-carbon. The ester of Compound A-2 is hydrolyzed under basic conditions to provide the carboxylic acid in Compound A-3. The unmasked carboxylic acid is converted into the activated ester Compound A-4, which reacts with amine Compound A-4A to afford the amide Compound A-5. Ring A in Compound A-5 is as defined in Formula (I). The ketal protecting group on Compound A-5 is hydrolyzed (treated with TsOH in acetone) to unmask the cyclic ketone in Compound A-6, which is further deprotected using TFA to remove the tert-butyloxycarbonyl (Boc) group and affords Compound A-7 of Formula (I). Alternatively, the free carboxylic acid in Compound A- 6 reacts with an alcohol Compound A-6A (R1OH) (e.g., hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU), diisopropylethylamine (DIEA) in acetonitrile) to provide an alpha-amino acid ester Compound A-8. The tert-butyloxycarbonyl (Boc) group on Compound A-8 is removed using TFA to afford Compound A-9 of Formula (I).

[0100] Scheme 1: Synthesis of Compounds A-7 and A-9WSGR Docket No.65728-703.601

[0101] The coupling reaction between Compound A-6 and Compound A-6A can be conductedunder different conditions, including but not limited to: (1) HATU, DIEA, dimethylformamide (DMF), at room temperature; (2) hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), DIEA, DMF, at room temperature; (3) 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), DIEA, DMF, at room temperature; (4) benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), TEA, DMF, at room temperature; and (5) benzotriazol-1- yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), TEA, DMF, at room temperature. Other conditions are possible.

[0102] As shown in Scheme 1, the chirality of the amino acid portion can be controlled by thechirality of the amine Compound A-4A. The chirality of ring A can be controlled by the chirality of Compound A-1. There are many synthetic methods to synthesize chiral alpha-amino acids. See (1) Y. Zou et al., Asymmetric Synthesis of Tailor-Made Amino Acids Using ChiralWSGR Docket No.65728-703.601 Ni(II) Complexes of Schiff Bases. An Update of the Recent Literature. Molecules 2020; 25(12):2739; (2) A.S. Saghyan & P. Langer, Asymmetric Synthesis of Non-proteinogenic Amino Acids (Wiley-VCH, 2016); (3) C. Nájera & J.M. Sansano, Catalytic asymmetric Chem. Rev.2007; 107, 4584–4671; (3) Y.-P. Xue et al., Enzymatic Asymmetric Synthesis of Chiral Amino Acids. Chem. Soc. Rev., 2018, 47: 1516-1561. A racemic amino acid product can be resolved by (1) diastereomeric salt formation with a pure chiral acid or base, or (2) enzymatic discrimination in the hydrolysis of amides using an enzyme, such as, for example, aminoacylase. Some commonly used chiral resolving agents for amino acid resolution include, but are not limited to, tartaric acid, camphorsulfonic acid, brucine, strychnine, and various synthetic chiral amines. There are also many methods to synthesize chiral compounds in general, either chemically or enzymatically or via resolution of racemic products.

[0103] The synthetic routes shown in Scheme 1 can be modified to synthesize the uAAcompounds disclosed herein, in particular, those uAAs shown in Embodiments 16-26.

[0104] Compounds A-7 and A-9 can be synthesized using other routes as well. For example, asshown in Scheme 2, the primary amine of Compound A-20 is coupled with carboxylic acid Compound A-21 (e.g., hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU), diisopropylethylamine (DIEA) in acetonitrile) to afford the amide Compound A-22. Ring A in Compound A-4 is as defined in Formula (I). Acidic treatment (H3PO4, toluene) removes the t- butyl ester, and hydrogenation condition (H2over Pd / C) removes the Cbz protecting group in Compound A-22 to afford Compound A-7. Alternatively, the t-butyl ester in Compound A-21 is selectively removed in the presence of the Boc protecting group, followed by a coupling reaction between the unmasked carboxylic acid and R1OH (Compound A-6A) and a hydrogenation reaction to provide the alpha-amino acid ester Compound A-9.

[0105] Scheme 2. Another synthesis of Compounds A-7 and A-9WSGR Docket No.65728-703.601

[0106] Other protection groups different than the t-butyl ester and Cbz group can be usedsimilarly to allow the primary amine in Compound A-20 to reaction with Compound A-21. The ketone group in Compound A-21 can be protected as a ketal similar to the strategy used in Scheme 1, then complete the route in Scheme 2 by adding a few deprotection steps. For example, see Scheme 3 below.

[0107] Scheme 3. Still another synthesis of Compounds A-7 and A-9

[0108] Scheme 4:

[0109] WSGR Docket No.65728-703.601

[0110] 1,2-Ethanediol (1.2 eq.) and TsOH (0.05 eq.) are dissolved in benzene and the solutionwas stirred under reflux using a Dean–Stark apparatus to remove H2O from the reagents. After 1.5 h, LUX 102-1 (1 eq.) is added and the stirring is continued under reflux for 2.5 h. After cooling to rt, the solution is diluted with EtOAc, poured into NaHCO3saturated solution, and the two phases are separated. The organic layer is rinsed with brine, dried over anhydrous Na2SO4, and filtered. Evaporation of solvent followed by silica gel chromatography purification of the residue affords the LUX 102-2.

[0111] Step 2: LUX 102-3

[0112] LUX 102-2 (1.0 eq.) is stirred in a mixture of THF / water (10:1, v / v) and NaOH (0.05eq.) overnight. The mixture is quenched with NH4Cl aqueous solution, adjusting the pH to about 2 with hydrochloric acid (1 M), and extract the mixture with EtOAc (3×). Combined organic layers are washed with brine, dried over anhydrous MgSO4, filtered, and concentrated. The crude is purified by silica gel chromatography purification to provide the product LUX 102-3.

[0113] STEP 3: LUX 102-4

[0114] A solution of LUX 102-3 (1.0 eq.), Et3N (1.1 eq.), DMAP (0.1 eq.) and N,N'-disuccinimidyl carbonate (DSC, 1.1 eq.) in DCM is stirred at ambient temperature for more than two hours. Subsequently, the reaction mixture is admixed with EtOAc, with hydrochloric acid (0.1 N), water, aqueous saturated NaHCO3, and brine. Drying over anhydrous MgSO4, filtration, concentration, and purification by silica gel chromatography provides the product LUX 102-4.

[0115] Step 4: LUX 102-6

[0116] Ain DMF isstirred overnight. The mixture is concentrated. The residue is partitioned between EtOAc andWSGR Docket No.65728-703.601 hydrochloric acid (0.1 N). The organic layer is separated, washed by water, aqueous NaHCO3, and brine, then dried, filtered, and concentrated. The crude product is purified by silica gel chromatography to afford the desired LUX 102-6.

[0117] Step 5: LUX 102-7The The organic layer is separated and washed with brine, then dried, filtered, and concentrated. The crude product is purified by silica gel chromatography to afford the desired LUX 102-7.

[0119] Step 6: LUX 102-8

[0120] A mixture of LUX 102-7 in TFA / DCM (1:1, v / v) is stirred for 4 hr. The mixture isconcentrated. The residue is partitioned between EtOAc and water. The organic layer is separated, washed with aqueous NaHCO3, brine, then dried, filtered, and concentrated. The crude product is purified by silica gel chromatography to afford the desired LUX 102-8.

[0121] Additional compounds of Formula (I) can be made using similar methods. For example,according to Scheme 6, another compound LUX 103-8 is made. Analytical analysis / data confirm the structure of LUX 102-8.

[0122] Scheme 5:WSGR Docket No.65728-703.601anester LUX 103-2 without affecting the stereochemistry at the alpha-carbon. The ester of LUX 103-2 is hydrolyzed under basic conditions to provide the carboxylic acid in LUX 103-3. The unmasked carboxylic acid is converted into the activated ester LUX 103-4, which reacts with amine LUX 102-5 to afford the amide LUX 103-6. The ketal protecting group on LUX 103-6 is hydrolyzed (treated with TsOH in acetone) to unmask the cyclic ketone in LUX 103-7, which is further deprotected using TFA to remove the tert-butyloxycarbonyl (Boc) group and affords LUX 103-8 of Formula (I). Analytical analysis / data confirm the structure of LUX 103-8.

[0124] While preferred embodiments of the present invention have been shown and describedherein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No.65728-703.601 CLAIMS WHAT IS CLAIMED IS:

1. A compound of Formula (I):Formula (I) or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, wherein: R1is H, C1-C6alkyl, C3-C10cycloalkyl, or benzyl, wherein each C1-C6alkyl, C3-C10cycloalkyl, and benzyl is optionally substituted with 1 or 2 R9; is a 4-, 5-, 6-, or 7-membered ring consisting of C4-C7cycloalkyl and aconsisting of 4-6 carbon atoms and one or two heteroatoms or groups selected from –NH–, –NR2–, –O–, and –S–, wherein the 4-, 5-, 6-, or 7-membered ring is optionally substituted with 1 or 2 R9; R2is C1-C6alkyl, C1-C6haloalkyl, –C1-C6alkoxy, Linker is #–(L3)o–(L2)p–(L1)q–; # denotes a connection to ; each of L1, L2, and L3is independently a bond, –O–, –NH–, C1-C6alkylene, C1-C6haloalkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C10 aryl, or 5- to 10- membered heteroarylene, wherein each C1-C6alkylene, C1-C6haloalkylene, C1-C6alkoxy, C1-C6heteroalkylene, C2-C6alkenylene, C2-C6alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C10cycloalkylene, 3- to 10- membered heterocycloalkylene, C6-C10aryl, and 5- to 10-membered heteroarylene is optionally substituted with 1, 2, or 3 R9; each o and p is independently an integer of 0-6; q is an integer of 1-6; andWSGR Docket No.65728-703.601 R9is independently halogen, C1-C4alkyl, C2-C4alkenyl, C2-C4alkynyl, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C10cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C10aryl, 5- to 10-membered heteroaryl, –C(O)(C1-C4alkyl), –C(O)O(C1-C4alkyl), –C(O)NH2, –C(O)NH(C1-C4alkyl), –C(O)N(C1-C4alkyl)2, –NH2, –NH(C1-C4alkyl), –N(C1-C4alkyl)2, –NH(C2-C4alkylene)-OH, –NH(C2-C4alkylene)-O-(C1-C4alkyl), –OH, –O(C1-C4alkyl), –O(C1-C4haloalkyl), –O(C2-C4alkylene)-NH2, –O(C2-C4alkylene)-NH-(C1-C4alkyl), –O(C2-C4alkylene)-N-(C1-C4 alkyl)2, –O(C1-C4 alkylene)-C(O)OH, –O(C1-C4 alkylene)-C(O)O- (C1-C4alkyl), –O(C2-C4alkenyl), –O(C1-C4alkylene)-(C6-C10aryl), –O(C1-C4alkylene)- (5- to 10-membered heteroaryl),–O(C6-C10aryl),–SH, S(O)2OH, –S(O)2(C1-C4alkyl), – S(O)2NH2, –S(O)2NH(C1-C4alkyl), or –S(O)2N(C1-C4alkyl)2; or two R9, together with atoms to which they are attached, form a C3-C10cycloalkyl or a 3- to 10-membered heterocycloalkyl ring.

2. The compound of claim 1, wherein the compound is Formula (II):or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein the compound is Formula (III):or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein the compound is Formula (IV):or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof.

5. The compound of any one of claims 1-4, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof, wherein: R1is H.

6. The compound of any one of claims 1-4, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof, wherein:WSGR Docket No.65728-703.601 R1is C1-C6alkyl.

7. The compound of any one of claims 1-6, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof, wherein: is C4-C7cycloalkyl wherein the C4-C7cycloalkyl is optionally substituted 2 R9.

8. The compound of any one of claims 1-6, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof, wherein: is a heteroalkyl consisting of 3-6 carbon atoms and one or two heteroatoms or from –NH–, –NR2–, –O–, and –S–, wherein the heteroalkyl is optionallysubstituted with 1 or 2 R9.

9. The compound of any one of claims 1-6, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof,,10. The compound of any one of claims 1-6, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof, is:WSGR Docket No.65728-703.601 ,pharmaceutically acceptable salt thereof,,12. The compound of any one of claims 1-11, or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, wherein: Linker is #–(L3)o–(L2)p–(L1)q–; # denotes a ; each of L1, L2, and L3a bond, –O–, –NH–, C1-C6alkylene, C1-C6haloalkylene, C1-C6alkoxy, C1-C6heteroalkylene, alkoxycarbonyl, C3-C10cycloalkylene, or 3- to 10-membered heterocycloalkylene, wherein each C1-C6alkylene, C1-C6haloalkylene, C1-C6alkoxy, C1-C6heteroalkylene, alkoxycarbonyl, C3-C10cycloalkylene, 3- to 10-membered heterocycloalkylene is optionally substituted with 1, 2, or 3 R9; each o and p is independently an integer of 0-6; and q is an integer of 1-6.

13. The compound of any one of claims 1-11, or an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, wherein: Linker is methylene, –CF2–, –CH(CH3)–, ethylene, #–CH(CH3)–CH2–, #–CH2– CH(CH3)–, n-propylene, –CH2–CH(CH3)–CH2–, #–CH(CH3)–CH2–CH2–, #–CH2–CH2–WSGR Docket No.65728-703.601 CH(CH3)–, n-butylene, n-pentylene, –CH2–O–CH2–, #–CH2–O–CH2–CH2–, #–CH2– CH2–O–CH2–, –CH2–O–CH2–O–CH2–, or an enantiomer or a diastereomer thereof; and # denotes a .

14. The compound of any 1-11, or an enantiomer, a diastereomer, or a pharmaceutically acceptablewherein: Linker is methylene,–CH(CH3)–, ethylene, #–CH(CH3)–CH2–, #–CH2–CH(CH3)–, n- propylene, –CH2–CH(CH3)–CH2–, #–CH(CH3)–CH2–CH2–, #–CH2–CH2–CH(CH3)–, n- butylene, n-pentylene, –CH2–O–CH2–, #–CH2–O–CH2–CH2–, #–CH2–CH2–O–CH2–, or an enantiomer or a diastereomer thereof; and # denotes a .

15. The compound of any 1-11, or an enantiomer, a diastereomer, or apharmaceutically acceptable salt thereof, wherein: Linker is n-butylene, #–CH2–O–CH2–CH2–, #–CH2–CH2–O–CH2–, or an enantiomer or a diastereomer thereof; and # denotes a .

16. The compound ofa diastereomer, or a pharmaceutically acceptable salt thereof, wherein the compound is: ,,WSGR Docket No.65728-703.601,WSGR Docket No.65728-703.601 orthereof, wherein the,compound is: ,WSGR Docket No.65728-703.601 ,,compound is:WSGR Docket No.65728-703.601 ,compound is: ,WSGR Docket No.65728-703.601 22. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: ,23. A compound selected from the group consisting ,,WSGR Docket No.65728-703.601 24. The compound of claim 23, or an enantiomer, a diastereomer, or a pharmaceutically . the, thereof, wherein theorwherein each of the one or more non-natural amino acids is independently a compound of any one of claims 1-26.

28. The mutant protein of claim 27, wherein the mutant protein comprises one compound of any one of claims 1-26.

29. The mutant protein of claim 27, wherein the mutant protein comprises two or more non- natural amino acids, and wherein each of the two or more non-natural amino acids is the same compound of any one of claims 1-26.

30. Use of a compound of any one of claims 1-26 in the manufacture of a mutant protein comprising one or more non-natural amino acids, wherein at least one of the one or more non- natural amino acids is the compound of any one of claims 1-26.

31. The use of claim 30, wherein at least two of the one or more non-natural amino acids is independently the compound of any one of claims 1-26.