Modulators of the sestrin-gator2 interaction and uses thereof

Abstract

This invention provides compounds, compositions thereof, and methods of using thereof.

Description

Technical Field

[0001] This invention relates to compounds and methods suitable for modulating Sestrin-GATOR2 interactions, thereby indirectly and selectively regulating mTORC1 activity. The invention also provides pharmaceutically acceptable compositions comprising the compounds of this invention and methods for using said compositions to treat various conditions. Background Technology

[0002] Rapamycin complex 1 (mTORC1) protein kinase is a mechanistic target of master growth regulators that sense diverse environmental cues, such as growth factors, cellular stress, and nutrient and energy levels. Upon activation, mTORC1 phosphorylates substrates that enhance anabolic processes such as mRNA translation and lipid synthesis and restricts catabolic processes such as autophagy. Aberrant regulation of mTORC1 has been observed in a wide range of diseases, including, in particular, diabetes, epilepsy, neurodegeneration, immune responses, skeletal muscle growth inhibition, and cancer (Howell et al., (2013) Biochemical Society Transactions 41, 906-912; Kim et al., (2013) Molecules and Cells 35, 463-473; Laplante and Sabatini, (2012) Cell 149, 274-293).

[0003] Many upstream inputs, including growth factors and energy levels, signal to mTORC1 via the TSC complex. The TSC complex regulates Rheb, a small GTPase, which is a fundamental activator of mTORC1 (Brugarolas et al., (2004) Genes & Development 18, 2893-2904; Garami et al., (2003) Molecular Cell 11, 1457-1466; Inoki et al., (2003) Genes & Development 17, 1829-1834; Long et al., (2005) Current Biology Biology) 15, 702-713; Sancak et al., (2008) Science (NY, New York) 320, 1496-1501; Saucedo et al., (2003) Nature Cell Biology 5, 566-571; Stocker et al., (2003) Nature Cell Biology 5, 559-565; Tee et al., (2002) Proceedings of the National Academy of Sciences of the United States of America 99, 13571-13576. Amino acids do not appear to signal to mTORC1 via the TSC-Rheb axis, but rather via heterodimeric Rag GTPases composed of RagA or RagB bound to RagC or RagD, respectively (Hirose et al., (1998) Journal of Cell Science 111(Pt 1), 11-21; Kim et al., (2008) Nature Cell Biology 10, 935-945; Nobukuni et al., (2005) Proceedings of the National Academy of Sciences 102, 14238-14243; Roccio et al., (2005) Oncogene 25, 657-664; Sanjak et al., (2008) Science (NY, NY) 320, 1496-1501; Schürmann et al., (1995) The Journal of Biological Chemistry (Chemistry) 270, 28982-28988; Sekiguchi et al., (2001) Journal of Biochemistry 276, 7246-7257; Smith et al., (2005) Journal of Biochemistry 280, 18717-18727).Rag GTPases control the subcellular localization of mTORC1, and amino acids facilitate the recruitment of mTORC1 to the lysosomal surface. Rheb GTPases also exist on the lysosomal surface (Buerger et al., (2006) Biochemical and Biophysical Research Communications 344, 869-880; Dibble et al., (2012) Molecular Cell 47, 535-546; Saito et al., (2005) Journal of Biochemistry 137, 423-430; Sanjak et al., (2008) Science (NY, NY) 320, 1496-1501). Several positive components of the upstream Rag GTPase pathway have been identified. The Ragulator complex localizes Rag GTPase to the lysosomal surface and, together with vacuolar ATPase, facilitates the exchange of GTP for GDP on RagA / B (Bar-Peled et al., (2012) Cell 150, 1196-1208; Sanjak et al., (2010) Cell 141, 290-303; Zoncu et al., (2011) Science Signaling 334, 678-683). Different FLCN-FNIP complexes act on RagC / D and stimulate it to hydrolyze GTP to GDP (Tsun et al., 2013). When RagA / B is loaded with GTP and RagC / D is loaded with GDP, these heterodimers bind mTORC1 and recruit it to the lysosomal surface, where mTORC1 can initiate contact with its activator, Rheb GTPase.

[0004] Current research has identified the GATOR1 multiprotein complex as a major negative regulator of the amino acid sensing pathway, and the absence of the GATOR1 multiprotein complex results in complete insensitivity of mTORC1 signaling to amino acid starvation (Bar-Piled et al., (2013) Science 340, 1100-1106; Panchaud et al., (2013) Science Signaling 6, ra42). GATOR1 is composed of DEPDC5, Nprl2, and Nprl3 and is the GTPase activator (GAP) of RagA / B. The GATOR2 multiprotein complex, with five known subunits (WDR24, WDR59, Mios, Sec13, and Seh1L), is a positive component of the pathway and is upstream of or parallel to GATOR1, but its molecular function remained unknown until recently (Bar-Piled et al., (2013) Science 340, 1100-1106).

[0005] Recently, by identifying the binding of GATOR2 to one or more Sestrins and confirming that the resulting Sestrin-GATOR2 complex regulates the subcellular localization and activity of mTORC1, additional information has been provided regarding the mTORC1 pathway. Specifically, the presence of the Sestrin-GATOR2 complex inhibits the mTORC1 pathway and reduces mTORC1 activity by preventing mTORC1 translocation to the lysosomal membrane. The interaction of GATOR2 with Sestrin, and especially Sestrin1 and Sestrin2, is antagonized by amino acids, particularly leucine, and to a lesser extent by isoleucine, methionine, and valine. In the presence of leucine, GATOR2 does not interact with Sestrin1 or Sestrin2, and mTORC1 is able to migrate to the lysosomal membrane, where it is active. Sestrin1 and Sestrin2 bind directly to leucine and, to a lesser extent, to isoleucine and methionine (Chantranupong et al., (2014) Cell Reports; 9(1):1-8). Leucine binding of Sestrin1 or Sestrin2 is essential to disrupt their interaction with GATOR2 and subsequent mTORC1 activation. Sestrin2 mutants that cannot bind leucine cannot signal the presence of leucine to mTORC1, and cells depleted of Sestrin2 and its homologs make mTORC1 insensitive to the absence of leucine (Wolfson et al., (2015) Science pii:ab2674 [electronic version prior to print]).

[0006] Sestrin consists of three related proteins (Sestrin1, Sestrin2, and Sestrin3) whose molecular functional characteristics are poorly understood (Buckbinder et al., (1994) Proceedings of the National Academy of Sciences 91, 10640-10644; Budanov et al., (2002) Cell 134, 451-460; Peeters et al., (2003) Human Genetics 112, 573-580). Sestrin2 inhibits mTORC1 signaling and it has been proposed that Sestrin2 can activate AMPK upstream of TSC and interact with TSC (Budanov and Karin, (2008) Cell 134, 451-460). However, later studies have found that Sestrin2 inhibits mTORC1 in the absence of AMPK (Peng et al., (2014) Cell 159(1):122-33), further highlighting the important role of the GATOR2 complex in responding to Sestrin2 regulation of mTORC1.

[0007] Regulation of the Sestrin-GATOR2 complex represents a potential therapeutic target for indirectly and selectively modulating mTORC1 activity. Summary of the Invention

[0008] It has now been found that the compounds of the present invention and their pharmaceutically acceptable compositions are effective as Sestrin-GATOR2 modulators. The compounds have the general formula I:

[0009]

[0010] Or its pharmaceutically acceptable salt, wherein each variable is as defined and described herein.

[0011] The compounds of this invention and pharmaceutically acceptable compositions thereof are suitable for treating a variety of diseases, conditions, or illnesses associated with mTORC1. These diseases, conditions, or illnesses include diabetes, epilepsy, neurodegeneration, immune responses, skeletal muscle growth inhibition, and proliferative disorders (such as cancer), as described herein. Detailed Implementation

[0012] 1. General description of certain embodiments of the present invention:

[0013] The compounds and compositions thereof of the present invention are suitable as Sestrin-GATOR2 modifiers. In some embodiments, the present invention provides compounds of formula I:

[0014]

[0015] or a pharmaceutically acceptable salt thereof, wherein:

[0016] R 1 Is it H or C? 1-6 alkyl;

[0017] R 2 It is R, -(CH2) n -Phenyl, -C(O)R, -SO2R or -C(O)N(R)2;

[0018] n is 0, 1, or 2;

[0019] Each R is independently hydrogen, -CN, or a optionally substituted group selected from: saturated or unsaturated C 1-6 Aliphatic group, phenyl group, 4 to 7 membered saturated or partially unsaturated monocyclic carbon ring, 5 to 6 membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4 to 8 membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0020] R 3 It is a ring A, -C(O)R, -C(O)OR, -C(O)N(R)2, -SO3H, -SO2N(R)2, -S(O)R, -S(O)ring A, -OR, or -B(OR)2, wherein two OR groups on the same boron are attached to their intermediate atoms to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur in addition to the boron and two oxygen atoms, or R 3 and R 4 They link together to form a 5- or 6-membered ring with 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur, which are optionally substituted.

[0021] L is a covalently bonded or optionally substituted straight or branched C with 1 to 9 fluorine groups. 1-6 Alkylene chain;

[0022] Ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur;

[0023] R 4 It is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 3 and R 4 They link together to form a 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur, which are optionally substituted; and

[0024] R 5 Is it H or C? 1-4 alkyl.

[0025] 2. Compounds and definitions:

[0026] The compounds of this invention include those generally described herein, and further described by the classes, subclasses and species disclosed herein. Unless otherwise specified, the following definitions shall apply as used herein. For the purposes of this invention, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. Additionally, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999 and “March's Advanced Organic Chemistry”, 5th edition, edited by Smith MB and March J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0027] As used herein, the terms “aliphatic” or “aliphatic group” mean a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted, fully saturated or containing one or more unsaturated units, or a fully saturated or containing one or more unsaturated units, but not aromatic monocyclic or bicyclic hydrocarbon (also referred to herein as “carbocyclic,” “cycloaliphatic,” or “cycloalkyl”), having a single connection point to the rest of the molecule. Unless otherwise stated, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 4 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in other embodiments, an aliphatic group contains 1 to 2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclic” or “cycloalkyl”) means a fully saturated or containing one or more unsaturated units, but not aromatic monocyclic C3-C6 hydrocarbon, having a single connection point to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0028] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any quaternized form of basic nitrogen; or a substituted nitrogen of a heterocycle, such as N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or NR).+ (e.g., in pyrroleyl groups substituted at N).

[0029] As used in this article, the term "unsaturated" refers to a portion having one or more unsaturated units.

[0030] As used in this article, the term "divalent C" 1-8 (or C 1-6 "Saturated or unsaturated, straight or branched hydrocarbon chains" refers to straight or branched divalent alkylene, alkenyl, and ynylene chains as defined herein.

[0031] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n - where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. The substituted alkylene chain is a polymethylene in which one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents include those described below with respect to the substituted aliphatic group.

[0032] The term "alkenyl" refers to a divalent alkenyl group. A substituted alkenyl chain is a polymethylene chain containing at least one double bond, wherein one or more hydrogen atoms are replaced by substituents. Suitable substituents include those described below with respect to substituted aliphatic groups.

[0033] The term "halogen" means F, Cl, Br or I.

[0034] The term "aryl," used alone or as part of a larger portion of "aralkyl," "aralkyloxy," or "aryloxyalkyl," refers to a monocyclic or bicyclic system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and each ring in the system contains three to seven ring members. The term "aryl" may be used interchangeably with the term "aromatic ring." In some embodiments of the invention, "aryl" refers to an aromatic ring system, including but not limited to phenyl, biphenyl, naphthyl, anthracene, etc., which may have one or more substituents. Within the scope of the term "aryl" as used herein, it also includes groups fused to an aromatic ring with one or more non-aromatic rings, such as indanyl, phthalimide, naphthimidyl, phenidyl, or tetrahydronaphthyl.

[0035] The terms "heteroaryl" and "heteroary-" used alone or as part of a larger portion of, for example, "heteroarylalkyl" or "heteroarylalkoxy," refer to a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; sharing 6, 10, or 14 π electrons in the ring array; and having one to five heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrroleyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indoleazinyl, purine, naphridinyl, and pteridinyl. As used herein, the terms “heteroaryl” and “heteroary-” also include groups in which a heteroaryl ring is fused with one or more aryl, cycloaliphatic, or heterocyclic rings, wherein the linking group or linking point is located on the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothiopheneyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cenolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinazinyl, carbazolyl, acridineyl, benziazinyl, benzithiazolyl, benzioxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which includes an optionally substituted ring. The term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl moiety, wherein the alkyl and heteroaryl moieties are optionally substituted independently.

[0036] As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above, in addition to a carbon atom. When used as a ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (e.g., in 3,4-dihydro-2H-pyrroleyl), NH (e.g., in pyrroleyl), or... + NR (as in pyrroleyl groups substituted at N).

[0037] Heterocycles can be attached to their side groups at any heteroatom or carbon atom to produce a stable structure, and any ring atom can optionally be substituted. Examples of saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenylpyrrolyl, piperidinyl, pyrrololinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolyl, piperazine, dioxalyl, dioxopentyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quininecycloyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical” are used interchangeably herein and also include groups fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indololinyl, 3H-indolyl, chromanyl, phenadiazine, or tetrahydroquinolinyl. Heterocyclic groups can be monocyclic or bicyclic. The term “heterocyclic alkyl” refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl moiety and the heterocyclic moiety are optionally substituted independently.

[0038] As used herein, the term “partially unsaturated” refers to a ring portion that includes at least one double or triple bond. The term “partially unsaturated” is intended to cover rings having multiple unsaturated sites, but not necessarily aryl or heteroaryl portions as defined herein.

[0039] As described herein, the compounds of the present invention may contain "optionally substituted" portions. Generally, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified portion are replaced by suitable substituents. Unless otherwise specified, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure can be substituted by more than one substituent selected from the specified group. The combinations of substituents contemplated in this invention are preferably combinations that form stable or chemically viable compounds. The term "stable" as used herein means that the compound remains substantially unchanged when subjected to conditions for the production, detection, and, in some embodiments, recovery, purification, and use for one or more purposes disclosed herein.

[0040] The suitable monovalent substituent on the substituted carbon atom of the "optionally substituted" group is independently a halogen; -(CH2) 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o -O-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 CH(OR o )2;-(CH2) 0-4 SR o ; can be R o Substituted -(CH2) 0-4 Ph; -(CH2) that can be substituted by R° 0-4 O(CH2) 0-1 Ph; -CH=CHPh, which can be substituted by R°; can be substituted by R o Substituted -(CH2) 0-4 O(CH2) 0-1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2) 0-4 N(R o )C(O)NR o 2; -N(R) o )C(S)NR o 2;-(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2; -N(R) o )N(R o )C(O)OR o ;-(CH2) 0-4 C(O)R o ;-C(S)R o ;-(CH2) 0- 4C(O)OR o ;-(CH2) 0-4C(O)SR o ; -(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0-4 SR-;SC(S)SR o ; -(CH2) 0-4 SC(O)R o ; -(CH2) 0-4 C(O)NR o 2;-C(S)NR o 2;-C(S)SR o ;-SC(S)SR o ; -(CH2) 0-4 OC(O)NR o 2;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR o )R o ; -(CH2) 0-4 SSR o ; -(CH2) 0- 4S(O)2R o ; -(CH2) 0-4 S(O)2OR o ; -(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2; -(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2;-N(R o )S(O)2R o ;-N(OR o )R o ;-C(NH)NR o 2;-P(O)2R o ;-P(O)R o 2;-OP(O)R o 2; -OP(O)(OR o )2;-SiR o 3; -(C 1-4 (linear or branched alkylene)ON(R) o )2; or -(C 1-4 (straight-chain or branched alkylene)C(O)ON(R)o )2, where each R o It can be replaced as defined below and is independently hydrogen or C. 1-6 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (5 to 6-membered heteroaryl ring), or a 5 to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, or regardless of the above definition, two independently existing R o It is linked with its intermediate atom to form a 3-12 saturated, partially unsaturated or aryl monocyclic or bicyclic ring with 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur, which can be substituted as defined below.

[0041] R o (or by using two independent Rs) o The suitable monovalent substituents on the ring formed by its connection with the intermediate atom are independently halogens, -(CH2). 0-2 R ● -(halogenated R) ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● 、-(CH2) 0-2 CH(OR ● )2;-O(halogen R ● )、-CN、-N3、-(CH2) 0-2 C(O)R ● 、-(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● 、-(CH2) 0-2 SR ● 、-(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● 、-(CH2) 0-2 NR ● 2. -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● -(C 1-4 (straight-chain or branched alkylene)C(O)OR ● or -SSR ● , where each R ● When unsubstituted or when the preceding group is a "halogen group", it is substituted by only one or more halogens and is independently selected from C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2)0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur. R o Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0042] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following: =O, =S, =NNR * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * 、=NR * 、=NOR * 、-O(C(R * 2)) 2-3 O-or-S(C(R * 2)) 2-3 S-, where each independently existing R * Selected from hydrogen, C can be substituted as defined below. 1-6 An aliphatic group, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to the ortho-substituted carbon of the "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independently existing R * Selected from hydrogen, C can be substituted as defined below. 1-6 Aliphatic group, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0043] R * Suitable substituents on the aliphatic group include halogens, -R ● -(halogenated R) ● ), -OH, -OR ● 、-O(halogen R ● )、-CN、-C(O)OH、-C(O)OR ● 、-NH2、-NHR ● -NR ● 2 or -NO2, where each R ● When it is not substituted or when the preceding group is a "halogen group", it is substituted by only one or more halogens and is independently C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0044] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include or Each of them Independently hydrogen, C can be substituted as defined below. 1-6 Aliphatic group, unsubstituted -OPh, or unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur, or, regardless of the above definition, two independently existing... It is linked with its intermediate atom to form an unsubstituted 3-12 saturated, partially unsaturated or aryl monocyclic or bicyclic ring with 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur.

[0045] Suitable substituents on the aliphatic group are independently halogens, -R ● -(halogenated R) ● -OH, -OR ● -O(halogenated R) ● -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● -NR ● 2 or -NO2, where each R ● When it is not substituted or when the preceding group is a "halogen group", it is substituted by only one or more halogens and is independently C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0046] As used herein, the term "pharmaceutically acceptable salt" refers to salts that, to the extent of reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. described pharmaceutically acceptable salts in detail in the Journal of Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include salts derived from suitable inorganic and organic acids, as well as inorganic and organic bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, and organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, glucohepanoate, glyceryl phosphate, glucuronate, hemisulfate, heptahydrate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, p-pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0047] Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N salts. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, low-carbon alkyl sulfonate, and aryl sulfonate.

[0048] Unless otherwise specified, the structures described herein are also intended to include all isomers (e.g., enantiomers, diastereomers, and geometric isomers (or conformational isomers) of said structures; for example, R and S configurations, Z and E double bond isomers, and Z and E conformational isomers with respect to each asymmetry center. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and geometric isomers (or conformational isomers), are all within the scope of the present invention. Unless otherwise specified, all tautomers of the compounds of the present invention are within the scope of the present invention. Furthermore, unless otherwise specified, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, this includes compounds in which hydrogen is replaced by deuterium or tritium, or in compounds in which hydrogen is replaced by deuterium or tritium. 13 C or 14 Compounds having the structure of the present invention, which are carbon-enriched and have carbon-replaced by carbon, are within the scope of the present invention. Such compounds are suitable as, for example, analytical tools, as probes in bioanalysis, or as therapeutic agents according to the present invention.

[0049] As used herein, the term "leucine mimic" is defined as a compound that reduces the amount of Sestrin2 bound to GATOR2 by at least about 40% relative to leucine at 25 μM. In some embodiments, the "leucine mimic" reduces the amount of Sestrin2 bound to GATOR2 by at least about 100%, at least about 150%, or at least about 200%.

[0050] As used herein, the term "leucine antagonist" is defined as a compound that increases the amount of Sestrin2 bound to GATOR2 by at least about 40% (expressed as -40% of leucine activity) relative to leucine at 25 μM. In some embodiments, the "leucine antagonist" increases the amount of Sestrin2 bound to GATOR2 by at least about 100%, at least about 150%, or at least about 200%.

[0051] As used herein, the terms “measurable affinity” and “measurable inhibition” refer to a measurable change in the binding of Sestrin2 to GATOR2 between a sample containing the compounds of the present invention or compositions thereof, as well as Sestrin2, GATOR2 and leucine, and an equivalent sample containing Sestrin2, GATOR2 and leucine, but without the compounds or compositions thereof.

[0052] 3. Description of exemplary embodiments:

[0053] In some embodiments, the present invention provides compounds of formula I:

[0054]

[0055] or a pharmaceutically acceptable salt thereof, wherein:

[0056] R 1 Is it H or C? 1-6 alkyl;

[0057] R 2 It is R, -(CH2) n -Phenyl, -C(O)R, -SO2R or -C(O)N(R)2;

[0058] n is 0, 1, or 2;

[0059] Each R is independently hydrogen, -CN, or a optionally substituted group selected from: saturated or unsaturated C 1-6 Aliphatic group, phenyl group, 4 to 7 membered saturated or partially unsaturated monocyclic carbon ring, 5 to 6 membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4 to 8 membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0060] R 3 It is a ring A, -C(O)R, -C(O)OR, -C(O)N(R)2, -SO3H, -SO2N(R)2, -S(O)R, -S(O)ring A, -OR, or -B(OR)2, wherein two OR groups on the same boron are attached to their intermediate atoms to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur in addition to the boron and two oxygen atoms, or R 3 and R 4 They link together to form a 5- or 6-membered ring with 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur, which are optionally substituted.

[0061] L is a covalently bonded or optionally substituted straight or branched C with 1 to 9 fluorine groups. 1-6 Alkylene chain;

[0062] Ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur;

[0063] R 4 It is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 3 and R 4 They link together to form a 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur, which are optionally substituted; and

[0064] R 5 Is it H or C? 1-4 alkyl.

[0065] In some embodiments, the provided Formula I compounds are not those described in Table 2 below.

[0066] As generally defined above, R 1 Is it H or C? 1-6 In some embodiments, R 1 It is H. In other embodiments, R 1 It is C 1-6 In some embodiments, R 1 In some embodiments, R 1 It is isobutyl. In some embodiments, R 1 Selected from those depicted in Table 1 below. In some embodiments, R 1 Selected from those described in Table 2 below.

[0067] As generally defined above, R 2 It is R, -(CH2) n -Phenyl, -C(O)R, -SO2R, or -C(O)N(R)2. In some embodiments, R 2 It is R. In some embodiments, R 2 It is -(CH2) n -Phenyl. In some embodiments, R 2 It is -C(O)R. In some embodiments, R 2 It is -SO2R. In some embodiments, R 2 It is -C(O)N(R)2. In some embodiments, R 2 In some embodiments, R 2 It is -(CH2)-phenyl. In some embodiments, R 2 It is -C(O)CH3. In some embodiments, R 2 Selected from those depicted in Table 1 below. In some embodiments, R 2 Selected from those described in Table 2 below.

[0068] As generally defined above, n is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.

[0069] As generally defined above, R 3 It is a ring A, -C(O)R, -C(O)OR, -C(O)N(R)2, -SO3H, -SO2N(R)2, -S(O)R, -S(O)ring A, -OR, or -B(OR)2, wherein two -OR groups on the same boron are attached to their intermediate atoms to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur in addition to the boron and two oxygen atoms, or R3 and R 4 They link together to form a 5- or 6-membered ring with 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur, which are optionally substituted.

[0070] In some embodiments, R 3 It is -C(O)OH. In some embodiments, R 3 It is -C(O)N(R)2. In some embodiments, R 3 It is -SO3H. In some embodiments, R 3 It is -SO2N(R)2. In some embodiments, R 3 It is -B(OR)2, wherein two -OR groups on the same boron atom are linked together with their intermediate atoms to form a 5- to 8-membered monocyclic saturated, partially unsaturated, or heterocyclic ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur, in addition to the boron and two oxygen atoms. In some embodiments, R 3 and R 4 They link together to form 5- to 6-membered rings with 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur, which are optionally substituted.

[0071] In some embodiments, R 3 It is ring A. As generally defined above, ring A is an optionally substituted ring selected from phenyl or a optionally substituted 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, ring A is an optionally substituted phenyl ring. In some embodiments, ring A is a optionally substituted 5-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, ring A is a optionally substituted 5-membered heteroaryl ring selected from imidazolyl, isoxazolyl, 1H-pyrroleyl (e.g., maleimide), pyrazolyl, oxazolyl, tetrazolyl, thiazolyl, and triazolyl. In some embodiments, ring A is a optionally substituted 6-membered heteroaryl ring having 1 to 2 nitrogen atoms. In some embodiments, ring A is a optionally substituted 6-membered ring selected from pyridinyl and pyrimidinyl. In some embodiments, ring A is selected from those depicted in Table 1 below.

[0072] In some embodiments, R3 is (pinacol)boron. In some embodiments, R 3 Selected from those depicted in Table 1 below. In some embodiments, R 3 Selected from those described in Table 2 below.

[0073] As generally defined above, L is a covalently bonded or optionally substituted straight or branched C with 1 to 4 fluorine groups. 1-6 Alkylene chain. In some embodiments, L is a covalent bond. In some embodiments, L is a straight-chain or branched C group optionally substituted with 1 to 4 fluorine groups. 1-6Alkylene chain. In some embodiments, L is methylene. In some embodiments, L is n-butenyl. In some embodiments, L is vinyl. In some embodiments, L is n-propenyl. In some embodiments, L is selected from those depicted in Table 1 below. In some embodiments, L is selected from those depicted in Table 2 below.

[0074] In some embodiments, L is a branched C optionally substituted with 1 to 4 fluorine groups. 1-6 Alkylene chain. In some embodiments, L is -C(CH3)2-. In other embodiments, L is -C(CH3)(CF3)-.

[0075] As generally defined above, R 4 It is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 3 and R 4 They are linked together to form a optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur. In some embodiments, R 4 It is R. In some embodiments, R 4 It is -CF3. In some embodiments, R 4 Yes - OR. In some embodiments, R 4 It is -N(R)2. In some embodiments, R 4 It is -Si(R)3. In some embodiments, R 4 It is -SR. In some embodiments, R 4 It is isopropyl. In some embodiments, R 4 It is tert-butyl. In some embodiments, R 4 In some embodiments, R 4 It is cyclobutyl. In some embodiments, R 4 It is sec-butyl. In some embodiments, R 4 It is a methoxy group. In some embodiments, R 4 It is methylthioyl. In some embodiments, R 3 and R 4 They are linked together to form a optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur. In some embodiments, R 4 Selected from those depicted in Table 1 below. In some embodiments, R 4 Selected from those described in Table 2 below.

[0076] As generally defined above, R 5 Is it H or C? 1-4 Alkyl group. In some embodiments, R 5 is H. In some embodiments, R 5 It is C 1-4In some embodiments, R 5 In some embodiments, R 5 Selected from those depicted in Table 1 below. In some embodiments, R 5 Selected from those described in Table 2 below.

[0077] In some embodiments, the present invention provides compounds of formula II:

[0078]

[0079] Or a pharmaceutically acceptable salt thereof, wherein each variable, in individual and combined forms, is as defined above and as described in the examples provided herein.

[0080] In some embodiments, the present invention provides compounds of formula III:

[0081]

[0082] or a pharmaceutically acceptable salt thereof, wherein:

[0083] Q is -C(R')2- or -NH-;

[0084] R x and R y Each is hydrogen, or R x and R y Connected together, they form = O;

[0085] It is either a double bond or a single bond;

[0086] Each R is independently hydrogen, -CN, or a group selected from the following optionally substituted groups: C 1-6 Aliphatic group, phenyl group, 4 to 7 membered saturated or partially unsaturated monocyclic carbon ring, 5 to 6 membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4 to 8 membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0087] Each R' is independently hydrogen, halogen, -CN, or a group selected from the following optionally substituted groups: C 1-6 Aliphatic group, phenyl group, 4 to 7 membered saturated or partially unsaturated monocyclic carbon ring, 5 to 6 membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4 to 8 membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0088] L is a covalently bonded or optionally substituted straight or branched C with 1 to 9 fluorine groups. 1-6 Alkylene chain;

[0089] R 4'Is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR; and

[0090] R 5 Is it H, -OR, or C? 1-4 alkyl.

[0091] In some embodiments, Q is -NH-. In some embodiments, Q is -CH2-. In some embodiments, Q is -CHF-.

[0092] In some embodiments, L is -CH2-.

[0093] In some embodiments, R x and R y Each is hydrogen. In some embodiments, R x and R y When connected, they form =O.

[0094] In some embodiments, R 5 'Is H. In some embodiments, R 5 'Is -OH.

[0095] In some embodiments, It is a single key. In some embodiments, It is a double bond.

[0096] In some embodiments, the present invention provides compounds of formula IV-a, IV-b, or IV-c:

[0097]

[0098]

[0099] or a pharmaceutically acceptable salt thereof, wherein:

[0100] R 1 Is it H or C? 1-6 alkyl;

[0101] R 2 It is R, -(CH2) n -Phenyl, -C(O)R, -SO2R or -C(O)N(R)2;

[0102] Each R 4" It can be R, halogen, or -CF3 independently;

[0103] Each R is independently hydrogen, -CN, or a optionally substituted group selected from: saturated or unsaturated C 1-6Aliphatic group, phenyl group, 4 to 7 membered saturated or partially unsaturated monocyclic carbon ring, 5 to 6 membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4 to 8 membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen or sulfur; and

[0104] L 1 It is a straight or branched C-chain that is covalently bonded or optionally substituted with 1 to 9 fluorine groups. 1-6 Alkylene chain.

[0105] In some embodiments, R 1 is H. In some embodiments, R 1 It is C 1-6 alkyl.

[0106] In some embodiments, R 1 Selected from those described in Table 1 below.

[0107] In some embodiments, R 2 It is R. In some embodiments, R 2 It is -(CH2) n -Phenyl. In some embodiments, R 2 It is -C(O)R.

[0108] In some embodiments, R 2 Selected from those described in Table 1 below.

[0109] In some embodiments, each R 4" Independently, it is R, halogen, or -CF3. In some embodiments, R 4" It is R. In some embodiments, R 4" It is a halogen. In some embodiments, R 4" It is -CF3. In some embodiments, R 4" Selected from those described in Table 1 below.

[0110] In some embodiments, L 1 It is a straight or branched C-chain that is covalently bonded or optionally substituted with 1 to 9 fluorine groups. 1-6 Alkylene chain. In some embodiments, L 1 It is a covalent bond. In some embodiments, L 1 It is a straight-chain or branched C that is optionally substituted with 1 to 9 fluorine groups. 1-6 Alkylene chain. In some embodiments, L 1 Selected from those described in Table 1 below.

[0111] Exemplary compounds of the present invention are described in Table 1 below.

[0112] Table 1. Exemplary Compounds

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] Exemplary compounds of the present invention are described in Table 2 below.

[0138] Table 2. Exemplary Compounds

[0139]

[0140]

[0141] In some embodiments, the present invention provides a compound or a pharmaceutically acceptable salt thereof as described in Table 1 above. In some embodiments, the present invention provides a compound or a pharmaceutically acceptable salt thereof as described in Table 2 above.

[0142] 5. Uses, preparation, and administration of the medicine

[0143] Pharmaceutically acceptable compositions

[0144] According to another embodiment, the present invention provides a composition comprising a compound of the present invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or mediator. The amount of the compound in the composition of the present invention is an amount capable of effectively and measurably inhibiting or activating Sestrin-GATOR2 interactions in a biological sample or patient. In some embodiments, the amount of the compound in the composition of the present invention is an amount capable of effectively and measurably inhibiting or activating Sestrin-GATOR2 interactions in a biological sample or patient. In some embodiments, the composition of the present invention is formulated for administration to a patient requiring the composition. In some embodiments, the composition of the present invention is formulated for oral administration to a patient.

[0145] As used in this article, the term "patient" refers to an animal, preferably a mammal, and most preferably a human.

[0146] The term "pharmaceutically acceptable carrier, adjuvant, or catalyst" refers to a non-toxic carrier, adjuvant, or catalyst that does not impair the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, adjuvants, or catalysts that can be used in the compositions of this invention include, but are not limited to, ion exchangers; alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances, such as phosphates; glycine; sorbic acid; potassium sorbate; a mixture of partial glycerides of saturated vegetable fatty acids; water, salts, or electrolytes, such as protamine sulfate; disodium hydrogen phosphate; potassium hydrogen phosphate; sodium chloride; zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; cellulose-based substances; polyethylene glycol; sodium carboxymethyl cellulose; polyacrylates; waxes; polyethylene-polyoxypropylene block polymers; polyethylene glycol; and lanolin.

[0147] The compositions of this invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via implantable cartridges. As used herein, the term "parenterally" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheathic, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions of this invention can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injectable formulation can also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable mediators and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, non-volatile oils are conventionally used as solvents or suspension media.

[0148] For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. For example, fatty acids of oleic acid and their glyceride derivatives are suitable for preparing injectable formulations, as are pharmaceutically acceptable natural oils such as olive oil or castor oil, especially in their polyoxyethylated form. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, other commonly used surfactants, such as Tween and Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used.

[0149] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral administration, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with an emulsifier and a suspending agent. If necessary, certain sweeteners, flavoring agents, or coloring agents may also be added.

[0150] Alternatively, the pharmaceutically acceptable compositions of the present invention can be used for administration in the form of rectal suppositories. These suppositories can be prepared by mixing the pharmaceutical agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0151] The pharmaceutically acceptable compositions of the present invention can also be administered topically, particularly when the therapeutic target includes areas or organs easily accessible by topical application, including diseases of the eyes, skin, or lower intestine. Topical formulations suitable for each of these areas or organs are readily prepared.

[0152] Local administration of medication to the lower intestine can be achieved with rectal suppository formulations (see above) or with suitable enema formulations. Local transdermal patches may also be used.

[0153] For topical application, the pharmaceutically acceptable compositions provided may be formulated in a suitable ointment form containing an active ingredient suspended or dissolved in one or more carriers. Carriers for topical application of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the pharmaceutically acceptable compositions provided may be formulated in a suitable lotion or cream form containing an active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl wax, cetearyl alcohol, 2-octyldodecanool, benzyl alcohol, and water.

[0154] For ophthalmic use, the pharmaceutically acceptable composition provided may be formulated as a micronized suspension in pH-adjusted isotonic sterile saline, with or without a preservative such as benzylalkonium chloride, or preferably as a solution in pH-adjusted isotonic sterile saline. Alternatively, for ophthalmic use, the pharmaceutically acceptable composition may be formulated in an ointment such as petrolatum.

[0155] The pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or inhalation. The compositions are prepared according to techniques well-known in the field of pharmaceutical formulation and can be prepared in solution form in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers for improving bioavailability, fluorocarbons and / or other conventional solubilizers or dispersants.

[0156] Most preferably, the pharmaceutically acceptable composition of the present invention is formulated for oral administration. The formulation may be administered with or without food. In some embodiments, the pharmaceutically acceptable composition of the present invention is administered without food. In other embodiments, the pharmaceutically acceptable composition of the present invention is administered with food.

[0157] The amount of the compounds of the present invention that can be combined with a carrier material to produce a single-dose composition will vary depending on the host being treated and the specific administration modality. Preferably, the provided compositions should be formulated to administer an inhibitor to patients receiving these compositions at a dose between 0.01 and 100 mg / kg body weight / day.

[0158] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, overall health condition, sex, diet, timing of administration, excretion rate, drug combination, as well as the judgment of the treating physician and the severity of the specific disease being treated. The amount of the compound of the invention in the composition will also depend on the specific compound in the composition.

[0159] Uses of compounds and pharmaceutically acceptable compositions

[0160] The compounds and compositions described herein are generally suitable for inhibiting or activating Sestrin-GATOR2 interactions. In some embodiments, the provided compounds or compositions thereof are activators of Sestrin-GATOR2 interactions.

[0161] The activity of compounds used as inhibitors or activators of Sestrin-GATOR2 interaction in this invention can be analyzed in vitro, in vivo, or in cell lines. In vitro analysis includes assays to determine inhibition or activation of the Sestrin-GATOR2 interaction. Alternative in vitro assays quantify the ability of inhibitors to reduce the binding of Sestrin to GATOR2 or the ability of activators to increase the binding of Sestrin to GATOR2. Detailed analytical conditions for compounds used as inhibitors or activators of Sestrin-GATOR2 interaction in this invention are described in the following examples.

[0162] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing or alleviating a disease or condition or one or more of its symptoms as described herein, delaying its onset, or inhibiting its progression. In some embodiments, treatment may be administered after one or more symptoms have been present. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0163] The provided compounds are inhibitors or activators of Sestrin-GATOR2 interaction and are therefore suitable for treating one or more conditions associated with mTORC1 activity. Accordingly, in some embodiments, the present invention provides a method for treating mTORC1-mediated conditions, the method comprising the step of administering to a patient in need a compound of the present invention or a pharmaceutically acceptable combination thereof.

[0164] As used herein, the term "mTORC1-mediated" condition, disease, and / or illness means any disease or other harmful condition for which mTORC1 is known to play a role. Therefore, another embodiment of the invention relates to treating or reducing the severity of one or more diseases for which mTORC1 is known to play a role.

[0165] The methods described herein include methods for treating cancer in subjects. As used herein, "treatment" means improvement or modification of at least one symptom or clinical parameter of the cancer. For example, treatment can cause a reduction in tumor size or growth rate. In all subjects, treatment is not required to cure the cancer or achieve 100% remission.

[0166] As described herein, agents that activate Sestrin-GATOR2 interactions and thereby reduce mTORC1 activity, such as inhibitory nucleic acids or small molecules, can reduce cancer cell proliferation in subjects and thus treat cancer upon administration. Therefore, in some embodiments, the methods described herein include administering a therapeutically effective dose of one or more agents that activate Sestrin-GATOR2 interactions and thereby indirectly inhibit the mTORC1 pathway.

[0167] As used herein, the term “cancer” refers to an abnormal state or condition characterized by the autonomous growth of cells, i.e., rapidly proliferating cell growth. The term is intended to encompass all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. As used herein, the term “tumor” refers to multiple cancer cells, such as a mass of cancer cells.

[0168] Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as those affecting the lungs, breast, thyroid, lymph nodes, gastrointestinal tract, and genitourinary tract, as well as adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell lung cancer, small bowel cancer, and esophageal cancer.

[0169] In some embodiments, the methods described herein are used to treat or diagnose a cancer in a subject. The term "cancer" is known in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some embodiments, the cancer is kidney cancer or melanoma. Exemplary cancers include those arising from cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcomas, for example, malignant tumors comprising both cancerous and sarcomatous tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which tumor cells form identifiable glandular structures.

[0170] The term "sarcoma" is known in the art and refers to a malignant tumor derived from mesenchyme.

[0171] In some embodiments, the cancer treated by the methods described herein is a cancer with increased mTORC1 levels or increased mTORC1 expression or activity relative to normal tissue or other cancers in the same tissue; such cancers can be identified using methods known in the art and described herein. In some embodiments, the method includes: obtaining a sample containing cancer cells, measuring mTORC1 activity in the sample, and administering a treatment as described herein (e.g., a modulator of Sestrin-GATOR2 interaction). In some embodiments, the cancer is one shown herein to have increased levels of mTORC1 activity.

[0172] In some embodiments, the present invention provides a method for treating one or more conditions, diseases, and / or illnesses, wherein the conditions, diseases, or illnesses include, but are not limited to, proliferative diseases.

[0173] Cellular proliferative disorders

[0174] The present invention is characterized by methods and compositions for diagnosing and prognosing proliferative disorders (e.g., cancer) and for treating these disorders by indirectly and selectively regulating mTORC1 activity through modulation of Sestrin-GATOR2 interactions. Proliferative disorders described herein include, for example, cancer, obesity, and proliferation-dependent diseases. These disorders can be diagnosed using methods known in the art.

[0175] cancer

[0176] Cancers include, but are not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain diseases, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphoendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor). Tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma. In some embodiments, the cancer is melanoma or breast cancer.

[0177] Other proliferative diseases

[0178] Other proliferative disorders include, for example, obesity, benign prostatic hyperplasia, psoriasis, abnormal keratinization, lymphoproliferative disorders (such as disorders of abnormal proliferation of lymphocytes), chronic rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy. Proliferative disorders incorporated herein by reference include those described in U.S. Patents Nos. 5,639,600 and 7,087,648.

[0179] Other diseases

[0180] In some embodiments, mTORC1 activation methods are used to treat ribosomal lesions (e.g., Diamond-Blackfan anemia, 5q- syndrome, Shwachman-Diamond syndrome, X-linked dyskeratosis, chondrodysplasia, and Treacher-Collins syndrome). (See Payne et al., (2012) Blood. Sep 13; 120(11):2214-24; Efeyan et al., (2012) Trends in Molecular Medicine. Sep 18(9):524-533). Therefore, in some embodiments, the present invention provides a method for treating ribosomal lesions in a patient in need, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method for treating a patient in need of ribosomal lesions selected from Deutsche-Budd-Chiari anemia, 5q-syndrome, Shu-Deutsche-Chiari syndrome, X-linked dyskeratosis, chondrodysplasia, or Treacher-Collins syndrome, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient.

[0181] In some embodiments, methods activating mTORC1 activity are used to treat adhesion lesions (e.g., Roberts syndrome and Cornelia de Lange syndrome) (see Xu et al., (2016) BMC Genomics 17:25). Therefore, in some embodiments, the present invention provides a method for treating adhesion lesions (e.g., Roberts syndrome and Cornelia de Lange syndrome) in patients of need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient.

[0182] In some embodiments, the mTORC1 activation method is used to reverse or prevent muscle atrophy caused by: inactivity due to lifestyle, inactivity due to orthopedic surgery, immobilization, or the age of the subject or a disease or condition that the subject has or suffers from. (See Cuthbertson et al., (2005) FASEB J. March; 19(3):422-4. Electronic version December 13, 2004; Rennie, (2009) Applied Physiology, Nutrition and Metabolism (Appl. Physiol. Nutr. Metab.) 34:377-381; Ham et al., (2014) Clin Nutrition (ClinNutr.) December; 33(6):937-45). Therefore, in some embodiments, the present invention provides a method for reversing or preventing muscle atrophy in a patient in need caused by inactivity due to lifestyle, inactivity due to orthopedic surgery, immobilization, or a disease or condition that the subject has or suffers from, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient.

[0183] In some embodiments, the mTORC1 activation method is used to reverse or prevent muscle atrophy caused by bone fracture, severe burns, spinal cord injury, resection, degenerative diseases, conditions requiring bed rest, intensive care unit (ICU) or long-term hospitalization for recovery. (See Gordon et al., (2013) International Journal of Biochemistry and Cell Biology, October; 45(10):2147-57; Léger et al., (2009) Muscle and Nerve, July; 40(1):69-78). Therefore, in some embodiments, the present invention provides a method for reversing or preventing muscle atrophy in a patient in need caused by bone fracture, severe burns, spinal cord injury, resection, degenerative diseases, conditions requiring bed rest, ICU or long-term hospitalization for recovery, the method comprising the step of administering the provided compound or a pharmaceutically acceptable combination thereof to the patient.

[0184] In some embodiments, the mTORC1 activation approach is used to treat diseases, conditions, or symptoms that cause skeletal muscle atrophy, such as sarcopenia, muscle denervation, muscular dystrophy, inflammatory myopathy, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), or myasthenia gravis. (See Kye et al., (2014) Human Molecular Genetics, Dec 1; 23(23):6318-6331; Gurpur et al., (2009) American Journal of Pathology, Mar; 174(3):999-1008; Chauhan et al., (2013) Neuroscience Research, Sep-October; 77(1-2):102-9); Ching et al., (2013) Human Molecular Genetics, Mar 15; 22(6):1167-79). Therefore, in some embodiments, the present invention provides a method for treating a patient in need of a disease, condition, or symptom causing skeletal muscle atrophy, such as sarcopenia, muscle denervation, muscular dystrophy, inflammatory myopathy, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), or myasthenia gravis, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient.

[0185] In some embodiments, the mTORC1 activation method is used to prevent muscle loss in subjects preparing for space travel, to enable subjects participating in space travel to tolerate muscle loss, or to enhance the recovery of muscle loss in subjects recently returned from space travel. (See Stein et al., (1999) American Journal of Physiology; 276: E1014-21). Therefore, in some embodiments, the present invention provides a method for preventing muscle loss in subjects preparing for space travel, enabling subjects participating in space travel to tolerate muscle loss, or enhancing the recovery of muscle loss in subjects recently returned from space travel, in subjects in need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0186] In some embodiments, the mTORC1 activation method is used to enable subjects preparing for armed conflict or military training, participating in armed conflict or military training, or recently returning from armed conflict or military training to withstand excessive muscle stress and / or fatigue or enhance recovery. (See Pasiakos et al., (2011) American Journal of Clinical Nutrition, September; 94(3):809-18). Therefore, in some embodiments, the present invention provides a method for enabling subjects preparing for armed conflict or military training, participating in armed conflict or military training, or recently returning from armed conflict or military training to withstand excessive muscle stress and / or fatigue or enhance recovery in subjects in need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0187] In some embodiments, an mTORC1 activation method is used to prevent autophagy in a patient. In some embodiments, the patient has or suffers from cancer that is resistant to therapy in a manner dependent on the induction of autophagy. (See Kim and Guan, (2015) J Clin Invest. January; 125(1):25-32). Therefore, in some embodiments, the present invention provides a method for preventing autophagy in a patient in need who has or suffers from cancer that is resistant to therapy in a manner dependent on the induction of autophagy, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0188] In some embodiments, the mTORC activation method is used to treat or prevent depression. (See Ignácio et al., (2015) Br J Clin Pharmacol., November 27). Therefore, in some embodiments, the present invention provides a method for treating or preventing depression in a patient in need, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0189] In some embodiments, the mTORC1 activation method is used to induce rapid-acting antidepressant activity. Therefore, in some embodiments, the present invention provides a method for inducing rapid-acting antidepressant activity in a patient in need, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0190] In some embodiments, the mTORC1 activation method is used to treat or prevent jet lag caused by accelerated diurnal re-entrainment in response to the alternation of day and night cycles. (See Cao et al., (2013) Neuron. Aug 21; 79(4):712-24 10.1016). Therefore, in some embodiments, the present invention provides a method for treating or preventing jet lag in a patient in need caused by accelerated diurnal re-entrainment in response to the alternation of day and night cycles, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0191] In some embodiments, an mTORC1 activation method is used to prevent or reverse myocardial atrophy in a subject. In some embodiments, the subject has or has consistently had a disease or condition selected from heart attack, congestive heart failure, heart transplantation, heart valve repair, atherosclerosis, other major vascular diseases, and coronary artery bypass surgery. (See Song et al., (2010) American Journal of Physiology: Cell Physiology, Dec; 299(6):C1256-C1266). Therefore, in some embodiments, the present invention provides a method for preventing or reversing myocardial atrophy in a subject in need, wherein the subject has or has consistently had a disease or condition selected from heart attack, congestive heart failure, heart transplantation, heart valve repair, atherosclerosis, other major vascular diseases, and coronary artery bypass surgery, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0192] In some embodiments, the mTORC1 activation method is used to increase strength and / or increase post-exercise muscle mass. In some embodiments, the method is performed in conjunction with physical therapy, which is part of total parenteral nutrition or used to promote functional electrical stimulation. (See Nakamura et al., (2012) Geriatr Gerontol Int. January; 12(1):131-9). Therefore, in some embodiments, the present invention provides a method for increasing strength and / or increasing post-exercise muscle mass. In some embodiments, the method is performed in conjunction with physical therapy in a subject in need, which is part of total parenteral nutrition or used to promote functional electrical stimulation, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0193] In some embodiments, the mTORC1 activation method is used to reduce food intake. (See Pedroso et al., (2015) Nutrients. May 22; 7(5):3914-37). Therefore, in some embodiments, the present invention provides a method for reducing food intake in a subject in need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0194] In some embodiments, the mTORC1 activation method is used to treat obesity. Therefore, in some embodiments, the present invention provides a method for treating obesity in a subject of need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0195] In some embodiments, the mTORC1 activation method is used to improve productivity in the process of manufacturing therapeutic recombinant proteins from a bioreactor. (See McVey et al., (2016) Biotechnology & Bioengineering, Feb 16. doi:10.1002 / bit.25951). Therefore, in some embodiments, the present invention provides a method for improving productivity in the process of manufacturing therapeutic recombinant proteins from a bioreactor, the method comprising the step of adding the provided compound or a pharmaceutically acceptable composition thereof to the manufacturing process.

[0196] In some embodiments, mTORC1 activation methods are used in immune cells to promote and / or maintain their antitumor activity. This includes increasing mTORC1 in immune cells in vitro prior to adoptive transfer, and increasing mTORC1 in immune cells in vivo when co-administered with other targeted immunotherapy strategies. In some embodiments, immune cells include naive T cells, CD4+ or CD8+ T cells, Th1, Th2, and T3 cells. RegAnd Th17 cells, dendritic cells, NK cells and macrophages. (See Yang et al., (2011) Nature Immunology; 12:888-897; O'Brien et al., (2011) Eur J Immunol.; 41:3361-3370; Delgoffe et al., (2009) Immunology, 19 June; 30(6):832-44; Chi, (2012) Nature Rev Immunology, 20 April; 12(5):325-338; Pollizzi et al., (2015) Journal of Clinical Research; 125(5):2090-2108; Ali et al., (2015) Frontiers in Immunology) Immunol.; 6:355; Katholnig et al., (2013) Biochem Soc Trans., Aug; 41(4):927-33; Wang et al., (2013) Proceedings of the National Academy of Sciences, Dec 10; 110(50):E4894-903; Yang and Qi, (2013) Journal of Clinical Research, Dec; 123(12):5165-78. Therefore, in some embodiments, the present invention provides a method for activating mTORC1 in immune cells to promote and / or maintain its antitumor activity. In some embodiments, the present invention provides a method for increasing mTORC1 in immune cells in vitro prior to adoptive transfer. In some embodiments, the present invention provides a method for increasing mTORC1 in immune cells in vivo when co-administered with other targeted immunotherapy strategies. In some embodiments, immune cells include naive T cells, CD4+ or CD8+ T cells, Th1, Th2, T cells, etc. Reg And Th17 cells, dendritic cells, NK cells, and macrophages, comprising the step of adding the provided compound or a pharmaceutically acceptable composition thereof to the said immune cells.

[0197] In some embodiments, the mTORC1 activation method is used in the retina to treat retinitis pigmentosa and other forms of ocular neurodegeneration. (See Punzo et al., (2009) Nature Neuroscience, January; 12(1):44-52). Therefore, in some embodiments, the present invention provides a method for treating retinitis pigmentosa and other forms of ocular neurodegeneration in a subject of need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0198] In some embodiments, the mTORC1 activation method is used to increase central or peripheral axonal regeneration. (See Namiko et al., (2010) Journal of Biochemistry 285:28034-28043). Therefore, in some embodiments, the present invention provides a method for increasing central or peripheral axonal regeneration in a subject of need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0199] In some embodiments, following injury or in diseases characterized by demyelination, such as multiple sclerosis and Parkinson's disease, the mTORC1 activation approach is used to promote myelin regeneration and neuronal activity. (See Tyler et al., (2009) J Neurosci. May 13; 29(19):6367-78; Norrmén et al., (2014) Cell Reports October 23; 9(2):646-60; Love (2006). J Clin Pathol. November; 59(11):1151-1159). Therefore, in some embodiments, the present invention provides a method for promoting myelin regeneration and neuronal activity in a subject of need following injury or in a disease characterized by demyelination, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method for treating multiple sclerosis in a subject of need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method for treating Parkinson's disease in a subject of need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0200] In some embodiments, the mTORC1 activation method is used to treat multiple sclerosis. Therefore, in some embodiments, the present invention provides a method for treating multiple sclerosis or a variant thereof in a subject of need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method for treating Balo's concentric sclerosis, Schilder's disease, acute (Marburg type) multiple sclerosis, inflammatory demyelinative polyradiculoneuropathy, or mass-like multiple sclerosis in a subject of need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0201] In some embodiments, the mTORC1 activation method is used to treat Devic's disease, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, progressive multifocal leukoencephalopathy, and Niemann-Pick disease. (See Takikita et al., (2004) J Neuropathol Exp Neurol., June; 63(6):660-73). Therefore, in some embodiments, the present invention provides a method for treating a subject in need of Devic's disease, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, progressive multifocal leukoencephalopathy, and Niemann-Pick disease, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0202] In some embodiments, an activated mTORC1 approach is used to treat or prevent various forms of autism. (See Novarino et al., (2012) Science October 19, 338:6105, pp. 394-397). Therefore, in some embodiments, the present invention provides a method for treating or preventing a form of autism in a subject of need, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0203] In some embodiments, methods using mTORC1 activation are employed to treat neurodegenerative diseases. Therefore, in some embodiments, the present invention provides a method for treating a subject with a neurodegenerative disease, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0204] In some embodiments, methods using mTORC1 activation are employed to treat diseases associated with synaptic dysfunction. Therefore, in some embodiments, the present invention provides a method for treating a subject with a disease associated with synaptic dysfunction, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0205] In some embodiments, the use of mTORC1 activation in the central nervous system increases dendrite formation and synapsis in neurodegenerative diseases characterized by dendritic spine reduction and synaptic loss, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), stroke, and glaucoma. (See DiPolo et al., (2015) Neural Regen Res., April; 10(4):559-561). Therefore, in some embodiments, the present invention provides a method for increasing dendrite formation and synapsis in neurodegenerative diseases characterized by dendritic spine reduction and synaptic loss in a subject of need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject. In some embodiments, the present invention provides a method for treating Alzheimer's disease, ALS, stroke, or glaucoma in a subject of need, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0206] In some embodiments, methods activating mTORC1 are used to treat diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, schizophrenia, Rett syndrome, Fragile X syndrome, Parkinson's disease, Huntington's disease, stroke, and glaucoma. (See Lin et al., PLoS ONE 8(4):e62572, 2013; Lee et al., Neuron Jan 21, 2015; 85(2):303-315; Bowling et al., Sci Signal, Jan 14, 2014; 7(308):ra4). Therefore, in some embodiments, the present invention provides a method for treating a subject in need of diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, schizophrenia, Ritter syndrome, Fragile X syndrome, Parkinson's disease, Huntington's disease, stroke, and glaucoma, the method comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the subject.

[0207] The pharmaceutically acceptable compositions of the present invention may be administered to humans and other animals, depending on the severity of the infection being treated, via oral, rectal, parenteral, intracerebrospinal, vaginal, intraperitoneal, topical (e.g., by powder, ointment, or drops), buccal, as an oral spray, or nasal spray, etc. In some embodiments, the compounds of the present invention may be administered orally or parenterally at a dose level of about 0.01 mg / kg to about 50 mg / kg daily, and preferably about 1 mg / kg to about 25 mg / kg of the subject's body weight, once or more times daily to achieve the desired therapeutic effect.

[0208] Oral liquid dosage forms include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol fatty acid esters, and mixtures thereof. Besides inert diluents, oral compositions may also include adjuvants, such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0209] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in parenteral acceptable non-toxic diluents or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids, such as oleic acid, are used in the preparation of injectable formulations.

[0210] Injectable formulations can be sterilized, for example, by filtering with a bacterial retention filter or by incorporating a sterilizing agent, and are in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0211] To prolong the effects of the compounds of this invention, it is generally desirable to slow down the absorption of the compounds via subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of the compound then depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, absorption of the parenteral administration of the compound can be delayed by dissolving or suspending the compound in an oil-based medium. Injectable accumulation forms are prepared by forming the compound in a microcapsule matrix of a biodegradable polymer such as polylactide-polyglycolic acid. The release rate of the compound can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoester) and poly(anhydride). Injectable accumulation formulations are also prepared by retaining the compound in liposomes or microemulsions that are compatible with body tissues.

[0212] The composition administered rectally or vaginally is preferably a suppository prepared by mixing the compound of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at ambient temperature but liquid at body temperature and thus melts in the rectal or vaginal cavity and releases the active compound.

[0213] Oral solid dosage forms include capsules, tablets, pills, powders, and granules. In the solid dosage forms, the active compound is mixed with at least one pharmaceutically acceptable inert excipient or carrier, such as sodium citrate or dicalcium phosphate; and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) humectants, such as cetyl alcohol and glyceryl monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate; and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer.

[0214] Similar solid compositions can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or toffee and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. They may optionally contain emulsifiers and may also have compositions that optionally release, or preferentially release, the active ingredient in a portion of the intestine in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar solid compositions can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or toffee and high molecular weight polyethylene glycol.

[0215] The active compound can also be in a microencapsulated form with one or more excipients as noted above. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings well known in pharmaceutical formulation techniques. In said solid dosage forms, the active compound can be mixed with at least one inert diluent, such as sucrose, lactose, or starch. In normal practice, these dosage forms may also contain additional substances besides inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage forms may also contain buffers. They may optionally contain emulsifiers and may also have compositions that optionally release only or preferentially the active ingredient in a delayed manner in a portion of the intestine. Examples of encapsulation compositions that can be used include polymers and waxes.

[0216] Dosage forms for topical or transdermal administration of the compounds of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient may be mixed with a pharmaceutically acceptable carrier and any desired preservative or buffer under aseptic conditions, as needed. Ophthalmic formulations, ear drops, and eye drops are also covered within the scope of this invention. Additionally, this invention covers the use of transdermal patches, which have the added advantage of controlled delivery of the compound into the body. The dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0217] According to one embodiment, the present invention relates to a method for modulating Sestrin-GATOR2 interactions in a biological sample to indirectly and selectively modulate mTORC1 activity, the method comprising the step of contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0218] As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof; biopsy material obtained from mammals or extracts thereof; and blood, saliva, urine, feces, semen, tears or other bodily fluids or extracts thereof.

[0219] Another embodiment of the invention relates to a method for modulating Sestrin-GATOR2 interaction in a patient, thereby indirectly and selectively modulating mTORC1 activity, the method comprising the step of administering the compound of the invention or a composition comprising the compound to the patient.

[0220] According to another embodiment, the present invention relates to a method for modulating Sestrin-GATOR2 interactions in a patient, thereby indirectly and selectively modulating mTORC1 activity, the method comprising the step of administering to the patient a compound of the present invention or a composition comprising the compound. In other embodiments, the present invention provides a method for treating mTORC1-mediated conditions in patients in need, the method comprising the step of administering to the patient a compound of the present invention or a pharmaceutically acceptable composition thereof. The conditions are described in detail herein.

[0221] Depending on the specific condition or disease to be treated, additional therapeutic agents typically administered for treating said condition may also be present in the compositions of the invention. As used herein, additional therapeutic agents typically administered for treating a specific disease or condition are referred to as “the disease or condition suitable for treatment.”

[0222] The compounds of the present invention can also be advantageously used in combination with other antiproliferative compounds. These antiproliferative compounds include, but are not limited to, aromatase inhibitors; anti-estrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule-active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce cell differentiation; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; anti-proliferative antimetabolites; platinum compounds; compounds that target / reduce the activity of protein or lipid kinases and other anti-angiogenic compounds; compounds that target, reduce, or inhibit the activity of protein or lipid phosphatases; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparinase inhibitors; Ras oncogenic isoform inhibitors; telomerase inhibitors; proteasome inhibitors; compounds for the treatment of hematological malignancies; compounds that target, reduce, or inhibit the activity of Flt-3; and Hsp90 inhibitors, such as those from Conforma. Therapeutics' 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010; temozolomide Spindle kinesin inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine / chlorpromazine from CombinatoRx; MEK inhibitors, such as ARRY142886 from Array BioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer, and formyltetrahydrofolate. The term "aromatase inhibitor," as used herein, refers to a compound that inhibits estrogen production, for example, by inhibiting the conversion of substrates androstenedione and testosterone to estrone and estradiol, respectively. The terminology includes, but is not limited to, steroids, particularly atamestane, exemestane, and formestane; and particularly, nonsteroids, particularly aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole, and letrozole. Exemestane is marketed under the brand name Aromasin. TM For Sale. Formistan is sold under the trademark Lentaron. TM For sale. Faldrozol is marketed under the brand name Afema. TM For sale. Anastrozole is sold under the brand name Arimidex. TM For sale. Letrozole is sold under the brand name Femara. TM or Femar TM For sale. Ammonialumine is marketed under the trademark Orimeten. TM Available for sale. The combinations of the present invention, which contain chemotherapeutic agents as aromatase inhibitors, are particularly suitable for treating hormone receptor-positive tumors, such as breast tumors.

[0223] As used herein, the term "anti-estrogenic" refers to compounds that antagonize the effects of estrogen at the estrogen receptor level. This term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride. Tamoxifen is marketed under the brand name Novadex. TM For Sale. Raloxifene hydrochloride is marketed under the brand name Evista. TM For Sale. Fluvestralc is available under the brand name Faslodex. TMApplication. The combinations of the present invention, which contain chemotherapeutic agents as anti-estrogens, are particularly suitable for treating estrogen receptor-positive tumors, such as breast tumors.

[0224] As used herein, the term "anti-androgen" refers to any substance capable of inhibiting the biological effects of androgens, including but not limited to bicalutamide (Casodex). TM As used herein, the term "goserelin agonist" includes, but is not limited to, abarelix, goserelin, and goserelin acetate. Goserelin may be marketed under the brand name Zoladex. TM Invest.

[0225] As used herein, the term "topoisomerase I inhibitor" includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecian and its analogues, 9-nitrocamptothecian and the macromolecular camptothecian conjugate PNU-166148. Irinotecan may be marketed, for example, under the trademark Camptosar. TM It is offered for sale. Topotecan is marketed under the trademark Hycamptin. TM sell.

[0226] As used herein, the term "topoisomerase II inhibitor" includes, but is not limited to, anthracyclines such as doxorubicin (including liposome formulations such as Caelyx). TM The drug contains daunorubicin, epirubicin, idarubicin, and nemorubicin; anthraquinones, mitoxantrone, and losoxantrone; and podophyllotoxin, etoposide, and teniposide. Etoposide is marketed under the brand name Etopophos. TM For sale. Teniposide is sold under the brand name VM 26-Bristol. Doxorubicin is sold under the brand name Acriblastin. TM or Adriamycin TM For Sale. Farmorubicin is sold under the trademark name Farmorubicin. TM For Sale. Itabizin is sold under the trademark name Zavedos. TM For sale. Mitoxantrone is sold under the trademark Novantron.

[0227] The term "microtubule activator" refers to microtubule stabilizing, microtubule destabilizing, and microtubule polymerization inhibitors, including but not limited to taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vincaine or vinca sulfate, vincristine or vinca sulfate, and vinorelbine; discodermolide; colchicine and epothilone, and their derivatives. Paclitaxel is marketed under the trade name Taxol. TM For Sale. Dorcetaxis is sold under the trademark name Taxotere. TM For Sale. Vinblastin RP (Vinblastin Sulfate) TM For Sale. Vincristine sulfate, marketed under the brand name Farmistin. TM sell.

[0228] As used herein, the term "alkylating agent" includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide is marketed under the trade name Cyclostin. TM For Sale. Ifosfamide is marketed under the brand name Holoxan. TM sell.

[0229] The term "histone deacetylase inhibitor" or "HDAC inhibitor" refers to compounds that inhibit histone deacetylases and have antiproliferative activity. These include, but are not limited to, sialylaniline isohydroxamic acid (SAHA).

[0230] The term "anti-metabolic antimetabolite" includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folic acid antagonists such as pemetrexed. Capecitabine is marketed under the brand name Xeloda. TM For Sale. Gescitabine is marketed under the trademark Gemzar. TM sell.

[0231] As used herein, the term "platinum compound" includes, but is not limited to, carboplatin, cisplatin, cisplatinum, and oxaliplatin. Carboplatin may be marketed, for example, under the trademark Carboplatin TMOxaliplatin may be sold, for example, under the trademark Eloxatin. TM It is offered in the form of a sale.

[0232] As used herein, the term "compounds that target / reduce the activity of protein or lipid kinases or protein or lipid phosphatases, or other anti-angiogenic compounds" includes, but is not limited to, protein tyrosine kinase and / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, such as a) compounds that target, reduce, or inhibit the activity of platelet-derived growth factor receptor (PDGFR), such as compounds that target, reduce, or inhibit the activity of PDGFR, especially compounds that inhibit PDGF receptors, such as N-phenyl-2-pyrimidinylamine derivatives, such as imatinib. SU101, SU6668, and GFB-111; b) compounds that target, reduce, or inhibit the activity of fibroblast growth factor receptor (FGFR); c) compounds that target, reduce, or inhibit the activity of insulin-like growth factor receptor I (IGF-IR), such as compounds that target, reduce, or inhibit the activity of IGF-IR, especially compounds that inhibit the kinase activity of IGF-I receptor, or antibodies that target the extracellular domain of IGF-I receptor or its growth factor; d) compounds that target, reduce, or inhibit the activity of the Trk receptor tyrosine kinase family, or ephrin B4 inhibitors; e) compounds that target, reduce, or inhibit the activity of the AxI receptor tyrosine kinase family; f) compounds that target, reduce, or inhibit the activity of Ret receptor tyrosine kinases; g) compounds that target, reduce, or inhibit the activity of Kit / SCFR receptor tyrosine kinases, such as imatinib; h) compounds that target, reduce, or inhibit the activity of C-kit receptor tyrosine kinases, which are part of the PDGFR family, such as compounds that target, reduce, or inhibit the activity of the c-Kit receptor tyrosine kinase family, especially compounds that inhibit c-Kit receptors, such as imatinib; i) compounds that target, reduce, or inhibit the activity of c-Abl family members, their gene fusion products (e.g., BCR-Abl kinases), and mutants, such as compounds that target, reduce, or inhibit the activity of c-Abl family members and their gene fusion products, such as N-phenyl-2-pyrimidinylamine derivatives, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from Parke Davis; or dasatinib (BMS-354825); j) compounds that target, reduce or inhibit the activity of members of the Raf family of protein kinase C (PKC) and serine / threonine kinases, members of the MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and TEC families and / or members of the cyclin-dependent kinase family (CDK), including astrocytocin derivatives such as midostaurin;Examples of other compounds include UCN-01, safingol, BAY 43-9006, bryostatin 1, perifosine; ilmofosine; RO 318220 and RO 320432; GO 6976; lsis 3521; LY333531 / LY379196; isoquinoline compounds; FTIs; PD184352 or QAN697 (P13K inhibitors) or AT7519 (CDK inhibitors); k) compounds that target, reduce, or inhibit the activity of protein-tyrosine kinase inhibitors, such as compounds that target, reduce, or inhibit the activity of protein-tyrosine kinase inhibitors, including imatinib mesylate (Gleevec); TM Or tyrphostin, such as tyrphostin A23 / RG-50810; AG 99; tyrphostin AG213; tyrphostin AG 1748; tyrphostin AG 490; tyrphostin B44; tyrphostin B44(+) enantiomers; tyrphostin AG 555; AG 494; tyrphostin AG 556, AG957 and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-adamantane benzoate; NSC 680410, Adafustin); l) Compounds that target, reduce, or inhibit the activity of epidermal growth factor family (EGFR1, ErbB2, ErbB3, ErbB4, in homopolymer or heterodimer form) and their mutants, such as compounds that target, reduce, or inhibit the activity of epidermal growth factor receptor family, especially those that inhibit members of the EGF receptor tyrosine kinase family, such as EGF receptor, ErbB2, ErbB3, and ErbB4, or compounds, proteins, or antibodies that bind to EGF or EGF-associated ligands, CP358774, ZD 1839, ZM 105180; trastuzumab (Herceptin) TM ), cetuximab (Erbitux) TM(i) Iressa, Tarceva, OSI-774, Cl-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidine derivatives; m) compounds that target, reduce or inhibit the activity of c-Met receptors, such as compounds that target, reduce or inhibit the activity of c-Met, especially those that inhibit the kinase activity of c-Met receptors. Compounds that target the extracellular domain of c-Met or antibodies that bind to HGF; and compounds that target, reduce, or inhibit the kinase activity of one or more JAK family members (JAK1 / JAK2 / JAK3 / TYK2 and / or pan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG- 101348, tofacitinib and ruxolitinib; o) compounds that target, reduce or inhibit the kinase activity of PI3 kinase (PI3K), including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL- 719, dactolisib, XL-147, XL-765 and idelalisib; and; and q) compounds that target, reduce or inhibit the signaling effects of hedgehog protein (Hh) or the smooth receptor (SMO) pathway, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib and IPI-926 (saridegib).

[0233] As used herein, the term "PI3K inhibitor" includes, but is not limited to, compounds that have inhibitory activity against one or more enzymes in the phosphatidylinositol-3-kinase family, including but not limited to PI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87. Examples of PI3K inhibitors applicable to this invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, bupacicoside, petrocicoside, PF-4691502, BYL-719, dartocicoside, XL-147, XL-765, and edicoside.

[0234] As used herein, the term "Bcl-2 inhibitor" includes, but is not limited to, compounds with inhibitory activity against B-cell lymphoma-2 protein (Bcl-2), including but not limited to ABT-199, ABT-731, ABT-737, apogossypol, pan-Bcl-2 inhibitors of Ascenta, curcumin (and its analogues), dual Bcl-2 / Bcl-xL inhibitors (Infinity Pharmaceuticals / Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and its analogues; see WO2008118802), navitoclax (and its analogues; see US7390799), NH-1 (Shenyang Pharmaceutical University). Bcl-2 inhibitors include (University of Michigan), obatoclax (and its analogues, see WO2004106328), S-001 (Gloria Pharmaceuticals), TW series compounds (University of Michigan), and venetoclax. In some embodiments, Bcl-2 inhibitors are small molecule therapeutic agents. In some embodiments, Bcl-2 inhibitors are peptide mimics.

[0235] As used herein, the term "BTK inhibitor" includes, but is not limited to, compounds that have inhibitory activity against Bruton's tyrosine kinase (BTK), including but not limited to AVL-292 and ibrutinib.

[0236] As used herein, the term “SYK inhibitor” includes, but is not limited to, compounds that have inhibitory activity against spleen tyrosine kinase (SYK), including but not limited to PRT-062070, R-343, R-333, Excellair, PRT-062607, and fostamatinib.

[0237] Other examples of BTK inhibitory compounds and conditions that can be treated by combination of said compounds with compounds of the present invention can be found in WO2008039218 and WO2011090760, the entire contents of which are incorporated herein by reference.

[0238] Other examples of SYK inhibitory compounds and conditions that can be treated by combination of said compounds with compounds of the present invention can be found in WO2003063794, WO2005007623 and WO2006078846, the entire contents of which are incorporated herein by reference.

[0239] Other examples of PI3K inhibitory compounds and conditions that can be treated by combination of said compounds with compounds of the present invention can be found in WO2004019973, WO2004089925, WO2007016176, US8138347, WO2002088112, WO2007084786, WO2007129161, WO2006122806, WO2005113554 and WO2007044729, the entire contents of which are incorporated herein by reference.

[0240] Other examples of JAK inhibitory compounds and conditions that can be treated by combination of said compounds with compounds of the present invention can be found in WO2009114512, WO2008109943, WO2007053452, WO2000142246 and WO2007070514, the entire contents of which are incorporated herein by reference.

[0241] Other anti-angiogenic compounds include those with an alternative mechanism of activity, such as one independent of protein or lipid kinase inhibition, like thalidomide. TM ) and TNP-470.

[0242] Examples of proteasome inhibitors suitable for use in combination with the compounds of the present invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0243] Compounds that target, reduce, or inhibit the activity of protein or lipid phosphatases are, for example, phosphatase 1 inhibitors, phosphatase 2A inhibitors, or CDC25 inhibitors, such as okadaic acid or its derivatives.

[0244] Compounds that induce cell differentiation include, but are not limited to, retinoic acid, α-tocopherol, γ-tocopherol or δ-tocopherol or α-tocotrienol, γ-tocotrienol or δ-tocotrienol.

[0245] As used herein, cyclooxygenase inhibitors include, but are not limited to, Cox-2 inhibitors, 5-alkyl-substituted 2-arylaminophenylacetic acid and derivatives, such as celecoxib. TM rofecoxib (Vioxx) TM Etoricoxib, valdecoxib, or 5-alkyl-2-arylaminophenylacetic acid, such as 5-methyl-2-(2'-chloro-6'-fluoroaniline)phenylacetic acid, lumiracoxib.

[0246] As used herein, the term "bisphosphonate" includes, but is not limited to, etridonic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid, and zoledronic acid. Etidronic acid is marketed under the trade name Didronel. TM For Sale. Chlorphosphine is marketed under the brand name Bonefos. TM For Sale. Tiludronic acid is marketed under the brand name Skelid. TM For Sale. Pamidronate is marketed under the brand name Aredia. TM For sale. Alendronate is marketed under the brand name Fosamax. TMFor Sale. Ibandronic acid is marketed under the brand name Bondranat. TM For Sale. Risedronic acid is marketed under the brand name Actonel. TM For Sale. Zoledronic acid is marketed under the brand name Zometa. TM For Sale. The term "mTOR inhibitor" refers to compounds that inhibit the mammalian target of rapamycin (mTOR) and possess antiproliferative activity, such as sirolimus. everolimus (Certican) TM ), CCI-779 and ABT578.

[0247] As used herein, the term "heparinase inhibitor" refers to a compound that targets, reduces, or inhibits the degradation of heparin sulfate. This term includes, but is not limited to, PI-88. As used herein, the term "biological response modifier" refers to lymphokines or interferons.

[0248] As used in this article, "inhibitors of Ras oncogenic isoforms (such as H-Ras, K-Ras, or N-Ras)" refers to compounds that target, reduce, or inhibit the oncogenic activity of Ras; for example, "farnesyltransferase inhibitors," such as L-744832, DK8G557, or R115777 (Zarnestra). TM As used herein, the term "telomerase inhibitor" refers to a compound that targets, reduces, or inhibits telomerase activity. Compounds that target, reduce, or inhibit telomerase activity are particularly those that inhibit telomerase receptors, such as telomestatin.

[0249] As used herein, the term "methionine aminopeptidase inhibitor" refers to a compound that targets, reduces, or inhibits the activity of methionine aminopeptidase. Compounds that target, reduce, or inhibit methionine aminopeptidase activity include, but are not limited to, bengamide or its derivatives.

[0250] As used herein, the term "proteasome inhibitor" refers to compounds that target, reduce, or inhibit proteasome activity. Compounds that target, reduce, or inhibit proteasome activity include, but are not limited to, bortezomib (Velcade). TM ) and MLN341.

[0251] As used herein, the term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) includes, but is not limited to, collagen peptide mimicry and non-peptide mimicry inhibitors, tetracycline derivatives such as the oxime peptide mimicry inhibitor batimastat and its analogs with oral bioavailability, marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551), BMS-279251, BAY12-9566, TAA211, MMI270B, or AAJ996.

[0252] As used herein, the term "compounds for the treatment of hematological malignancies" includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, reduce or inhibit the activity of the FMS-like tyrosine kinase receptor (Flt-3R); interferons, 1-β-D-arasulfuran cytosine (ara-c) and bisulfan; and ALK inhibitors, which are compounds that target, reduce or inhibit anaplastic lymphoma kinases.

[0253] Compounds that target, reduce, or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R), especially compounds, proteins, or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, astrocytocin derivatives, SU11248, and MLN518.

[0254] As used herein, the term "HSP90 inhibitor" includes, but is not limited to, compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90; compounds that degrade, target, reduce, or inhibit HSP90 client proteins via the ubiquitin-proteasome pathway. Compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90, particularly compounds, proteins, or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino,17-demethoxygeldmycin (17AAG), a geldmycin derivative; other geldmycin-related compounds; radicicol; and HDAC inhibitors.

[0255] As used in this article, the term "antiproliferative antibody" includes, but is not limited to, trastuzumab (Herceptin). TM Trastuzumab-DM1, Erbitux, Bevacizumab (Avastin) TM ), rituximab PRO64553 (anti-CD40) and 2C4 antibody. Antibody refers to complete monoclonal antibody, polyclonal antibody, multispecific antibody formed by at least two complete antibodies, and antibody fragment, as long as it can exhibit the desired biological activity.

[0256] For the treatment of acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard leukemia therapies, particularly with therapies used to treat AML. Specifically, the compounds of the present invention can be administered in combination with, for example, farnesyltransferase inhibitors and / or other drugs suitable for the treatment of AML, such as doxorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatinum, and PKC412.

[0257] Other anti-leukemia compounds include, for example, Ara-C, a pyrimidine analogue, which is a 2'-α-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are purine analogues of hypoxanthine, 6-mercaptopurine (6-MP), and fludarabine phosphate. Compounds that target, reduce, or inhibit the activity of histone deacetylase (HDAC) inhibitors such as sodium butyrate and salicylanilide isohydroxamic acid (SAHA) inhibit the activity of enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), Trichostatin A, and compounds disclosed in US 6,552,065, including but not limited to N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-acrylamide or pharmaceutically acceptable salts thereof, and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-acrylamide or pharmaceutically acceptable salts thereof, especially lactate. Somatostatin receptor antagonists, as used herein, refer to compounds that target, treat, or inhibit somatostatin receptors, such as octreotide and SOM230. Tumor cell destruction methods refer to methods such as ionizing radiation. The term "ionizing radiation" as used in this context refers to ionizing radiation occurring in the form of electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiotherapy, and ionizing radiation is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, Principles and Practice of Oncology, eds. Devita et al., 4th ed., Vol. 1, pp. 248-275 (1993).

[0258] This also includes EDG binders and ribonucleotide reductase inhibitors. As used herein, the term "EDG binder" refers to a class of immunosuppressants that regulate lymphocyte recirculation, such as FTY720. The term "ribonucleotide reductase inhibitor" refers to pyrimidine or purine nucleoside analogs, including but not limited to fludarabine and / or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C for all antiviral purposes), and / or pentostatin. Ribonucleotide reductase inhibitors are particularly hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0259] This also includes, in particular, compounds, proteins, or monoclonal antibodies that contain VEGF, such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or its pharmaceutically acceptable salts, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate; Angiostatin TM Endostatin TM ; anthranilamide; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, such as rhuMAb and RHUFab, VEGF aptamers, such as Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgG1 antibodies, angiozyme (RPI4610) and bevacizumab (Avastin) TM ).

[0260] Photodynamic therapy, as used in this article, refers to the use of certain chemicals called photosensitive compounds to treat or prevent cancer. Examples of photodynamic therapy include the use of, for example, Visudyne. TM Treatment with compounds such as porfimer sodium.

[0261] As used in this article, angiogenesis-inhibiting steroids refer to compounds that block or inhibit angiogenesis, such as anecocave, triamcinolone, hydrocortisone, 11-α-epihydrocotisol, cortexolone, 17α-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone, and dexamethasone.

[0262] Implants containing corticosteroids refer to compounds such as fluocinolone and dexamethasone.

[0263] Other chemotherapy compounds include, but are not limited to, alkaloids, hormone compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or mixed compounds or compounds with other or unknown mechanisms of action.

[0264] The structure of an active compound identified by its serial number, generic name, or trademark name can be obtained from the current standard outline, "The Merck Index," or from databases such as Patents International (e.g., IMS World Publications).

[0265] The compounds of this invention can also be used in combination with known treatment methods, such as administration of hormones or radiation. In some embodiments, the provided compounds are used as radiosensitizers, particularly for treating tumors poorly sensitive to radiotherapy.

[0266] The compounds of this invention can be administered alone or in combination with one or more other therapeutic compounds. Possible combination therapies may be in a fixed combination, or administered alternately or independently of each other, or in combination with a fixed combination and one or more other therapeutic compounds. In addition, the compounds of this invention can be administered, in particular, with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or combinations thereof to treat tumors. As mentioned above, in the case of other treatment strategies, long-term therapy and adjuvant therapy are also possible. Other possible treatments are therapies for maintaining the patient's condition after tumor regression or even chemotherapy, for example, for at-risk patients.

[0267] These additional agents can be administered separately from the composition containing the compound of the invention as part of a multiple-dose regimen. Alternatively, these agents can be part of a single dosage form, mixed together with the compound of the invention in a single composition. If administered as part of a multiple-dose regimen, the two active agents can be provided simultaneously, sequentially, or at intervals, typically within 5 hours of each other.

[0268] As used herein, the terms “combination,” “combined,” and related terms refer to the simultaneous or sequential administration of a therapeutic agent according to the invention. For example, the compound of the invention may be administered simultaneously with another therapeutic agent, sequentially in separate unit dosage forms, or together in a single unit dosage form. Therefore, the present invention provides a single unit dosage form comprising the compound of the invention, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0269] The amounts of the compounds of the present invention and additional therapeutic agents that can be combined with carrier materials to produce a single dosage form (in those compositions comprising the additional therapeutic agents as described above) will vary depending on the host being treated and the specific administration modality. Preferably, the compositions of the present invention should be formulated to administer the compounds of the present invention at doses between 0.01 and 100 mg / kg body weight / day.

[0270] In those compositions that include an additional therapeutic agent, the additional therapeutic agent and the compound of the present invention can work synergistically. Therefore, the amount of the additional therapeutic agent in these compositions will be less than that required in a single therapy using only that therapeutic agent. In these compositions, the additional therapeutic agent can be administered at a dose between 0.01 and 1,000 micrograms / kg body weight / day.

[0271] The amount of additional therapeutic agent present in the composition of the present invention will not exceed the amount typically administered in a composition containing said therapeutic agent as the sole active agent. Preferably, the amount of additional therapeutic agent in the composition disclosed in the present invention will be in the range of about 50% to 100% of the amount typically present in a composition containing that agent as the sole active agent.

[0272] The compounds of the present invention or pharmaceutical compositions thereof can also be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, artificial blood vessels, stents, and catheters. Vascular stents have been used, for example, to overcome restenosis (the narrowing of the vessel wall after injury). However, patients using stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition containing a kinase inhibitor. Implantable devices coated with the compounds of the present invention are another embodiment of the invention.

[0273] illustration

[0274] As depicted in the following examples, in some exemplary embodiments, the compounds are prepared according to the following general procedure. It should be understood that while the general method describes the synthesis of some of the compounds of the present invention, the following general method and other methods known to those skilled in the art can be applied to all compounds as described herein and to the subclasses and species of each of these compounds.

[0275] List of abbreviations used in the experimental section.

[0276] 4A MS: Molecular sieve

[0277] AcOH: Acetic acid

[0278] ACN: acetonitrile

[0279] Anhyd: Waterless

[0280] Aq: Aqueous solution

[0281] Bn: benzyl

[0282] Boc: tert-butoxycarbonyl

[0283] CbzCl: Benzoic acid chloroformate

[0284] Cbz-OSU: N-(Benzyloxycarbonyloxy)succinimide

[0285] Cu(OAc)₂: Copper(II) acetate

[0286] d: sky

[0287] DAST: Diethylaminosulfonium trifluoride

[0288] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0289] DCE: 1,2-Dichloroethane

[0290] DCM: dichloromethane

[0291] DEA: Diethylamine

[0292] DIBAL-H: Diisobutylaluminum hydride

[0293] DIPEA: N,N-diisopropylethylamine

[0294] DMA: N,N-dimethylacetamide

[0295] DMAP: 4-Dimethylaminopyridine

[0296] DMF: N,N-dimethylformamide

[0297] DMSO-dimethyl sulfoxide

[0298] DPPA: Diphenyl azidophosphate

[0299] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0300] ee: enantiomer excess

[0301] ESI: Electrospray Ionization

[0302] Et3N: Triethylamine

[0303] Et2O: Diethyl ether

[0304] EtOAc: Ethyl acetate

[0305] EtOH: Ethanol

[0306] Fmoc: fluorenylmethoxycarbonyl

[0307] Fmoc-OSu: N-(9-fluorenylmethoxycarbonyloxy)succinimide

[0308] h: hours

[0309] HATU: 3-O-1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate

[0310] HCOONH4: Ammonium formate

[0311] HPLC: High Performance Liquid Chromatography

[0312] IBX: 2-Iodobenzoic acid

[0313] IPA: Isopropyl alcohol

[0314] KOAc: Potassium acetate

[0315] M: molar concentration

[0316] Me: Methyl

[0317] MeOH: Methanol

[0318] min: minutes

[0319] mL: milliliters

[0320] mM: millimolecular concentration

[0321] mmol: millimole

[0322] MTBE: Methyl tert-butyl ether

[0323] NaBH3CN: Sodium cyanoborohydride

[0324] Na2CO3: Sodium carbonate

[0325] NaHCO3: Sodium bicarbonate

[0326] NMP: N-methylpyrrolidine

[0327] NMR: Nuclear Magnetic Resonance

[0328] ℃: degrees Celsius

[0329] PBS: Phosphate-buffered saline

[0330] Pd / C: Palladium / Carbon

[0331] Pd(OH)2 / C: Pearlman's catalyst

[0332] PE: Petroleum ether

[0333] PhNH2: Aniline

[0334] PPh3: Triphenylphosphine

[0335] Rel: relative

[0336] rt: room temperature

[0337] sat: saturation

[0338] SFC: Supercritical Fluid Chromatography

[0339] SOCl2: thionyl chloride

[0340] TBAB: Tetra-n-Butylammonium Bromide

[0341] tBuOK: Potassium tert-butoxide

[0342] TEA: triethylamine

[0343] Tf: Trifluoromethanesulfonate

[0344] TfAA: Trifluoromethanesulfonic anhydride

[0345] TFA: trifluoroacetic acid

[0346] TIPS: Triisopropylsilane

[0347] THF: Tetrahydrofuran

[0348] TMSCN: Trimethylcyanosylsilane

[0349] pTSA: p-Toluenesulfonic acid

[0350] TsOH: p-Toluenesulfonic acid

[0351] The preparation of representative, non-limiting examples of the provided compounds is described below.

[0352] Example 1: (S)-2-(dimethylamino)-4-methylpentanoic acid [I-1].

[0353]

[0354] Reaction process:

[0355]

[0356] Procedures and representations:

[0357] Step 1: (S)-2-(dimethylamino)-4-methylpentanoic acid:

[0358] Formaldehyde (38%, 24.0 g) and Pd / C (10%, 500 mg) were added to a solution of (S)-2-amino-4-methylpentanoic acid (2.0 g, 15.24 mmol), and the resulting solution (60 mL) was filtered. The mixture was hydrogenated at room temperature for two days and filtered to remove the catalyst. The filtrate was concentrated to dryness, and EtOH (30 mL) was added to the residue. The mixture was stirred for 1 hour and filtered. The filtrate was concentrated to give (S)-2-(dimethylamino)-4-methylpentanoic acid (1.3 g, 8.16 mmol, 53%) as a white powder. ESI-MS (EI) + ,m / z):160.2[M+H] + . 1 H-NMR (400MHz, MeOD-d4): δ3.47 (dd, J = 4.4Hz, 10.0Hz, 1H), 2.85 (S, 6H), 1.89-1.74 (m, 2H), 1.62-1.55 (m, 1H), 1.00 (dd, J = 2.8Hz, 6.8Hz, 6H).

[0359] Examples 2 and 3: (S)-2-amino-7,7,7-trifluoroheptanoate [I-2] and (R)-2-amino-7,7,7-trifluoroheptanoate [I-3].

[0360]

[0361] Reaction process:

[0362]

[0363] Procedures and representations:

[0364] Step 1: 1,1,1-Trifluoro-5-iodopentane:

[0365] Under ice bath conditions, I₂ (4.45 g, 17.5 mmol) was added to a solution of 5,5,5-trifluoropentane-1-ol (2.0 g, 14.0 mmol), imidazole (1.48 g, 21.7 mmol), and PPh₃ (5.5 g, 21.0 mmol) in DCM (40 mL). The mixture was heated to room temperature and stirred overnight. Et₂O (50 mL) was added to the mixture, and then stirred for 10 minutes. The mixture was filtered, and the filtrate was evaporated at atmospheric pressure (65 °C) to remove the solvent. The residue was diluted with Et₂O (30 mL), the mixture was filtered again, and the filtrate was used for the next step.

[0366] Step 2: (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester and (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester:

[0367] At -10°C, KOH (50%, 20 mL) was added to a solution of 2-(diphenylmethyleneamino)acetic acid tert-butyl ester (2.0 g, 6.78 mmol) and TBAB (109 mg, 0.339 mmol) in toluene (35 mL) and DCM (15 mL). After 5 minutes, 1,1,1-trifluoro-5-iodopentane from Et₂O (30 mL) was added dropwise to the above solution over a period of 5 minutes. The resulting mixture was stirred at -10°C to 0°C for 1 hour. The solution was diluted with water (200 mL) and extracted with EA (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was then purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 10) and chiral preparative HPLC [column: R,R-whelk-ol 4.6*250 mm 5 μm; solvent: MeOH (0.2% methanol ammonia)] to obtain (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%) and (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%).

[0368] (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%). ESI-MS (EI+, m / z): 243.1 [M+H]+. 1H-NMR (500MHz, CDCl3): δ8.64 (d, J = 8.0Hz, 2H), 7.43-7.46 (m, 3H), 7.38-7.39 (m, 1H), 7.31-7.34 (m, 2H), 7 .15-7.17(m,2H),3.91(dd,J=5.5Hz,7.5Hz,1H),2.00-2.05(m,2H),1.88-1.92(m,2H),1.31-1.52(m,13H).

[0369] (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%). ESI-MS (EI+, m / z): 243.1 [M+H]+. 1 H-NMR (500MHz, CDCl3): δ8.64 (d, J = 7.0Hz, 2H), 7.43-7.46 (m, 3H), 7.38-7.39 (m, 1H), 7.31-7.34 (m, 2H), 7 .15-7.17(m,2H),3.92(dd,J=5.5Hz,7.5Hz,1H),2.00-2.05(m,2H),1.88-1.92(m,2H),1.31-1.52(m,13H).

[0370] Step 3: (S)-2-amino-7,7,7-trifluoroheptanoate salt [I-2]:

[0371] A solution of (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol) in 6M HCl (10 mL) and dioxane (5 mL) was heated to 100 °C for 17 hours. The solution was extracted with Et₂O (10 mL × 2), and the aqueous phase was concentrated to dryness to give (S)-2-amino-7,7,7-trifluoroheptanoate salt (I-2) (82.7 mg, 0.35 mmol, 74%) as a white solid. ESI-MS (EI+, m / z): 200.1 [M+H]+. ¹H NMR (500 MHz, D₂O) δ 3.93 (t, J = 6.0 Hz, 1H), 2.10–2.15 (m, 2H), 1.83–1.90 (m, 2H), 1.40–1.56 (m, 4H).

[0372] Step 4: (R)-2-amino-7,7,7-trifluoroheptanoate salt [I-3]:

[0373] A solution of (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol) in 6 M HCl (10 mL) and dioxane (5 mL) was heated to 100 °C for 17 hours. The solution was extracted with Et₂O (10 mL × 2), and the aqueous phase was concentrated to dryness to give (R)-2-amino-7,7,7-trifluoroheptanoate salt (I-3) (91.6 mg, 0.39 mmol, 82%) as a white solid. ESI-MS (EI+, m / z): 200.1 [M+H]+. ¹H NMR (500 MHz, D₂O) δ 3.92 (t, J = 6.0 Hz, 1H), 2.09–2.14 (m, 2H), 1.82–1.89 (m, 2H), 1.39–1.55 (m, 4H).

[0374] Examples 4 and 5: (S)-2-amino-4,4,4-trifluorobutyric acid [I-4] and (R)-2-amino-4,4,4-trifluorobutyric acid [I-5].

[0375]

[0376] Reaction process:

[0377]

[0378] Procedures and representations:

[0379] Step 1: (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid

[0380] N-(benzyloxycarbonyloxy)succinimide (1.75 g, 7.01 mmol) was slowly added to a solution of 2-amino-4,4,4-trifluorobutyric acid (1.0 g, 6.36 mmol) and NaHCO3 (589 mg, 7.01 mmol) in acetone (60 mL), and the resulting solution (60 mL) was filtered at 0 °C. The mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with CH2Cl2 (2 × 100 mL), and the aqueous layer was acidified to approximately pH 4 with HCl (3 M), followed by extraction with EtOAc (3 × 150 mL). The organic phase was dried with Na2SO4, and the solvent was evaporated under vacuum. The crude product was purified by chiral preparative HPLC (column: AY-H 4.6*250mm 5μm; solvent: EtOH) to obtain (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol, 37.8%) and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol, 37.8%) as white solids. ESI-MS (EI+, m / z): 314.0 [M+Na]+.

[0381] (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid. 1 H-NMR (500MHz, DMSO-d6): δ13.20 (s, 1H), 7.84 (d, J = 9.0Hz, 1H), 7.40-7.30 (m, 5H), 5.06 (s, 2H), 4.31-4.27 (m, 1H), 2.85-2.58 (m, 2H).

[0382] (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid. 1 H-NMR (500MHz, DMSO-d6): δ13.21(s,1H),7.85(d,J=8.5Hz,1H),7.38-7.30(m,5H),5.06(s,2H),4.31-4.27(m,1H),2.83-2.59(m,2H).

[0383] Step 2: (S)-2-amino-4,4,4-trifluorobutyric acid [I-4].

[0384] A mixture of (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol) and Pd / C (10%) (200 mg) in MeOH (50 mL) was stirred at room temperature under a hydrogen atmosphere for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (S)-2-amino-4,4,4-trifluorobutyric acid (I-4) (250 mg, 1.59 mmol, 66.3%) as a white solid. ESI-MS (EI+, m / z): 158.1 [M+H]+. 1H-NMR (500 MHz, DMSO-d6+1 drop TFA+1 drop D2O): δ 4.32 (t, J = 6.0 Hz, 1H), 3.03–2.82 (m, 2H).

[0385] Step 3: (R)-2-amino-4,4,4-trifluorobutyric acid [I-5].

[0386] A mixture of (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol) and Pd / C (10%) (200 mg) in MeOH (50 mL) was stirred for 2 hours at room temperature under a hydrogen atmosphere. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (R)-2-amino-4,4,4-trifluorobutyric acid (I-5) (250 mg, 1.59 mmol, 66.3%) as a white solid. ESI-MS (EI) + ,m / z):158.1[M+H] + . 1 ¹H-NMR (500MHz, DMSO-d⁶ + 1 drop TFA + 1 drop D₂O): δ 4.31 (t, J = 6.0 Hz, 1H), 3.03-2.83 (m, 2H).

[0387] Examples 6 and 7: (S)-2-amino-5,5,5-trifluorovaleric acid [I-6] and (R)-2-amino-5,5,5-trifluorovaleric acid [I-7].

[0388]

[0389] Reaction process:

[0390]

[0391] Procedures and representations:

[0392] Step 1: 4,4,4-Trifluorobutyraldehyde:

[0393] Under ice bath conditions, IBX (13.0 g, 46.9 mmol) was added to a solution of 4,4,4-trifluorobut-1-ol (4.0 g, 31.3 mmol) in DMSO (80 mL). The mixture was heated to room temperature and stirred overnight. The reaction mixture was poured into water (200 mL) and extracted with Et₂O (100 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was used for the next step.

[0394] Step 2: 2-(phenylmethylamino)-5,5,5-trifluoropentadienonitrile:

[0395] Under ice bath conditions, benzylamine (4 mL), AcOH (3.0 mL), and then TMSCN (3.5 mL) were added to a solution of 4,4,4-trifluorobutyraldehyde in Et₂O (200 mL). The mixture was heated to room temperature and stirred overnight. The solution was diluted with water (200 mL) and extracted with EtOAc (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 6.7 g (crude) 2-(phenylmethylamino)-5,5,5-trifluoropentadienonitrile as a brown solid, which was used in the next step. ESI-MS (EI+, m / z): 243.1 [M+H]+.

[0396] Step 3: 2-(phenylmethylamino)-5,5,5-trifluorovaleric acid:

[0397] A solution of 2-(phenylmethylamino)-5,5,5-trifluoropentanoic acid (6.7 g, crude) in concentrated HCl (80 mL) and AcOH (30 mL) was heated to 95 °C for 17 hours. The solution was concentrated to dryness, filtered (100 mL), diluted with ACN (50 mL), and the pH was adjusted to 3-4 with saturated NaHCO3 solution. The mixture was filtered and dried to give 2-(phenylmethylamino)-5,5,5-trifluoropentanoic acid (3.5 g, 13.4 mmol, 43%, 3 steps) as a white solid. ESI-MS (EI) + ,m / z):262.1[M+H] + .

[0398] Step 4: 2-Amino-5,5,5-trifluorovaleric acid:

[0399] A mixture of 2-(benzylamino)-5,5,5-trifluorovaleric acid (3.3 g, 12.6 mmol) and Pd(OH)₂ / C (20%, 400 mg) in AcOH (60 mL) was stirred at 30 °C for 17 hours. The mixture was filtered, and the filtrate was concentrated to dryness to give 2-amino-5,5,5-trifluorovaleric acid (3.0 g, crude) as a brown solid. ESI-MS (EI+, m / z): 172.2 [M+H]+.

[0400] Step 5: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid:

[0401] Under ice bath conditions, Cbz-OSu (3.45 g, 13.9 mmol) was added to a solution of 2-amino-5,5,5-trifluorovaleric acid (3.0 g, crude) in saturated NaHCO3 solution (100 mL) and acetone (100 mL). After 2 hours, the mixture was adjusted to pH 3 with 6 M HCl, extracted with EtOAc (50 mL × 2), washed with water (50 mL) and brine (100 mL), dried (Na2SO4), and concentrated under vacuum. The crude product was purified successively by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 2) and chiral preparative HPLC [column: AY-H 4.6*250mm 5μm; solvent: MeOH (0.5% NH4OH)] to obtain (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps) as white solids.

[0402] (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps). ESI-MS (EI+, m / z): 328.0 [M+Na]+. 1H-NMR (500 MHz, DMSO-d6): δ 12.86 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.31–7.39 (m, 5H), 5.05 (s, 2H), 4.05–4.10 (m, 1H), 2.34–2.41 (m, 1H), 2.21–2.29 (m, 1H), 1.84–1.97 (m, 2H).

[0403] (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps) ESI-MS (EI+, m / z): 328.0 [M+Na]+. 1H-NMR (500 MHz, DMSO-d6): δ 12.85 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.30–7.39 (m, 5H), 5.05 (s, 2H), 4.05–4.10 (m, 1H), 2.34–2.41 (m, 1H), 2.21–2.29 (m, 1H), 1.84–1.97 (m, 2H).

[0404] Step 6: (S)-2-amino-5,5,5-trifluorovaleric acid [I-6]:

[0405] A mixture of (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (500 mg, 1.64 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred at room temperature under hydrogen for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (S)-2-amino-5,5,5-trifluorovaleric acid (I-6) (200 mg, 1.17 mmol, 71%) as a white solid. ESI-MS (EI) + ,m / z):172.1[M+H] + . 1 H-NMR (400MHz, DMSO-d6): δ8.38 (s, 3H), 4.05 (d, J = 4.4Hz, 1H), 2.34-2.55 (m, 2H), 1.95-20.9 (m, 2H).

[0406] Step 7: (R)-2-amino-5,5,5-trifluorovaleric acid [I-7]:

[0407] A mixture of (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (500 mg, 1.64 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred at room temperature under hydrogen for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (R)-2-amino-5,5,5-trifluorovaleric acid (I-7) (160 mg, 0.94 mmol, 57%) as a white solid. ESI-MS (EI+, m / z): 172.1 [M+H]+. 1H-NMR (400 MHz, DMSO-d6): δ 8.38 (s, 3H), 4.05 (d, J = 4.4 Hz, 1H), 2.34–2.55 (m, 2H), 1.95–20.9 (m, 2H).

[0408] Examples 8 and 9: (S)-2-amino-6,6,6-trifluorohexanoic acid [I-8] and (R)-2-amino-6,6,6-trifluorohexanoic acid [I-9].

[0409]

[0410] Reaction process:

[0411]

[0412] Procedures and representations:

[0413] Step 1: (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid and (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid

[0414] At 0 °C, methyl chloroformate (554 mg, 3.25 mmol) was slowly added to a solution of 2-amino-6,6,6-trifluorohexanoic acid (556 mg, 2.5 mmol) and 1 M NaOH (25 mL, 25 mmol) in THF (25 mL), and the mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with DCM (2 × 100 mL), and the aqueous layer was acidified to approximately pH 4 with HCl (3 M), followed by extraction with EtOAc (3 × 50 mL). The organic phase was dried with Na₂SO₄ and the solvent was evaporated under vacuum. The crude product was purified by chiral preparative HPLC (column: AY-H (250*4.6mm 5μm); mobile phase: n-hexane (0.1% DEA):EtOH (0.1% DEA) = 90:10) to obtain (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (232 mg, 0.73 mmol, 29%) and (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (250 mg, 0.78 mmol, 31.3%) as white solids. ESI-MS (EI+, m / z): 342.0 [M+Na]+.

[0415] (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid, 1H-NMR (500MHz, DMSO-d6): δ 12.68 (s, 1H), 7.66 (d, J = 7.5Hz, 1H), 7.38–7.32 (m, 5H), 5.04 (s, 2H), 4.00–3.96 (m, 1H), 2.28–2.19 (m, 2H), 1.80–1.51 (m, 4H).

[0416] (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid, 1H-NMR (500MHz, DMSO-d6): δ 12.68 (s, 1H), 7.67 (d, J = 8.5Hz, 1H), 7.38–7.30 (m, 5H), 5.04 (s, 2H), 4.00–3.96 (m, 1H), 2.33–2.15 (m, 2H), 1.82–1.51 (m, 4H).

[0417] Step 2: (S)-2-amino-6,6,6-trifluorohexanoic acid [I-8].

[0418] A mixture of (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (200 mg, 0.63 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred at room temperature under a hydrogen atmosphere for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (S)-2-amino-6,6,6-trifluorohexanoic acid (I-8) (56.2 mg, 0.30 mmol, 48.2%) as a white solid. ESI-MS (EI... + ,m / z):186.1[M+H] + . 1 ¹H-NMR (500MHz, DMSO-d⁶ + 1 drop TFA + 1 drop D₂O): δ 3.99 (t, J = 5.5 Hz, 1H), 2.32–2.30 (m, 2H), 1.91–1.83 (m, 2H), 1.70–1.57 (m, 2H).

[0419] Step 3: (R)-2-amino-6,6,6-trifluorohexanoic acid [I-9].

[0420] A mixture of (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (250 mg, 0.78 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred at room temperature under a hydrogen atmosphere for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (R)-2-amino-6,6,6-trifluorohexanoic acid (I-9) (48.8 mg, 0.26 mmol, 33.8%) as a white solid. ESI-MS (EI) + ,m / z):186.1[M+H] + . 1¹H-NMR (500MHz, DMSO-d⁶ + 1 drop TFA + 1 drop D₂O): δ 3.98 (t, J = 6.5 Hz, 1H), 3.33–2.28 (m, 2H), 1.93–1.81 (m, 2H), 1.71–1.54 (m, 2H).

[0421] Example 11: (S)-2-(phenylmethylamino)-4-methylpentanoic acid [I-11].

[0422]

[0423] Reaction process:

[0424]

[0425] Procedures and representations:

[0426] Step 1: (S)-2-(phenylmethylamino)-4-methylpentanoic acid benzoate:

[0427] Benzaldehyde (0.26 g, 2.4 mmol) and potassium acetate (0.4 g, 4.1 mmol) were added to a stirred solution of L-leucine benzyl ester p-toluenesulfonate (800 mg, 2.0 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 min. Then, sodium cyanoborohydride (0.2 g, 3.0 mmol) was added, and the mixture was stirred at room temperature for another 5 h. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL × 2), filtered (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21*250 mm × 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(phenylmethylamino)-4-methylpentanoic acid benzyl ester (200 mg, 0.64 mmol, 32%) as a colorless oil. MS(EI+,m / z):312.3[M+H]+. 1H-NMR (500MHz, MeOD): δ7.41~7.49(m,10H),5.34(dd,J=12.0Hz,45.0Hz,2H),4.23(q ,J=12.0Hz,2H),4.07~4.09(m,3H),1.68~1.85(m,3H),0.94(dd,J=8.5Hz,20.5Hz,6H).

[0428] Step 2: (S)-2-(phenylmethylamino)-4-methylpentanoic acid [I-11]:

[0429] Add 1M NaOH (0.5 mL) to a stirred solution of (S)-2-(phenylmethylamino)-4-methylpentanoic acid methyl ester (50 mg, 0.16 mmol) in MeOH (5 mL). Stir the reaction mixture at room temperature for 4 hours. Concentrate the resulting solution and purify the residue by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(phenylmethylamino)-4-methylpentanoic acid (I-11) (21 mg, 0.095 mmol, 58%) as a white solid. MS (EI+, m / z): 222.2 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ9.32 (s, 1H), 7.43~7.50 (m, 5H), 4.17 (dd, J=13.0 Hz, 44.0Hz, 2H), 3.82 (t, J = 6.5Hz, 1H), 1.68 ~ 1.76 (m, 3H), 0.85 ~ 0.90 (m, 6H).

[0430] Example 12: (S)-4-methyl-2-(2-phenylacetamido)valerate [I-12]:

[0431]

[0432] Reaction process:

[0433]

[0434] Procedures and representations:

[0435] Step 1: (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid methyl ester:

[0436] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), 2-phenylacetic acid (260 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid benzyl ester (300 mg, 0.88 mmol, 70%) as a white solid. MS (EI+, m / z): 340.2 [M+H]+.

[0437] Step 2: (S)-4-methyl-2-(2-phenylacetamido)valerate [I-12]:

[0438] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid methyl ester (250 mg, 0.74 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid (I-12) (100 mg, 0.40 mmol, 54%) as a white solid. MS (EI+, m / z): 250.2 [M+H]+. 1H-NMR (500MHz, MeOD): δ7.24-7.32 (m, 5H), 4.44 (t, J = 7.5 Hz, 1H), 3.58 (s, 2H), 1.64-1.68 (m, 3H), 0.96 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H).

[0439] Example 13: (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]:

[0440]

[0441] Reaction process:

[0442]

[0443] Procedures and representations:

[0444] Step 1: (S)-2-(isopropylamino)-4-methylpentanoic acid benzoate:

[0445] Acetone (177 mg, 3.05 mmol) and potassium acetate (0.5 g, 5.08 mmol) were added to a stirred solution of L-leucine benzyl ester p-toluenesulfonate (1.0 g, 2.53 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 min. Then, sodium cyanoborohydride (0.24 g, 3.81 mmol) was added, and the mixture was stirred at room temperature for another 3 h. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL × 2), filtered (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21*250 mm × 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(isopropylamino)-4-methylpentanoic acid benzyl ester (200 mg, 0.76 mmol, 30%) as a colorless oil. MS(EI+,m / z):264.3[M+H]+. 1H-NMR (500MHz, MeOD): δ7.22~7.29(m,5H),5.07(dd,J=11.5Hz,17.0Hz,2H),3.33( dd, J=6.5Hz, 8.5Hz, 1H), 2.54~2.59(m,1H), 1.30~1.48(m,3H), 0.72~0.94(m,12H).

[0446] Step 2: (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]:

[0447] A catalytic amount of Pd / C (10%, 50 mg) was added to a stirred solution of (S)-2-(isopropylamino)-4-methylpentanoic acid benzoate (200 mg, 0.76 mmol) in MeOH (10 mL). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 24 hours. The resulting solution was filtered and concentrated to give (S)-2-(isopropylamino)-4-methylpentanoic acid (I-13) (100 mg, 0.57 mmol, 76%) as a white solid. MS (EI+, m / z): 174.3 [M+H]+. 1H-NMR (500MHz, MeOD): δ3.56 (dd, J=6.0Hz, 8.5Hz, 1H), 3.33~3.40 (m, 1H) ,1.75~1.86(m,2H),1.53~1.58(m,1H),1.31~1.36(m,6H),0.96~1.02(m,6 H).3.85(dd,J=5.5Hz,8.5Hz,1H),2.87(q,J=6.0Hz,1H),2.68(dd,J=7.5H z,12.0Hz,1H),1.92~1.99(m,1H),1.65~1.78(m,3H),0.88~0.96(m,12H).

[0448] Example 14: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0449]

[0450] Reaction process:

[0451]

[0452] Procedures and representations:

[0453] Step 1: (S)-2-(isobutylamino)-4-methylpentanoic acid benzoate:

[0454] Isobutyraldehyde (0.22 g, 3.05 mmol) and potassium acetate (0.5 g, 5.08 mmol) were added to a stirred solution of L-leucine benzyl ester p-toluenesulfonate (1.0 g, 2.53 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 minutes, followed by the addition of sodium cyanoborohydride (0.24 g, 3.81 mmol). The mixture was stirred at room temperature for another 5 hours. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL × 2), filtered (50 mL) and washed with brine (50 mL) with the resulting solution. The concentrated organic phase was purified by preparative HPLC (Boston C1821*250mm 10μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain a colorless oily form of (S)-2-(isobutylamino)-4-methylpentanoic acid methyl ester (300 mg, 1.08 mmol, 50%). MS (EI+, m / z): 278.2 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ9.16 (s, 1H), 9.14 (d, J = 17.5Hz, 2H), 7.42-7.43 (m, 5H), 5.28 (q, J = 12.0Hz, 2H), 4.0 8-4.09(m,1H),2.87-2.89(m,1H),2.65-2.66(m,1H),1.91-1.95(m,1H),1.62~1.71(m,3H),0.88~0.94(m,12H).

[0455] Step 2: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0456] A catalytic amount of Pd / C (10%, 50 mg) was added to a stirred solution of (S)-2-(isobutylamino)-4-methylpentanoic acid methyl ester (300 mg, 1.08 mmol) in MeOH (10 mL). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 24 hours. The resulting solution was filtered and the filtrate was concentrated to give (S)-2-(isobutylamino)-4-methylpentanoic acid (I-14) (150 mg, 0.8 mmol, 74%) as a white solid. MS (EI+, m / z): 188.3 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ8.82(s,2H),3.85(dd,J=5.5Hz,8.5Hz,1H),2.87(q,J=6.0Hz,1H ), 2.68 (dd, J=7.5Hz, 12.0Hz, 1H), 1.92~1.99 (m, 1H), 1.65~1.78 (m, 3H), 0.88~0.96 (m, 12H).

[0457] Example 15: (S)-2-benzamido-4-methylpentanoic acid [I-15]:

[0458]

[0459] Reaction process:

[0460]

[0461] Procedures and representations:

[0462] Step 1: (S)-2-benzamido-4-methylpentanoic acid methyl ester:

[0463] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), benzoic acid (223 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-benzamido-4-methylpentanoic acid benzyl ester (300 mg, 0.92 mmol, 73%) as a white solid. MS (EI+, m / z): 326.2 [M+H]+.

[0464] Step 2: (S)-2-benzamido-4-methylpentanoic acid [I-15]:

[0465] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-benzamido-4-methylpentanoic acid methyl ester (100 mg, 0.46 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-benzamido-4-methylpentanoic acid (I-15) (100 mg, 0.42 mmol, 65%) as a white solid. MS (EI+, m / z): 236.2 [M+H]+. 1H-NMR (400MHz, MeOD): δ7.87 (t, J = 6.5 Hz, 2H), 7.47-7.57 (m, 3H), 4.69 (dd, J = 4.0 Hz, 11.0 Hz, 1H), 1.75-1.84 (m, 3H), 1.01 (dd, J = 6.5 Hz, 10.5 Hz, 6H).

[0466] Example 16: (S)-2-isobutyramido-4-methylpentanoic acid [I-16]:

[0467]

[0468] Reaction process:

[0469]

[0470] Procedures and representations:

[0471] Step 1: (S)-2-isobutyramido-4-methylpentanoic acid benzoate:

[0472] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), isobutyric acid (168 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-isobutyramido-4-methylpentanoic acid benzyl ester (300 mg, 1.03 mmol, 81%) as a white solid. MS (EI+, m / z): 292.2 [M+H]+.

[0473] Step 2: (S)-2-isobutyramido-4-methylpentanoic acid [I-16]:

[0474] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-(cyclohexylformamido)-4-methylpentanoic acid benzyl ester (200 mg, 0.69 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-isobutyramamido-4-methylpentanoic acid (100 mg, 0.50 mmol, 73%) as a white solid. MS (EI+, m / z): 202.2 [M+H]+. 1H-NMR (400MHz, MeOD): δ4.43 (t, J = 6.4 Hz, 1H), 2.49-2.56 (m, 1H), 1.60-1.74 (m, 3H), 1.12 (dd, J = 2.4Hz, 6.8Hz, 6H), 0.96 (dd, J = 6.4Hz, 16.0Hz, 6H).

[0475] Example 17: (S)-2-(cyclohexanesulfonamido)-4-methylpentanoic acid [I-17]:

[0476]

[0477] Reaction process:

[0478]

[0479] Procedures and representations:

[0480] Step 1: (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid methyl ester:

[0481] Cyclohexanesulfonyl chloride (278.53 mg, 1.52 mmol) was added to a solution of (S)-2-amino-4-methylpentanoic acid benzoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and Et3N (642.89 mg, 6.35 mmol) in DMF (3 mL) under ice bath cooling. The mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid benzoate (200 mg, 0.544 mmol, 98%) as a white solid. ESI-MS(EI+,m / z):368.3[M+H]+. 1H-NMR (500MHz, DMSO-d6) δ7.70 (d, J=9.0Hz, 1H), 7.38 (t, J=6.5Hz, 4H), 7.37-7. 32(m,1H),5.14(q,J=12.5Hz,2H),3.91(td,J=5.0Hz,9.5Hz,1H),2.69-2.74(m,1 H),2.05(d,J=12.5Hz,1H),1.97(d,J=12.5Hz,1H),1.74-1.67(m,2H),1.57-1.51 (m,2H),1.50-1.44(m,1H),1.36-0.99(m,5H),0.87(dt,J=10.5Hz,J=20.5Hz,6H).

[0482] Step 2: (S)-2-(cyclohexanesulfonamido)-4-methylpentanoic acid [I-17]:

[0483] Pd / C (20 mg, 10%) was added to a solution of (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid benzoate (192 mg, 0.552 mmol) in EtOH (3 mL). The reaction mixture was stirred at 50 °C under hydrogen for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid (I-17) (23.3 mg, 0.084 mmol, 100%) as a white solid. ESI-MS (EI) + ,m / z):300.2[M+Na] + . 1 H NMR(500MHz,DMSO-d6)δ12.75(s,1H),7.47(d,J=9.0Hz,1H),3.76(td,J=5.0Hz,9.5Hz,1H),2.82-2.69(m,1H),2.1 8-1.97(m,2H),1.82-1.69(m,3H),1.61(d,J=12.5Hz,1H),1.54-1.40(m,2H),1.39-1.07(m,5H),0.95-0.80(m,6H).

[0484] Example 18: (S)-4-methyl-2-(phenylmethanesulfonamide)valerate [I-18]:

[0485]

[0486] Reaction process:

[0487]

[0488] Procedures and representations:

[0489] Step 1: (S)-4-methyl-2-(phenylmethanesulfonamide)pentanoic acid methyl ester:

[0490] Benzoyl chloride (290.71 mg, 1.52 mmol) was added to a solution of (S)-2-amino-4-methylpentanoic acid benzoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and Et3N (642.89 mg, 6.35 mmol) in DMF (3 mL) under ice bath cooling. The mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-4-methyl-2-(phenylmethanesulfonamide)pentanoic acid benzoate (149 mg, 0.396 mmol, 90%) as a white solid. ESI-MS(EI+,m / z):398.0[M+Na]+. 1H-NMR(500MHz,DMSO-d6)δ7.81(d,J=8.5Hz,1H),7.52-7.18(m,9H),5.15(s,2H),4.28(dd,J=13.5 Hz, 44.5Hz, 2H), 3.87 (dd, J = 8.0Hz, 15.0Hz, 1H), 1.57-1.15 (m, 4H), 0.82 (dd, J = 4.5Hz, 6.0Hz, 6H).

[0491] Step 2: (S)-4-methyl-2-(phenylmethanesulfonamide)valerate [I-18]:

[0492] Pd / C (20 mg, 10%) was added to a solution of (S)-4-methyl-2-(phenylmethanesulfonyl)valerate benzyl ester (121 mg, 0.322 mmol) in EtOH (3 mL). This reaction mixture was stirred at 50 °C under hydrogen for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(phenylmethanesulfonyl)valerate (I-18) (41.2 mg, 0.144 mmol, 100%) as a white solid. ESI-MS (EI+, m / z): 308.0 [M+Na]+. 1H NMR(500MHz,DMSO-d6)δ12.77(s,1H),7.59(d,J=8.5Hz,1H),7.47-7.25(m,5H),4.30(dd,J=13.5Hz,37.0Hz,2H),3. 75(dd,J=7.5Hz,15.5Hz,1H),1.65(dt,J=6.5Hz,13.5Hz,1H),1.45(t,J=7.2Hz,2H),0.85(dd,J=1.5Hz,6.5Hz,6H).

[0493] Example 19: (S)-4-methyl-2-(methanesulfonamide)valerate [I-19]:

[0494]

[0495] Reaction process:

[0496]

[0497] Procedures and representations:

[0498] Step 1: (S)-4-methyl-2-(methanesulfonamide)pentanoic acid methyl ester:

[0499] Methanesulfonyl chloride (290.71 mg, 1.52 mmol) was added to a solution of (S)-2-amino-4-methylpentanoic acid benzoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and Et3N (642.89 mg, 6.35 mmol) in DMF (3 mL) under ice bath cooling, and the mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-4-methyl-2-(methanesulfonamide)pentanoic acid benzoate (192 mg, 0.641 mmol, 98%) as a white solid. ESI-MS (EI + ,m / z):323.0[M+Na] + . 1 H-NMR(500MHz,DMSO-d6)δ7.79(d,J=8.8Hz,1H),7.42-7.36(m,4H),7.37-7.32(m,1H),5.16(s,2H),3.97(t d, J=6.0Hz, 9.0Hz, 1H), 2.85 (s, 3H), 1.68 (dq, J=6.5Hz, 13.0Hz, 1H), 1.54-1.46 (m, 2H), 0.91-0.82 (m, 6H).

[0500] Step 2: (S)-4-methyl-2-(methanesulfonamide)valerate [I-19]:

[0501] Pd / C (20 mg, 10%) was added to a solution of (S)-4-methyl-2-(methanesulfonamide)pentanoic acid benzoate (149 mg, 0.497 mmol) in EtOH (3 mL). This reaction mixture was stirred at 50 °C under a hydrogen atmosphere for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(methanesulfonamide)pentanoic acid (I-19) (31.4 mg, 0.150 mmol, 100%) as a white solid. ESI-MS (EI...) + ,m / z):232.1[M+Na] + . 1H NMR(500MHz,DMSO-d6)δ12.82(s,1H),7.56(d,J=9.0Hz,1H),3.82(dd,J=8.0Hz,15.5Hz,1H) ,2.88(s,3H),1.72(dt,J=6.5Hz,13.0Hz,1H),1.48(t,J=7.0Hz,2H),0.89(t,J=7.0Hz,6H).

[0502] Example 20: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0503]

[0504] Reaction process:

[0505]

[0506] Procedures and representations:

[0507] Step 1: (S)-4-methyl-1-oxo-1-(phenylamino)pent-2-ylcarbamate:

[0508] At room temperature, aniline (702 mg, 7.55 mmol), HATU (1.72 g, 4.52 mmol), and Et3N (1.14 g, 11.31 mmol) were added to a solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 3.77 mmol) in DMF (20 mL). After 2 hours, the solution was diluted with EtOAc (80 mL), filtered (80 mL × 3), washed with brine (80 mL), dried (Na2SO4), filtered again, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 3) to give (S)-4-methyl-1-oxo-1-(phenylamino)pent-2-ylcarbamate (350 mg, 1.03 mmol, 27%) as a white solid. ESI-MS (EI+, m / z): 341.1 [M+H]+.

[0509] Step 2: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0510] A mixture of (S)-4-methyl-1-oxo-1-(phenylamino)pentan-2-ylcarbamate (350 mg, 1.03 mmol) and Pd / C (10%, 50 mg) in MeOH (10 mL) was stirred under hydrogen at room temperature for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-amino-4-methyl-N-phenylpentanamide (I-20) (100 mg, 0.49 mmol, 47%) as a white solid. ESI-MS (EI+, m / z): 207.2 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ9.86 (s, 1H), 7.63 (dd, J = 1.0Hz, 8.5Hz, 2H), 7.31-7.27 (m, 2H), 7.03 (t, J = 7.5Hz, 1H), 3. 31(dd,J=5.0Hz,8.5Hz,1H),1.80-1.71(m,1H),1.50-1.44(m,1H),1.35-1.29(m,1H),0.90(dd,J=6.5Hz,14.0Hz,6H).

[0511] Example 21: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0512]

[0513] Reaction process:

[0514]

[0515] Procedures and representations:

[0516] Step 1: (S)-4-methyl-1-(methylamino)-1-oxopent-2-ylcarbamate:

[0517] At 25 °C, a solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 3.77 mmol) in DMF (20 mL) was added with MeNH2·HCl (509 mg, 7.54 mmol), HATU (1.72 g, 4.52 mmol), and Et3N (1.14 g, 11.31 mmol). After 2 hours, the solution was diluted with EtOAc (80 mL), filtered (80 mL × 3), washed with brine (80 mL), dried (Na2SO4), filtered again, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 3) to give (S)-4-methyl-1-(methylamino)-1-oxopentan-2-ylcarbamate (550 mg, 1.98 mmol, 52%) as a colorless oil. ESI-MS(EI+,m / z):279.2[M+H]+.

[0518] Step 2: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0519] A mixture of (S)-4-methyl-1-(methylamino)-1-oxopentan-2-ylcarbamate (300 mg, 1.08 mmol) and Pd / C (10%) (50 mg) in MeOH (10 mL) was stirred at room temperature under hydrogen for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-amino-N,4-dimethylpentanamide (I-21) (152 mg, 1.05 mmol, 98%) as a colorless oil. ESI-MS (EI+, m / z): 145.3 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ7.80 (s, 1H), 3.10 (dd, J=5.0Hz, 9.0Hz, 1H), 2.57 (dd, J=3.0Hz, 5.0H z,3H),1.81(s,2H),1.66-1.69(m,1H),1.34-1.39(m,1H),1.16-1.22(m,1H),0.81-0.87(m,6H).

[0520] Example 22: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-22]:

[0521]

[0522] Reaction process:

[0523]

[0524] Procedures and representations:

[0525] Step 1: (S)-2-(cyclohexylformamido)-4-methylpentanoic acid benzoate:

[0526] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), cyclohexanecarboxylic acid (244 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-(cyclohexylformamido)-4-methylpentanoic acid benzyl ester (300 mg, 0.91 mmol, 71%) as a white solid. MS (EI+, m / z): 332.3 [M+H]+.

[0527] Step 2: (S)-2-(cyclohexylformamido)-4-methylvaleric acid [I-22]:

[0528] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-(cyclohexylformamido)-4-methylpentanoic acid benzyl ester (200 mg, 0.60 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-(cyclohexylformamido)-4-methylpentanoic acid (I-22) (100 mg, 0.41 mmol, 69%) as a white solid. MS (EI+, m / z): 242.3 [M+H] + . 1H-NMR (500MHz, CD3OD): δ4.43 (t, J=7.5Hz, 1H), 2.29 (td, J=8.0Hz, 11.0Hz, 1H), 1.74-1.85 (m ,4H),1.63-1.72(m,4H),1.43-1.49(m,2H),1.26-1.36(m,3H),0.96(dd,J=6.0Hz,20.5Hz,6H).

[0529] Example 25: (S)-4-methyl-2-(phenylsulfonamido)valerate [I-25]:

[0530]

[0531] Reaction process:

[0532]

[0533] Procedures and representations:

[0534] Step 1: (S)-4-methyl-2-(phenylsulfonamido)pentanoic acid methyl ester:

[0535] Benzenesulfonyl chloride (148.12 mg, 0.838 mmol) was added to a solution of (S)-2-amino-4-methylpentanoate benzoate 4-methylbenzenesulfonate (300 mg, 0.762 mmol) and Et3N (385.73 mg, 3.81 mmol) in DMF (3 mL) under ice bath cooling. The mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), filtered (10 mL × 3) and washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product (280 mg, purity: 85%, yield: 74%) was used directly for the next step. ESI-MS (EI+, m / z): 384.1 [M+Na]+.

[0536] Step 2: (S)-4-methyl-2-(phenylsulfonamido)valerate [I-25]:

[0537] Pd / C (20 mg, 10%) was added to a solution of (S)-4-methyl-2-(phenylsulfonamido)pentanoic acid methyl ester (200 mg, 0.553 mmol) in EtOH (3 mL). This reaction mixture was stirred at 50 °C under a hydrogen atmosphere for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(phenylsulfonamido)pentanoic acid (I-25) (63.7 mg, 0.234 mmol, 100%) as a white solid. ESI-MS (EI...) + ,m / z):294.0[M+Na] + . 1 H-NMR (500MHz, DMSO-d6) δ12.61(s,1H),8.16(d,J=8.6Hz,1H),7.79-7.73(m,2H),7.62(t,J=7.3Hz,1H),7.56(t,J=7.4Hz,2H), 3.63(dd,J=8.5Hz,14.5Hz,1H),1.53(td,J=6.5Hz,13.5Hz,1H),1.41-1.31(m,2H),0.79(d,J=6.6Hz,3H),0.66(d,J=6.5Hz,3H).

[0538] Example 26: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]:

[0539]

[0540] Reaction process:

[0541]

[0542] Procedures and representations:

[0543] Step 1: (S)-4-methyl-2-(phenylamino)pentanoic acid methyl ester:

[0544] To a mixture of L-leucine benzyl ester p-toluenesulfonate (200 mg, 0.51 mmol), phenylboronic acid (186 mg, 1.52 mmol), and Cu(OAc)₂ (462 mg, 2.54 mmol) in DCM (10 mL), 4A MS (1.0 g) and Et₃N (155 mg, 1.52 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The mixture was filtered through a filter (50 mL), quenched, extracted with EtOAc (50 mL × 2), filtered through a filter (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 20) to give (S)-4-methyl-2-(phenylamino)pentanoic acid benzyl ester (100 mg, 0.34 mmol, 66%) as a colorless oil. MS (EI+, m / z): 298.2 [M+H]⁺.

[0545] Step 2: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]:

[0546] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-4-methyl-2-(phenylamino)pentanoic acid methyl ester (100 mg, 0.34 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 2 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-4-methyl-2-(phenylamino)pentanoic acid (I-26) (30 mg, 0.15 mmol, 43%) as a white solid. MS (EI+, m / z): 208.1 [M+H]+. 1H-NMR (400MHz, CDCl3): δ7.23(t,J=8.0Hz,2H),6.83(t,J=7.6Hz,1H),6.66(d,J=8.0Hz,2H),3.9 9(d,J=8.4Hz,1H), 2.87(q,J=6.0Hz,1H), 1.72~1.86(m,2H), 1.62~1.68(m,1H), 0.85~1.03(m,6H).

[0547] Example 36: (S)-2-acetamido-4-methylvaleric acid [I-36]:

[0548]

[0549] Reaction process:

[0550]

[0551] Procedures and representations:

[0552] Step 1: (S)-2-acetamido-4-methylpentanoic acid benzoate:

[0553] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), acetic acid (114 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-acetamito-4-methylpentanoic acid benzyl ester (300 mg, 1.14 mmol, 89%) as a white solid. MS (EI+, m / z): 264.2 [M+H]+.

[0554] Step 2: (S)-2-acetamido-4-methylpentanoic acid [I-36]:

[0555] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-acetamido-4-methylpentanoic acid methyl ester (250 mg, 0.74 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-acetamido-4-methylpentanoic acid (I-36) (100 mg, 0.57 mmol, 81%) as a white solid. MS (EI+, m / z): 174.2 [M+H] + . 1 H-NMR (500MHz, MeOD): δ4.43 (dd, J = 6.0 Hz, 9.5 Hz, 1H), 2.00 (s, 3H), 1.61-1.73 (m, 3H), 0.97 (dd, J = 6.0 Hz, 17.5 Hz, 6H).

[0556] Example 45: (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid [I-45]:

[0557]

[0558] Reaction process:

[0559]

[0560] Procedures and representations:

[0561] Step 1: (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid [I-45]:

[0562] SOCl2 (1.83 g, 15.38 mmol) and DMF (0.1 mL) were successively added to a solution of (E)-4-methoxy-4-oxobut-2-enoic acid (1.0 g, 7.69 mmol) in DCM (30 mL). The solution was heated to 40 °C for 4 hours. The solution was concentrated to dryness to obtain an oil. The oil was diluted with DCM (10 mL). A solution of (S)-2-amino-4-methylpentanoic acid (1.0 g, 7.62 mmol) cooled in an ice bath in acetone (20 mL) and saturated Na2CO3 (20 mL) was added dropwise. After 1 hour, the solution was adjusted to pH 2 with 6 M HCl solution, extracted with EtOAc (40 × 2), filtered (80 mL × 3), washed with brine (80 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, MeOH / DCM = 1 / 20) to give (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid (I-45) (1.0 g, 4.11 mmol, 53%) as a yellow oil. ESI-MS (EI + ,m / z):244.2[M+H] + . 1 H-NMR (400MHz, CDCl3): δ7.32(d,J=15.2Hz,1H),7.05(d,J=15.2Hz,1H),6.85-6.8 9(m,2H),7.30-7.46(m,1H),3.82(s,1H),1.63-1.78(m,3H),0.97(d,J=4.8Hz,6H).

[0563] Examples 46 and 47: (R)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-46] and (S)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-47]:

[0564]

[0565] Reaction process:

[0566]

[0567] Procedures and representations:

[0568] Step 1: Ethyl 2,2-difluoro-3-methylbutyrate:

[0569] A mixture of ethyl 3-methyl-2-oxobutyrate (10 g, 0.069 mol) and DAST (16.8 g, 0.10 mol) was stirred at room temperature for 12 hours. After TLC analysis, the reaction mixture was slowly added dropwise to a cold, saturated aqueous solution of sodium bicarbonate. The mixture was extracted with Et₂O (300 mL × 2), and the organic layer was washed with brine, dried, and concentrated to give crude ethyl 2,2-difluoro-3-methylbutyrate (8.3 g), which was used directly in the next step.

[0570] Step 2: 2,2-Difluoro-3-methylbutanal:

[0571] Under argon atmosphere at -78°C, a solution of DIBAL-H in hexane (1.0 M, 69 mL, 69.0 mmol) was added dropwise to a solution of crude ethyl 2,2-difluoro-3-methylbutyrate (8.3 g) in CH₂Cl₂ (200 mL), and the mixture was stirred at -78°C for 30 min. After TLC examination, the reaction mixture was quenched with saturated citric acid and extracted with Et₂O. The extract was washed with saturated citric acid and brine, dried over Na₂SO₄, and concentrated under reduced pressure to give the oily aldehyde 2,2-difluoro-3-methylbutyraldehyde (4.2 g), which was used immediately in the next step without purification.

[0572] Step 3: 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide:

[0573] A solution of crude 2,2-difluoro-3-methylbutanal (4.2 g) in 50 mL of MeOH was cooled to 0 °C. Acetic acid (ice-cold, 2.1 mL) was added dropwise, maintaining the temperature at approximately 0 °C, followed by the addition of trimethylcyanosilane (4.2 mL) over 15 minutes. The reaction mixture was heated to 25 °C and stirred overnight. The resulting cold solution (200 mL) was filtered and added to the reaction mixture, which was then extracted with dichloromethane (2 x 200 mL). The dichloromethane layer was subsequently washed with the resulting solution (2 x 100 mL) and brine (2 x 50 mL). The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (2.8 g), which was used immediately in the next step without purification. ESI-MS (EI+, m / z): 238.2 [M+H]+.

[0574] Step 4: 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid:

[0575] A solution of crude 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (2.8 g) in 50 mL concentrated hydrochloric acid and 10 mL HOAc was stirred at 90 °C for 24 hours and then concentrated. The residue was purified by preparative HPLC to obtain 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (513 mg) as a white solid. The pure product was purified by chiral HPLC to obtain (R)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (80 mg) and (S)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (63 mg), both white solids. ESI-MS (EI+, m / z): 258.2 [M+H]+.

[0576] Step 5-A: (R)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-46]:

[0577] At room temperature, HCOONH4 (98 mg, 1.56 mmol) and Pd / C (100 mg) were added to a solution of (R)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid (80 mg, 0.31 mmol) in 20 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give (R)-2-amino-3,3-difluoro-4-methylpentanoic acid (I-46) (23 mg, 44%) as a white solid; 1H-NMR (500 MHz, D2O): δ 4.27 (dd, J = 24.0, 3.5 Hz, 1H), 2.55–2.42 (m, 1H), 1.04 (d, J = 7.0 Hz, 3H), 0.993 (d, J = 6.5 Hz, 3H).

[0578] Step 5-B: (S)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-47]:

[0579] At room temperature, HCOONH4 (77 mg, 1.22 mmol) and Pd / C (100 mg) were added to a solution of (S)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid (63 mg, 0.24 mmol) in 15 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give (S)-2-amino-3,3-difluoro-4-methylpentanoic acid (I-47) (14 mg, 34%) as a white solid; 1H-NMR (500 MHz, D2O): δ 4.27 (dd, J = 24.0, 3.5 Hz, 1H), 2.55–2.42 (m, 1H), 1.04 (d, J = 7.0 Hz, 3H), 0.993 (d, J = 6.5 Hz, 3H).

[0580] Example 147: (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147].

[0581]

[0582] Reaction process:

[0583]

[0584] Procedures and representations:

[0585] Step 1: (S)-4-methyl-1-(methanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0586] TEA (1.3 g, 12.9 mmol) was added to a solution of (S)-2-(tert-butoxycarbonylamino)-4-methylpentanoic acid (1.0 g, 4.32 mmol), methanesulfonamide (452 ​​mg, 4.75 mmol), and HATU (1.8 g, 4.75 mmol) in DMF (30 mL), and the solution was stirred at room temperature for 17 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain a white solid (S)-4-methyl-1-(methanesulfonamide)-1-oxopentan-2-ylcarbamate tert-butyl ester (130 mg, 0.42 mmol, 8.9%). MS (EI-, m / z): 307.0 [MH] - .

[0587] Step 2: (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147]:

[0588] Add 4M HCl / dioxane (5 mL) to a solution of (S)-4-methyl-1-(methanesulfonyl)-1-oxopentan-2-ylcarbamate (130 mg, 0.42 mmol) in Et2O (15 mL) and stir for 3 hours at room temperature. Filter off the solid to give (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147] (32 mg, 0.13 mmol, 31%) as a white solid. ESI-MS (EI+, m / z): 209.1 [M+H] + . 1H NMR (500MHz, CD3OD) δ3.96 (t, J = 3.0Hz, 1H), 3.32 (s, 3H), 1.74-1.79 (m, 3H), 1.02-1.05 (m, 6H).

[0589] Example 193: (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193].

[0590]

[0591] Reaction process:

[0592]

[0593] Procedures and representations:

[0594] Step 1: (S)-4,4-dimethyl-1-(N-methylmethanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0595] HATU (900 mg, 2.36 mmol) was added to a solution of (S)-2-(tert-butoxycarbonylamino)-4,4-dimethylvaleric acid (500 mg, 1.97 mmol) in DCM (60 mL), and the mixture was stirred at room temperature for 2 hours. Then, Cs₂CO₃ (1.92 g, 5.91 mmol) and N-methylmethanesulfonamide (322 mg, 2.95 mmol) were added to the mixture, and the mixture was stirred overnight at room temperature. The solution was diluted with water (200 mL) and extracted with DCM (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 5) to give a yellow oily (S)-4,4-dimethyl-1-(N-methylmethanesulfonamide)-1-oxopentan-2-ylcarbamate tert-butyl ester (420 mg, 1.25 mmol, 63%). ESI-MS (EI+, m / z): 359.1 [M+Na] + .

[0596] Step 2: (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193]:

[0597] A solution of (S)-4,4-dimethyl-1-(N-methylmethanesulfonamide)-1-oxopentan-2-ylcarbamate tert-butyl ester (420 mg, 1.25 mmol) in Et2O (20 mL) was added with 4 M HCl / dioxane (10 mL) and stirred at room temperature for 17 hours. The solid was filtered off to give (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193] (250 mg, 0.13 mmol, 71%) as a white solid. ESI-MS (EI+, m / z): 237.1 [M+H] + . 1H NMR (500MHz, DMSO) δ8.55(s,3H),4.59(s,1H),3.50(s,3H),3.26(s,3H),1.81-1.85(m,1H),1.63-1.67(m,1H),0.95(s,9H).

[0598] Example 192: 2-Amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192].

[0599]

[0600] Reaction process:

[0601]

[0602] Procedures and representations:

[0603] Step 1: 4-Fluoro-4-methyl-1-(methanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0604] HATU (451 mg, 1.19 mmol) was added to a solution of 4-fluoro-4-methyl-1-(methanesulfonamide)-1-oxopentan-2-ylcarbamate tert-butyl ester (270 mg, 1.08 mmol) in DCM (50 mL), and the mixture was stirred at room temperature for 2 hours. Then, Cs₂CO₃ (1.06 g, 3.24 mmol) and methanesulfonamide (206 mg, 2.17 mmol) were added to the mixture, and the mixture was stirred overnight at room temperature. The solution was diluted with water (200 mL) and extracted with DCM (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 5) to give a yellow oily (S)-4,4-dimethyl-1-(N-methylmethanesulfonamide)-1-oxopentan-2-ylcarbamate tert-butyl ester (200 mg, 0.6 mmol, 55%). ESI-MS (EI+, m / z): 344.1 [M+NH₄] + .

[0605] Step 2: 2-Amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192].

[0606] A solution of (S)-4,4-dimethyl-1-(N-methylmethanesulfonamide)-1-oxopentan-2-ylcarbamate tert-butyl ester (200 mg, 0.6 mmol) in Et2O (20 mL) was added with 4 M HCl / dioxane (10 mL) and stirred at room temperature for 17 hours. The solid was filtered off to give 2-amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192] (89.8 mg, 0.34 mmol, 57%) as a white solid. ESI-MS (EI+, m / z): 227.1 [M+H] + . 1H NMR (500MHz, DMSO) δ8.44(s,3H),4.02(s,1H),3.25(s,3H),2.16-2.25(m,1H),2.03-2.10(m,1H),1.43(s,3H),1.38(s,3H).

[0607] Example 190: (S)-2-((S)-2-amino-4,4-dimethylpentamido)-4-methylpentanoic acid methyl ester hydrochloride [I-190].

[0608]

[0609] Reaction process:

[0610]

[0611] Procedures and representations:

[0612] Step 1: Methyl (S)-2-((S)-2-(tert-Butoxycarbonylamino)-4,4-dimethylpentamido)-4-methylpentanoate:

[0613] HATU (900 mg, 2.3 mmol) was added to a solution of (S)-2-(tert-butoxycarbonylamino)-4,4-dimethylvalerate (500 mg, 2.0 mmol) in DCM (80 mL), and the mixture was stirred at room temperature for 2 hours. Then, Cs₂CO₃ (1.95 g, 6.0 mmol) and (S)-2-amino-4-methylvalerate methyl hydrochloride (555 mg, 3.0 mmol) were added to the mixture, and the mixture was stirred overnight at room temperature. The solution was diluted with water (200 mL) and extracted with DCM (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 5) to give methyl (S)-2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentamido)-4-methylpentanoate (500 mg, 1.34 mmol, 67%) as a white solid. ESI-MS (EI+, m / z): 317.2 [M-56] + .

[0614] Step 2: (S)-2-((S)-2-amino-4,4-dimethylpentamido)-4-methylpentanoic acid methyl ester hydrochloride [I-190].

[0615] A solution of (S)-2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentamido)-4-methylpentanoate methyl ester (500 mg, 1.34 mmol) in Et₂O (20 mL) was added to 4 M HCl / dioxane (10 mL), and the mixture was stirred at room temperature for 17 hours. The solid was filtered off to give a white solid of (S)-2-(S)-2-amino-4,4-dimethylpentamido)-4-methylpentanoate methyl hydrochloride [I-190] (300 mg, 0.97 mmol, 73%). ESI-MS (EI+, m / z): 273.2 [M+H] + . 1H NMR(500MHz,DMSO)δ9.07-9.09(d,J=7.5Hz,1H),8.42(s,3H),4.29-4.34(m,1H),3 .82(m,1H),3.60(s,3H),1.72-1.83(m,2H),1.50-1.62(m,3H),0.86-0.91(m,15H).

[0616] Example 122: (S)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-122].

[0617]

[0618] Reaction process:

[0619]

[0620] Procedures and representations:

[0621] Step 1: (S)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-122]:

[0622] A solution of (S)-2-amino-4,4-dimethylvalerate (100 mg, 0.69 mmol) in MeOH (10 mL) was added with 4 M HCl / dioxane (10 mL) and stirred at 80 °C for 24 h. The mixture was concentrated and the residue was knocked off with Et₂O to give a white solid of (S)-2-amino-4,4-dimethylvalerate methyl hydrochloride [I-122] (23.6 mg, 0.12 mmol, 20%). ESI-MS (EI+, m / z): 160.1 [M+H]+. ¹H-NMR (500 MHz, CD₃OD): δ 4.02–4.04 (m, ¹H), 3.86 (s, ³H), 1.97–2.02 (m, ¹H), 1.64–1.68 (m, ¹H), 1.03–1.05 (d, ⁹H).

[0623] Example 123: (R)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-123].

[0624]

[0625] Reaction process:

[0626]

[0627] Procedures and representations:

[0628] Step 1: (R)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-123]:

[0629] SOCl2 (0.5 mL) was added to a mixture of (R)-2-amino-4,4-dimethylvalerate (50 mg, 0.34 mmol) and anhydrous MeOH (10 mL), and the mixture was stirred at room temperature for 17 hours. The mixture was concentrated and the residue was knocked off with Et2O to give a white solid of (R)-2-amino-4,4-dimethylvalerate methyl hydrochloride [I-123] (34.2 mg, 0.17 mmol, 50%). ESI-MS (EI+, m / z): 160.1 [M+H]+. 1H-NMR (500 MHz, CD3OD): δ 4.02–4.04 (m, 1H), 3.86 (s, 3H), 1.97–2.02 (m, 1H), 1.64–1.68 (m, 1H), 1.03 (s, 9H).

[0630] Example 205: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205].

[0631]

[0632] Reaction process:

[0633]

[0634] Procedures and representations:

[0635] Step 1: 2-(tert-Butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid:

[0636] A mixture of 2-amino-5,5,5-trifluoro-4-methylpentanoic acid (250 mg, 1.35 mmol), Boc₂O (353 mg, 1.62 mmol), and NaOH (80 mg, 2.0 mmol) was dissolved in dioxane (10 mL) and H₂O (2 mL). The mixture was stirred at room temperature for 3 hours. The solution was diluted with water (200 mL) and extracted with DCM (50 mL). The organic phase was washed with water (20 mL × 2) and brine (10 mL), dried (Na₂SO₄), filtered, and concentrated to give crude 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (385 mg) as a colorless oil. ESI-MS (EI) + ,m / z):307.9[M+Na] + .

[0637] Step 2: 2-(tert-Butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid 2,5-dioxopyrrolidine-1-yl ester:

[0638] A mixture of 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (385 mg, 1.35 mmol), 1-hydroxypyrrolidine-2,5-dione (197 mg, 1.71 mmol), and DCC (353 mg, 1.71 mmol) was dissolved in DCM (15 mL). The mixture was stirred at room temperature for 17 hours. The mixture was filtered and the filtrate was washed with brine (20 mL), dried (Na2SO4), filtered again, and concentrated to give crude 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid 2,5-dioxopyrrolidine-1-yl ester (400 mg) as a white solid. ESI-MS (EI) + ,m / z):282.9[M-100] + .

[0639] Step 3: 1-Cyanamido-5,5,5-trifluoro-4-methyl-1-oxopent-2-ylcarbamate tert-butyl ester:

[0640] A mixture of 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid 2,5-dioxopyrrolidine-1-yl ester (300 mg, 0.78 mmol), cyanamide (66 mg, 1.57 mmol), and NaOH (156 mg, 3.9 mmol) was dissolved in THF (16 mL). The mixture was stirred at 0 °C for 0.5 h and then at room temperature for 17 h. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give 1-cyanoamido-5,5,5-trifluoro-4-methyl-1-oxopentan-2-ylcarbamate tert-butyl ester (45 mg, 0.14 mmol) as a white solid. MS (EI+, m / z): 310.3 [M+H] + .

[0641] Step 4: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205]:

[0642] To a solution of 1-cyanoamido-5,5,5-trifluoro-4-methyl-1-oxopentan-2-ylcarbamate tert-butyl ester (45 mg, 0.14 mmol) in Et2O (20 mL), 4 M HCl / dioxane (10 mL) was added, and the mixture was stirred at room temperature for 24 hours. The solution was purified by preparative HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain a white solid, 2-amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205] (12.3 mg, 0.05 mmol, 27%). MS (EI+, m / z): 210.1 [M+H] + . 1H NMR (500MHz, CD3OD) δ4.06-4.09(m,1H), 2.43-2.65(m,1H), 1.67-1.85(m,2H), 1.18-1.22(m,3H).

[0643] Example 206: 2-Amino-3-(1-methylcyclobutyl)propionic acid [I-206].

[0644]

[0645] Reaction process:

[0646]

[0647] Procedures and representations:

[0648] Step 1: N-methoxy-N,1-dimethylcyclobutane formamide:

[0649] TEA (30.3 g, 0.3 mol) was added to a solution of 1-methylcyclobutanecarboxylic acid (11.6 g, 0.1 mol), N,O-dimethylhydroxylamine hydrochloride (19.5 g, 0.2 mol), and HATU (42 g, 0.11 mol) in DMF (300 mL), and the solution was stirred at room temperature for 17 hours. The solution was diluted with water (600 mL) and extracted with EtOAc (400 mL × 2). The organic phase was washed with 1N HCl, saturated NaHCO3, and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give N-methoxy-N,1-dimethylcyclobutanecarboxamide (12.2 g, 0.07 mol, 75%) as a colorless oil. ESI-MS (EI) + ,m / z):158.2[M+H] + .

[0650] Step 2: 1-Methylcyclobutane formaldehyde:

[0651] At 0 °C under N2, 1 M LiAlH4 (19 mL, 19 mmol) was added dropwise to a solution of N-methoxy-N,1-dimethylcyclobutaneformamide (2.0 g, 12.7 mmol) in anhydrous THF (20 mL). The mixture was heated to room temperature and stirred for 2 hours. The solution was slowly quenched with saturated seignette salt and extracted with Et2O (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, and used for the next step.

[0652] Step 3: (Z)-2-(tert-Butoxycarbonylamino)-3-(1-Methylcyclobutyl)acrylate:

[0653] At 0 °C, t-BuONa (844 mg, 8.79 mmol) was added to a solution of Witting reagent (2.15 g, 5.86 mmol) in anhydrous THF (80 mL), and the mixture was stirred for 1 hour. Then, a solution of 1-methylcyclobutaneformaldehyde was added, and the mixture was stirred at room temperature for 17 hours. The solution was extracted with EtOAc (100 mL × 2). The organic phase was washed with brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 30) to give (Z)-2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)acrylate tert-butyl ester (700 mg, 2.2 mmol) as a colorless oil. ESI-MS (EI) + ,m / z):200.2[M-56*2] + .

[0654] Step 4: 2-(tert-Butoxycarbonylamino)-3-(1-Methylcyclobutyl)propionate tert-butyl ester:

[0655] A mixture of (Z)-2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)acrylate tert-butyl ester (700 mg, 2.2 mmol) and Pd / C (10%, 100 mg) in MeOH (100 mL) was stirred at 30 °C for 17 hours. The mixture was filtered, and the filtrate was concentrated to dryness to give tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propionate tert-butyl ester (600 mg, crude) as a colorless oil. ESI-MS (EI...) + ,m / z):158.2[M-156] + .

[0656] Step 5: 2-Amino-3-(1-methylcyclobutyl)propionic acid [I-206]:

[0657] Add 4M HCl / dioxane (10 mL) to a solution of 600 mg crude tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propionate in Et2O (20 mL) and stir for 17 hours at room temperature. Concentrate the solution to give 2-amino-3-(1-methylcyclobutyl)propionic acid. MS (EI) + ,m / z):158.0[M+H] + .

[0658] 1 H NMR (500MHz, D2O) δ3.91 (t, J = 7.5Hz, 1H), 2.06-2.02 (m, 1H), 1.88-1.64 (m, 7H), 1.15 (s, 3H).

[0659] Example 93: S-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93].

[0660]

[0661] Reaction process:

[0662]

[0663] Procedures and representations:

[0664] The procedure for 2-amino-3-(1-methylcyclobutyl)propionic acid is the same as in Example 8.

[0665] Step 6: 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid:

[0666] A mixture of 2-amino-3-(1-methylcyclobutyl)propionic acid (300 mg, crude), CbzOSu (714 mg, 2.8 mmol), and acetone (10 mL) and saturated NaHCO3 (3 mL) was stirred at room temperature for 5 hours. The solution was purified by preparative HPLC (Boston C1821*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (160 mg, 0.54 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + .

[0667] Step 7: (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid:

[0668] 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (160 mg, 0.54 mmol) was purified by chiral HPLC to give (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (50 mg, 0.17 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + .

[0669] Step 8: (S)-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93]:

[0670] A mixture of (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (50 mg, 0.17 mmol) and Pd / C (10%, 10 mg) in MeOH (10 mL) was stirred at room temperature for 1 hour. The solution was purified by preparative HPLC (Boston C1821*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93] (2 mg, 0.01 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + . 1H NMR(500MHz,D2O)δ3.76-3.79(t,1H),1.96-2.00(m,1H),1.61-1.86(m,7H),1.11(s,3H).

[0671] Example 204: 2-Amino-3-(trimethylsilyl)propionate [I-204]

[0672]

[0673] Reaction process:

[0674]

[0675] Procedures and representations:

[0676] Step 1: 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester:

[0677] A solution of 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester (2.5 g, 8.47 mmol) in THF (20 mL) was cooled to -78 °C, and then LiHMDS (8.47 mL, 8.47 mmol) was added dropwise under N2. The solution was stirred at -78 °C for 1 hour. (iodomethyl)trimethylsilane (1.8 g, 8.47 mmol) was added dropwise. The solution was stirred overnight at -78 °C to room temperature. The solution was washed with brine (25 mL x 2), dried (Na2SO4), concentrated, and purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 30) to give 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester (2.3 g, 6.04 mmol, 71%) as a yellow solid. ESI-MS (EI+, m / z): 382.3 [M+H] + .

[0678] Step 2: 2-Amino-3-(trimethylsilyl)propionate [I-204]:

[0679] A solution of tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate (500 mg, 1.31 mmol) in 4 M HCl / dioxane (6 mL) was stirred at room temperature for 17 hours. DCM (80 mL) was added. The solid was filtered off to give a white solid of 2-amino-3-(trimethylsilyl)propionate [I-204] (113 mg, 0.57 mmol, 44%). ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500MHz, CD3OD) δ13.78(br,1H),8.33(br,1H),3.75(m,1H),1.00-1.14(m,2H),0.06(s,9H).

[0680] Example 201: (S)-2-amino-3-(trimethylsilyl)propionate [I-201].

[0681]

[0682] Reaction process:

[0683]

[0684] Procedures and representations:

[0685] Step 1: (S)-2-amino-3-(trimethylsilyl)propionate [I-201]:

[0686] A solution of (S)-2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester (300 mg, 0.79 mmol) in 4 M HCl / dioxane (3 mL) was stirred at room temperature for 17 hours. DCM (40 mL) was added. The solid was filtered off to give (S)-2-amino-3-(trimethylsilyl)propionate salt [I-201] (92 mg, 0.47 mmol, 62%) as a white solid. ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500MHz, CD3OD) δ13.76(br,1H),8.38(br,1H),3.76(m,1H),1.02-1.16(m,2H),0.06(s,9H).

[0687] Example 200: (R)-2-amino-3-(trimethylsilyl)propionate [I-200].

[0688]

[0689] Reaction process:

[0690]

[0691] Procedures and representations:

[0692] Step 1: (R)-2-amino-3-(trimethylsilyl)propionate [I-200]:

[0693] A solution of (R)-2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester (300 mg, 0.79 mmol) in 4 M HCl / dioxane (3 mL) was stirred at room temperature for 17 hours. DCM (40 mL) was added. The solid was filtered off to give (R)-2-amino-3-(trimethylsilyl)propionate salt [I-200] (80 mg, 0.41 mmol, 52%) as a white solid. ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500MHz, CD3OD) δ13.77(br,1H),8.33(br,1H),3.76(m,1H),1.02-1.14(m,2H),0.06(s,9H).

[0694] Example 194: (S)-2-amino-4-fluoro-4-methylvaleric acid [I-194].

[0695]

[0696] Reaction process:

[0697]

[0698] Procedures and representations:

[0699] Step 1: (S)-2-amino-4-fluoro-4-methylpentanoic acid [I-194]:

[0700] A mixture of (S)-2-amino-4-fluoro-4-methylpentanoic acid ethyl ester hydrochloride (65 mg, 0.31 mmol), LiOH·H₂O (29 mg, 0.69 mmol), and H₂O (2 mL) was stirred at room temperature for 2.5 hours. Then, 1 N HCl was added to adjust the pH to 3. The mixture was purified directly by reversed-phase HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-amino-4-fluoro-4-methylpentanoic acid [I-194] (40 mg, 0.27 mmol, 87%) as a white solid. MS (EI+, m / z): 150.3 [M+H] + . 1H NMR (500MHz, CD3OD) δ 8.10 (br, 2H), 3.79 (m, 1H), 2.19-2.26 (m, 1H), 1.97-2.05 (m, 1H), 1.42 (d, Jz = 3.5Hz, 3H), 1.37 (d, Jz = 4.0Hz, 3H).

[0701] Example 94: (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)but-1-amine [I-94].

[0702]

[0703] Reaction process:

[0704]

[0705] Procedures and representations:

[0706] Step 1: (S)-1-cyano-3,3-dimethylbutylcarbamate tert-butyl ester:

[0707] Cyanuric chloride (450 mg, 2.5 mmol) was added to a solution of (S)-1-amino-4,4-dimethyl-1-oxopentan-2-ylcarbamate (500 mg, 2.1 mmol) in DMF (10 mL), and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with brine (100 mL), extracted with ethyl acetate (50 mL), dried (Na₂SO₄), and concentrated to give crude (S)-1-cyano-3,3-dimethylbutylcarbamate (500 mg) as a yellow viscous substance. ESI-MS (EI+, m / z): 249.2 [M+Na] + .

[0708] Step 2: (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate tert-butyl ester:

[0709] A mixture of (S)-1-cyano-3,3-dimethylbutylcarbamate (crude 500 mg), ZnBr2 (900 mg, 4.0 mmol), and NaN3 (260 mg, 4.0 mmol) in DMF (20 mL) was stirred at 100 °C for 17 hours. The mixture was then diluted with brine (200 mL), extracted with ethyl acetate (60 mL), dried over (Na2SO4), and concentrated to give crude (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate (400 mg) as a yellow viscous substance. ESI-MS (EI+, m / z): 214.3 [M+H-56] + .

[0710] Step 3: ((S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)but-1-amine [I-94]:

[0711] A solution of (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate tert-butyl ester (crude, 300 mg) in 4M HCl / dioxane (3.5 mL) was stirred at room temperature for 17 hours. The solution was then concentrated and purified directly by reversed-phase HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butyl-1-amine 2,2,2-trifluoroacetate [I-94] as a white solid [I-94] (30 mg, 0.11 mmol, 9%, 3 steps). MS (EI+, m / z): 170.2 [M+H] +. 1H NMR (500MHz, CD3OD) δ 8.18 (br, 3H), 4.48 (m, 1H), 2.14 (m, 1H), 1.73 (dd, Jz = 3.5, 16.5Hz 1H), 0.72 (s, 9H).

[0712] Example 175: Synthesis of 2-amino-5,5,5-trifluoro-4-methoxyvalerate [I-175]:

[0713]

[0714] Reaction process:

[0715]

[0716] Procedures and representations:

[0717] Step 1: (S)-4-methyl-2-(phenylmethanesulfonamide)pentanoic acid methyl ester:

[0718] IBX (20.2 g, 72.29 mmol) was added to a solution of 3-(benzyloxy)prop-1-ol (10.0 g, 60.24 mmol) in DMSO (100 mL) under ice bath conditions. The mixture was heated to room temperature and stirred at this temperature for 17 hours. The reaction mixture was poured into water (300 mL) and extracted with EA (200 mL × 2). The organic phase was washed with water (200 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was concentrated. The crude product was purified by SGC to give a pale yellow liquid (8.0 g, 81%).

[0719] 1H NMR (500MHz, CDCl3) δ9.77(s,1H),7.36-7.26(m,5H),4.53(s,2H),3.8-3.83(m,2H),2.71-2.68(m,2H).

[0720] Step 2: (4-(benzoxy)-1,1,1-trifluorobut-2-yloxy)trimethylsilane:

[0721] At room temperature, trimethyl(trifluoromethyl)silane (10.4 g, 73.2 mmol) was added to a solution of 3-(benzoxy)propionaldehyde (4.0 g, 24.4 mmol) in THF (50 mL), followed by the addition of CsF (0.37 g, 2.44 mmol). The resulting solution was stirred at room temperature for 2 hours. The solution was then quenched with water (100 mL) and extracted with EA (100 mL × 2). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated. The crude product was purified by ISCO biotage to obtain a colorless liquid (4.5 g, 60%) of (4-(benzoxy)-1,1,1-trifluorobut-2-yloxy)trimethylsilane.

[0722] 1H NMR (500MHz, CDCl3) δ7.38-7.29(m,5H),4.51(t,J=12Hz,2H),4.23-4.19(m ,1H),3.59-3.57(m,2H),2.04-2.01(m,1H),1.78-1.73(m,1H),0.13(s,9H).

[0723] Step 3: 4-(benzooxy)-1,1,1-trifluorobut-2-ol:

[0724] A solution of 4-(benzoxy)-1,1,1-trifluorobut-2-ol (4.5 g, 14.7 mmol) in HCl solution (3 M, in MeOH, 50 mL) was stirred at room temperature for 2 hours. The solution was then concentrated and purified by ISCO biotage to give 2.75 g, 80%, a colorless liquid of 4-(benzoxy)-1,1,1-trifluorobut-2-ol.

[0725] Step 4: ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene:

[0726] At 0 °C, t-BuOK (1.58 g, 14.1 mmol) was added to a solution of 4-(benzyloxy)-1,1,1-trifluorobut-2-ol (2.75 g, 11.75 mmol) in THF (100 mL), and the mixture was stirred at this temperature for 30 min. Then, MeI (2.17 g, 15.28 mmol) was added, and the mixture was stirred again at room temperature for 1 h. The reaction mixture was quenched with water (100 mL) and extracted with EA (100 mL × 2). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated. The crude product was purified by ISCObiotage to obtain ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene (2.04 g, 70%) as a colorless liquid.

[0727] 1H NMR (500MHz, CDCl3) δ7.38-7.29(m,5H),4.53(t,J=12Hz,2H),3.78-3.74(m ,1H),3.66-3.57(m,2H),3.5(s,3H),2.03-1.96(m,1H),1.78-1.57(m,1H).

[0728] Step 5: 4,4,4-Trifluoro-3-methoxybut-1-ol:

[0729] A solution of ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene (2.04 g, 8.23 ​​mmol) and Pd / C (0.5 g) in MeOH (30 mL) was stirred at room temperature for 2 hours, then filtered and concentrated to obtain a colorless liquid of 4,4,4-trifluoro-3-methoxybut-1-ol. This crude product was used directly in the next step.

[0730] Step 6: 4,4,4-Trifluoro-3-methoxybutyraldehyde:

[0731] Under ice bath conditions, IBX (2.76 g, 9.88 mmol) was added to a solution of 4,4,4-trifluoro-3-methoxybut-1-ol (1.3 g, from the crude product of the last step) in DMSO (20 mL). The mixture was heated to room temperature and stirred at this temperature for 17 hours. The reaction mixture was poured into water (80 mL) and extracted with Et2O (80 mL × 2). The organic phase was washed with water (80 mL × 3) and brine (80 mL), and the solution was used directly for the next step.

[0732] Step 7: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentadienonitrile:

[0733] Under ice bath conditions, benzylamine (2 mL), AcOH (2.0 mL), and then TMSCN (3 mL) were added to a solution of 4,4,4-trifluoro-3-methoxybutyraldehyde in Et₂O (160 mL). The mixture was heated to room temperature and stirred at this temperature for 17 hours. The solution was diluted with water (200 mL) and extracted with EA (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentadienonitrile (2.0 g, crude), which was used in the next step. ESI-MS (EI + (m / z):

[0734] Step 8: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxyvalerate:

[0735] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentanilic acid (2.0 g, crude) in concentrated HCl (30 mL) and AcOH (10 mL) was heated to 100 °C for 17 hours. The solution was concentrated to dryness, diluted with H₂O (100 mL) and ACN (50 mL), and the pH was adjusted to 3-4 with saturated NaHCO₃ solution. The mixture was filtered and dried to obtain 0.8 g, 35%, 4 steps, as a brown solid of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentanilic acid. ESI-MS (EI + ,m / z):[M+H] + .

[0736] Step 9: 2-Amino-5,5,5-trifluoro-4-methoxyvalerate [I-175]:

[0737] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxyvalerate (300 mg, 1.03 mmol) and HCOONH4 (650 mg, 10.3 mmol) in MeOH (10 mL) was stirred at 60 °C for 2 hours, then filtered and concentrated. The crude product was purified by reverse-phase biotage to obtain 2-amino-5,5,5-trifluoro-4-methoxyvalerate [I-175] as a white solid.

[0738] 1H NMR (500MHz, methanol-d4) δ 4.23-4.19 (m, 1H), 3.96-3.88 (m, 1H), 3.64-3.6 (m, 3H), 2.29-2.22 (m, 1H), 2.04-1.97 (m, 1H).

[0739] Example 176: 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0740]

[0741] Reaction process:

[0742]

[0743] Procedures and representations:

[0744] Step 1: Diethyl 2-(2,3-dimethylbut-2-yl)malonate:

[0745] A solution of diethyl 2-(propane-2-yl)malonate (2 g, 10.0 mmol) in THF (60 mL) was cooled to 0 °C, and then copper iodide (I) (2.9 g, 15.0 mmol) was added. The mixture was stirred at 0 °C for 0.5 h. Then, at 0 °C, magnesium isopropyl bromide (1 mol / L, 30.0 mL, 30.0 mmol) was added dropwise to the mixture. The mixture was stirred at 0 °C for 2 h. The mixture was quenched with HCl (1 mol / L) and extracted with EtOAc (60 mL x 2). The organic phase was separated, washed with water (100 mL x 2) and brine (130 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give diethyl 2-(2,3-dimethylbutane-2-yl)malonate (2.4 g, 10.0 mmol, 98%) as a yellow solid. ESI-MS (EI) + ,m / z):245.3[M+H] + .

[0746] Step 2: 2-(2,3-Dimethylbut-2-yl)malonic acid:

[0747] A mixture of diethyl 2-(2,3-dimethylbut-2-yl)malonate acetamide (2.4 g, 10.0 mmol) and hydrated lithium hydroxide (2.1 g, 50.0 mmol) in DMSO (50 mL) and water (10 mL) was heated to 98 °C and maintained for 20 hours. The mixture was cooled, acidified with HCl (1 mol / L), and partitioned between EtOAc (30 mL) and water (30 mL). The organic phase was separated, washed with water (50 mL × 2) and brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(2,3-dimethylbut-2-yl)malonate (1.8 g, 10.0 mmol, 95%) as a yellow oil. ESI-MS (EI) + ,m / z):212.2[M+H] + .

[0748] Step 3: 3,3,4-Trimethylvaleric acid:

[0749] A solution of 2-(2,3-dimethylbut-2-yl)malonic acid (1.8 g, 10.0 mmol) in DMSO (30 mL) was heated to 120 °C and maintained for 12 hours. The mixture was cooled and partitioned between EtOAc (50 mL) and water (60 mL). The organic phase was separated, washed with water (60 mL × 2) and brine (60 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 3,3,4-trimethylvaleric acid (1.4 g, 10.0 mmol, 95%) as a yellow oil. ESI-MS (EI-, m / z): 143.2 [MH]+ .

[0750] Step 4: N-methoxy-N,3,3,4-tetramethylpentanamide:

[0751] At 20 °C, N,O-dimethylhydroxylamine hydrochloride (1.2 g, 12.0 mmol) and DIEA (3.8 g, 30.0 mmol) were successively added to a solution of 3,3,4-trimethylpentanoic acid (1.4 g, 10.0 mmol) in 30 mL of DMF. Then HATU (5.8 g, 15.0 mmol) was added. The mixture was heated to 25 °C with stirring and maintained for 18 hours. The reaction mixture was quenched successively with water and methyl tert-butyl ether (50 mL x 2). Phase separation was performed, the organic layer was washed with brine (80 mL x 3), dried with Na₂SO₄, filtered, and concentrated under vacuum to give N-methoxy-N,3,3,4-tetramethylpentanoamide (1.5 g, 90%) as a brown oil. ESI-MS (EI) + ,m / z):188.2[M+H] + .

[0752] Step 4: 3,3,4-Trimethylpentanal:

[0753] At 0°C, LiAlH4 (1 g, 0.03 mol) was added to a solution of N-methoxy-N,3,3,4-tetramethylpentanamide (1.9 g, 0.01 mol) in 30 mL of THF. The mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched successively with water and methyl tert-butyl ether (50 mL x 2). The phases were separated, the organic layer was washed with brine (80 mL x 3), dried with Na2SO4, and filtered. The filtrate contained 3,3,4-trimethylpentanal (1.3 g, 95%) as a colorless solution, which was used directly in the next step.

[0754] Step 5: 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile:

[0755] Under ice bath conditions, benzylamine (1.6 mL), AcOH (1.0 mL), and TMSCN (1.8 mL) were successively added to a solution of 3,3,4-trimethylpentanal in methyl tert-butyl ether (120 mL). The mixture was heated to 25 °C and stirred overnight. The solution was diluted with water (60 mL) and extracted with EtOAc (30 mL). The organic phase was washed with water (50 mL × 2) and brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give a brown oily 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile (2 g, crude), which was used in the next step. ESI-MS (EI+, m / z): 245.4 [M+H] + .

[0756] Step 6: 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid:

[0757] A solution of 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile (2 g, crude) in concentrated HCl (60 mL) and AcOH (10 mL) was heated to 95 °C for 18 hours. The solution was cooled to 15 °C, the pH was adjusted to 3-4 with saturated NaHCO3 solution, the mixture was filtered and dried to obtain 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid (0.6 g, 2.3 mmol, 30%, 3 steps) as a white solid. ESI-MS (EI...) + ,m / z):264.4[M+H] + .

[0758] 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0759] At room temperature, HCOONH4 (0.13 g, 2.0 mmol) and Pd / C (30 mg) were added to a solution of 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid (78 mg, 0.3 mmol) in 8 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give 2-amino-6,6,6-trifluoro-4-methylhexanoic acid [I-176] (40 mg, 90%) as a white solid; ESI-MS (EI) + ,m / z):174.3[M+H] + ;1H NMR (500MHz, MeOD) δ3.56 (dd, J = 7.2, 4.9 Hz, 1H), 2.12 (dd, J = 14.7, 4.9 Hz, 1H), 1.66-1.51 (m, 2H), 0.97 (d, J = 14.9 Hz, 6H), 0.92 (dd, J = 6.8, 3.6 Hz, 6H).

[0760] Example 178: 2-Amino-4,4-dimethylheptanoic acid [I-178]

[0761]

[0762] Reaction process:

[0763]

[0764] Procedures and representations:

[0765] The program is the same as that used in Example 176.

[0766] 2-Amino-4,4-dimethylheptanoic acid [I-178]:1 H NMR (500MHz, MeOD-d4) δ3.77 (t, J = 6Hz, 1H), 2.09-2.05 (m, 1H), 1.6-1.56 (m, 1H), 1.37-1.26 (m, 4H), 1.01-0.92 (m, 9H).

[0767] Example 195: 2-Amino-4,4-dimethylhexanoic acid [I-195], (S)-2-amino-4,4-dimethylhexanoic acid [I-120], (R)-2-amino-4,4-dimethylhexanoic acid [I-191].

[0768]

[0769] Reaction process:

[0770]

[0771] Procedures and representations:

[0772] The program is the same as that used in Example 176.

[0773] 2-Amino-4,4-dimethylheptanoic acid [I-195]: 1 H NMR(500MHz,D2O)δ3.87(t,J=6.0Hz,1H),1.93(dd,J=15.0Hz,J=5.5Hz,1H),1.57(dd,J=1 5.0Hz, J=6.5Hz, 1H), 1.22-1.26 (m, 2H), 0.86 (d, (dd, J=2.0Hz, 6H), 0.76 (t, J=7.5Hz, 3H).

[0774] (S)-2-amino-4,4-dimethylhexanoic acid [I-120]: 1 H NMR (500MHz, MeOD-d4) δ3.43 (dd, J=7.0Hz, J=5.0Hz, 1H), 1.95 (dd, J=15.0Hz, J=5.0Hz, 1H), 1.42 ( dd,J=15.0Hz,J=7.0Hz,1H),1.23-1.28(m,2H),0.87(d,(dd,J=4.5Hz,6H),0.80(t,J=7.5Hz,3H).

[0775] (R)-2-amino-4,4-dimethylhexanoic acid [I-191]: 1H NMR (500MHz, MeOD-d4) δ3.43 (dd, J=7.0Hz, J=5.0Hz, 1H), 1.95 (dd, J=15.0Hz, J=5.0Hz, 1H), 1.42 ( dd,J=15.0Hz,J=7.0Hz,1H),1.23-1.28(m,2H),0.87(d,(dd,J=4.5Hz,6H),0.80(t,J=7.5Hz,3H).

[0776] Example 177: 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0777]

[0778] Reaction process:

[0779]

[0780] Procedures and representations:

[0781] Step 1: N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide:

[0782] A mixture of 2-chloro-N-methoxy-N-methylacetamide (13.7 g, 0.1 mol) and triphenylphosphine (26.2 g, 0.1 mol) in acetonitrile (200 mL) was heated to 80 °C and maintained for 20 hours. The mixture was cooled below 40 °C and concentrated to remove the solvent. The residue was successively dissolved in dichloromethane (200 mL) and 2N KOH (100 mL). The resulting mixture was stirred at 20 °C for 1 hour. Phase separation was performed, the organic layer was washed with brine (200 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated under vacuum to give N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide (36 g, 0.1 mol, 98%) as a yellow solid. ESI-MS (EI) + ,m / z):364.4[M+H] + .

[0783] Step 2: (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide:

[0784] A mixture of N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide (36.3 g, 0.1 mol) and 4,4,4-trifluorobutyl-2-one (25.2 g, 0.2 mol) in tetrahydrofuran (500 mL) was heated to 70 °C and maintained for 7 days. The mixture was cooled under vacuum below 40 °C and concentrated to remove the solvent. The residue was purified by silica gel column chromatography (200 g, 200–300 mesh, UV 254 nm) eluting with 0–35% ethyl acetate / petroleum ether to give (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide (6 g, 0.03 mol, 28%) as a yellow oil. ESI-MS (EI) + ,m / z):212.2[M+H] + .

[0785] Step 3: 5,5,5-Trifluoro-N-methoxy-N,3-dimethylpentanamide:

[0786] A mixture of (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanylamide (6 g, 0.03 mol) and Pd / C (10%, 400 mg) in THF (100 mL) was stirred at 30 °C for 18 hours. The mixture was filtered, and the filtrate was concentrated under vacuum to dryness to give 5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanylamide (6 g, 0.03 mol, 98%) as a yellow oil. ESI-MS (EI+, m / z): 214.2 [M+H] + .

[0787] Step 4: 5,5,5-Trifluoro-3-methylpentanal:

[0788] At 0°C, LiAlH4 (1 g, 0.03 mol) was added to a solution of 5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanamide (6 g, 0.03 mol) in 100 mL of THF. The mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched successively with water and methyl tert-butyl ether (60 mL x 2). The phases were separated, the organic layer was washed with brine (80 mL x 3), dried with Na2SO4, and filtered. The filtrate was retained to obtain a colorless solution of 5,5,5-trifluoro-3-methylpentanal (4.5 g, 95%), which was used directly in the next step.

[0789] Step 5: 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanenitrile:

[0790] Under ice bath conditions, benzylamine (5 mL), AcOH (4.0 mL), and TMSCN (5 mL) were successively added to a solution of 5,5,5-trifluoro-3-methylpentanal in methyl tert-butyl ether (200 mL). The mixture was heated to 20 °C and stirred overnight. The solution was diluted with water (100 mL) and extracted with EtOAc (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give a brown oily 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanenitrile (6 g, crude), which was used in the next step. ESI-MS (EI+, m / z): 271.3 [M+H] + .

[0791] Step 6: 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanoic acid:

[0792] A solution of 3 g crude 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanonitrile (CQN) in concentrated HCl (100 mL) and AcOH (20 mL) was heated to 100 °C for 17 hours. The solution was cooled to 15 °C, the pH was adjusted to 3-4 with saturated NaHCO3 solution, the mixture was filtered and dried to give 1 g, 13.4 mmol, 33%, 3 steps, as a white solid. ESI-MS (EI...) + ,m / z):290.3[M+H] + .

[0793] 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0794] At room temperature, HCOONH4 (0.13 g, 2.0 mmol) and Pd / C (30 mg) were added to a solution of 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanoic acid (88 mg, 0.31 mmol) in 8 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give 2-amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177] (45 mg, 84%) as a white solid; ESI-MS (EI) + ,m / z):200.2[M+H] +;1H NMR (500MHz, DMSO) δ3.15(d,J=5.7Hz,1H),2.39-2.24(m,1H),2.19-1.96(m,2H),1.82-1.66(m,1H),1.63-1.35(m,1H),0.98(dd,J=16.5,6.2Hz,3H).

[0795] Example 179: (S)-2-amino-5-fluoro-4-(fluoromethyl)valerate [I-179]

[0796]

[0797] Reaction process:

[0798]

[0799] Procedures and representations:

[0800] Step 1: 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane:

[0801] At 0 °C, NaH (60%, in oil, 0.12 g, 3.0 mmol) was added to a solution of (2,2-dimethyl-1,3-dioxane-5-yl)methanol (0.29 g, 2.0 mmol) in DMF (10 mL). The mixture was stirred at 0 °C for 0.2 h. Then (bromomethyl)benzene (0.45 g, 2.6 mmol) was added. The mixture was heated to 10 °C for 3 h and maintained for 18 h. The reaction mixture was quenched successively with ice water and EtOAc (60 mL). The phases were separated, the organic layer was washed with brine (60 mL x 3), dried with Na2SO4, and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (20 g, UV 254 nm, elution with 10% to 50% EtOAc / PE) to give 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane (1) (0.46 g, 0.2 mol, 95%) as a colorless oil. ESI-MS (EI + ,m / z):237.3[M+H] + .

[0802] Step 2: 2-(benzyloxymethyl)propane-1,3-diol:

[0803] To a solution of 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane (930 mg, 3.94 mmol) in MeOH (20 mL), 2 mL of 3N HCl aqueous solution was added. The mixture was stirred at 50 °C for 2 hours. The reaction mixture was concentrated and diluted with DCM (20 mL), washed with brine (15 mL), dried, and evaporated to give a crude colorless oil (780 mg, 100%). ESI-MS (EI) + ,m / z):197[M+H] + .

[0804] Step 3: ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene:

[0805] DAST (1.9 g, 11.8 mmol) was added dropwise to a pre-cooled solution of 2-(benzyloxymethyl)propane-1,3-diol (780 mg, 3.94 mmol) in DCM (20 mL) at -78 °C. The mixture was stirred at 20 °C for 24 h. The reaction mixture was quenched with a saturated aqueous solution of NaHCO3 (10 mL) at -78 °C. The DCM phase was separated and washed with brine, dried over MgSO4, filtered through a short silica gel pad, and then concentrated to give a crude colorless oil (800 mg, 100%). ESI-MS (EI) + ,m / z):223[M+Na] + . 1 H NMR (500MHz, CDCl3) δ7.37-7.28(m,5H), 4.65-4.57(m,2H), 4.55-4.48(m,4H), 3.57(d,J=6.2Hz,2H), 2.50-2.34(m,1H).

[0806] Step 4: 3-Fluoro-2-(fluoromethyl)prop-1-ol:

[0807] At -78°C, BCl3 / toluene (1M, 6 mL, 6.0 mmol) was added dropwise to a pre-cooled solution of ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene (800 mg, 3.94 mmol) in DCM (20 mL). The mixture was stirred at -78 to 0°C for 2 hours. The reaction mixture was quenched with H2O (0.5 mL) at -78°C. The DCM phase was dried with MgSO4, filtered, and the solution (approximately 20 mL) was used directly for the next step.

[0808] Step 5: 3-Fluoro-2-(fluoromethyl)propyl trifluoromethanesulfonate:

[0809] At -40°C, py (380 mg, 4.8 mmol) and Tf₂O (1.36 g, 4.8 mmol) were added dropwise sequentially to a pre-cooled solution of 3-fluoro-2-(fluoromethyl)prop-1-ol (8 mL solution, from step 4, 1.6 mmol). The mixture was stirred at -30°C for 1 hour. The reaction mixture was quenched with brine (20 mL) at -40°C. The DCM phase was separated and dried over MgSO₄, filtered, and then concentrated to give a crude brown oil (200 mg, 51%), which was used directly in the next step.

[0810] Step 6: 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)pentanoic acid tert-butyl ester:

[0811] At -78°C, over 25 minutes, LDA (2.5 M, in THF / toluene / hexane, 1.28 mL, 3.2 mmol) was added to a pre-cooled solution of tert-butyl 2-(diphenylmethyleneamino)acetate (944 mg, 3.2 mmol) in THF (20 mL). The mixture was stirred at this temperature for 10 minutes. 3-fluoro-2-(fluoromethyl)propyl trifluoromethanesulfonate (200 mg, 0.82 mmol) was added dropwise to a solution of THF (2 mL) at -78°C. The reaction mixture was placed directly above a cooling bath and stirred for another 1 hour. The reaction mixture was quenched with saturated NH4Cl aqueous solution (20 mL), extracted with MTBE (30 mL x 2), washed with H2O and brine (50 mL each time), dried, and concentrated to give a crude product. The crude product was purified twice by chromatography (silica gel, PE to 5% EA / PE) to give the desired product (22 mg, 6.9%) as a white solid. ESI-MS (EI) + ,m / z):388[M+H] + . 1 H NMR (500MHz, DMSO) δ7.56-7.45(m,6H),7.41(t,J=7.4Hz,2H),7.18(d,J=6.3Hz,2H),4.50-4.17( m, 4H), 3.91 (dd, J = 7.7, 5.5Hz, 1H), 2.11-1.97 (m, 1H), 1.87 (dd, J = 12.7, 5.5Hz, 2H), 1.38 (s, 9H).

[0812] Step 7: (S)-2-amino-5-fluoro-4-(fluoromethyl)valerate:

[0813] A solution of tert-butyl 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)valerate (55 mg, 0.14 mmol) in 3N HCl / MeOH (2 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated and washed with Et₂O to give a crude solid, which was dissolved in DCM / TFA (1:1, 2 mL) and stirred at room temperature for 20 hours. The reaction mixture was evaporated and washed with Et₂O to give a crude solid, which was dissolved in 6N HCl (1 mL) and stirred at 80 °C for 2 hours. The reaction mixture was evaporated and lyophilized to give a crude product, which was purified by RP-biotage using 3 mM HCl / H₂O to give the desired product (8.3 mg, 29%) as a white solid. ESI-MS (EI) + ,m / z):168[M+H] + . 1 H NMR (500MHz, DMSO) δ7.85 (bs, 3H), 4.48 (dd, J = 48.3, 14.2Hz, 4H), 3.46-3.36 (m,1H),2.47-2.26(m,1H),1.78(dt,J=14.3,7.3Hz,1H),1.63-1.53(m,1H).

[0814] Example 187: (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0815]

[0816] Reaction process:

[0817]

[0818] Procedures and representations:

[0819] Step 1: (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0820] Concentrated HCl (1 mL) and SOCl2 (0.2 mL) were added to a round-bottom flask containing (S)-2-amino-4-methylpentan-4-enoic acid (100 mg). The mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated and washed with Et2O to give a crude solid, which was purified by RP-biotage using 0.025% TFA / H2O / MeCN to give the desired product (20.2 mg, 11.4%) as a white solid. ESI-MS (EI) + ,m / z):130.1[M+H] + . 1H NMR (500MHz, DMSO) δ8.80 (bs, 3H), 4.58 (dd, J = 11.2, 9.3Hz, 1H), 2.53-2.48 (m, 1H), 2.13 (t, J = 11.7Hz, 1H), 1.45 (s, 3H), 1.40 (s, 3H).

[0821] Example 90: Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-90]:

[0822]

[0823] Reaction process:

[0824]

[0825] Procedures and representations:

[0826] Step 1: Diethyl 2-(1,1,1-trifluoropropionic-2-ylidene)malonate:

[0827] Over 20 minutes, add TiCl4 (65.8 mL, 600 mmol) dropwise to THF (1 L) in an ice bath, followed by CCl4 (30 mL). Add diethyl malonate (48.0 g, 300 mmol) and 1,1-difluoroprop-2-one (56.4 g, 600 mmol) to the mixture. Warm the mixture to room temperature and stir overnight. Add pyridine (200 mL) dropwise over 20 minutes in an ice bath. The reaction mixture was poured into water (2 L), filtered, and the filtrate was extracted with EtOAc (500 mL × 2). The organic phase was washed with water (600 mL), 1 M HCl (600 mL × 2), water (600 mL), saturated NaHCO3 (600 mL), and brine (600 mL). The mixture was dried (Na2SO4), filtered, concentrated under vacuum, and purified by chromatography (silica gel, 0% to 5% ethyl acetate / petroleum ether) to give diethyl 2-(1,1-difluoropropionic-2-yl)malonate (60.9 g, 258 mmol, 86%) as a colorless liquid. ESI-MS (EI) + ,m / z):237.0[M+H] + . 1 H-NMR (500MHz, CDCl3): δ6.97 (t, J = 55.5Hz, 1H), 4.25-4.33 (m, 4H), 2.03 (s, 3H), 1.29-1.34 (m, 6H).

[0828] Step 2: Diethyl 2-(1,1-difluoro-2-methylprop-2-yl)malonate:

[0829] At -20°C, over 1 hour, MeMgI (42.3 mL, 130.5 mmol) was added dropwise to a mixture of 2-(1,1-difluoro-2-methylpropion-2-yl)malonate (10.0 g, 42.3 mmol) and CuI (12.1 g, 63.5 mmol) in DCM (100 mL) and THF (25 mL). The solution was poured into ice water (200 mL) and treated with saturated NH4Cl solution (100 mL). The mixture was stirred for 30 minutes and filtered. The filtrate was extracted with DCM (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give a brown liquid 2-(1,1-difluoro-2-methylpropion-2-yl)malonate (10.1 g, 40.2 mmol, 95%), which was used in the next step. ESI-MS (EI + ,m / z):253.1[M+H] + . 1 H-NMR (500MHz, CDCl3): δ6.05 (t, J=57.5Hz, 1H), 4.17-4.23 (m, 4H), 3.49 (s, 1H), 1.22-1.28 (m, 6H), 1.20 (s, 6H).

[0830] Step 3: 4,4-Difluoro-3,3-dimethylbutyric acid:

[0831] A mixture of diethyl 2-(1,1-difluoro-2-methylprop-2-yl)malonate (6.1 g, 24.2 mmol) and LiOH·H₂O (5.1 g, 121 mmol) in DMSO (50 mL) and H₂O (0.5 mL) was heated to 90 °C for 17 hours. The mixture was diluted with water (200 mL), extracted with DCM (100 mL), and the pH of the aqueous phase was adjusted to 3–4 with 6 M HCl solution. The mixture was then extracted with DCM (100 mL × 2), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 3.6 g (crude) 4,4-difluoro-3,3-dimethylbutyric acid as a brown liquid. ESI-MS (EI) was then used to analyze the final product. + m / z): 151.1 [MH] - .

[0832] Step 4: 4,4-Difluoro-N-methoxy-N,3,3-trimethylbutyramide:

[0833] Et3N (7.18 g, 71.1 mmol) was added to a solution of 4,4-difluoro-3,3-dimethylbutyric acid (3.6 g, crude), N,O-dimethylhydroxylamine hydrochloride (4.6 g, 47.4 mmol), and HATU (10.8 g, 28.4 mmol) in DMF (50 mL), and the mixture was stirred at room temperature for 17 hours. The mixture was filtered, and the filtrate was diluted with water (200 mL), extracted with Et2O (100 mL × 2), washed with water (100 mL), 1 M HCl (100 mL), and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give 4,4-difluoro-N-methoxy-N,3,3-trimethylbutyramide (3.1 g, 15.9 mmol, 66%, 2 steps) as a brown liquid. ESI-MS (EI) + ,m / z):196.0[M+H] + . 1 H-NMR (500MHz, CDCl3): δ5.95 (t, J=57.5Hz, 1H), 3.69 (s, 3H), 3.17 (s, 3H), 2.51 (s, 2H), 1.12 (s, 6H).

[0834] Step 5: 4,4-Difluoro-3,3-dimethylbutanal:

[0835] Under ice bath conditions, LiAlH4 (24 mL, 24 mmol) was added dropwise to a solution of 4,4-difluoro-N-methoxy-N,3,3-trimethylbutyramide (3.1 g, 15.9 mmol) in THF (80 mL). After 1 hour, the mixture was quenched with citric acid solution (100 mL), extracted with Et2O (100 mL × 2), washed with brine (100 mL), dried (Na2SO4), and the solution was used for the next step.

[0836] Step 6: 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanilonitrile:

[0837] Under ice bath conditions, benzylamine (3 mL), AcOH (3 mL), and TMSCN (3 mL) were successively added to a solution of 4,4-difluoro-3,3-dimethylbutanal in Et₂O (200 mL). The solution was stirred at 0–room temperature for 17 hours, and then diluted with EtOAc (100 mL). The solution was washed with H₂O (100 mL × 2) and then concentrated to obtain 3.2 g (crude) of 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanilide, a brown liquid. ESI-MS (EI) + ,m / z):253.0[M+H] + .

[0838] Step 7: 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanoic acid:

[0839] A solution of 1.8 g crude 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanoic acid in concentrated HCl (50 mL) and AcOH (10 mL) was heated to 100 °C for 64 hours. The mixture was concentrated to remove the solvent, the pH was adjusted to 12 with 1 M NaOH solution, extracted with PE (100 mL), and the aqueous phase was adjusted to pH 5-6 with 6 M HCl. A white solid was formed, filtered, and the filter cake was washed with water (50 mL) and dried under vacuum to give 1.3 g, 4.80 mmol, 54%, 3 steps, as a white solid. ESI-MS (EI) + ,m / z):272.0

[0840] Step 8: 2-Amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0841] A mixture of 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (1.3 g, 4.80 mmol), HCOONH4 (1.51 g, 24 mmol), and Pd / C (10%, 200 mg) in MeOH (50 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated to give 1.0 g (crude) of 2-amino-5,5-difluoro-4,4-dimethylpentanoic acid as a white solid. ESI-MS (EI...) + ,m / z):182.0

[0842] Step 9: 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid

[0843] CbzOSu (2.39 g, 9.6 mmol) was added to a solution of 2-amino-5,5-difluoro-4,4-dimethylvaleric acid (1.0 g, crude) and NaHCO3 (1.27 g, 14.4 mmol) in acetone (30 mL) and H2O (30 mL) under ice bath conditions. After stirring for 17 hours, the mixture was adjusted to pH 3-4 with 1M HCl solution, and the extract was obtained by extraction with EtOAc (50 mL × 2). The extract was washed with brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was then purified by reversed-phase silica gel chromatography and chiral preparative HPLC [column: CC4 4.6*250 mm 5 μm; solvent: MeOH (0.2% methanol-ammonia)] to give (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid (400 mg, 1.27 mmol, 26%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid (380 mg, 1.21 mmol, 25%, 2 steps), both as colorless oils. ESI-MS (EI) + ,m / z):316.0

[0844] Step 10: (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0845] A solution of (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid (400 mg, 1.27 mmol) and Pd / C (10%, 50 mg) in MeOH (30 mL) was stirred under hydrogen at room temperature for 2 hours. The mixture was filtered, concentrated under vacuum, and purified by reversed-phase silica gel chromatography to give (S)-2-amino-5,5-difluoro-4,4-dimethylvaleric acid (115.7 mg, 0.64 mmol, 50%). ESI-MS (EI... + ,m / z):182.0 1 H-NMR (500MHz, MeOD-d4): δ5.60(t,J=56.5Hz,1H),3.97(t,J=6.0Hz,1H),2.07(dd , J=15.5Hz, J=5.5Hz, 1H), 1.77 (dd, J=15.5Hz, J=6.5Hz, 1H), 0.96 (d, J=9.5Hz, 6H).

[0846] Example 88: Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-88]:

[0847]

[0848] Reaction process:

[0849]

[0850] Procedures and representations:

[0851] Step 1: (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide:

[0852] Over 5 minutes, concentrated H2SO4 (10 mL) was added dropwise to a solution of 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanonitrile (1.2 g, 4.76 mmol) in DCM (20 mL). The mixture was then heated to room temperature and stirred for 6 hours. The mixture was poured into ice water (100 mL), and the solution was adjusted to pH 8–9 with 10% NaOH solution. It was then extracted with EtOAc (100 mL × 2), and the organic phase was washed with water (100 mL) and brine (100 mL). The mixture was dried (Na₂SO₄), filtered, concentrated under vacuum, and purified successively by chromatography (0% to 5% MeOH / DCM) and chiral preparative HPLC [column: CC44.6*250 mm 5 μm; solvent: MeOH (0.2% methanol-ammonia)] to obtain (S)-2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanamide (400 mg, 1.48 mmol, 31%) and (R)-2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanamide (380 mg, 1.41 mmol, 30%), both as colorless liquids. ESI-MS (EI + ,m / z):253.0[M+H] + .

[0853] Step 2: (S)-2-amino-5,5-difluoro-4,4-dimethylpentanamide:

[0854] A mixture of (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide (200 mg, 0.74 mmol), HCOONH4 (233 mg, 3.7 mmol), and Pd / C (10%, 40 mg) in MeOH (15 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give (S)-2-amino-5,5-difluoro-4,4-dimethylpentanamide trifluoroacetic acid (128 mg, 0.44 mmol, 59%) as a white solid. ESI-MS (EI) + ,m / z):181.0[M+H] + . 1H-NMR (500MHz, MeOD-d4): δ5.66 (t, J=56.5Hz, 1H), 3.95 (dd, J=8.0Hz, J=5.0Hz, 1H), 2.1 4(dd, J=10.0Hz, J=8.0Hz, 1H), 1.83 (dd, J=14.5Hz, J=5.5Hz, 1H), 1.12 (d, J=15.0Hz, 6H).

[0855] Example 185: Synthesis of methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate [I-185]:

[0856]

[0857] Reaction process:

[0858]

[0859] Procedures and representations:

[0860] The procedure for 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid is the same as in Example 90.

[0861] Step 1: Methyl (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate:

[0862] A solution of (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (150 mg, 0.476 mmol), HATU (199 mg, 0.524 mmol), (S)-2-amino-4-methylpentanoic acid methyl ester hydrochloride (104 mg, 0.571 mmol), and DIPEA (123 mg, 0.952 mmol) was stirred at room temperature for 1 hour, then quenched with ice water (20 mL), extracted with EA (2 × 30 mL), dried, filtered, and concentrated. The crude product was purified by reversed-phase silica gel chromatography using Biotage to obtain (95 mg, 45%) of (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid methyl ester (95 mg, 45%) as a white solid. ESI-MS (EI) + (m / z):443.0

[0863] Step 2: Methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate:

[0864] A solution of (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate (95 mg, 0.215 mmol) and Pd / C (30 mg) in THF (5 mL) was stirred at room temperature for 2 hours, then filtered and concentrated. The crude product was purified by reversed-phase silica gel chromatography using Biotage to obtain (45 mg, 69%) methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate as a white solid. ESI-MS (EI) + ,m / z):309.0

[0865] 1 H-NMR (500MHz, DMSO-d6):9.11(d,J=7Hz,1H),8.41(s,3H),5.81(t,J=56.5Hz,1H),4.34-4.31(m,1H),3.8 9-3.81(m,1H),3.62(s,3H),1.98-1.93(m,1H),1.76-1.73(m,1H),1.65-1.54(m,3H),0.93-0.81(m,12H).

[0866] Example 184: Synthesis of methyl (S)-2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate [I-184]:

[0867]

[0868] The program is the same as examples 90 and 185.

[0869] Methyl (S)-2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentanoyl)-4-methylpentanoate: ESI-MS (EI + ,m / z):309.0

[0870] 1 H-NMR (500MHz, DMSO-d6):9.18(d,J=7Hz,1H),8.38(s,3H),5.79(t,J=56.5Hz,1H),4.37-4.32(m,1H),3.8 5-3.78(m,1H),3.58(s,3H),1.97-1.92(m,1H),1.77-1.72(m,1H),1.61-1.51(m,3H),0.94-0.82(m,12H).

[0871] Example 145: Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid, (2R,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid, (2R,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid and (2S,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid: [3d; I-145]; [3c; I-146]; [3a; I-167]; [3b; I-250]

[0872]

[0873] Reaction process:

[0874]

[0875] Procedures and representations:

[0876] Step 1: Synthesis of (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid, (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid, (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid and (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid:

[0877] CbzOSu (970 mg, 3.9 mmol) was added to a solution of 2-amino-5,5,5-trifluoro-4-methylpentanoic acid (600 mg, 3.2 mmol) in acetone (10 mL) and saturated aqueous solution of NaHCO3 (10 mL). The mixture was stirred at room temperature for 3 hours. Then, EtOAc (20 mL) and H2O (20 mL) were added, the aqueous solution was separated and further extracted with EtOAc (2 x 20 mL), the extracts were combined and washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated, and the residue was purified by preparative HPLC to give 2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (750 mg) as a white solid. The product was purified by chiral HPLC to yield four isomers: (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (150 mg, 15%), (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (40 mg, 3.9%), (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (50 mg, 4.9%), and (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (80 mg, 7.8%), all as white solids. ESI-MS (EI+, m / z): 342.0 [M+Na]+.

[0878] Step 2-A: Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0879] A solution of (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (150 mg, 0.47 mmol) and Pd / C (75 mg) in MeOH (15 mL) was stirred at room temperature for 3 hours. The reaction mixture was filtered and concentrated to give (2S,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (51.7 mg, 59%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + . 1 H-NMR (500MHz, MeOD): δ 3.64-3.60 (m, 1H), 2.77-2.71 (br, 1H), 2.24-2.18 (m, 1H), 1.76-1.69 (m, 1H), 1.25 (d, J = 7.0Hz, 3H).

[0880] Step 2-B: Synthesis of (2R,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0881] A solution of (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (40 mg, 0.12 mmol) and Pd / C (20 mg) in MeOH (4 mL) was stirred at room temperature for 3 hours. The reaction mixture was filtered and concentrated to give (2R,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (13.3 mg, 60%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + . 1 H-NMR (500MHz, MeOD): δ3.51-3.47(m,1H), 2.64-2.58(br,1H), 2.12-2.06(m,1H), 1.63-1.57(m,1H), 1.13(d,J=7.0Hz,3H).

[0882] Step 2-C: Synthesis of (2R,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0883] A solution of (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (50 mg, 0.16 mmol) and Pd / C (25 mg) in MeOH (5 mL) was stirred at room temperature for 3 hours. The reaction mixture was filtered and concentrated to give (2R,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (18.0 mg, 61%) as a white solid. ESI-MS (EI) + ,m / z):186.1[M+H] + . 1 H-NMR (500MHz, MeOD): δ3.51-3.47(m,1H), 2.46-2.44(br,1H), 1.95-1.87(m,2H), 1.11(d,J=7.0Hz,3H).

[0884] Step 2-D: Synthesis of (2S,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0885] A solution of (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (80 mg, 0.25 mmol) and Pd / C (40 mg) in MeOH (8 mL) was stirred at room temperature for 3 hours. The reaction mixture was filtered and concentrated to give (2S,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (38.1 mg, 82%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + .1 H-NMR (500MHz, MeOD): δ3.51-3.47(m,1H), 2.46-2.44(br,1H), 1.95-1.87(m,2H), 1.11(d,J=7.0Hz,3H).

[0886] Example 128: (S)-2-amino-5,5,5-trifluoro-4,4-dimethylvaleric acid (I-128):

[0887]

[0888] Reaction process:

[0889]

[0890] Procedures and representations:

[0891] The program is the same as that used in Example 187.

[0892] (S)-2-amino-5,5,5-trifluoro-4,4-dimethylvaleric acid: ESI-MS (EI + ,m / z):200.1 1 H-NMR (500MHz, D2O): δ3.94 (t, J=5.5Hz, 1H), 2.23 (dd, J=15.5Hz, J=5.5Hz, 1H), 1.90 (dd, J=15.5Hz, J=6.0Hz, 1H), 1.13 (d, J=8.5Hz, 6H).

[0893] Example 188: (S)-2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate methyl ester [I-188]:

[0894]

[0895] Reaction process:

[0896]

[0897] Procedures and representations:

[0898] The procedure for 2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylvaleric acid is the same as in Example 90.

[0899] Step 1: Methyl (S)-2-((R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate:

[0900] A solution of (S)-2-(benzyloxycarbonylamino)-5,5,5-difluoro-4,4-dimethylpentanoic acid (150 mg, 0.45 mmol), HATU (188 mg, 0.495 mmol), (S)-2-amino-4-methylpentanoic acid methyl ester hydrochloride (123 mg, 0.675 mmol), and DIPEA (175 mg, 1.35 mmol) was stirred at room temperature for 1 hour, then quenched with ice water (20 mL), extracted with EA (2 × 30 mL), dried, filtered, and concentrated. The crude product was purified by reversed-phase silica gel chromatography using Biotage to obtain (120 mg, 58%) methyl (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid)-4-methylpentanoic acid methyl ester as a white solid. ESI-MS (EI) + ,m / z):461.0

[0901] Step 2: Methyl (S)-2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate:

[0902] A solution of (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate (120 mg, 0.26 mmol) and Pd / C (30 mg) in THF (10 mL) was stirred at room temperature for 2 hours, then filtered and concentrated. The crude product was purified by reversed-phase silica gel chromatography using Biotage to obtain (49 mg, 57%) methyl (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate as a white solid. ESI-MS (EI) + ,m / z):326.0

[0903] 1 H-NMR(500MHz,MeOD-d4):4.47(t,J=7.5Hz,1H),3.99-3.97(m,1H),3.77(s,3H),2.33-2 .28(m,1H),1.95-1.91(m,1H),1.69-1.68(m,3H),1.24-1.17(m,6H),1.00-0.94(m,6H).

[0904] Example 189: (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate methyl ester [I-189]:

[0905]

[0906] Reaction process:

[0907] The program is the same as that used in Example 188.

[0908] Procedures and representations:

[0909] Example 189: (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate methyl ester [I-189]: 1 H-NMR(500MHz,MeOD-d4):4.52(t,J=7.5Hz,1H),4.02-3.99(m,1H),3.73(s,3H),2.35-2.30 (m,1H),1.95-1.91(m,1H),1.78-1.67(m,3H),1.25(s,3H),1.17(s,3H),1.01-0.97(m,6H).

[0910] Example 108: (S)-2-amino-6-fluorohexanoic acid [I-108].

[0911]

[0912] Example 109: (R)-2-amino-6-fluorohexanoic acid [I-109].

[0913]

[0914] Reaction process:

[0915]

[0916] Procedures and representations:

[0917] Step 1: 2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester:

[0918] A mixture of 1-fluoro-4-iodobutane (2.0 g, 9.90 mmol), 2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester (2.43 g, 8.25 mmol), TBAB (266 mg, 0.83 mmol), and KOH (aqueous solution, 50%) (10 mL) in DCM (10 mL) and toluene (25 mL) was stirred at 50 °C for 16 hours. The solution was purified by SGC (silica gel, ethyl acetate / petroleum ether = 1 / 5) to give a colorless oily tert-butyl 2-(diphenylmethyleneamino)-6-fluorohexanoate (0.91 g, 2.47 mmol, 30%). MS (EI+, m / z): 370.2 [M+H] + .

[0919] Step 2: (S)-2-amino-6-fluorohexanoic acid [I-108]:

[0920] A solution of (S)-2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester (360 mg, 0.97 mmol) in dioxane (10 mL) and HCl (aqueous solution, 6 M) was stirred at room temperature for 16 hours. The mixture was extracted with ether and water. After adjusting the pH to 3–4, the aqueous layer was extracted with EA. The organic layer was concentrated to give (S)-2-amino-6-fluorohexanoic acid [I-108] (125 mg, 0.84 mmol, 86%) as a white solid. ESI-MS (EI+, m / z): 150.3 [M+H] + . 1H NMR(500MHz,D2O)δ4.469(t,J=6.0Hz,1H),4.351(t,J=6.0Hz,1H),3.950(t,J =6.0Hz,1H),1.904-1.820(m,2H),1.690-1.588(m,2H),1.456-1.388(m,2H).

[0921] Step 2: (R)-2-amino-6-fluorohexanoic acid [I-109]:

[0922] A solution of (R)-2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester (300 mg, 0.81 mmol) in dioxane (10 mL) and HCl (aqueous solution, 6 M) was stirred at room temperature for 16 hours. The mixture was extracted with ether and water. After adjusting the pH to 3–4, the aqueous layer was extracted with EA. The organic layer was purified by HPLC to give (R)-2-amino-6-fluorohexanoic acid [I-109] (35 mg, 0.23 mmol, 29%) as a white solid. ESI-MS (EI+, m / z): 150.2 [M+H] + . 1H NMR(500MHz,D2O)δ4.505(t,J=6.0Hz,1H),4.410(t,J=6.0Hz,1H),3.823(t,J =6.0Hz,1H),1.906-1.827(m,2H),1.722-1.639(m,2H),1.485-1.399(m,2H).

[0923] Example 198: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (I-198):

[0924]

[0925] Reaction process:

[0926]

[0927] Procedures and representations:

[0928] Step 1: 4,4,4-Trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide:

[0929] Over one hour, H₂SO₄ (100 mL, concentrated) was slowly added dropwise to a stirred solution of hexafluoroacetone trihydrate (30 g, 136 mmol), and gaseous hexafluoroacetone was introduced into a solution of N-methoxy-N-methyl-2-(triphenylphosphine)-acetamide (10 g, 27.5 mmol) in THF (200 mL). The mixture was stirred at room temperature for 16 hours. Then, petroleum ether (200 mL) was added, and a white precipitate was filtered off. The filtrate was concentrated, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 5 / 1 to 3 / 1) to give 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide (6.2 g, 24.7 mmol, 90%) as a light oil. ESI-MS (EI) + ,m / z):252.1[M+H] + . 1 H-NMR (500MHz, CDCl3): δ7.15(s,1H),3.67(s,3H),3.26(s,3H).

[0930] Step 2: 4,4,4-Trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butyramide:

[0931] A mixture of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide (4.5 g, 17.9 mmol), Pd(OH)₂ / C (620 mg), and MeOH (100 mL) was stirred at room temperature under a hydrogen atmosphere for 16 hours. The mixture was then filtered and concentrated to give 1.8 g, 7.1 mmol, 40%, of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butanamide as a light oil. ESI-MS (EI) + ,m / z):254.1[M+H] + .

[0932] Step 3: 4,4,4-Trifluoro-3-(trifluoromethyl)butanal:

[0933] Under ice bath conditions, LiAlH4 (8.5 mL, 8.5 mmol) was added dropwise to a solution of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butyramide (1.8 g, 7.1 mmol) in THF (50 mL). After 1 hour, the mixture was quenched with citric acid solution (100 mL), the solution was extracted with Et2O (100 mL × 2), the organic phase was washed with brine (100 mL), dried (Na2SO4), and the solution was used for the next step.

[0934] Step 4: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanilonitrile:

[0935] Under ice bath conditions, benzylamine (2 mL), AcOH (2 mL), and TMSCN (2 mL) were successively added to a solution of 4,4,4-trifluoro-3-(trifluoromethyl)butanal in Et₂O (200 mL). The solution was stirred at 0–room temperature for 17 hours, and then diluted with EtOAc (100 mL). The solution was washed with H₂O (100 mL × 2) and then concentrated to obtain 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanilonitrile (2.1 g, crude) as a brown liquid. ESI-MS (EI + ,m / z):311.2[M+H] + .

[0936] Step 5: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0937] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (2.1 g, crude) in concentrated HCl (50 mL) and AcOH (10 mL) was heated to 100 °C for 40 hours. The mixture was concentrated to remove the solvent, the pH was adjusted to 12 with 1 M NaOH solution, extracted with PE (100 mL), and the pH of the aqueous phase was adjusted to 5-6 with 6 M HCl to form a white solid. The solid was filtered, and the filter cake was washed with water (50 mL) and dried under vacuum to give 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (1.0 g, 3.0 mmol, 42%, 3 steps) as a white solid. ESI-MS (EI) + ,m / z):272.0

[0938] Step 6: Methyl 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0939] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (800 mg, 2.4 mmol) in HCl / MeOH (50 mL, 2 M) was heated to 75 °C for 17 hours. The concentrated solution was then analyzed by preparative HPLC (Boston C1821*250 mm). Mobile phase: A: 0.1% TFA; B: ACN) Purification yielded methyl 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (120 mg, 0.35 mmol, 15%) as a colorless oil. ESI-MS (EI + ,m / z):344.1[M+H] + .

[0940] Step 7: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate trifluoroacetic acid:

[0941] A mixture of methyl 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (100 mg, 0.30 mmol), HCOONH4 (92 mg, 1.5 mmol), and Pd / C (10%, 20 mg) in MeOH (10 mL) was heated to 65 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to obtain a white solid methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate trifluoroacetic acid (76 mg, 0.21 mmol, 70%). ESI-MS (EI) + ,m / z):254.1[M+H] + . 1 H NMR (500MHz, MeOD-d4) δ4.26 (dd, J=7.5Hz, J=6.0Hz, 1H), 3.91 (m, 4H), 2.49 (dd, J=8.5Hz, J=5.0Hz, 1H), 2.33-2.37 (m, 1H).

[0942] Example 164: (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (I-164):

[0943]

[0944] Reaction process:

[0945]

[0946] Procedures and representations:

[0947] Step 1: 2-Amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0948] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (480 mg, 1.46 mmol) and Pd(OH)₂ / C (20%, 100 mg) in AcOH (15 mL) was stirred at 35 °C under hydrogen atmosphere for 17 hours. The mixture was filtered and the filtrate was concentrated under vacuum to give 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (460 mg, crude) as a white solid. ESI-MS (EI) + ,m / z):240.2[M+H] + .

[0949] Step 2: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0950] CbzOSu (727 mg, 2.92 mmol) was added to a solution of 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (460 mg, crude) and NaHCO3 (368 mg, 4.38 mmol) in acetone (30 mL) and H2O (30 mL) under ice bath conditions. Seventeen hours later, the reaction mixture was adjusted to pH 3-4 with 1M HCl solution, and the extract was obtained with EtOAc (50 mL × 2). The extract was washed with brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was then purified by reversed-phase silica gel chromatography and chiral preparative HPLC [column: CC4 4.6*250 mm 5 μm; solvent: MeOH (0.2% methanol-ammonia)] to give (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valeric acid (27 mg, 0.072 mmol, 5%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valeric acid (22 mg, 0.059 mmol, 4%, 2 steps), both as colorless oils. ESI-MS (E I+ ,m / z):396.0[M+Na] + .

[0951] Step 3: (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0952] A mixture of (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (27 mg, 0.072 mmol) and Pd / C (10%, 5 mg) in MeOH (10 mL) was stirred at room temperature for 1 hour. The solution was filtered and purified by reversed-phase silica gel chromatography to give (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate [I-164] (8.5 mg, 0.036 mmol, 49%) as a white solid. MS (EI) + ,m / z):240.2[M+H] + . 1 H NMR(500MHz,D2O)δ3.74-3.80(m,2H),2.88-2.31(m,1H),1.91-2.20(m,1H).

[0953] Example 203: 2-Amino-4-cyclopentylbutyric acid [I-203]:

[0954]

[0955] Reaction process:

[0956]

[0957] Procedures and representations:

[0958] Step 1: 2-Cyclopentylacetaldehyde:

[0959] Under ice bath conditions, IBX (7.35 g, 26.3 mmol) was added to a solution of 3-cyclopentylprop-1-ol (2.0 g, 17.5 mmol) in DMSO (40 mL). The mixture was heated to room temperature and stirred overnight. The reaction mixture was poured into water (200 mL) and extracted with Et₂O (100 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was used for the next step.

[0960] Step 2: (Z)-2-(tert-Butoxycarbonylamino)-4-cyclopentylbut-2-enoic acid tert-butyl ester:

[0961] Under ice bath conditions, NaOt-Bu (785 mg, 8.2 mmol) was added to a solution of Wittig reagent (2.5 g, 6.8 mmol) in THF (50 mL). After 1 hour, a solution of the above-mentioned 2-cyclopentylacetaldehyde in Et2O (200 mL) was added. The mixture was heated to room temperature and stirred overnight. The solution was diluted with water (200 mL) and extracted with EA (100 mL × 2). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, concentrated under vacuum, and purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 20) to give (Z)-2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoic acid tert-butyl ester (1.0 g, 3.1 mmol, 45%, 2 steps) as a colorless liquid. ESI-MS (EI) + ,m / z):326.2[M+H] + .

[0962] Step 3: 2-(tert-Butoxycarbonylamino)-4-cyclopentylbutyrate tert-butyl ester:

[0963] A mixture of (Z)-2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoate tert-butyl ester (240 mg, 0.74 mmol), HCOONH4 (233 mg, 3.7 mmol), and Pd / C (10%, 30 mg) in MeOH (15 mL) was heated to reflux for 4 hours. The mixture was filtered and concentrated, diluted with Et2O (50 mL), washed with water (50 mL) and brine (50 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give 2-(tert-butoxycarbonylamino)-4-cyclopentylbutyrate tert-butyl ester (224 mg, 0.69 mmol, 93%) as a colorless liquid. ESI-MS (EI) + ,m / z):328.2[M+H] + .

[0964] Step 4: 2-Amino-4-cyclopentylbutyric acid:

[0965] A solution of tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbutyrate (224 mg, 0.69 mmol) in 6 M HCl (20 mL) and dioxane (10 mL) was heated to 70 °C for 2 hours. The mixture was concentrated under vacuum, diluted with water (30 mL), extracted with Et₂O (20 mL × 2), and the filtrate was concentrated to dryness to give 2-amino-4-cyclopentylbutyric acid (114.9 mg, 0.52 mmol, 81%) as a white solid. ESI-MS (EI) + ,m / z):172.3[M+H] + . 1H-NMR (500MHz, D2O): δ3.91 (t, J=6.0Hz, 1H), 1.82-1.89 (m, 2H), 1.66-1.72 (m, 3H), 1.28-1.52 (m, 6H), 1.00-1.01 (m, 2H).

[0966] Example 202: 2-Amino-5-cyclopentylpentanoic acid [I-202]:

[0967]

[0968] Reaction process:

[0969]

[0970] Procedures and representations:

[0971] Step 1: 3-Cyclopentylpropionaldehyde:

[0972] Under ice bath conditions, IBX (3.28 g, 11.7 mmol) was added to a solution of 3-cyclopentylprop-1-ol (1.0 g, 7.8 mmol) in DMSO (20 mL). The mixture was heated to room temperature and stirred overnight. The reaction mixture was poured into water (100 mL) and extracted with Et₂O (60 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was used for the next step.

[0973] Step 2: (E)-2-(tert-Butoxycarbonylamino)-5-cyclopentylpent-2-enoic acid tert-butyl ester:

[0974] Under ice bath conditions, NaOt-Bu (157 mg, 1.63 mmol) was added to a solution of Wittig reagent (500 mg, 1.36 mmol) in THF (15 mL). After 1 hour, a solution of the above-mentioned 3-cyclopentylpropionaldehyde in Et2O (100 mL) was added. The mixture was heated to room temperature and stirred overnight. The solution was diluted with water (200 mL) and extracted with EtOAc (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, concentrated under vacuum, and purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 20) to give (E)-2-(tert-butoxycarbonylamino)-5-cyclopentylpent-2-enoic acid tert-butyl ester (250 mg, 0.74 mmol, 9.5%, 2 steps) as a colorless liquid. ESI-MS (EI) + ,m / z):340.2[M+H] + .

[0975] Step 3: 2-(tert-Butoxycarbonylamino)-5-cyclopentylvaleric acid tert-butyl ester:

[0976] A mixture of 2-(tert-butoxycarbonylamino)-5-cyclopentylpent-2-enoate (250 mg, 0.74 mmol) and Pd / C (10%, 30 mg) in MeOH (15 mL) was stirred under hydrogen at room temperature for 17 hours. The mixture was filtered and concentrated to give tert-butyl 2-(tert-butoxycarbonylamino)-5-cyclopentylpentanoate (250 mg, 0.73 mmol, 99%) as a colorless liquid. ESI-MS (EI) + ,m / z):342.2[M+H] + .

[0977] Step 4: 2-Amino-5-cyclopentylpentanoic acid:

[0978] A solution of 2-(tert-butoxycarbonylamino)-5-cyclopentylpentanoic acid ester (250 mg, 0.73 mmol) in 6M HCl (20 mL) and dioxane (10 mL) was heated to 80 °C for 5 hours. The mixture was concentrated under vacuum, diluted with water (30 mL), extracted with Et₂O (20 mL × 2), and the filtrate was concentrated to dryness to give 2-amino-5-cyclopentylpentanoic acid (115 mg, 0.52 mmol, 71%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + . 1 H-NMR (400MHz, D2O): δ3.84 (t, J=6.0Hz, 1H), 1.79-1.84 (m, 2H), 1.61-1.67 (m, 3H), 1.25-1.49 (m, 8H), 0.95-0.99 (m, 2H).

[0979] Example 197: Synthesis of 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide [I-197]:

[0980]

[0981] Reaction process:

[0982]

[0983] Procedures and representations:

[0984] Step 1: 2-(phenylmethylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0985] A mixture of 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid (80 mg, 0.31 mmol), N-methylcyclopentanamine (62 mg, 0.62 mmol), HATU (141 mg, 0.37 mmol), and Et3N (94 mg, 0.93 mmol) in DMF (2 mL) was stirred at room temperature for 3 hours. The mixture was then analyzed by preparative HPLC (Boston C18 21*250 mm). Mobile phase: A: 0.1% TFA; B: ACN) The purified mixture yielded 2-(phenylmethylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (45 mg, 0.13 mmol, 43%) as a white solid. ESI-MS (EI + ,m / z):339.0

[0986] Step 2: 2-Amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0987] A mixture of 2-(phenylmethylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (45 mg, 0.13 mmol), HCOONH4 (41 mg, 0.65 mmol), and Pd / C (10%, 10 mg) in MeOH (5 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (16.3 mg, 0.066 mmol, 49%) as a white solid. ESI-MS (EI) + ,m / z):249.21H NMR (500MHz, MeOD-d4) δ5.26 (dd, J=15.5Hz, J=6.0Hz, 0.5H), 5.08 (dd, J=16.5Hz, J=5.0Hz, 1H), 4.28-4. 31(m,0.5H),2.97(d,J=48.5Hz,3H),2.38(m,1H),1.65-1.99(m,8H),11.16(dt,J=6.5Hz,J=3.0Hz,6H).

[0988] Example 196: 2-Amino-5-fluoro-4,4-dimethylvaleric acid [I-196].

[0989]

[0990] Reaction process:

[0991]

[0992] Procedures and representations:

[0993] Step 1: 3-Hydroxy-N-methoxy-N,2,2-trimethylpropionamide:

[0994] A mixture of 3-hydroxy-2,2-dimethylpropionic acid (10 g, 84.7 mmol), N,O-dimethylhydroxylamine hydrochloride (16.4 g, 101.7 mmol), EDCI (24.4 g, 127.1 mmol), HOBT (17.2 g, 127.1 mmol), and DIPEA (28 mL, 169.5 mmol) in DMF (200 mL) was stirred at room temperature for 16 hours. The reaction mixture was extracted with EtOAc (200 mL × 3) and water (100 mL), the organic layers were combined, washed with 1N HCl (30 mL × 2), 1N NaHCO3 (30 mL × 2), and brine (50 mL), dried, concentrated, and the residue was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 2) to give 3-hydroxy-N-methoxy-N,2,2-trimethylpropionamide (6.9 g, 50%) as a colorless oil. ESI-MS (EI) + ,m / z):162.2[M+H] + .

[0995] Step 2: 3-Fluoro-N-methoxy-N,2,2-trimethylpropionamide:

[0996] DAST (7.4 mL, 55.9 mmol) was added dropwise to a mixture of 3-hydroxy-N-methoxy-N,2,2-trimethylpropionamide (4.5 g, 27.9 mmol) cooled to -78 °C in DCM (40 mL). The mixture was then stirred at room temperature for 1–2 hours, cooled again to -78 °C, and DAST (4 mL, 27.9 mmol) was added dropwise. The reaction mixture was stirred at room temperature for another 1 hour. The reaction mixture was cooled to -78 °C, and saturated NH4Cl (15 mL) was slowly added, followed by DCM (50 mL). The organic layer was separated, washed with saturated NH4Cl (30 mL) and brine (30 mL × 2), dried, and concentrated to obtain the residue. The residue was purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 4) to give 3-fluoro-N-methoxy-N,2,2-trimethylpropionamide (1.9 g, 28%) as a colorless oil. ESI-MS (EI) + ,m / z):164.2[M+H] + .

[0997] Step 3: 3-Fluoro-2,2-Dimethylpropionaldehyde:

[0998] LiAlH4 (6.1 mL, 61.3 mmol, 1 M, in THF) was added dropwise to a mixture of 3-fluoro-N-methoxy-N,2,2-trimethylpropionamide (1.0 g, 61.3 mmol) cooled to 0 °C in 10 mL of THF. The mixture was then stirred at this temperature for 0.5–1 hour. Saturated NH4Cl (10 mL) was slowly added, and the mixture was extracted with Et2O (20 mL × 3), washed with water (15 mL × 2) and brine (15 mL), dried, and used directly for the next step. ESI-MS (EI) was then performed. + ,m / z): No MS.

[0999] Step 4: (Z)-2-(tert-Butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoic acid tert-butyl ester:

[1000] A mixture of 3-fluoro-2,2-dimethylpropionaldehyde (approximately 630 mg, 6.1 mmol, from the Et₂O solution from the previous step), 2-(tert-butoxycarbonylamino)-2-diethoxyphosphoryl-tert-butyl acetate (2.25 g, 6.1 mmol), and t-BuONa (1.2 g, 12.3 mmol) in THF (15 mL) was stirred at room temperature for 16 hours. Saturated NH₄Cl (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The organic layer was combined, washed with water (15 mL) and brine (15 mL), dried, concentrated, and the residue was purified by chromatography (silica gel, petroleum ether / DCM) to give (Z)-2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoic acid tert-butyl ester (190 mg, 0.60 mmol, 8%) as a white solid. ESI-MS (EI+, m / z): 206 [M-111] + .

[1001] Step 5: 2-(tert-Butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoate tert-butyl ester:

[1002] A mixture of (Z)-2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentan-2-enoate tert-butyl ester (190 mg, 0.60 mmol) and Pd / C (10%, 30 mg) in IPA (15 mL) was stirred under hydrogen at room temperature for 17 hours. The mixture was filtered and concentrated to give 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanate tert-butyl ester (200 mg, crude) as a colorless liquid. ESI-MS (EI... + ,m / z):342.2[M+Na] + .

[1003] Step 6: 2-Amino-5-fluoro-4,4-dimethylpentanoic acid trifluoroacetic acid:

[1004] A solution of 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoic acid tert-butyl ester (200 mg, crude) in 6M HCl (20 mL) and dioxane (10 mL) was heated to 50 °C for 17 hours. The mixture was concentrated under vacuum, diluted with water (30 mL), extracted with Et₂O (20 mL × 2), and the filtrate was concentrated under vacuum and purified by reversed-phase silica gel chromatography to give 2-amino-5-cyclopentylpentanoic acid trifluoroacetic acid (31.7 mg, 0.11 mmol, 19%) as a white solid. ESI-MS (EI) + ,m / z):164.2[M+H] + . 1 H-NMR (500MHz, D2O): δ4.16 (d, J = 47.5Hz, 1H), 3.97 (t, J = 5.5Hz, 1H), 2.03 (dd, J = 15.5 Hz, J=5.5Hz, 1H), 1.71 (dd, J=15.5Hz, J=6.0Hz, 1H), 0.91 (dd, J=15.0Hz, J=2.0Hz, 6H).

[1005] Example 186: Synthesis of 2,4-diamino-4-methylpentanoic acid [I-186]:

[1006]

[1007] Reaction process:

[1008]

[1009] Procedures and representations:

[1010] Step 1: 4-(methoxy(methyl)amino)-2-methyl-4-oxobutyl-2-ylcarbamate tert-butyl ester:

[1011] DIPEA (1.49 g, 11.53 mmol) was added to a solution of 3-(tert-butoxycarbonylamino)-3-methylbutyric acid (1 g, 4.61 mmol), N,O-dimethylhydroxylamine hydrochloride (536 mg, 5.53 mmol), and HATU (2.26 g, 5.99 mmol) in DMF (15 mL). The solution was stirred at room temperature for 2 hours, then diluted with brine (100 mL) and extracted with EtOAc (50 mL × 2). The organic layers were combined, concentrated, and purified by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 3) to give tert-butyl 4-(methoxy(methyl)amino)-2-methyl-4-oxobut-2-ylcarbamate (1.0 g, 3.8 mmol, 82%) as a colorless oil. ESI-MS (EI) + ,m / z):261.2[M+H]+ .

[1012] Step 2: 2-Methyl-4-oxobutyl-2-ylcarbamate tert-butyl ester:

[1013] At room temperature, LiAlH4 (16 mL, 1 M, in THF) was added to a solution of 3.8 g (14.6 mmol) of 4-(methoxy(methyl)amino)-2-methyl-4-oxobut-2-ylcarbamate in 50 mL of THF. The solution was stirred at room temperature for 2 hours, quenched with Na2SO4·10H2O, filtered, and washed with THF to give a yellow solution of 2-methyl-4-oxobut-2-ylcarbamate (approximately 14 mmol, in 110 mL of THF). MS (EI) + ,m / z):146.3[M+H-56] + .

[1014] Step 3: 4-(benzylamino)-4-cyano-2-methylbut-2-ylcarbamate tert-butyl ester:

[1015] To a solution of tert-butyl 2-methyl-4-oxobut-2-ylcarbamate (crude, about 14 mmol, in 110 mL THF), BnNH2 (2.2 mL) and AcOH (2.2 mL) were added. The solution was stirred at room temperature for 10 minutes. TMSCN (2.2 mL) was added. The mixture was stirred at room temperature for 17 hours. The reaction mixture was then concentrated and passed by chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 4) to give tert-butyl 4-(benzylamino)-4-cyano-2-methylbut-2-ylcarbamate (670 mg, 2.11 mmol, 15%) as a yellow viscous substance. MS (EI) + ,m / z):318.3[M+H] + .

[1016] Step 4: tert-butyl 5-amino-4-(benzylamino)-2-methyl-5-oxopent-2-ylcarbamate:

[1017] 30% H₂O₂ (0.64 mL, 5.67 mmol) was added to a mixture of 4-(phenylmethylamino)-4-cyano-2-methylbut-2-ylcarbamate tert-butyl ester (640 mg, 2.00 mmol), K₂CO₃ (550 mg, 3.98 mmol) in DMSO (16 mL), and the mixture was stirred at room temperature for 17 hours. The reaction mixture was then diluted with H₂O (200 mL) and extracted with EtOAc (100 mL × 2). The organic layers were combined and concentrated to give 2-(phenylmethylamino)-4-(tert-butyloxycarbonylamino)-4-methylpentanoic acid (crude, 890 mg) as a yellow viscous substance. MS (EI+, m / z): 336.0 [M+H] + .

[1018] Step 5: 2-(phenylmethylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid:

[1019] A mixture of tert-butyl 5-amino-4-(phenylmethylamino)-2-methyl-5-oxopentan-2-ylcarbamate (crude, 890 mg, approx. 2.0 mmol), KOH (406 mg, 7.25 mmol), and ethane-1,2-diol (9 mL) and H₂O (9 mL) was stirred at 100 °C for 5 hours. The reaction mixture was then diluted with brine (200 mL), extracted with THF / EA = 2:1 (90 mL × 5), the organic layers were combined, concentrated, and purified by reversed-phase HPLC (Boston C18 21*250 mm 10 μm; mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give 2-(phenylmethylamino)-4-(tert-butyloxycarbonylamino)-4-methylpentanoic acid (120 mg, 0.36 mmol, 18%) as a white solid. MS (EI+, m / z): 337.3 [M+H] + .

[1020] Step 6: 2-Amino-4-(tert-Butoxycarbonylamino)-4-methylpentanoic acid:

[1021] A mixture of 2-(phenylmethylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (140 mg, 0.42 mmol), HCOONH4 (132 mg, 2.1 mmol), and Pd / C (10%, 20 mg) in MeOH (15 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give 2-amino-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (60 mg, 0.24 mmol, 58%) as a white solid. ESI-MS (EI) + ,m / z):247.2

[1022] Step 7: 2,4-Diamino-4-methylpentanoic acid:

[1023] A solution of 2-amino-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (60 mg, 0.24 mmol) in 6 M HCl (10 mL) and dioxane (0 mL) was stirred at room temperature for 17 hours. The solution was concentrated under vacuum to give 2,4-diamino-4-methylpentanoic acid (51.8 mg, 0.236 mmol, 97%) as a white solid. ESI-MS (EI) + ,m / z):147.1 1H NMR (500MHz, D2O) δ4.04 (dd, J = 9.5Hz, J = 3.5Hz, 1H), 2.32 (dd, J = 15.0Hz, J = 9.5Hz, 1H), 1.94 (dd, J = 15.0Hz, J = 3.0Hz, 1H), 1.38 (dd, J = 9.5Hz, J = 5.0Hz, 6H).

[1024] Example 199: Synthesis of 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine [I-199]:

[1025]

[1026] Reaction process:

[1027]

[1028] Procedures and representations:

[1029] Step 1: N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide:

[1030] A mixture of 2-chloro-N-methoxy-N-methylacetamide (13.7 g, 0.1 mol) and triphenylphosphine (26.2 g, 0.1 mol) in acetonitrile (200 mL) was heated to 80 °C and maintained for 20 hours. The mixture was cooled below 40 °C and concentrated to remove the solvent. The residue was successively dissolved in dichloromethane (200 mL) and 2N KOH (100 mL). The resulting mixture was stirred at 20 °C for 1 hour. The organic layer was washed with brine (200 mL × 3), dried over Na₂SO₄, and filtered. The filtrate was concentrated under vacuum to give N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide (36 g, 0.1 mol, 98%) as a yellow solid. ESI-MS (EI) + ,m / z):364.4[M+H] + .

[1031] Step 2: (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide:

[1032] A mixture of N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide (36.3 g, 0.1 mol) and 1,1,1-trifluoroprop-2-one (22.4 g, 0.2 mol) in tetrahydrofuran (500 mL) was heated to 20 °C and maintained for 20 hours. The mixture was cooled under vacuum at below 40 °C and concentrated to remove the solvent. The residue was purified by silica gel column chromatography (200 g, 200–300 mesh, UV 254 nm) eluting with 0–25% ethyl acetate / petroleum ether to give (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide (19.5 g, 0.1 mol, 98%) as a yellow oil. ESI-MS (EI...) + ,m / z):198.2[M+H] + .

[1033] Step 3: 4,4,4-Trifluoro-N-methoxy-N,3-dimethylbutyramide:

[1034] A mixture of (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbutyr-2-enamide (2 g, 0.01 mol) and Pd / C (10%, 200 mg) in THF (50 mL) was stirred at 26 °C for 18 hours. The mixture was filtered, and the filtrate was concentrated under vacuum to dryness to give 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutyramide (2 g, 0.01 mol, 98%) as a yellow oil. ESI-MS (EI...) + ,m / z):200.2[M+H] + .

[1035] Step 4: 4,4,4-Trifluoro-3-methylbutanal:

[1036] At 0°C, LiAlH4 (0.4 g, 0.01 mol) was added to a solution of 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutyramide (2 g, 0.01 mol) in 40 mL of THF. The mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched successively with water and methyl tert-butyl ether (30 mL × 2). The organic layer was washed with brine (50 mL × 3), dried over Na2SO4, and filtered. The filtrate was retained to obtain a colorless solution of 4,4,4-trifluoro-3-methylbutyraldehyde (1.4 g, crude), which was used directly in the next step.

[1037] Step 5: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanilide:

[1038] Under ice bath conditions, benzylamine (1.5 mL), AcOH (1.0 mL), and TMSCN (1.5 mL) were successively added to a solution of 4,4,4-trifluoro-3-methylbutanal in methyl tert-butyl ether (100 mL). The mixture was heated to 20 °C and stirred overnight. The solution was diluted with water (30 mL) and extracted with EtOAc (30 mL). The organic phase was washed with water (30 mL × 2) and brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give a brown oily 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanilide (2.6 g, crude), which was used in the next step. ESI-MS (EI + ,m / z):257.3[M+H] + .

[1039] Step 6: N-Benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine:

[1040] A solution of 0.3 g crude 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanilitonium (DMF) in 10 mL DMF was added with 0.15 g NH4Cl and 0.003 mol NaN3, and heated to 95 °C for 18 hours. The solution was cooled to 15 °C and extracted with EtOAc (20 mL). The organic phase was washed with water (20 mL × 2) and brine (20 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give N-phenylmethyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine (0.1 g, 0.5 mmol, 33%, 3 steps) as a white solid. ESI-MS (EI) + ,m / z):300.3[M+H] + .

[1041] 4,4,4-Trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-aminetrifluoroacetic acid:

[1042] At room temperature, HCOONH4 (0.17 g, 2.7 mmol) and Pd / C (30 mg) were added to a solution of N-benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine (160 mg, 0.54 mmol) in MeOH (15 mL). The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-aminetrifluoroacetic acid (72.8 mg, 0.23 mmol, 42%) as a white solid; ESI-MS (EI)+ ,m / z):210.2[M+H] + ; 1H NMR (500MHz, DMSO-d6) δ4.67-4.93 (m, 1H), 2.31-2.41 (m, 1H), 2.00-2.12 (m, 2H), 0.99 (dd, J = 16.8, J = 6.4Hz, 6H).

[1043] Example 210: Western blot assay

[1044] This screening analysis measured the in vitro activity of the test compounds against the GATOR2 / Sestrin2 complex purified by immunoprecipitation of FLAG-WDR24 stably expressed from HEK293T cells. HEK293T cells (293T) were engineered to stably express the N-terminal labeled FLAG-WDR24 via lentiviral transduction. Lentiviral transmission was generated by co-transfecting the lentiviral transfer vector pLJM60 with the ΔVPR envelope and the CMV VSV-G packaging plasmid into HEK-293T cells using XTremeGene 9 transfection reagent (Roche Diagnostics). Twenty-four hours post-transfection, the medium was changed to Dulbecco's Modified Eagle's media (DMEM) supplemented with 30% inactivated fetal serum. Virus-containing supernatants were collected at 48 and 72 hours post-transfection and passed through a 0.45 μm filter to remove cells. Target cells in 6-well tissue culture plates were infected with a medium containing 8 μg / mL polybrene and infected by rotational centrifugation at 2,200 rpm for 1 hour. Twenty-four hours post-infection, the virus was removed and cells were selected with appropriate antibiotics. Cells were then grown in DMEM supplemented with 10% fetal bovine serum and antibiotics.

[1045] To screen for leucine mimicry compounds, 2,000,000 FLAG-WDR24-expressing 293T cells were plated in 10 cm tissue culture plates. After 72 hours, the cells were placed in standard RPMI medium (-AA RPMI, US Biological Life Sciences) without amino acid formulation and supplemented with 5 mM glucose (-AA RPMI) for 1 hour, and then lysed in lysis buffer (40 mM HEPES, 1% Triton, 10 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 2.5 mM MgCl2, and protease inhibitors). To isolate the FLAG-WDR24 / endogenous Sestrin2 complex, 1 ml of crude lysate (equivalent to 2-4 mg of total protein) was immunoprecipitated with 30 μl of anti-flag resin (SIGMA) at 4 °C for 2 hours. The sample was washed twice with 0.5 M NaCl in cold lysis buffer and resuspended in 1 ml of cold cytoplasmic buffer (40 mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100). Then, a different concentration of the test compound or control (filtered solution or leucine) was added to each immunoprecipitated sample, and the sample was incubated at 4 °C for 60 minutes. After the incubation period, the samples were centrifuged to allow the FLAG-WDR24 / endogenous Sestrin2 complex bound to the anti-flag resin to clump together. The supernatant was completely removed, and the resin was resuspended in SDS-PAGE sample buffer and boiled for 5 minutes. The samples were then processed by SDS-PAGE and blotted with anti-FLAG (Sigma) and anti-Sestrin2 (Cell Signaling Technology) antibodies, as described in L. Qian et al., Cell Reports 9:1-8 (2014).

[1046] Scan the obtained protein blot and use Imaging platforms quantified the band intensities corresponding to Sestrin2 and FLAG-WDR24. To determine the amount of Sestrin2 bound to GATOR2 in each condition, the band intensity of Sestrin2 was normalized to that of FLAG-WDR24. For each batch of test compounds, a negative control (filtered solution) and a positive control (leucine, 25 μM, sigma) were also performed. The consumption of endogenous Sestrin2 bound to FLAG-WDR24 by leucine was normalized to represent 100% activity. Compounds were analyzed in duplicate, and the activity of each compound was quantified as a percentage of leucine activity and averaged. Repeated attempts at analysis were performed, and the mean activity of leucine was found to have a standard deviation of 20% relative to water; therefore, test compounds that reduced the amount of Sestrin2 bound to GATOR2 by at least 40% in duplicate at 25 μM were considered statistically significant and characterized as leucine mimics. Some compounds increase the amount of Sestrin2 bound to FLAG-WDR24. Compounds that increase the amount of Sestrin2 bound to GATOR2 by more than 40% (expressed as less than -40% of leucine activity) are characterized as leucine antagonists.

[1047] Example 211. A method for identifying compounds that mimic or antagonize the activity of leucine in relation to Sestrin2 and the Sestrin2 / GATOR2 interaction.

[1048] introduction

[1049] In the presence of insufficient leucine, Sestrin1 and Sestrin2 interact with GATOR2 via the GATOR2 components WDR24 and Seh1L. Under conditions of sufficient leucine, leucine directly binds to Sestrin2, inducing the dissociation of Sestrin2 from GATOR2. The aim of the following method is to identify compounds that mimic the binding of leucine to Sestrin2 and disrupt the Sestrin2 / GATOR2 interaction. Furthermore, the method identifies compounds that antagonize leucine binding to Sestrin2 and prevent Sestrin2 from reacting with leucine and dissociating from GATOR2.

[1050] Method 1 (In vitro PPI analysis)

[1051] This screening analysis measured the in vitro activity of compounds against the GATOR2 / Sestrin2 complex purified by immunoprecipitation of Flag-WDR24 stably expressed from HEK293T cells. HEK293T cells (293T) were engineered to stably express the N-terminal labeled Flag-WDR24 via lentiviral transduction. Lentiviral cells were generated by co-transfecting the lentiviral transfer vector pLJM60 with the ΔVPR envelope and the CMV VSV-G packaging plasmid into HEK-293T cells using XTremeGene9 transfection reagent. Twenty-four hours post-transfection, the medium was changed to Dalberg Modified Eagle Medium (DMEM) supplemented with 30% inactivated fetal serum. Virus-containing supernatants were collected at 48 and 72 hours post-transfection and passed through a 0.45 μm filter to remove cells. Target cells were infected in 6-well tissue culture plates in medium containing 8 μg / mL polybrene and rotated for infection by centrifugation at 2,200 rpm for 1 hour. Twenty-four hours after infection, the virus was removed and cells were selected with appropriate antibiotics. The cells were then grown in DMEM supplemented with 10% fetal bovine serum and antibiotics.

[1052] To screen for leucine mimicry compounds, 2,000,000 293T cells expressing Flag-WDR24 were plated in 10 cm tissue culture plates. After 72 hours, the cells were placed in standard RPMI medium (AARPMI, BioLife Sciences, USA) without amino acid formulation and supplemented with 5 mM glucose for 1 hour, followed by lysis in lysis buffer (40 mM HEPES, 1% Triton, 10 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 2.5 mM MgCl2, and protease inhibitors). The Flag-WDR24 / endogenous Sestrin2 complex was isolated as follows: 1 ml of crude lysate (equivalent to 2-4 mg of total protein) was immunoprecipitated (IP) for 2 hours at 4°C with 30 μl of anti-flag resin (Sigma). The sample was washed twice with 0.5 M NaCl in cold lysis buffer and resuspended in 1 ml of cold cytoplasmic buffer (40 mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100). The compound was then added to each sample at a specified concentration of 25 μM and incubated at 4°C for 30 minutes. After incubation, the samples were centrifuged to solidify the Flag-WDR24 / endogenous Sestrin2 complex bound to the anti-flag resin. The supernatant was completely removed, and the resin was resuspended in SDS-PAGE sample buffer and boiled for 5 minutes. The samples were then processed by SDS-PAGE and blotted with anti-Flag (Sigma) and anti-Sestrin2 (Cycintron Technologies) antibodies, as described in L. Qian et al., Cell Reports 9:1-8 (2014).

[1053] Scan the obtained protein blot and use Imaging platform quantification corresponded to the band intensities of Sestrin2 and Flag-WDR24. To determine the amount of Sestrin2 bound to GATOR2 in each condition, the band intensity of Sestrin2 was normalized to the band intensity of Flag-WDR24. For each batch of test compounds, negative controls (water) and positive controls (leucine, 25 μM, sigma) were also performed. The consumption of endogenous Sestrin2 bound to Flag-WDR24 by leucine was normalized to represent 100% activity. Compounds were analyzed in duplicate, and the activity of each compound was quantified as a percentage of leucine activity and averaged. A table listing the quantitative data of the test compounds is presented in Table 3. Repeated trial analyses yielded a mean activity of leucine with a standard deviation of 20% compared to water; therefore, test compounds that reduced the amount of Sestrin2 bound to GATOR2 by at least 40% in both copies at 25 μM were considered statistically significant and referred to as leucine mimics. Some compounds increased the amount of Sestrin2 binding to Flag-WDR24 (shown as a negative percentage of leucine activity in Table 3). Compounds showing less than -40% leucine activity were also considered hits and referred to as leucine antagonists.

[1054] Method 2 (based on mTORC1 activation in cells)

[1055] To demonstrate the efficacy of compounds identified as leucine mimics in intact cells, mTORC1 signaling in response to compound treatment following leucine starvation was measured by Western blotting. Following leucine starvation, and 10 to 90 minutes after leucine addition, the addition of exogenous leucine activated mTORC1 when signaling was measured, as described in Wang, S., Tu, Z. et al., Science 347(6218):188-194 (2015). Therefore, a similar assay was designed to test whether compounds identified as leucine mimics could activate mTORC1 in a similar manner. In short, 800,000 HEK293T cells were plated in each well of a 6-well plate in DMEM supplemented with 10% fetal bovine serum and antibiotics. The next day, the cells were placed in modified DMEM without leucine (Thermo Scientific) or serum for 1 hour, followed by the addition of a specified concentration of leucine mimic (n=3) for a period exceeding 10 minutes. Cells were then lysed, treated with SDS-PAGE, and subjected to Western blotting using antibodies against mTORC1 substrates phosphorylated S6 kinase (Thr389) and phosphorylated 4EBP1 (Thr37 / 46) (Cycintron Technologies) and a loading control (β-actin, Santa Cruz Biotechnology), as described in Kang, SA et al., Science 341(6144):364-374 (2013). Then, [the following was used]... The imaging platform normalized the intensity of the actin bands to correspond to the phosphorylated substrates. Compounds that significantly increased mTORC1 signaling compared to untreated leucine-starved cells (Student's t-test, p < 0.05) were considered cellularly active. As a positive control, 100 μM leucine was added to leucine-starved cells for 60 minutes.

[1056] Method 3 (based on mTORC1 activation in cells)

[1057] To demonstrate the efficacy of compounds identified as leucine antagonists or to determine whether weak leucine mimics enhance leucine activity in intact cells, the same paradigm was repeated as above, but with the following changes: Cells were placed in DMEM medium (as described in Method 3) without leucine for 60 minutes, followed by the compound (n=3) for a period of 60 minutes or longer. After compound treatment, cells were stimulated with 30 and 100 μM leucine for 60 minutes. mTORC1 signaling was measured by Western blotting as described in Method 2. Compounds that responded statistically (Student's t-test, p<0.05) to reduce the level of actin-normalized phosphorylated substrates of mTORC1 in response to 30 μM or 100 μM leucine were considered active in cells. Compounds that increased the level of actin-normalized phosphorylated substrates in mTORC1 in response to 30 μM or 100 μM leucine were considered leucine enhancers in cells in a statistically significant manner (Student's t-test, p < 0.05). As a control, leucine-starved cells were pretreated with water before leucine addition. Alternatively, potential leucine antagonists in HEK293T cells were analyzed in the same manner as described above, but without leucine starvation and stimulation. Western blotting was performed to determine whether baseline mTORC1 signaling was attenuated under replete culturing conditions after compound treatment.

[1058] Method 4

[1059] The ability of compounds to modulate the interaction between Sestrin2 and GATOR2 in cells was measured by repeating the analyses described in Methods 2 and 3 in HEK293T cells engineered to stably express Flag-WDR24 and plated in 10 cm tissue culture dishes. The interaction between endogenous Sestrin2 and Flag-WDR24 was measured from lysates obtained from cells treated with the compounds as described in Method 1 (n=3). Briefly, to measure the amount of endogenous Sestrin2 binding to Flag-WDR24 after cell treatment, immunoprecipitation with anti-flag resin and processing of the resulting samples for SDS-PAGE and Western blotting were performed to measure the amount of endogenous Sestrin2 binding to Flag-WDR24. Compounds that modulated the amount of Sestrin2 binding to GATOR2 in a statistically significant manner (Student's t-test, p<0.05) were considered hits.

[1060] Method 5 (ALPHALISA cell-based analysis)

[1061] To demonstrate the efficacy of the compound identified as a leucine mimic in intact cells in a plate-based format, mTORC1 signaling in response to compound treatment following leucine starvation was measured by AlphaLISA. In short, 1,000,000 HEK293T cells were plated in DMEM supplemented with 10% fetal bovine serum in T-75 cell culture flasks. After the cells reached confluence, they were placed in modified DMEM containing 10% dialyzed fetal bovine serum (10%) and leucine-free (Thermo Scientific) for 1 hour. The cells were then trypsinized and re-plated at 50,000 cells / well in leucine-free DMEM containing 10% dialyzed fetal bovine serum in 96-well plates with a black, clear bottom. Cells were allowed to attach to the plate for 2 hours, and then the specified concentration of the compound (n=4) was added for a period exceeding 1 hour. After reaching the time point, cells were lysed and analyzed using the p-p70S6K(Thr389) SureFire Ultra AlphaLISA kit according to the manufacturer's instructions (http: / / www.perkinelmer.com / CMSResources / Images / 44-176283MAN_SureFire_TGR70S_p70_pT389.pdf). Compounds that significantly increased mTORC1 signaling compared to untreated leucine-starved cells (Student's t-test, p<0.05) were considered mTORC1 activators. Compounds that significantly decreased mTORC1 signaling compared to untreated leucine-starved cells (Student's t-test, p<0.05) were considered inhibitors in the cells. As a positive control, 100 μM leucine was added to leucine-starved cells for the same duration as compound treatment.

[1062] Method 6, Thermal Shift Protocol (Tm Shift):

[1063] The codon-optimized full-length human Sestrin2 was fused to the N-terminus of a His-MBP tag and cloned into the pMAL6H-C5XT bacterial expression vector. This vector was transformed into *E. coli* LOBSTR(DE3) cells (Kerafast). Cells were grown at 37°C to 0.6 OD, and protein production was induced at 18°C ​​with 0.2 mM IPTG for 12–14 hours. Cells were collected by centrifugation at 6,000 g, resuspended in lysis buffer (50 mM potassium phosphate pH 8.0, 500 mM NaCl, 30 mM imidazole, 1 mM DTT, 10 μg / ml nuclease, and 1 mM PMSF) and lysed by acoustic treatment. The lysate was cleaned by centrifugation at 10,000 g for 20 min. The Sestrin2 protein was isolated from the soluble fraction with near 100% purity by affinity capture of the His tag, followed by ion exchange and size exclusion chromatography. For thermal migration analysis, Sestrin2 protein was diluted to 2 mg / mL in dilution buffer (10 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA). Prior to thermal migration analysis, 2 μL of Sestrin2 protein was combined with 8 μL of ROX dye (Thermo Fisher), 1 μL of mediator or compound, and 14 μL of dilution buffer for each well of a 96-well plate and incubated on ice for 1 hour to allow compound binding. Thermal migration analysis was then performed on an Agilent MX3005p, with each compound analyzed three times at 10 μM, 100 μM, and 1000 μM. Incubation with leucine shifted the melting temperature of Sestrin2 by 2.16 to 11.61 degrees Celsius in a dose-dependent manner. Based on the CV% rate of change of repeated thermal offset measurements of Sestrin2 grown with the medium, a positive offset of 2 degrees or greater was considered statistically significant.

[1064] Method 7, Indirect Ligand Binding Analysis (ILBA)

[1065] The binding of Sestrin2 to leucine or other ligands can be detected in vitro in intact cells or immunoassay using purified protein via rabbit monoclonal anti-Sestrin2 antibody from CST Technologies (Catalog No. 8487). The binding of CST antibodies to native (non-denatured) Sestrin2 is modulated by leucine binding, with antibody affinity decreasing upon binding. Similarly, the affinity of CST antibodies for native Sestrin2 decreases in a similar manner to that for leucine upon binding to a compound. Conversely, compounds that destabilize Sestrin2, as measured by thermal shift analysis, increase the affinity of CST antibodies for non-denatured Sestrin2. Therefore, various forms of this indirect ligand binding assay (ILBA) have been developed to measure the affinity of CST anti-Sestrin2 antibodies upon binding to leucine or other compounds. In one version, crude lysates produced from human cell lines after a 1-hour amino acid starvation period (cells lysed in 1% Triton, 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 40 mM HEPES [pH 7.4], 150 mM NaCl, and 2.5 mM MgCl2) were analyzed. The lysates were then incubated on ice or at room temperature for 1 hour with leucine or other compounds. After compound incubation, samples were immunoprecipitated with CST anti-Sestrin2 antibody for 1.5 hours, followed by incubation with a protein A agarose gel for 30 minutes, as described in L. Qian et al., Cell Reports 9:1-8 (2014). The agarose gel-bound antibody-protein complex was precipitated by centrifugation and a second round of immunoprecipitation was performed on the flow-through with rabbit polyclonal anti-Sestrin2 antibody (ProteinTech, #10795-1-AP) to determine that the total Sestrin2 protein levels were equal between samples. SDS-PAGE was performed on the immunoprecipitated samples, followed by Western blotting with a mouse monoclonal anti-Sestrin2 antibody from Sigma (catalog number WH0083667M3). On the immunoblot of samples immunoprecipitated with the CST anti-Sestrin2 antibody, leucine binding induced a significant decrease in the intensity of the band corresponding to Sestrin2 by 50% or more, but on the immunoblot of samples immunoprecipitated with the protein technology antibody, leucine binding did not cause any change in the Sestrin2 band. This version of the analysis also measured increased Sestrin2 instability induced by co-culturing with the compound. Analysis was performed in the same manner, but compounds that destabilized Sestrin2 (as measured by thermal migration analysis) resulted in increased intensity of the immunoblot band corresponding to Sestrin2 immunoprecipitation using the CST antibody.

[1066] This analysis was also performed in cultured human cells overexpressing Sestrin2 fused to the N-terminus of the Flag tag. In this version of the analysis, the procedure remained the same, but immunoblotting was performed using a mouse anti-Flag antibody (#F3165, Sigma). When ILBA was performed using a point-mutated form of Sestrin2 that cannot bind leucine, no decrease in CST antibody affinity was observed after leucine or γ-methylleucine binding.

[1067] In another version of the analysis, cultured human cells were subjected to a combination of amino acid starvation for 1 hour, followed by stimulation with leucine or a compound. Cells were lysed one hour after stimulation and treated as described above, except for a 1-hour ligand binding step.

[1068] Indirect ligand binding analysis was also performed in a porous configuration using ALPHAlisa technology (PerkinElmer). This version of the analysis required coupling of a biotin-labeled anti-Sestrin2 antibody, an anti-streptolysin donor bead (PerkinElmer), and an anti-Flag receptor bead (PerkinElmer) to detect overexpressed Flag-Sestrin2, or coupling with a mouse anti-Sestrin2 antibody (Sigma) and an anti-mouse receptor bead (PerkinElmer) to detect endogenous Sestrin2.

[1069] The analysis was performed as described above, but with the following modifications: For the leucine or compound binding moiety being analyzed, crude lysates produced from cells transiently or stably overexpressing human Flag-Sestrin2 after 1 hour of amino acid starvation were diluted in lysis buffer to 0.8 mg / ml of total protein and arranged in multi-well plates such as 96-well plates. For the detection of endogenous Sestrin2, the crude lysates were diluted in lysis buffer to 4 mg / ml of total protein. Leucine or the compound was added to each well and the plate was incubated on ice or at room temperature with gentle stirring for 1 hour. During the ligand binding step, biotin-labeled anti-Sestrin2 antibody (CST) was diluted to 5 nM in ALPH-ALISA immunoassay buffer (PerkinElmer), and 5 nM mouse anti-Sestrin2 antibody (Sigma) was combined with 4× stock solution (40 μg / ml) of anti-mouse receptor beads for the analysis of endogenous Sestrin2. For Flag-Sestrin2 detection, a 4× stock solution (40 μg / ml) of anti-Flag receptor beads was prepared in immunoassay buffer. After the ligand binding step, 5 μL of lysate was combined with 10 μL of biotin-labeled anti-Sestrin2 antibody, 12.5 μL of a mixture of mouse Sestrin2 antibody / anti-mouse receptor beads or anti-Flag receptor beads, and 10 μL of ALPHAISA immunoassay buffer and incubated at room temperature for 1 hour. Finally, 12.5 μL of streptavidin donor beads (160 μg / ml in immunoassay buffer) was added, and the mixture was incubated in the dark for another hour before being read on an Envision plate reader.

[1070] The ALPHALISA analysis was performed as described above, but instead of the purified Sestrin2 protein, it was diluted in immunoassay buffer to a final reaction concentration of 3 ng / ml.

[1071] Finally, prior to lysis, ALPHAISA was performed on lysates of cells treated with leucine or a compound under amino acid starvation conditions. Cell-based processing was performed in multi-well plates, and each ALPHAISA reaction used 15 μL of lysate (1 mg / ml total protein) along with 10 μL of biotin-labeled antibody, 12.5 μL of antibody / receptor bead mixture, and 12.5 μL of streptavidin donor bead mixture.

[1072] Indirect ligand binding analysis is also performed using capture-based methods, such as sandwich ELISA used in this field. In one version of the analysis, the method developed by Meso-Scale Discovery (MSD) was used. ILBA was performed using the MSD system, which is based on electrochemiluminescence detection using antibodies that bind to the analyte. ILBA was performed using crude lysates expressing endogenous Sestrin2 or overexpressing Flag-Sestrin2, with leucine treatment performed in vitro or in cells prior to lysis. For in vitro ILBA of endogenous Sestrin2, crude lysates (0.8 mg / ml total protein) were prepared and leucine was bound in the same manner as described for ALPHAISA ILBA. After complete ligand binding, biotin-labeled anti-Sestrin2 antibody from CST was added to each well to a final concentration of 0.25 μg / ml, and the plates were incubated at 4°C for 1 hour with gentle stirring. Capture of each sample into the wells of a 96-well plate was achieved in one of the following ways: by coating an MSD plate coated with streptavidin-coated MSD plate or a bare MSD plate with mouse anti-Sestrin2 antibody from Sigma. Capture required 25 μL of sample per well, followed by incubation at 350 rpm for 1 hour. After sample capture, wash the wells three times with Tris-buffered saline and 0.1% Tween (TBS-T). If the sample was captured onto a plate coated with anti-Sestrin2 antibody, add mouse monoclonal anti-Sestrin2 antibody (Sigma) to a final concentration of 1 μg / ml and shake at 350 rpm for 1 hour. Wash the wells again with TBS-T and add anti-mouse second SULFO-TAG antibody (MSD) to a final concentration of 1 μg / ml, shaking at 350 rpm for 1 hour. Finally, wash the wells three times with TBS-T and add 2× read buffer (MSD), and read the plate immediately on the MSD instrument. If the sample was captured using a bare plate coated with mouse anti-Sestrin2 antibody, after washing, add anti-Sestrin2 antibody (MSD) to a final concentration of 1 μg / ml, shake for 1 hour, then wash and incubate with read buffer before analysis.

[1073] In another version of this analysis, crude lysates overexpressing Flag-Sestrin2 were analyzed and captured or detected with mouse monoclonal anti-Flag antibody (Sigma) using the same MSD-based protocol as described above.

[1074] For all analyses, compounds that significantly reduced the signal corresponding to Sestrin2 immunoreactivity were considered leucine mimics, while compounds that significantly increased the signal were considered potential leucine antagonists.

[1075] Table 3 shows the activities of the selected compounds of the present invention. The compound numbers correspond to the compound numbers in Tables 1 and 2. Compounds with activity indicated as “A” provide ≥40% activity relative to leucine, compounds with activity indicated as “B” provide ≤-40% activity relative to leucine, compounds with activity indicated as “C” provide activity between -40% and 40% relative to leucine. At specified concentrations, compounds with activity indicated as “D” provide a shift of 0.5 to 2 times relative to the DMSO control, compounds with activity indicated as “E” provide a shift of 2.1 to 5 times relative to DMSO, compounds with activity indicated as “F” provide a shift of 5.1 to 10 times relative to DMSO, and compounds with activity indicated as “G” provide a shift of 10.1 to 14 times relative to DMSO.

[1076] Activity was determined as a percentage of activity relative to leucine using analytical method 1. Activity was determined as a cell-based mTORC1 activation assay using analytical method 2.

[1077] Table 3. Analytical data of exemplary compounds

[1078]

[1079]

[1080]

[1081] Table 4 shows the selected compounds of the present invention that are active in ALPHALISA cell-based analysis (Method 5). The compound numbers correspond to the compound numbers in Tables 1 and 2. The compounds listed in Table 4 are mTORC1 activators and have >2-fold activity compared to the positive leucine control.

[1082] Table 4. Exemplary compounds active in ALPHALISA cell-based analyses

[1083]

[1084]

[1085] Table 5 shows the selected compounds of the present invention that are active in thermal migration analysis (Method 6). The compound numbers correspond to the compound numbers in Tables 1 and 2. The compounds listed in Table 5 exhibit a positive migration of 2 degrees or greater.

[1086] Table 5. Exemplary compounds showing activity in thermal migration analysis

[1087]

[1088]

Claims

1. A compound, said compound being selected from: Or its pharmaceutically acceptable salt.

2. The compound according to claim 1, wherein the compound is Or its pharmaceutically acceptable salt.

3. The compound according to claim 1, wherein the compound is 4. A compound selected from: Or its pharmaceutically acceptable salt.

5. A pharmaceutically acceptable composition comprising the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or mediator.

6. The composition according to claim 5, in combination with an additional therapeutic agent.

7. The composition of claim 6, wherein the additional therapeutic agent is an antiproliferative compound.

Images