Modulators of sestrin-gator2 interaction and uses thereof

Abstract

The present application relates to modulators of the SESTRIN-GATOR2 interaction and uses thereof. The present invention provides compounds, compositions thereof, and methods of using the same.

Description

[0001] This application is a divisional application of the invention patent application with the application date of 21 October 2016, the application number 201680069916.X, and the title "Modulators of Sestrin-GATOR2 interaction and uses thereof". TECHNICAL FIELD

[0002] The present invention relates to compounds and methods useful for modulating Sestrin-GATOR2 interaction, thereby indirectly selectively modulating mTORCl activity. The present invention also provides pharmaceutically acceptable compositions comprising the compounds of the present invention and methods of using the same to treat various disorders. BACKGROUND

[0003] The mechanistic target of rapamycin complex 1 (mTORCl) protein kinase is a master growth regulator that senses diverse environmental cues such as growth factors, cellular stress, and nutrient and energy levels. Upon activation, mTORCl phosphorylates substrates that potentiate anabolic processes such as mRNA translation and lipid synthesis and limit catabolic processes such as autophagy. Aberrations in mTORCl regulation occur in a wide range of diseases, including, among others, diabetes, epilepsy, neurodegeneration, immune responses, inhibition of skeletal muscle growth, 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).

[0004] Many upstream inputs, including growth factors and energy levels, signal to mTORCl through the TSC complex, which regulates Rheb, a small GTPase that is a fundamental activator of mTORCl (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 15, 702-713; Sancak et al., (2008) Science (New York, N.Y.) 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) Proc Natl Acad Sci U S A 99, 13571-13576). Amino acids do not appear to signal to mTORCl through the TSC-Rheb axis, but rather through a heterodimeric Rag GTPase 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) Proc Natl Acad Sci U S A 102, 14238-14243; Roccio et al., (2005) Oncogene 25, 657-664; Sancak et al., (2008) Science (New York, N.Y.) 320, 1496-1501; Schürmann et al., (1995) The Journal of biological chemistry 270, 28982-28988; Sekiguchi et al., (2001) The Journal of biological chemistry 276, 7246-7257; Smith et al., (2005) The Journal of biological chemistry 280, 18717-18727).Rag GTPases control the subcellular localization of mTORCl and amino acids favor recruitment of mTORCl to the lysosomal surface, where Rheb GTPase is also present (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; Sancak et al., (2008) Science (New York, N.Y.) 320, 1496-1501). Several positive components of the Rag GTPase upstream pathway have been identified. The Ragulator complex localizes Rag GTPases to the lysosomal surface and, together with vacuolar ATPases, favors GDP exchange for GTP on Rag A / B (Bar-Peled et al., (2012) Cell 150, 1196-1208; Sancak et al., (2010) Cell 141, 290-303; Zoncu et al., (2011) Science Signaling 334, 678-683). A distinct FLCN-FNIP complex acts on Rag C / D and stimulates its GTP hydrolysis to GDP (Tsun et al., 2013). When Rag A / B are loaded with GTP and Rag C / D with GDP, these heterodimers bind mTORCl and recruit it to the lysosomal surface, where it can start contacting its activator Rheb GTPase.

[0005] Current studies have identified the GATOR1 multiprotein complex as the primary negative regulator of the amino acid sensing pathway, and deletion of the GATOR1 multiprotein complex renders mTORCl signaling completely insensitive to amino acid starvation (Bar-Peled 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 a GTPase-activating protein (GAP) for RagA / B. The GATOR2 multiprotein complex, with five known subunits (WDR24, WDR59, Mios, Sec13, and SehIL), is a positive component of the pathway and is upstream or parallel to GATOR1, but its molecular function remained unknown until recently (Bar-Peled et al., (2013) Science 340, 1100-1106).

[0006] Recently, additional information about the mTORCl pathway was elucidated by identifying the binding of GATOR2 to one or more Sestrins and demonstrating that the resulting Sestrin-GATOR2 complex regulates the subcellular localization and activity of mTORCl. Specifically, the presence of the Sestrin-GATOR2 complex inhibits the mTORCl pathway and reduces mTORCl activity by preventing translocation of mTORCl to the lysosomal membrane. The interaction of GATOR2 with Sestrins, and particularly Sestrinl and Sestrin2, is antagonized by amino acids, particularly leucine, and to a lesser extent, isoleucine, methionine, and valine. In the presence of leucine, GATOR2 does not interact with Sestrinl or Sestrin2 and mTORCl is able to migrate to the lysosomal membrane, where it is active. Sestrinl and Sestrin2 directly bind leucine and, to a lesser extent, isoleucine and methionine (Chantranupong et al., (2014) Cell Rep. 9(1): 1-8). Leucine binding by Sestrinl or Sestrin2 is necessary to disrupt their interaction with GATOR2 and subsequent activation of mTORCl. A mutant of Sestrin2 that cannot bind leucine is unable to signal the presence of leucine to mTORCl, and cells depleted of Sestrin2 and its homolog are insensitive to the absence of leucine for mTORCl (Wolfson et al., (2015) Science pii: ab2674 [Epub ahead of print]).

[0007] Sestrins are three related proteins (Sestrin 1, Sestrin 2, and Sestrin 3) of which little is known about their molecular function (Buckbinder et al., (1994) Proc Natl Acad Sci U S A 91, 10640-10644; Budanov et al., (2002) Cell 134, 451-460; Peeters et al., (2003) Human genetics 112, 573-580). Sestrin 2 inhibits mTORCl signaling and it was proposed that Sestrin 2 can activate AMPK upstream of TSC and interact with TSC (Budanov and Karin, (2008) Cell 134, 451-460), but later studies found that Sestrin 2 inhibits mTORCl in the absence of AMPK (Peng et al., (2014) Cell 159(1): 122-33), further highlighting the important role of the GATOR2 complex in regulating mTORCl in response to Sestrin 2.

[0008] Modulation of the Sestrin-GATOR2 complex represents a potential therapeutic target for indirectly and selectively modulating mTORCl activity. SUMMARY

[0009] It has now been discovered that the compounds of the present application and pharmaceutically acceptable compositions thereof are effective as Sestrin-GATOR2 modulators. The compounds have the general formula I:

[0010]

[0011] or pharmaceutically acceptable salts thereof, wherein each variable is as defined and described herein.

[0012] The compounds of the present application and pharmaceutically acceptable compositions thereof are useful for treating various diseases, disorders, or conditions associated with mTORCl. These diseases, disorders, or conditions include diabetes, epilepsy, neurodegeneration, immune responses, inhibition of skeletal muscle growth, and cell proliferative disorders (e.g., cancer), such as those described herein. DETAILED DESCRIPTION

[0013] 1. General Description of Certain Embodiments of the Invention:

[0014] The compounds of the present application and compositions thereof are useful as Sestrin-GATOR2 modulators. In certain embodiments, the present application provides compounds of Formula I:

[0015]

[0016] or a pharmaceutically acceptable salt thereof, wherein:

[0017] R 1 is H or C 1-6 alkyl;

[0018] R 2 is R, -(CH2) n -phenyl, -C(O)R, -SO2R, or -C(O)N(R)2;

[0019] n is 0, 1, or 2;

[0020] each R is independently hydrogen, -CN, or an optionally substituted group selected from saturated or unsaturated C 1-6 aliphatic, phenyl, 4- to 7-membered saturated or partially unsaturated carbocyclic ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

[0021] R 3 is 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 the two OR groups on the same boron are taken together with the intervening atom to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur in addition to the boron and two oxygens, or R 3 and R 4 are taken together to form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur;

[0022] L is a covalent bond or a straight or branched C 1-6 alkylene chain optionally substituted with 1 to 9 fluoro groups;

[0023] 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;

[0024] R 4 is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 3 and R 4 are taken together to form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur; and

[0025] R 5 is H or C 1-4 alkyl.

[0026] 2. Compounds and Definitions:

[0027] The compounds of the present application include those generally described herein and are further illustrated by the classes, sub-classes, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this application, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5thEd., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0028] The term "aliphatic" or "aliphatic group", as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted, completely saturated or containing one or more units of unsaturation, hydrocarbon chain, or a monocyclic, or bicyclic hydrocarbon moiety (also referred to herein as "carbocyclic", "cycloaliphatic" or "cycloalkyl") which is completely saturated or contains one or more units of unsaturation, but not aromatic, having a single point of attachment to the rest of the molecule. Unless otherwise specified, 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") refers to a monocyclic, or bicyclic hydrocarbon moiety which is completely saturated or contains one or more units of unsaturation, but not aromatic, having a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl groups.

[0029] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or an unsubstituted nitrogen of a heterocyclic ring, such as N (as in 3,4-dihydro-2H-pyranyl), NH (as in pyrrolidinyl), or NR+ (as in pyrrolidinyl substituted on the N).

[0030] The term "unsaturated" as used herein means a moiety having one or more units of unsaturation.

[0031] The term "divalent C 1-8 (or C 1-6 ) saturated or unsaturated, straight or branched hydrocarbon chain" refers to straight-chain or branched-chain, bivalent alkylenic, alkenylenic and alkynylenic chains as defined herein.

[0032] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene, i.e., -(CH2) n wherein n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkylene chain is a polymethylene in which one or more of the methylene hydrogens is replaced by a substituent. Suitable substituents include those described below with respect to substituted aliphatic groups.

[0033] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene containing at least one double bond in which one or more of the hydrogens is replaced by a substituent. Suitable substituents include those described below with respect to substituted aliphatic groups.

[0034] The term "halogen" means F, CI, Br, or I.

[0035] The term "aryl" used alone or as part of a larger moiety, such as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic or bicyclic ring system having from five to fourteen ring members in which at least one ring is aromatic and in which each ring in the system contains from 3 to 7 ring members. The term "aryl" can be used interchangeably with the term "aromatic ring." In certain embodiments of the application, "aryl" refers to an aromatic ring system, including but not limited to phenyl, biphenyl, naphthyl, anthryl, and the like, which can have one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, benzimidazolyl, or tetrahydronaphthyl, etc.

[0036] The terms "heteroaryl" and "heteroar-," used alone or as part of, for example, "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; sharing six, ten, or fourteen pi electrons in the ring array; and having, in addition to carbon atoms, between one and five heteroatoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroar-," as used herein also include groups in which the heteroaryl ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is to the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. The heteroaryl group can be mono- or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring", "heteroaryl group", or "heteroaromatic", any of which includes rings that are optionally substituted.

[0037] The terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring," as used herein, can be used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety, which is either saturated or partially unsaturated and which, additionally to carbon atoms, has one or more, preferably one to four, heteroatoms as set forth in the Summary as members of its ring skeleton. The term "nitrogen" when used as a ring atom of a heterocycle includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, a nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR (as in pyrrolidinyl substituted on the N). + ​

[0038] Heterocycles can be attached to their side groups at any heteroatom or carbon atom, resulting in stable structures, and any ring atom can be optionally substituted. Examples of the saturated or partially unsaturated heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H- indolyl, chromanyl, benzoxazinonyl, or tetrahydroquinolinyl. The heterocyclyl group can be mono- or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group, wherein the alkyl portion and the heterocyclyl portion are independently optionally substituted.

[0039] The term "partially unsaturated" as used herein means that the ring moiety includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings that have multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0040] As described herein, the compounds of the application can contain "optionally substituted" moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position. Combinations of substituents envisioned by this application are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions of production, detection, and in certain embodiments, recovery and purification, and formulation for use in one or more of the disclosed uses.

[0041] 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; can be R o Substituted -(CH2) 0-4 O(CH2) 0-1 Ph; can be R o The substituted -CH=CHPh can be replaced 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) 0-4 C(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 1-4straight or branched alkylene)C(O)O-N(R o )2, wherein each R o may be substituted as defined herein and is independently hydrogen, C 1-6 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or two independent occurrences of R o together with the intervening atoms form a 3-12 membered saturated, partially unsaturated, or fully unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which ring can be substituted as defined herein.

[0042] R o (or the ring formed by taking together two independent occurrences of R o ) is independently halogen, -(CH2) 0-2 R ● , -(haloR ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2; -O(haloR ● ), -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 or branched alkylene)C(O)OR ● , or -SSR ● , wherein each R ● is unsubstituted or, if substituted, is substituted only with one or more halogens and is independently selected from C 1-4aliphatic, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R o Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include =0 and =S.

[0043] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =0, =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 occurring R * is selected from hydrogen, C 1-6 aliphatic which can be substituted as defined herein below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents bound to a carbon in ortho position to an "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independently occurring R * is selected from hydrogen, C 1-6 aliphatic which can be substituted as defined herein below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0044] Suitable substituents on an aliphatic group of R * include halogen, -R ● , -(haloR ● ), -OH, -OR ● , -O(haloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0045] Suitable substituents on the optionally substituted nitrogen of an "optionally substituted" group include or wherein each is independently hydrogen, C 1-6 aliphatic, unsubstituted -OPh, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent independently selected from nitrogen, oxygen, or sulfur, are combined together to form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring.

[0046] Suitable substituents on the aliphatic of an "optionally substituted" group are independently halogen, -R ● , -(haloR ● ), -OH, -OR ● , -O(haloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, wherein each R ● is unsubstituted or, if preceded by "halo," substituted only with one or more halogens, and is independently C 1-4 aliphatic, -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.

[0047] The term "pharmaceutically acceptable salt" as used herein, refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid; with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid; or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bicarbonate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0048] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0049] Unless otherwise specified, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational) isomeric) forms of the structures; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers are included. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the application. Unless otherwise specified, all tautomeric forms of the compounds of the application are within the scope of the application. Additionally, unless otherwise specified, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or replacement of a carbon by a 13C- or 14C- enriched carbon are within the scope of this application. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present application. 13 C or 14 C-enriched carbon for a carbon of the present structures are within the scope of this application. Such compounds are useful as, for example, analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present application.

[0050] The term "leucine mimetic" as used herein is defined as a compound that reduces the amount of Sestrin2 bound to GATOR2 by at least about 40% relative to leucine at 25 μΜ. In certain embodiments, a "leucine mimetic" reduces the amount of Sestrin2 bound to GATOR2 by at least about 100%, at least about 150%, or at least about 200%.

[0051] The term "leucine antagonist" as used herein is defined as a compound that increases the amount of Sestrin2 bound to GATOR2 by at least about 40% relative to leucine at 25 μΜ (expressed as -40% of leucine activity). In certain embodiments, a "leucine antagonist" increases the amount of Sestrin2 bound to GATOR2 by at least about 100%, at least about 150%, or at least about 200%.

[0052] The terms "measurable affinity" and "measurably inhibits" as used herein mean a measurable change in Sestrin2 binding to GATOR2 between a sample comprising a compound of the application or a composition thereof and Sestrin2, GATOR2, and leucine, and an equivalent sample comprising Sestrin2, GATOR2, and leucine, but in the absence of the compound or composition thereof.

[0053] 3. Description of Exemplary Embodiments:

[0054] In certain embodiments, the present application provides a compound of Formula I:

[0055]

[0056] or a pharmaceutically acceptable salt thereof, wherein:

[0057] R 1 is H or C 1-6 alkyl;

[0058] R 2 is R, -(CH2) n -phenyl, -C(O)R, -SO2R, or -C(O)N(R)2;

[0059] n is 0, 1, or 2;

[0060] each R is independently hydrogen, -CN, or an optionally substituted group selected from saturated or unsaturated C 1-6 aliphatic, phenyl, 4- to 7-membered saturated or partially unsaturated carbocyclic ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

[0061] R 3 is 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 the two OR groups on the same boron are taken together with the intervening atom to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur in addition to the boron and two oxygens, or R 3 and R 4 are taken together to form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur;

[0062] L is a covalent bond or a straight or branched C 1-6 alkylene chain optionally substituted with 1 to 9 fluoro groups;

[0063] 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;

[0064] R 4 is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 3 and R 4 are taken together to form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur; and

[0065] R 5 is H or C 1-4 alkyl.

[0066] In some embodiments, the provided Formula I compounds are not those described in Table 2 below.

[0067] As generally defined above, R 1 Is it H or C? 1-6 Alkyl group. In some embodiments, R 1 It is H. In other embodiments, R 1 It is C 1-6 Alkyl group. In some embodiments, R 1 It is methyl. 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.

[0068] 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 It is methyl. 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.

[0069] 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.

[0070] 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 together form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0071] In some embodiments, R 3 is -C(O)OH. In some embodiments, R 3 is -C(O)N(R)2. In some embodiments, R 3 is -SO3H. In some embodiments, R 3 is -SO2N(R)2. In some embodiments, R 3 is -B(OR)2, wherein the two -OR groups on the same boron are taken together with the middle atom to form a 5- to 8-membered monocyclic saturated, partially unsaturated, or heterocyclic ring having, in addition to the boron and two oxygens, 0 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 and R 4 together form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0072] In some embodiments, R 3 is ring A. As generally defined above, ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, ring A is an optionally substituted phenyl. In some embodiments, ring A is an optionally substituted 5-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, ring A is an optionally substituted 5-membered heteroaryl ring selected from imidazolyl, isoxazolyl, 1H-pyrrolyl (e.g., maleimido), pyrazolyl, oxazolyl, tetrazolyl, thiazolyl, and triazolyl. In some embodiments, ring A is an optionally substituted 6-membered heteroaryl ring having 1 to 2 nitrogen atoms. In some embodiments, ring A is an optionally substituted 6-membered ring selected from pyridyl and pyrimidinyl. In some embodiments, ring A is selected from those depicted in Table 1 below.

[0073] In some embodiments, R3is (pinacolato)boron. In some embodiments, R 3 is selected from those depicted in Table 1 below. In some embodiments, R 3 is selected from those depicted in Table 2 below.

[0074] As generally defined above, L is a covalent bond or a straight- or branched-chain C 1-6 alkylene chain optionally substituted with 1 to 4 fluoro groups. In some embodiments, L is a covalent bond. In some embodiments, L is a straight- or branched-chain C 1-6alkylene chain. In some embodiments, L is methylene. In some embodiments, L is n-butenyl. In some embodiments, L is ethenyl. 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.

[0075] In some embodiments, L is a branched C 1-6 alkylene chain. In certain embodiments, L is -C(CH3)2-. In other embodiments, L is -C(CH3)(CF3)-.

[0076] R is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 4 and R 3 are taken together to form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R 4 is R. In some embodiments, R 4 is -CF3. In some embodiments, R 4 is -OR. In some embodiments, R 4 is -N(R)2. In some embodiments, R 4 is -Si(R)3. In some embodiments, R 4 is -SR. In some embodiments, R 4 is isopropyl. In some embodiments, R 4 is t-butyl. In some embodiments, R 4 is cyclopropyl. In some embodiments, R 4 is cyclobutyl. In some embodiments, R 4 is sec-butyl. In some embodiments, R 4 is methoxy. In some embodiments, R 4 is methylthio. In some embodiments, R 4 and R 3 are taken together to form an optionally substituted 5- to 6-membered ring having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R 4 is selected from those depicted in Table 1 below. In some embodiments, R 4 is selected from those depicted in Table 2 below.

[0077] R is R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR, or R 4 is H or C 5 alkyl. In some embodiments, R 1-4 is H. In some embodiments, R 5 is C 5 alkyl. In some embodiments, R 1-4alkyl. In some embodiments, R 5 is methyl. In some embodiments, R 5 is selected from those depicted in Table 1 below. In some embodiments, R 5 is selected from those depicted in Table 2 below.

[0078] In certain embodiments, the present application provides a compound of Formula II:

[0079]

[0080] or a pharmaceutically acceptable salt thereof, wherein each variable, both alone and in combination, is as defined above and described in embodiments provided herein.

[0081] In certain embodiments, the present application provides a compound of Formula III:

[0082]

[0083] or a pharmaceutically acceptable salt thereof, wherein:

[0084] Q is -C(R')2- or -NH-;

[0085] R x and R y are each hydrogen, or R x and R y are taken together to form =0;

[0086] is a double bond or a single bond;

[0087] each R is independently hydrogen, -CN, or an optionally substituted group selected from C 1-6 aliphatic, phenyl, a 4- to 7-membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or a 4- to 8-membered saturated or partially saturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

[0088] each R' is independently hydrogen, halogen, -CN, or an optionally substituted group selected from C 1-6 aliphatic, phenyl, a 4- to 7-membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or a 4- to 8-membered saturated or partially saturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

[0089] L is a covalent bond or a straight or branched C 1-6 alkylene chain optionally substituted with 1 to 9 fluoro groups;

[0090] R 4'R, -CF3, -OR, -N(R)2, -Si(R)3, or -SR; and

[0091] R 5' is H, -OR, or C 1-4 alkyl.

[0092] In some embodiments, Q is -NH-. In some embodiments, Q is -CH2-. In some embodiments, Q is -CHF-.

[0093] In some embodiments, L is -CH2-.

[0094] In some embodiments, R x and R y are each hydrogen. In some embodiments, R x and R y are taken together to form =0.

[0095] In some embodiments, R 5' is H. In some embodiments, R 5' is -OH.

[0096] In some embodiments, is a single bond. In some embodiments, is a double bond.

[0097] In certain embodiments, the present application provides a compound of Formula IV-a, IV-b, or IV-c:

[0098]

[0099]

[0100] or a pharmaceutically acceptable salt thereof, wherein:

[0101] R 1 is H or C 1-6 alkyl;

[0102] R 2 is R, -(CH2) n -phenyl, -C(O)R, -SO2R, or -C(O)N(R)2;

[0103] each R 4" is independently R, halogen, or -CF3;

[0104] each R is independently hydrogen, -CN, or an optionally substituted group selected from saturated or unsaturated C 1-6aliphatic, phenyl, 4- to 7-membered saturated or partially unsaturated monocyclic carbocyclic ring, 5- to 6-membered monocyclic heteroaryl ring having 1- to 4-heteroatoms, or 4- to 8-membered saturated or partially saturated heterocyclic ring having 1- to 2-heteroatoms independently selected from nitrogen, oxygen, or sulfur; and

[0105] L 1 is a covalent bond or a straight- or branched-chain C 1-6 alkylene chain optionally substituted with 1- to 9-fluoro groups.

[0106] In some embodiments, R 1 is H. In some embodiments, R 1 is C 1-6 alkyl.

[0107] In some embodiments, R 1 is selected from those depicted in Table 1 below.

[0108] In some embodiments, R 2 is R. In some embodiments, R 2 is -(CH2) n -phenyl. In some embodiments, R 2 is -C(O)R.

[0109] In some embodiments, R 2 is selected from those depicted in Table 1 below.

[0110] In some embodiments, each R 4" is independently R, halo, or -CF3. In some embodiments, R 4" is R. In some embodiments, R 4" is halo. In some embodiments, R 4" is -CF3. In some embodiments, R 4" is selected from those depicted in Table 1 below.

[0111] In some embodiments, L 1 is a covalent bond or a straight- or branched-chain C 1-6 alkylene chain optionally substituted with 1- to 9-fluoro groups. In some embodiments, L 1 is a covalent bond. In some embodiments, L 1 is a straight- or branched-chain C 1-6 alkylene chain optionally substituted with 1- to 9-fluoro groups. In some embodiments, L 1 is selected from those depicted in Table 1 below.

[0112] Exemplary compounds of the present application are set forth in Table 1 below.

[0113] Table 1. Exemplary Compounds

[0114] Table 1. Exemplary Compounds

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Exemplary compounds of the invention are set forth in Table 2, below.

[0139] Table 2. Exemplary Compounds

[0140]

[0141]

[0142] In some embodiments, the present application provides a compound set forth in Table 1 above, or a pharmaceutically acceptable salt thereof. In some embodiments, the present application provides a compound set forth in Table 2 above, or a pharmaceutically acceptable salt thereof.

[0143] 5. Uses, Formulations, and Administration

[0144] Pharmaceutically Acceptable Compositions

[0145] According to another embodiment, the present application provides a composition comprising a compound of the present application or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of compound in the compositions of the present application is an amount that is effective to measurably inhibit or activate a Sestrin-GATOR2 interaction in a biological sample or in a patient. In certain embodiments, the amount of compound in the compositions of the present application is an amount that is effective to measurably inhibit or activate a Sestrin-GATOR2 interaction in a biological sample or in a patient. In certain embodiments, the compositions of the present application are formulated for administration to a patient in need of the composition. In some embodiments, the compositions of the present application are formulated for oral administration to a patient.

[0146] The term "patient" as used herein means an animal, preferably a mammal, and most preferably a human.

[0147] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" means a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of this application include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures 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, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0148] The compositions of the present application can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenterally" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this application can be aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0149] For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. These solutions or suspensions can also contain long chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the art for making pharmaceutical solutions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other similar emulsifying agents or bioavailability enhancers which are commonly used in manufacturing pharmaceuticals, can also be used for the purposes of formulation.

[0150] The pharmaceutical compositions of this application can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents can also be added.

[0151] Alternatively, the pharmaceutical compositions of this application can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0152] The pharmaceutically acceptable compositions of the present application can also be administered topically, especially when the target of treatment includes areas or organs readily accessible by such administration, including diseases of the eye, the skin, or the lower intestinal tract. Suitable formulations for each of these areas or organs are readily prepared and do not differ from those already established for use with the compounds of the present application.

[0153] Topical administration of the pharmaceutical compositions of this application can also be accomplished by using local transdermal patches, which can be worn on the skin, or by using local iontophoresis devices.

[0154] For topical administration, the pharmaceutically acceptable compositions provided can be in suitable ointment formulations suspending or dissolving the active component in one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, provided pharmaceutically acceptable compositions can be in the form of a suitable lotion or cream formulated in one or more of the following: mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0155] For ophthalmic use, the pharmaceutically acceptable compositions provided can be formulated in the form of micronized suspensions in an isotonic, pH adjusted, sterile saline or, preferably, as solutions in isotonic, pH adjusted, sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions can be formulated in an ointment such as petrolatum.

[0156] The pharmaceutically acceptable compositions of this application can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0157] Most preferably, the pharmaceutically acceptable compositions of this application are formulated in a form suitable for oral use. The pharmaceutical compositions can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present application are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present application are administered with food.

[0158] The amount of a compound of the application that can be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, the composition to be provided should be formulated to allow the proper dosage for a patient being treated to be administered.

[0159] It will also be appreciated that the specific dose regimen and treatment period will depend on a number of factors, including the specific compound employed, the age, body weight, general health condition, sex, diet, time of administration, rate of excretion, drug combination and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the application in the composition will also depend on the particular compound in the composition.

[0160] Uses of Compounds and Pharmaceutically Acceptable Compositions

[0161] The compounds and compositions described herein are generally useful for inhibiting or activating Sestrin-GATOR2 interactions. In some embodiments, the provided compounds or compositions thereof are activators of Sestrin-GATOR2 interactions.

[0162] The activity of a compound used in the present application as an inhibitor or activator of Sestrin-GATOR2 interactions can be analyzed in vitro, in vivo, or in a cell line. In vitro analysis includes assays that determine inhibition or activation of Sestrin-GATOR2 interactions. Alternative in vitro assays quantify the ability of an inhibitor to decrease binding of Sestrin to GATOR2 or the ability of an activator to increase binding of Sestrin to GATOR2. Detailed assay conditions for compounds used in the present application as inhibitors or activators of Sestrin-GATOR2 interactions are set forth in the Examples below.

[0163] The terms "treatment," "treat," and "treating," as used herein, refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0164] The compounds provided are inhibitors or activators of Sestrin-GATOR2 interactions and are therefore useful in the treatment of one or more conditions associated with the activity of mTORCl. Accordingly, in certain embodiments, the present application provides a method for treating an mTORCl-mediated condition, the method comprising the step of administering to a patient in need thereof a compound of the present application or a pharmaceutically acceptable composition thereof.

[0165] As used herein, the term "mTORCl-mediated" condition, disease and / or disorder as used herein means any disease or other deleterious condition in which mTORCl is known to play a role. Accordingly, another embodiment of the present application relates to the treatment of one or more diseases in which mTORCl is known to play a role or to reduce the severity thereof.

[0166] The methods described herein include methods for treating cancer in a subject. As used in this context, "treatment" means amelioration or improvement of at least one symptom or clinical parameter of cancer. For example, treatment can result in a decrease in tumor size or growth rate. In all subjects, treatment need not cure cancer or result in remission 100% of the time.

[0167] As described herein, administration of an agent, such as an inhibitory nucleic acid or small molecule, that activates Sestrin-GATOR2 interactions and thereby reduces mTORCl activity can reduce cancer cell proliferation in a subject and thus treat cancer. Accordingly, 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 mTORCl pathway.

[0168] The term "cancer" as used herein refers to a cell having the ability to grow autonomously, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. The term "tumor" as used herein refers to a plurality of cancer cells, e.g., a mass of cancer cells.

[0169] Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic, gastrointestinal, and genitourinary tracts, as well as adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, small bowel cancer, and esophageal cancer.

[0170] In some embodiments, the methods described herein are used to treat or diagnose a carcinoma in a subject. The term "carcinoma" is known in the art and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is a renal carcinoma or a melanoma. Exemplary carcinomas include those formed from uterine cervical, lung, prostate, breast, head and neck, colon, and ovarian tissues. The term also includes carcinosarcomas, e.g., malignant tumors composed of both carcinoma and sarcoma tissue. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the neoplastic cells form recognizable glandular structures.

[0171] The term "sarcoma" is known in the art and refers to a mesenchymal-derived malignant tumor.

[0172] In some embodiments, the cancer treated by the methods described herein is a cancer having increased levels of mTORCl or increased expression or activity of mTORCl relative to normal tissue or other cancers of the same tissue; those cancers can be identified using methods known in the art and described herein. In some embodiments, the methods comprise obtaining a sample comprising cancer cells, assaying the sample for mTORCl activity, and administering a treatment as described herein (e.g., a modulator of Sestrin-GATOR2 interaction). In some embodiments, the cancer is a cancer shown herein to have increased levels of mTORCl activity.

[0173] In some embodiments, the present application provides a method for treating one or more disorders, diseases, and / or conditions, wherein the disorder, disease, or condition includes, but is not limited to, a cell proliferative disorder.

[0174] Cell Proliferative Disorders

[0175] The present application features methods and compositions for diagnosing and prognosing cell proliferative disorders, such as cancer, and treating these disorders by modulating Sestrin-GATOR2 interactions, thereby indirectly selectively modulating mTORCl activity. Cell proliferative disorders described herein include, for example, cancer, obesity, and proliferation-dependent diseases. The disorders can be diagnosed using methods known in the art.

[0176] Cancer

[0177] Cancers include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, 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, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some embodiments, the cancer is melanoma or breast cancer.

[0178] Other proliferative diseases

[0179] Other proliferative diseases include, for example, obesity, benign prostatic hyperplasia, psoriasis, abnormal keratinization, lymphoproliferative disorders (e.g., disorders in which cells of the lymphoid system proliferate abnormally), chronic rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy. Proliferative diseases described in U.S. Patents Nos. 5,639,600 and 7,087,648, which are incorporated herein by reference, include those described therein.

[0180] Other disorders

[0181] In some embodiments, methods of activating mTORCl are used to treat ribosomopathies (e.g., Diamond-Blackfan anemia, 5q- syndrome, Shwachman-Diamond syndrome, X-linked dyskeratosis, chondro-ectodermal dysplasia, and Treacher Collins syndrome). (See Payne et al. (2012) Blood. Sep 13; 120(11):2214-24; Efeyan et al. (2012) Trends Mol Med. Sep; 18(9):524-533). Accordingly, in some embodiments, the application provides a method of treating a ribosomopathy in a patient in need thereof, comprising the step of administering to the patient a provided compound, or a pharmaceutically acceptable composition thereof. In certain embodiments, the application provides a method of treating a ribosomopathy selected from Diamond-Blackfan anemia, 5q- syndrome, Shwachman-Diamond syndrome, X-linked dyskeratosis, chondro-ectodermal dysplasia, or Treacher Collins syndrome in a patient in need thereof, comprising the step of administering to the patient a provided compound, or a pharmaceutically acceptable composition thereof.

[0182] In some embodiments, methods of activating mTORCl activity are used to treat cohesinopathies (e.g., Roberts syndrome and Cornelia de Lange syndrome). (See Xu et al. (2016) BMC Genomics 17:25). Accordingly, in some embodiments, the application provides a method of treating a cohesinopathy (e.g., Roberts syndrome and Cornelia de Lange syndrome) in a patient in need thereof, comprising the step of administering to the patient a provided compound, or a pharmaceutically acceptable composition thereof.

[0183] In some embodiments, the mTORCl activation method is used to reverse or prevent muscle atrophy due to inactivity from lifestyle, inactivity from orthopedic surgery, immobilization, or the age of the subject or a disease or condition the subject has or has suffered from. (See Cuthbertson et al., (2005) FASEB J. Mar; 19(3):422-4. Epub 2004 Dec 13; Rennie, (2009) Appl. Physiol. Nutr. Metab. 34:377-381; Ham et al., (2014) Clin Nutr. Dec; 33(6):937-45). Accordingly, in some embodiments, the present application provides a method of reversing or preventing muscle atrophy in a patient in need thereof due to inactivity from lifestyle, inactivity from orthopedic surgery, immobilization, or the age of the subject or a disease or condition the subject has or has suffered from, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0184] In some embodiments, the mTORCl activation method is used to reverse or prevent muscle atrophy due to a bone fracture, severe burns, spinal cord injury, amputation, degenerative disease, a condition requiring the subject to rest in bed, stay in an intensive care unit, or long-term hospitalization to recover. (See Gordon et al., (2013) Int J Biochem Cell Biol. Oct; 45(10):2147-57; Léger et al., (2009) Muscle Nerve. Jul; 40(1):69-78). Accordingly, in some embodiments, the present application provides a method of reversing or preventing muscle atrophy in a patient in need thereof due to a bone fracture, severe burns, spinal cord injury, amputation, degenerative disease, a condition requiring the subject to rest in bed, stay in an intensive care unit, or long-term hospitalization to recover, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0185] In some embodiments, the mTORCl activation approach is used to treat a disease, condition, or disorder that causes 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) Hum Mol Genet. Dec 1 ;23(23):6318-6331 ; Gurpur et al. (2009) Am J Pathol. Mar;174(3):999-1008; Chauhan et al. (2013) Neurosci Res. Sep-Oct;77(l-2): 102-9); Ching et al. (2013) Hum Mol Genet. Mar 15;22(6): 1167-79). Accordingly, in some embodiments, the present application provides a method of treating a disease, condition, or disorder that causes skeletal muscle atrophy, such as sarcopenia, muscle denervation, muscular dystrophy, inflammatory myopathy, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), or myasthenia gravis, in a patient in need thereof, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0186] In some embodiments, the mTORCl activation approach is used to prevent muscle loss in a subject preparing for space travel, enable a subject to withstand muscle loss while participating in space travel, or enhance recovery from muscle loss in a subject who has recently returned from space travel. (See Stein et al. (1999) Am J Physiol. ;276:E1014-21). Accordingly, in some embodiments, the present application provides a method of preventing muscle loss in a subject preparing for space travel, enabling a subject to withstand muscle loss while participating in space travel, or enhancing recovery from muscle loss in a subject who has recently returned from space travel, in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0187] In some embodiments, the mTORC1 activation method is used to enable subjects preparing for armed conflict or military training, subjects participating in armed conflict or military training, or subjects who have recently returned from armed conflict or military training to withstand excessive muscle stress and / or fatigue or to enhance recovery. (See Pasiakos et al. (2011) Am J Clin Nutr. Sep; 94(3): 809-18). Accordingly, in some embodiments, the present application provides a method of enabling a subject preparing for armed conflict or military training, a subject participating in armed conflict or military training, or a subject who has recently returned from armed conflict or military training to withstand excessive muscle stress and / or fatigue or to enhance recovery in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0188] In some embodiments, the mTORC1 activation method is used to prevent autophagy in a patient. In some embodiments, the patient has or has a cancer that is resistant to therapy in a manner that depends on the induction of autophagy. (See Kim and Guan (2015) J Clin Invest. Jan; 125(1): 25-32). Accordingly, in some embodiments, the present application provides a method of preventing autophagy in a patient in a patient in need thereof who has or has a cancer that is resistant to therapy in a manner that depends on the induction of autophagy, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0189] In some embodiments, the mTORC activation method is used to treat or prevent depression. (See Ignácio et al. (2015) Br J Clin Pharmacol. Nov 27). Accordingly, in some embodiments, the present application provides a method of treating or preventing depression in a patient in need thereof, 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 elicit fast-acting antidepressant activity. Accordingly, in some embodiments, the present application provides a method of eliciting fast-acting antidepressant activity in a patient in need thereof, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0191] In some embodiments, the mTORCl activation methods are used to treat or prevent jet lag caused by accelerated circadian entrainment in response to day-night cycle alternation. (See Cao et al., (2013) Neuron. Aug 21 ;79(4):712-24 10.1016). Accordingly, in some embodiments, the present application provides a method of treating or preventing jet lag caused by accelerated circadian entrainment in response to day-night cycle alternation in a patient in need thereof, the method comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0192] In some embodiments, the mTORCl activation methods are used to prevent or reverse cardiac muscle atrophy in a subject. In some embodiments, the subject has or has had a disease or condition selected from the group consisting of heart attack, congestive heart failure, heart transplant, heart valve repair, atherosclerosis, other major blood vessel disease, and heart bypass surgery. (See Song et al., (2010) Am J Physiol Cell Physiol. Dec;299(6):C1256-C1266). Accordingly, in some embodiments, the present application provides a method of preventing or reversing cardiac muscle atrophy in a subject in need thereof, wherein the subject has or has had a disease or condition selected from the group consisting of heart attack, congestive heart failure, heart transplant, heart valve repair, atherosclerosis, other major blood vessel disease, and heart bypass surgery, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0193] In some embodiments, the mTORCl activation methods are used to increase strength and / or increase muscle mass after exercise. In some embodiments, the methods are performed in conjunction with physical therapy, either as part of total parenteral nutrition or to facilitate functional electrical stimulation. (See Nakamura et al., (2012) Geriatr Gerontol Int. Jan;12(l):131-9). Accordingly, in some embodiments, the present application provides a method of increasing strength and / or increasing muscle mass after exercise. In some embodiments, the methods are performed in conjunction with physical therapy, either as part of total parenteral nutrition or to facilitate functional electrical stimulation, in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0194] In some embodiments, mTORCl activation methods are used to reduce food intake. (See Pedroso et al. (2015) Nutrients. May 22;7(5):3914-37). Accordingly, in some embodiments, the present application provides a method of reducing food intake in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0195] In some embodiments, mTORCl activation methods are used to treat obesity. Accordingly, in some embodiments, the present application provides a method of treating obesity in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0196] In some embodiments, mTORCl activation methods are used to improve productivity in the manufacture of therapeutic recombinant proteins from bioreactors. (See McVey et al. (2016) Biotechnol Bioeng. Feb 16. doi: 10.1002 / bit.25951). Accordingly, in some embodiments, the present application provides a method of improving productivity in the manufacture of therapeutic recombinant proteins from bioreactors, the method comprising the step of adding to the manufacture a provided compound or a pharmaceutically acceptable composition thereof.

[0197] In some embodiments, mTORCl activation methods are used in immune cells to promote and / or sustain their anti-tumor activity. This includes increasing mTORCl in immune cells in vitro prior to adoptive transfer, and in vivo when co-administered with other targeted immunotherapy strategies. In some embodiments, immune cells include naive T cells, CD4+ or CD8+ T cells, Thl, Th2, T Regand macrophages. (See Yang et al. (2011) Nat Immunol. ; 12:888-897; O'Brien et al. (2011) Eur J Immunol. ; 41 :3361-3370; Delgoffe et al. (2009) Immunity. June 19; 30(6):832-44; Chi, (2012) Nat Rev Immunol. April 20; 12(5):325-338; Pollizzi et al. (2015) J Clin Invest. ; 125(5):2090-2108; Ali et al. (2015) Front Immunol. ; 6:355; Katholnig et al. (2013) Biochem Soc Trans. August; 41(4):927-33; Wang et al. (2013) Proc Natl Acad Sci U S A. December 10; 110(50):E4894-903; Yang and Chi, (2013) J Clin Invest. December; 123(12):5165-78). Accordingly, in some embodiments, the present application provides a method of activating mTORCi in immune cells to promote and / or maintain their anti-tumor activity. In some embodiments, the present application provides a method of increasing mTORCi in immune cells in vitro prior to adoptive transfer. In some embodiments, the present application provides a method of increasing mTORCi in immune cells in vivo when co-administered with other targeted immunotherapy strategies. In certain embodiments, the immune cells include naive T cells, CD4+or CD8+T cells, Th1, Th2, T Reg and macrophages, comprising the step of adding the provided compound or a pharmaceutically acceptable composition thereof to the immune cells.

[0198] In some embodiments, the mTORCi activation method is used in the retina to treat retinitis pigmentosa and other forms of ocular neurodegeneration. (See Punzo et al. (2009) Nat Neurosci. January; 12(1):44-52). Accordingly, in some embodiments, the present application provides a method of treating retinitis pigmentosa and other forms of ocular neurodegeneration in a subject in need thereof, comprising the step of administering to the subject the provided compound or a pharmaceutically acceptable composition thereof.

[0199] In some embodiments, mTORCl activation methods are used to increase central or peripheral axon regeneration. (See Namiko et al. (2010) J Biol Chem. 285:28034-28043). Accordingly, in some embodiments, the present application provides a method of increasing central or peripheral axon regeneration in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0200] In some embodiments, mTORCl activation methods are used to promote remyelination and neuronal activity after injury or in diseases characterized by demyelination, such as multiple sclerosis and Parkinson's disease. (See Tyler et al. (2009) J Neurosci. May 13; 29(19):6367-78; Norrmén et al. (2014) Cell Rep. Oct 23; 9(2):646-60; Love (2006). J Clin Pathol. Nov; 59(11): 1151-1159). Accordingly, in some embodiments, the present application provides a method of promoting remyelination and neuronal activity in a subject in need thereof after 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 application provides a method of treating multiple sclerosis in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present application provides a method of treating Parkinson's disease in a subject in need thereof, the method comprising the step of administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0201] In some embodiments, the mTORCl activation approach is used to treat multiple sclerosis. Accordingly, in some embodiments, the present application provides a method of treating multiple sclerosis, or a variant thereof, in a subject in need thereof, the method comprising the step of administering to the subject a provided compound, or a pharmaceutically acceptable composition thereof. In some embodiments, the present application provides a method of treating Balo's concentric sclerosis, Schilder's disease, acute (Marburg type) multiple sclerosis, inflammatory demyelinative polyradiculoneuropathy, or tumefactive multiple sclerosis in a subject in need thereof, the method comprising the step of administering to the subject a provided compound, or a pharmaceutically acceptable composition thereof.

[0202] In some embodiments, the mTORCl activation approach 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. Jun;63(6):660-73). Accordingly, in some embodiments, the present application provides a method of treating Devic's disease, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, progressive multifocal leukoencephalopathy, and Niemann-Pick disease in a subject in need thereof, the method comprising the step of administering to the subject a provided compound, or a pharmaceutically acceptable composition thereof.

[0203] In some embodiments, the mTORCl activation approach is used to treat or prevent various forms of autism. (See Novarino et al., (2012) Science Oct 19, 338:6105, pp. 394-397). Accordingly, in some embodiments, the present application provides a method of treating or preventing a form of autism in a subject in need thereof, the method comprising the step of administering to the subject a provided compound, or a pharmaceutically acceptable composition thereof.

[0204] In some embodiments, the mTORCl activation approach is used to treat neurodegenerative diseases. Accordingly, in some embodiments, the present application provides a method of treating a neurodegenerative disease in a subject in need thereof, the method comprising the step of administering to the subject a provided compound, or a pharmaceutically acceptable composition thereof.

[0205] 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.

[0206] 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) NeuralRegen 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.”

[0223] 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 sold under the brand name Afema. TM For sale. Anastrozole is sold under the brand name Arimidex. TM For sale. Letrozole is marketed under the brand name Femara. TM or Femar TM For sale. Ammonialumine is sold 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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).

[0231] 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.

[0232] 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.

[0233] 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 AG 213; 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).

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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, BAY 12-9566, TAA211, MMI270B, or AAJ996.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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).

[0259] 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.

[0260] 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 (RPI 4610) and bevacizumab (Avastin) TM ).

[0261] 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.

[0262] 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.

[0263] Implants containing corticosteroids refer to compounds such as fluocinolone and dexamethasone.

[0264] 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.

[0265] 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).

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] illustration

[0275] 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.

[0276] List of abbreviations used in the experimental section.

[0277] 4A MS: Molecular sieve

[0278] AcOH: Acetic acid

[0279] ACN: Acetonitrile

[0280] Anhyd: Waterless

[0281] Aq: Aqueous solution

[0282] Bn: benzyl

[0283] Boc: tert-butoxycarbonyl

[0284] CbzCl: Benzoic acid chloroformate

[0285] Cbz-OSU: N-(Benzyloxycarbonyloxy)succinimide

[0286] Cu(OAc)₂: Copper(II) acetate

[0287] d: sky

[0288] DAST: Diethylaminosulfonium trifluoride

[0289] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0290] DCE: 1,2-Dichloroethane

[0291] DCM: Dichloromethane

[0292] DEA: Diethylamine

[0293] DIBAL-H: Diisobutylaluminum hydride

[0294] DIPEA: N,N-Diisopropylethylamine

[0295] DMA: N,N-dimethylacetamide

[0296] DMAP: 4-Dimethylaminopyridine

[0297] DMF: N,N-Dimethylformamide

[0298] DMSO-dimethyl sulfoxide

[0299] DPPA: Diphenyl azidophosphate

[0300] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0301] ee: enantiomer excess

[0302] ESI: Electrospray Ionization

[0303] Et3N: Triethylamine

[0304] Et2O: Diethyl ether

[0305] EtOAc: Ethyl acetate

[0306] EtOH: Ethanol

[0307] Fmoc: fluorenemethyloxycarbonyl

[0308] Fmoc-OSu: N-(9-fluorenylmethoxycarbonyloxy)succinimide

[0309] h: hours

[0310] HATU: 3-O-1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 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; PE: Petroleum ether

[0332] PhNH2: Aniline

[0333] PPh3: Triphenylphosphine

[0334] Rel: relative

[0335] rt: room temperature

[0336] sat: saturation

[0337] SFC: Supercritical Fluid Chromatography

[0338] SOCl2: thionyl chloride

[0339] TBAB: Tetra-n-butylammonium bromide; tBuOK: Potassium tert-butoxide

[0340] TEA: Triethylamine

[0341] Tf: Trifluoromethanesulfonate

[0342] TfAA: Trifluoromethanesulfonic anhydride

[0343] TFA: Trifluoroacetic acid

[0344] TIPS: Triisopropylsilane

[0345] THF: Tetrahydrofuran

[0346] TMSCN: Trimethylcyanosylsilane

[0347] pTSA: p-Toluenesulfonic acid

[0348] TsOH: p-Toluenesulfonic acid

[0349] The preparation of representative, non-limiting examples of the provided compounds is described below.

[0350] Example 1: (S)-2-(dimethylamino)-4-methylpentanoic acid [I-1].

[0351]

[0352] Reaction process:

[0353]

[0354] Procedures and representations:

[0355] Step 1: (S)-2-(dimethylamino)-4-methylpentanoic acid:

[0356] 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).

[0357] Examples 2 and 3: (S)-2-amino-7,7,7-trifluoroheptanoate [I-2] and (R)-2-amino-7,7,7-trifluoroheptanoate [I-3].

[0358]

[0359] Reaction process:

[0360]

[0361] Procedures and representations:

[0362] Step 1: 1,1,1-Trifluoro-5-iodopentane:

[0363] 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.

[0364] Step 2: (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester and (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester:

[0365] 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%).

[0366] (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).

[0367] (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).

[0368] Step 3: (S)-2-amino-7,7,7-trifluoroheptanoate salt [I-2]:

[0369] 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).

[0370] Step 4: (R)-2-amino-7,7,7-trifluoroheptanoate salt [I-3]:

[0371] 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).

[0372] 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].

[0373]

[0374] Reaction process:

[0375]

[0376] Procedures and representations:

[0377] Step 1: (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid

[0378] 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]+.

[0379] (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).

[0380] (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).

[0381] Step 2: (S)-2-amino-4,4,4-trifluorobutyric acid [I-4].

[0382] 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).

[0383] Step 3: (R)-2-amino-4,4,4-trifluorobutyric acid [I-5].

[0384] 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).

[0385] 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].

[0386]

[0387] Reaction process:

[0388]

[0389] Procedures and representations:

[0390] Step 1: 4,4,4-Trifluorobutyraldehyde:

[0391] 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.

[0392] Step 2: 2-(phenylmethylamino)-5,5,5-trifluoropentadienonitrile:

[0393] 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]+.

[0394] Step 3: 2-(phenylmethylamino)-5,5,5-trifluorovaleric acid:

[0395] 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] + .

[0396] Step 4: 2-Amino-5,5,5-trifluorovaleric acid:

[0397] 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]+.

[0398] Step 5: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid:

[0399] 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.

[0400] (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).

[0401] (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).

[0402] Step 6: (S)-2-amino-5,5,5-trifluorovaleric acid [I-6]:

[0403] 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).

[0404] Step 7: (R)-2-amino-5,5,5-trifluorovaleric acid [I-7]:

[0405] 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).

[0406] 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].

[0407]

[0408] Reaction process:

[0409]

[0410] Procedures and representations:

[0411] Step 1: (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid and (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid

[0412] 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]+.

[0413] (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).

[0414] (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).

[0415] Step 2: (S)-2-amino-6,6,6-trifluorohexanoic acid [I-8].

[0416] 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).

[0417] Step 3: (R)-2-amino-6,6,6-trifluorohexanoic acid [I-9].

[0418] 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).

[0419] Example 11: (S)-2-(phenylmethylamino)-4-methylpentanoic acid [I-11].

[0420]

[0421] Reaction process:

[0422]

[0423] Procedures and representations:

[0424] Step 1: (S)-2-(phenylmethylamino)-4-methylpentanoic acid benzoate:

[0425] 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).

[0426] Step 2: (S)-2-(phenylmethylamino)-4-methylpentanoic acid [I-11]:

[0427] 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).

[0428] Example 12: (S)-4-methyl-2-(2-phenylacetamido)valerate [I-12]:

[0429]

[0430] Reaction process:

[0431]

[0432] Procedures and representations:

[0433] Step 1: (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid methyl ester:

[0434] 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]+.

[0435] Step 2: (S)-4-methyl-2-(2-phenylacetamido)valerate [I-12]:

[0436] 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).

[0437] Example 13: (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]:

[0438]

[0439] Reaction process:

[0440]

[0441] Procedures and representations:

[0442] Step 1: (S)-2-(isopropylamino)-4-methylpentanoic acid benzoate:

[0443] 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).

[0444] Step 2: (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]:

[0445] 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).

[0446] Example 14: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0447]

[0448] Reaction process:

[0449]

[0450] Procedures and representations:

[0451] Step 1: (S)-2-(isobutylamino)-4-methylpentanoic acid benzoate:

[0452] 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).

[0453] Step 2: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0454] 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).

[0455] Example 15: (S)-2-benzamido-4-methylpentanoic acid [I-15]:

[0456]

[0457] Reaction process:

[0458]

[0459] Procedures and representations:

[0460] Step 1: (S)-2-benzamido-4-methylpentanoic acid methyl ester:

[0461] 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]+.

[0462] Step 2: (S)-2-benzamido-4-methylvaleric acid [I-15]:

[0463] 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).

[0464] Example 16: (S)-2-isobutyramido-4-methylpentanoic acid [I-16]:

[0465]

[0466] Reaction process:

[0467]

[0468] Procedures and representations:

[0469] Step 1: (S)-2-isobutyramido-4-methylpentanoic acid benzoate:

[0470] 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]+.

[0471] Step 2: (S)-2-isobutyramido-4-methylpentanoic acid [I-16]:

[0472] 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).

[0473] Example 17: (S)-2-(cyclohexanesulfonamido)-4-methylpentanoic acid [I-17]:

[0474]

[0475] Reaction process:

[0476]

[0477] Procedures and representations:

[0478] Step 1: (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid methyl ester:

[0479] 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).

[0480] Step 2: (S)-2-(cyclohexanesulfonamido)-4-methylpentanoic acid [I-17]:

[0481] 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).

[0482] Example 18: (S)-4-methyl-2-(phenylmethanesulfonamide)valerate [I-18]:

[0483]

[0484] Reaction process:

[0485]

[0486] Procedures and representations:

[0487] Step 1: (S)-4-methyl-2-(phenylmethanesulfonamide)pentanoic acid methyl ester:

[0488] 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).

[0489] Step 2: (S)-4-methyl-2-(phenylmethanesulfonamide)valerate [I-18]:

[0490] 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).

[0491] Example 19: (S)-4-methyl-2-(methanesulfonamide)valerate [I-19]:

[0492]

[0493] Reaction process:

[0494]

[0495] Procedures and representations:

[0496] Step 1: (S)-4-methyl-2-(methanesulfonamide)pentanoic acid methyl ester:

[0497] 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).

[0498] Step 2: (S)-4-methyl-2-(methanesulfonamide)valerate [I-19]:

[0499] 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).

[0500] Example 20: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0501]

[0502] Reaction process:

[0503]

[0504] Procedures and representations:

[0505] Step 1: (S)-4-methyl-1-oxo-1-(phenylamino)pent-2-ylcarbamate:

[0506] 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]+.

[0507] Step 2: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0508] 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).

[0509] Example 21: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0510]

[0511] Reaction process:

[0512]

[0513] Procedures and representations:

[0514] Step 1: (S)-4-methyl-1-(methylamino)-1-oxopent-2-ylcarbamate:

[0515] 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]+.

[0516] Step 2: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0517] 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).

[0518] Example 22: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-22]:

[0519]

[0520] Reaction process:

[0521]

[0522] Procedures and representations:

[0523] Step 1: (S)-2-(cyclohexylformamido)-4-methylpentanoic acid benzoate:

[0524] 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]+.

[0525] Step 2: (S)-2-(cyclohexylformamido)-4-methylvaleric acid [I-22]:

[0526] 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).

[0527] Example 25: (S)-4-methyl-2-(phenylsulfonamido)valerate [I-25]:

[0528]

[0529] Reaction process:

[0530]

[0531] Procedures and representations:

[0532] Step 1: (S)-4-methyl-2-(phenylsulfonamido)pentanoic acid methyl ester:

[0533] Benzenesulfonyl chloride (148.12 mg, 0.838 mmol) was added to a solution of (S)-2-amino-4-methylpentanoic acid 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]+.

[0534] Step 2: (S)-4-methyl-2-(phenylsulfonamido)valerate [I-25]:

[0535] 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).

[0536] Example 26: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]:

[0537]

[0538] Reaction process:

[0539]

[0540] Procedures and representations:

[0541] Step 1: (S)-4-methyl-2-(phenylamino)pentanoic acid methyl ester:

[0542] 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]⁺.

[0543] Step 2: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]:

[0544] 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).

[0545] Example 36: (S)-2-acetamido-4-methylvaleric acid [I-36]:

[0546]

[0547] Reaction process:

[0548]

[0549] Procedures and representations:

[0550] Step 1: (S)-2-acetamido-4-methylpentanoic acid benzoate:

[0551] 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]+.

[0552] Step 2: (S)-2-acetamido-4-methylpentanoic acid [I-36]:

[0553] 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).

[0554] Example 45: (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid [I-45]:

[0555]

[0556] Reaction process:

[0557]

[0558] Procedures and representations:

[0559] Step 1: (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid [I-45]:

[0560] 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).

[0561] 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]:

[0562]

[0563] Reaction process:

[0564]

[0565] Procedures and representations:

[0566] Step 1: Ethyl 2,2-difluoro-3-methylbutyrate:

[0567] 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.

[0568] Step 2: 2,2-Difluoro-3-methylbutanal:

[0569] 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.

[0570] Step 3: 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide:

[0571] 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]+.

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

[0573] 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]+.

[0574] Step 5-A: (R)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-46]:

[0575] 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).

[0576] Step 5-B: (S)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-47]:

[0577] 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).

[0578] Example 147: (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147].

[0579]

[0580] Reaction process:

[0581]

[0582] Procedures and representations:

[0583] Step 1: (S)-4-methyl-1-(methanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0584] 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] - .

[0585] Step 2: (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147]:

[0586] 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).

[0587] Example 193: (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193].

[0588]

[0589] Reaction process:

[0590]

[0591] Procedures and representations:

[0592] Step 1: (S)-4,4-dimethyl-1-(N-methylmethanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0593] 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] + .

[0594] Step 2: (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193]:

[0595] 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).

[0596] Example 192: 2-Amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192].

[0597]

[0598] Reaction process:

[0599]

[0600] Procedures and representations:

[0601] Step 1: 4-Fluoro-4-methyl-1-(methanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0602] 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] + .

[0603] Step 2: 2-Amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192].

[0604] 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).

[0605] Example 190: (S)-2-((S)-2-amino-4,4-dimethylpentamido)-4-methylpentanoic acid methyl ester hydrochloride [I-190].

[0606]

[0607] Reaction process:

[0608]

[0609] Procedures and representations:

[0610] Step 1: Methyl (S)-2-((S)-2-(tert-Butoxycarbonylamino)-4,4-dimethylpentamido)-4-methylpentanoate:

[0611] 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] + .

[0612] Step 2: (S)-2-((S)-2-amino-4,4-dimethylpentamido)-4-methylpentanoic acid methyl ester hydrochloride [I-190].

[0613] 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).

[0614] Example 122: (S)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-122].

[0615]

[0616] Reaction process:

[0617]

[0618] Procedures and representations:

[0619] Step 1: (S)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-122]:

[0620] 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).

[0621] Example 123: (R)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-123].

[0622]

[0623] Reaction process:

[0624]

[0625] Procedures and representations:

[0626] Step 1: (R)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-123]:

[0627] 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).

[0628] Example 205: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205].

[0629]

[0630] Reaction process:

[0631]

[0632] Procedures and representations:

[0633] Step 1: 2-(tert-Butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid:

[0634] 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] + .

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

[0636] 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] + .

[0637] Step 3: 1-Cyanamido-5,5,5-trifluoro-4-methyl-1-oxopent-2-ylcarbamate tert-butyl ester:

[0638] 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] + .

[0639] Step 4: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205]:

[0640] 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).

[0641] Example 206: 2-Amino-3-(1-methylcyclobutyl)propionic acid [I-206].

[0642]

[0643] Reaction process:

[0644]

[0645] Procedures and representations:

[0646] Step 1: N-methoxy-N,1-dimethylcyclobutaneformamide:

[0647] 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] + .

[0648] Step 2: 1-Methylcyclobutane formaldehyde:

[0649] 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.

[0650] Step 3: (Z)-2-(tert-Butoxycarbonylamino)-3-(1-Methylcyclobutyl)acrylate:

[0651] 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] + .

[0652] Step 4: 2-(tert-Butoxycarbonylamino)-3-(1-Methylcyclobutyl)propionate tert-butyl ester:

[0653] 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] + .

[0654] Step 5: 2-Amino-3-(1-methylcyclobutyl)propionic acid [I-206]:

[0655] 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] + .

[0656] 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).

[0657] Example 93: S-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93].

[0658]

[0659] Reaction process:

[0660]

[0661] Procedures and representations:

[0662] The procedure for 2-amino-3-(1-methylcyclobutyl)propionic acid is the same as in Example 8.

[0663] Step 6: 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid:

[0664] 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] + .

[0665] Step 7: (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid:

[0666] 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] + .

[0667] Step 8: (S)-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93]:

[0668] 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).

[0669] Example 204: 2-Amino-3-(trimethylsilyl)propionate [I-204]

[0670]

[0671] Reaction process:

[0672]

[0673] Procedures and representations:

[0674] Step 1: 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester:

[0675] 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] + .

[0676] Step 2: 2-Amino-3-(trimethylsilyl)propionate [I-204]:

[0677] 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).

[0678] Example 201: (S)-2-amino-3-(trimethylsilyl)propionate [I-201].

[0679]

[0680] Reaction process:

[0681]

[0682] Procedures and representations:

[0683] Step 1: (S)-2-amino-3-(trimethylsilyl)propionate [I-201]:

[0684] 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).

[0685] Example 200: (R)-2-amino-3-(trimethylsilyl)propionate [I-200].

[0686]

[0687] Reaction process:

[0688]

[0689] Procedures and representations:

[0690] Step 1: (R)-2-amino-3-(trimethylsilyl)propionate [I-200]:

[0691] 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).

[0692] Example 194: (S)-2-amino-4-fluoro-4-methylvaleric acid [I-194].

[0693]

[0694] Reaction process:

[0695]

[0696] Procedures and representations:

[0697] Step 1: (S)-2-amino-4-fluoro-4-methylpentanoic acid [I-194]:

[0698] 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).

[0699] Example 94: (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)but-1-amine [I-94].

[0700]

[0701] Reaction process:

[0702]

[0703] Procedures and representations:

[0704] Step 1: (S)-1-cyano-3,3-dimethylbutylcarbamate tert-butyl ester:

[0705] 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] + .

[0706] Step 2: (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate tert-butyl ester:

[0707] 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] + .

[0708] Step 3: ((S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)but-1-amine [I-94]:

[0709] 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).

[0710] Example 175: Synthesis of 2-amino-5,5,5-trifluoro-4-methoxyvalerate [I-175]:

[0711]

[0712] Reaction process:

[0713]

[0714] Procedures and representations:

[0715] Step 1: (S)-4-methyl-2-(phenylmethanesulfonamide)pentanoic acid methyl ester:

[0716] 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%).

[0717] 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).

[0718] Step 2: (4-(benzoxy)-1,1,1-trifluorobut-2-yloxy)trimethylsilane:

[0719] 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.

[0720] 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).

[0721] Step 3: 4-(benzooxy)-1,1,1-trifluorobut-2-ol:

[0722] 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.

[0723] Step 4: ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene:

[0724] 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.

[0725] 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).

[0726] Step 5: 4,4,4-Trifluoro-3-methoxybut-1-ol:

[0727] 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.

[0728] Step 6: 4,4,4-Trifluoro-3-methoxybutyraldehyde:

[0729] 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.

[0730] Step 7: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentadienonitrile:

[0731] 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):

[0732] Step 8: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxyvalerate:

[0733] 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] + .

[0734] Step 9: 2-Amino-5,5,5-trifluoro-4-methoxyvalerate [I-175]:

[0735] 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.

[0736] 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).

[0737] Example 176: 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0738]

[0739] Reaction process:

[0740]

[0741] Procedures and representations:

[0742] Step 1: Diethyl 2-(2,3-dimethylbut-2-yl)malonate:

[0743] 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] + .

[0744] Step 2: 2-(2,3-Dimethylbut-2-yl)malonic acid:

[0745] 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] + .

[0746] Step 3: 3,3,4-Trimethylvaleric acid:

[0747] 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]+ .

[0748] Step 4: N-methoxy-N,3,3,4-tetramethylpentanamide:

[0749] 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] + .

[0750] Step 4: 3,3,4-Trimethylpentanal:

[0751] 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.

[0752] Step 5: 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile:

[0753] 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] + .

[0754] Step 6: 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid:

[0755] 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] + .

[0756] 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0757] 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).

[0758] Example 178: 2-Amino-4,4-dimethylheptanoic acid [I-178]

[0759]

[0760] Reaction process:

[0761]

[0762] Procedures and representations:

[0763] The program is the same as that used in Example 176.

[0764] 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).

[0765] 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].

[0766]

[0767] Reaction process:

[0768]

[0769] Procedures and representations:

[0770] The program is the same as that used in Example 176.

[0771] 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).

[0772] (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).

[0773] (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).

[0774] Example 177: 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0775]

[0776] Reaction process:

[0777]

[0778] Procedures and representations:

[0779] Step 1: N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide:

[0780] 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] + .

[0781] Step 2: (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide:

[0782] 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] + .

[0783] Step 3: 5,5,5-Trifluoro-N-methoxy-N,3-dimethylpentanamide:

[0784] 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] + .

[0785] Step 4: 5,5,5-Trifluoro-3-methylpentanal:

[0786] 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.

[0787] Step 5: 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanenitrile:

[0788] 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] + .

[0789] Step 6: 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanoic acid:

[0790] 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] + .

[0791] 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0792] 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).

[0793] Example 179: (S)-2-amino-5-fluoro-4-(fluoromethyl)valerate [I-179]

[0794]

[0795] Reaction process:

[0796]

[0797] Procedures and representations:

[0798] Step 1: 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane:

[0799] 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] + .

[0800] Step 2: 2-(benzyloxymethyl)propane-1,3-diol:

[0801] 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] + .

[0802] Step 3: ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene:

[0803] 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).

[0804] Step 4: 3-Fluoro-2-(fluoromethyl)prop-1-ol:

[0805] 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.

[0806] Step 5: 3-Fluoro-2-(fluoromethyl)propyl trifluoromethanesulfonate:

[0807] 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.

[0808] Step 6: 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)pentanoic acid tert-butyl ester:

[0809] At -78°C, LDA (2.5 M, in THF / toluene / hexane, 1.28 mL, 3.2 mmol) was added over 25 minutes to a pre-cooled solution of tert-butyl 2-(diphenylmethyleneamino)acetate (944 mg, 3.2 mmol) in 20 mL of THF. 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 2 mL of THF 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 20 mL of saturated NH4Cl aqueous solution, 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).

[0810] Step 7: (S)-2-amino-5-fluoro-4-(fluoromethyl)valerate:

[0811] 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).

[0812] Example 187: (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0813]

[0814] Reaction process:

[0815]

[0816] Procedures and representations:

[0817] Step 1: (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0818] 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).

[0819] Example 90: Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-90]:

[0820]

[0821] Reaction process:

[0822]

[0823] Procedures and representations:

[0824] Step 1: Diethyl 2-(1,1,1-trifluoropropionic-2-ylidene)malonate:

[0825] 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).

[0826] Step 2: Diethyl 2-(1,1-difluoro-2-methylprop-2-yl)malonate:

[0827] 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).

[0828] Step 3: 4,4-Difluoro-3,3-dimethylbutyric acid:

[0829] 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] - .

[0830] Step 4: 4,4-Difluoro-N-methoxy-N,3,3-trimethylbutyramide:

[0831] 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).

[0832] Step 5: 4,4-Difluoro-3,3-dimethylbutanal:

[0833] 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.

[0834] Step 6: 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanilonitrile:

[0835] 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] + .

[0836] Step 7: 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanoic acid:

[0837] 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

[0838] Step 8: 2-Amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0839] 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

[0840] Step 9: 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid

[0841] 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

[0842] Step 10: (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0843] 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).

[0844] Example 88: Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-88]:

[0845]

[0846] Reaction process:

[0847]

[0848] Procedures and representations:

[0849] Step 1: (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide:

[0850] 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. Extraction was then performed with EtOAc (100 mL × 2). The organic phase was washed with water (100 mL) and brine (100 mL), 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] + .

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

[0852] 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).

[0853] Example 185: Synthesis of methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate [I-185]:

[0854]

[0855] Reaction process:

[0856]

[0857] Procedures and representations:

[0858] The procedure for 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid is the same as in Example 90.

[0859] Step 1: Methyl (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate:

[0860] 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

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

[0862] 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

[0863] 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).

[0864] Example 184: Synthesis of methyl (S)-2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentamido)-4-methylpentanoate [I-184]:

[0865]

[0866] The program is the same as in examples 90 and 185.

[0867] Methyl (S)-2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentanoyl)-4-methylpentanoate: ESI-MS (EI + ,m / z):309.0

[0868] 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).

[0869] 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]

[0870]

[0871] Reaction process:

[0872]

[0873] Procedures and representations:

[0874] 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:

[0875] 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]+.

[0876] Step 2-A: Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0877] 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).

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

[0879] 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).

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

[0881] 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).

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

[0883] 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).

[0884] Example 128: (S)-2-amino-5,5,5-trifluoro-4,4-dimethylvaleric acid (I-128):

[0885]

[0886] Reaction process:

[0887]

[0888] Procedures and representations:

[0889] The program is the same as that used in Example 187.

[0890] (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).

[0891] Example 188: (S)-2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate methyl ester [I-188]:

[0892]

[0893] Reaction process:

[0894]

[0895] Procedures and representations:

[0896] The procedure for 2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylvaleric acid is the same as in Example 90.

[0897] Step 1: Methyl (S)-2-((R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate:

[0898] 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

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

[0900] 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

[0901] 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).

[0902] Example 189: (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentamido)-4-methylpentanoate methyl ester [I-189]:

[0903]

[0904] Reaction process:

[0905] The program is the same as that used in Example 188.

[0906] Procedures and representations:

[0907] 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).

[0908] Example 108: (S)-2-amino-6-fluorohexanoic acid [I-108].

[0909]

[0910] Example 109: (R)-2-amino-6-fluorohexanoic acid [I-109].

[0911]

[0912] Reaction process:

[0913]

[0914] Procedures and representations:

[0915] Step 1: 2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester:

[0916] 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] + .

[0917] Step 2: (S)-2-amino-6-fluorohexanoic acid [I-108]:

[0918] 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).

[0919] Step 2: (R)-2-amino-6-fluorohexanoic acid [I-109]:

[0920] 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).

[0921] Example 198: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (I-198):

[0922]

[0923] Reaction process:

[0924]

[0925] Procedures and representations:

[0926] Step 1: 4,4,4-Trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide:

[0927] 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).

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

[0929] 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] + .

[0930] Step 3: 4,4,4-Trifluoro-3-(trifluoromethyl)butanal:

[0931] 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.

[0932] Step 4: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanilonitrile:

[0933] 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] + .

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

[0935] 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

[0936] Step 6: Methyl 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0937] 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] + .

[0938] Step 7: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate trifluoroacetic acid:

[0939] 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).

[0940] Example 164: (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (I-164):

[0941]

[0942] Reaction process:

[0943]

[0944] Procedures and representations:

[0945] Step 1: 2-Amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0946] 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] + .

[0947] Step 2: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0948] 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] + .

[0949] Step 3: (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0950] 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)δ

[0951] 3.74-3.80(m,2H),2.88-2.31(m,1H),1.91-2.20(m,1H).

[0952] Example 203: 2-Amino-4-cyclopentylbutyric acid [I-203]:

[0953]

[0954] Reaction process:

[0955]

[0956] Procedures and representations:

[0957] Step 1: 2-Cyclopentylacetaldehyde:

[0958] 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.

[0959] Step 2: (Z)-2-(tert-Butoxycarbonylamino)-4-cyclopentylbut-2-enoic acid tert-butyl ester:

[0960] 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] + .

[0961] Step 3: 2-(tert-Butoxycarbonylamino)-4-cyclopentylbutyrate tert-butyl ester:

[0962] 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] + .

[0963] Step 4: 2-Amino-4-cyclopentylbutyric acid:

[0964] 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).

[0965] Example 202: 2-Amino-5-cyclopentylpentanoic acid [I-202]:

[0966]

[0967] Reaction process:

[0968]

[0969] Procedures and representations:

[0970] Step 1: 3-Cyclopentylpropionaldehyde:

[0971] 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.

[0972] Step 2: (E)-2-(tert-Butoxycarbonylamino)-5-cyclopentylpent-2-enoic acid tert-butyl ester:

[0973] 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] + .

[0974] Step 3: 2-(tert-Butoxycarbonylamino)-5-cyclopentylvaleric acid tert-butyl ester:

[0975] 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] + .

[0976] Step 4: 2-Amino-5-cyclopentylpentanoic acid:

[0977] 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).

[0978] Example 197: Synthesis of 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide [I-197]:

[0979]

[0980] Reaction process:

[0981]

[0982] Procedures and representations:

[0983] Step 1: 2-(phenylmethylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0984] 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

[0985] Step 2: 2-Amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0986] 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).

[0987] Example 196: 2-Amino-5-fluoro-4,4-dimethylvaleric acid [I-196].

[0988]

[0989] Reaction process:

[0990]

[0991] Procedures and representations:

[0992] Step 1: 3-Hydroxy-N-methoxy-N,2,2-trimethylpropionamide:

[0993] 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] + .

[0994] Step 2: 3-Fluoro-N-methoxy-N,2,2-trimethylpropionamide:

[0995] 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] + .

[0996] Step 3: 3-Fluoro-2,2-Dimethylpropionaldehyde:

[0997] 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.

[0998] Step 4: (Z)-2-(tert-Butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoic acid tert-butyl ester:

[0999] 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] + .

[1000] Step 5: 2-(tert-Butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoate tert-butyl ester:

[1001] 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] + .

[1002] Step 6: 2-Amino-5-fluoro-4,4-dimethylpentanoic acid trifluoroacetic acid:

[1003] 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).

[1004] Example 186: Synthesis of 2,4-diamino-4-methylpentanoic acid [I-186]:

[1005]

[1006] Reaction process:

[1007]

[1008] Procedures and representations:

[1009] Step 1: 4-(methoxy(methyl)amino)-2-methyl-4-oxobutyl-2-ylcarbamate tert-butyl ester:

[1010] 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]+ .

[1011] Step 2: 2-Methyl-4-oxobutyl-2-ylcarbamate tert-butyl ester:

[1012] 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] + .

[1013] Step 3: 4-(benzylamino)-4-cyano-2-methylbut-2-ylcarbamate tert-butyl ester:

[1014] 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] + .

[1015] Step 4: tert-butyl 5-amino-4-(benzylamino)-2-methyl-5-oxopent-2-ylcarbamate:

[1016] 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] + .

[1017] Step 5: 2-(phenylmethylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid:

[1018] 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] + .

[1019] Step 6: 2-Amino-4-(tert-Butoxycarbonylamino)-4-methylpentanoic acid:

[1020] 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

[1021] Step 7: 2,4-Diamino-4-methylpentanoic acid:

[1022] 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).

[1023] Example 199: Synthesis of 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine [I-199]:

[1024]

[1025] Reaction process:

[1026]

[1027] Procedures and representations:

[1028] Step 1: N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide:

[1029] 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] + .

[1030] Step 2: (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide:

[1031] 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] + .

[1032] Step 3: 4,4,4-Trifluoro-N-methoxy-N,3-dimethylbutyramide:

[1033] 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] + .

[1034] Step 4: 4,4,4-Trifluoro-3-methylbutanal:

[1035] 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.

[1036] Step 5: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanilide:

[1037] 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] + .

[1038] Step 6: N-Benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine:

[1039] 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] + .

[1040] 4,4,4-Trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-aminetrifluoroacetic acid:

[1041] 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).

[1042] Example 210: Western blot assay

[1043] 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.

[1044] 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 (AARPMI, USBiological Life Sciences) 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). 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).

[1045] 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.

[1046] Example 211. A method for identifying compounds that mimic or antagonize the activity of leucine in relation to Sestrin2 and the Sestrin2 / GATOR2 interaction.

[1047] INTRODUCTION

[1048] 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.

[1049] Method 1 (in vitro PPI assay)

[1050] 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.

[1051] 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 supplemented with 5 mM glucose (-AA RPMI, BioLife Sciences, USA) without amino acid formulation and kept in this medium 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).

[1052] 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.

[1053] Method 2 (cell-based mTORC1 activation)

[1054] 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.

[1055] Method 3 (cell-based mTORC1 activation)

[1056] 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.

[1057] Method 4

[1058] 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.

[1059] Method 5 (ALPHA LI SA cell-based assay)

[1060] 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-p70 S6K(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.

[1061] Method 6, Thermal shift protocol (Tm shift):

[1062] 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 mordant 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 from 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.

[1063] Method 7, Indirect ligand binding assay (ILBA)

[1064] 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.

[1065] 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.

[1066] 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.

[1067] 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.

[1068] 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.

[1069] 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.

[1070] 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.

[1071] 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.

[1072] 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.

[1073] 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.

[1074] 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.

[1075] 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.

[1076] Table 3. Analytical data for exemplary compounds

[1077]

[1078]

[1079]

[1080] 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.

[1081] Table 4. Exemplary compounds active in ALPHALISA cell-based analyses

[1082]

[1083]

[1084] 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.

[1085] Table 5. Exemplary compounds showing activity in thermal migration analysis

[1086]

[1087]

Claims

1. Use in the preparation of a medicament by means of a compound or a pharmaceutically acceptable salt thereof, or a composition comprising said compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or mediator. The compound is selected from: The drug is used to treat mTORC1-mediated conditions in patients in need, wherein the mTORC1-mediated conditions are: I) Cancer, selected from leukemia (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 (Hodgkin's disease or non-Hodgkin's disease), multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphoendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor). Tumors, including leiomyosarcoma, rhabdomyosarcoma, 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 gland carcinoma, cystic adenocarcinoma, 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. II) Proliferative disorders, selected from obesity, psoriasis, abnormal keratinization, lymphoproliferative disorders, chronic rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy; or III) Ribosomal disorders (Diamond-Blackfan anemia, 5q- syndrome, Shwachman-Diamond syndrome, X-linked dyskeratosis, chondrodysplasia, or Treacher-Collins syndrome), adhesive disorders (Roberts syndrome or Cornelia de Lange syndrome), muscular atrophy, autophagy, or depression.

2. The use according to claim 1, wherein the mTORC1-mediated condition is depression.

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