Modulators of sestrin-gator2 interaction and uses thereof

Abstract

To provide compounds, compositions thereof, and methods of using the same.SOLUTION: The present invention provides compounds, compositions thereof, and methods of using the same. The present invention relates to compounds and methods useful for modulating the Sestrin-GATOR2 interaction thereby selectively modulating mTORC1 activity indirectly. The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders. Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful for treating a variety of diseases, disorders or conditions, associated with mTORC1. Such diseases, disorders, or conditions include diabetes, epilepsy, neurodegeneration, immune response, suppressed skeletal muscle growth, and cellular proliferative disorders (e.g., cancer) such as those described herein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical field of the invention The present invention relates to compounds and methods useful for modulating Sestrin-GATOR2 interaction, thereby indirectly and selectively modulating mTORC1 activity. The present invention also provides pharmaceutically acceptable compositions comprising the compounds of the present invention and methods of using the compositions in the treatment of various disorders. [Background technology]

[0002] Background of the Invention The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that senses diverse environmental cues, including growth factors, cellular stress, and nutrient and energy levels. When activated, mTORC1 phosphorylates substrates that stimulate anabolic processes, such as mRNA translation and lipid synthesis, while limiting catabolic processes, such as autophagy. Dysregulation of mTORC1 occurs in a wide range of diseases, including diabetes, epilepsy, neurodegeneration, immune responses, suppressed skeletal muscle growth, and cancer, among others (Howell et al., (2013) Biochemical Society Transactions 41:906-912; Kim et al., (2013) Molecules and Cells 35:463-473; Laplante and Sabatini, (2012) Cell 149:274-293).

[0003] Multiple upstream inputs, including growth factors and energy levels, signal to mTORC1 through the TSC complex, which regulates Rheb, a small GTPase that is an essential activator of mTORC1 (Brugarolas et al., (2004) Genes & Development 18:2893-2904; Garami et al., (2003) Molecular Cell 11:1457-1466; Inoki et al., (2003) Genes & Development 17:1829-1834; Long et al., (2005) Current Biology 15:702-713; Sancak et al., (2008) Science (New York, NY) 320:1496-1501; Saucedo et al., (2003) Nature Cell Biology 5, pp. 566-571; Stocker et al. (2003) Nature cell biology 5, pp. 559-565; Tee et al. (2002) Proc Natl Acad Sci USA 99, pp. 13571-13576). Amino acids do not appear to signal to mTORC1 through the TSC-Rheb axis, but instead act through heterodimeric Rag GTPases consisting of RagA or RagB bound to RagC or RagD, respectively (Hirose et al., (1998) Journal of cell science 111(1):11-21; Kim et al., (2008) Nature cell biology 10:935-945; Nobukuni et al., (2005) Proc Natl Acad Sci USA 102:14238-14243; Roccio et al., (2005) Oncogene 25:657-664; Sancak et al., (2008) Science (New York, NY) 320:1496-1501; Schuermann et al., (1995) The Journal of biological chemistry). 270, pp. 28982-28988; Sekiguchi et al. (2001) The Journal of biological chemistry, vol. 276, pp. 7246-7257; Smith et al. (2005) The Journal of biological chemistry, vol. 280, pp. 18717-187 Rag GTPases regulate the subcellular localization of mTORC1, and amino acids promote the recruitment of mTORC1 to the lysosomal surface, where Rheb GTPases also reside (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 (NY) Vol. 320, pp. 1496-1501). The Ragulator complex localizes Rag GTPases to the lysosomal surface and, together with vacuolar ATPases, promotes the exchange of GDP for GTP on RagA / B (Bar-Peled et al., (2012) Cell 15 0, pp. 1196-1208; Sancak et al. (2010) Cell 141, pp. 290-303; Zoncu et al. (2011) Science Signaling 334, pp. 678-683) A separate FLCN-FNIP complex acts on RagC / D to stimulate the hydrolysis of GTP to GDP (Tsun et al., 2013). Once RagA / B is loaded with GTP and RagC / D with GDP, the heterodimers bind and recruit mTORC1 to the lysosomal surface, where it can access its activator, the Rheb GTPase.

[0004] Recent studies have identified the GATOR1 multiprotein complex as a key negative regulator of the amino acid sensing pathway, and its loss renders mTORC1 signaling completely unresponsive to amino acid starvation (Bar-Peled et al., (2013) Science 340:1100-1106; Panchaud et al., (2013) Science Signaling 6:ra42). GATOR1, consisting of DEPDC5, Nprl2, and Nprl3, is a GTPase that activates RagA / B GAPs. The GATOR2 multiprotein complex has five known subunits (WDR24, WDR59, Mios, Sec13, and Seh1L) and is a positive component of the GATOR1 pathway and upstream or parallel pathways to GATOR1, but its molecular function was unknown until recently (Bar-Peled et al., (2013) Science 340:1100-1106).

[0005] The recent identification of GATOR2 binding to one or more Sestrins has provided additional information about the mTORC1 pathway, demonstrating that the resulting Sestrin-GATOR2 complex regulates the subcellular localization and activity of mTORC1. Specifically, the presence of Sestrin-GATOR2 complexes inhibits the mTORC1 pathway and reduces mTORC1 activity by preventing mTORC1 translocation to the lysosomal membrane. The interaction of GATOR2 with Sestrins, particularly Sestrin 1 and Sestrin 2, is antagonized by amino acids, particularly leucine, and to a lesser extent by isoleucine, methionine, and valine. In the presence of leucine, GATOR2 does not interact with Sestrin 1 or Sestrin 2, allowing mTORC1 to translocate to the lysosomal membrane, where it is active. Sestrin1 and Sestrin2 bind directly to leucine and, to a lesser extent, isoleucine and methionine (Chantranupong et al., (2014) Cell Rep.; 9(1):1-8). Binding of leucine by Sestrin 1 or 2 is required for interaction with GATOR2 and subsequent disruption of mTORC1 activation. Sestrin 2 mutants unable to bind leucine are unable to signal the presence of leucine to mTORC1, and cells lacking Sestrin 2 and its homologs render mTORC1 unresponsive to the absence of leucine (Wolfson et al. (2015) Science pii:ab2674 [Epub ahead of print]).

[0006] The sestrins are three related proteins (sestrin 1, 2, and 3) whose molecular functions are not well characterized (Buckbinder et al., (1994) Proc Natl Acad Sci USA 91, 10640-10644; Budanov et al., (2002) Cell 134, 10640-10644). (Peeters et al., (2003) Human Genetics 112, 573-580). Sestrin2 has been proposed to interact with TSC by inhibiting mTORC1 signaling and activating AMPK upstream of TSC (Budanov and Karin, (2008) Cell 134, 451-460). However, later studies have shown that AMPK It was found that mTORC1 was inhibited by Sestrin2 in the absence of GATOR2 (Peng et al. (2014) Cell 159(1):122-33), further highlighting the important role that the GATOR2 complex plays in modulating mTORC1 in response to Sestrin2. Modulation of the Sestrin-GATOR2 complex indirectly represents a potential therapeutic target for selectively modulating mTORC1 activity. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Howell et al. (2013) Biochemical Society transactions 41, 906-912 [Non-patent document 2] Kim et al. (2013) Molecules and cells 35, 463-473 [Non-patent document 3] Laplante and Sabatini, (2012) Cell 149, 274-293 [Non-patent document 4] Brugarolas et al. (2004) Genes & Development 18, 2893-2904 [Non-patent document 5] Garami et al. (2003) Molecular Cell 11, 1457-1466 [Non-patent document 6] Inoki et al. (2003) Genes & Development, 17, 1829-1834 [Non-Patent Document 7] Long et al. (2005) Current Biology 15, 702-713 [Non-patent document 8] Sancak et al. (2008) Science (New York, NY) 320, 1496-1501 [Non-Patent Document 9] Saucedo et al. (2003) Nature cell biology 5, 566-571 [Non-Patent Document 10] Stocker et al. (2003) Nature Cell Biology 5, 559-565 [Non-Patent Document 11] Tee et al. (2002) Proc Natl Acad Sci USA 99, 13571-13576 [Non-Patent Document 12] Hirose et al. (1998) Journal of cell science, vol. 111 (part 1), pp. 11-21 [Non-Patent Document 13] Kim et al. (2008) Nature Cell Biology 10, 935-945 [Non-Patent Document 14] Nobukuni et al. (2005) Proc Natl Acad Sci USA 102, 14238-14243 [Non-Patent Document 15] Roccio et al. (2005) Oncogene 25, 657-664 [Non-Patent Document 16] Sancak et al. (2008) Science (New York, NY) 320, 1496-1501 [Non-Patent Document 17] Schuermann et al. (1995) The Journal of biological chemistry, vol. 270, pp. 28982-28988 [Non-Patent Document 18] Sekiguchi et al. (2001) The Journal of biological chemistry, vol. 276, pp. 7246-7257 [Non-Patent Document 19] Smith et al. (2005) The Journal of biological chemistry, vol. 280, pp. 18717-18727 [Non-Patent Document 20] Buerger et al. (2006) Biochemical and Biophysical Research Communications 344, 869-880 [Non-Patent Document 21] Dibble et al. (2012) Molecular Cell 47, 535-546 [Non-Patent Document 22] Saito et al. (2005) Journal of Biochemistry, Vol. 137, pp. 423-430 [Non-Patent Document 23] Sancak et al. (2008) Science (New York, NY) 320, 1496-1501 [Non-Patent Document 24] Bar-Peled et al. (2012) Cell 150, 1196-1208 [Non-Patent Document 25] Sancak et al. (2010) Cell vol. 141, pp. 290-303 [Non-Patent Document 26] Zoncu et al. (2011) Science Signaling 334, 678-683 [Non-Patent Document 27] Bar-Peled et al. (2013) Science 340, 1100-1106 [Non-patent document 28] Panchaud et al. (2013) Science Signaling, Vol. 6, ra42 [Non-Patent Document 29] Chantranupong et al. (2014) Cell Rep.; Volume 9(Issue 1): Pages 1-8 [Non-Patent Document 30] Wolfson et al. (2015) Science pii:ab2674 [Epub ahead of print] [Non-Patent Document 31] Buckbinder et al. (1994) Proc Natl Acad Sci USA 91, 10640-10644 [Non-Patent Document 32] Budanov et al. (2002) Cell 134, 451-460 [Non-Patent Document 33] Peeters et al. (2003) Human Genetics 112, 573-580 [Non-Patent Document 34] Budanov and Karin, (2008) Cell 134, 451-460 [Non-Patent Document 35] Peng et al. (2014) Cell 159(1):122-33 Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention It has now been found that compounds of the present invention and pharmaceutically acceptable compositions thereof are effective as Sestrin-GATOR2 modulators. Such compounds have the general formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein each variable is as defined and described herein.

[0009] The compounds of the present invention and pharmaceutically acceptable compositions thereof are useful for treating various diseases, disorders, or conditions associated with mTORC1, including diabetes, epilepsy, neurodegeneration, immune response, suppression of skeletal muscle growth, and cell proliferative disorders (e.g., cancer), such as those described herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] Detailed Description of Certain Embodiments 1. General Description of Certain Embodiments of the Invention: The compounds of the present invention and compositions thereof are useful as Sestrin-GATOR2 modulators. In certain embodiments, the present invention provides compounds of formula I: [ka] or a pharmaceutically acceptable salt thereof, R 1 is H or C 1~6 is alkyl, R 2 is R, -(CH2) n -phenyl, -C(O)R, -SOR or -C(O)N(R), n is 0, 1 or 2; Each R is independently hydrogen, -CN, or a saturated or unsaturated C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms, or a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R 3is ring A, -C(O)R, -C(O)OR, -C(O)N(R), -SOH, -SON(R), -S(O)R, -S(O)R, -S(O)R, -OR, or -B(OR), where two OR groups on the same boron together with their intervening atoms form a 5-8 membered saturated or partially unsaturated monocyclic ring having, in addition to the boron and two oxygens, 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 3 and R 4 together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen or sulfur; L is a covalent bond or a straight or branched C optionally substituted with 1 to 9 fluoro groups. 1~6 is an alkylene chain, Ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R 4 is R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4 together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen or sulfur; R 5 is H or C 1~4 alkyl) to provide. 2. Compounds and definitions:

[0011] The compounds of the present invention include those generally described herein and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, chemical elements are defined as defined in the CAS Handbook of Chemistry and Physics, 75th Edition, Elements. Further general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th ed., Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. and is incorporated herein by reference.

[0012] The terms "aliphatic" or "aliphatic group," as used herein, mean a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation but is not aromatic (also referred to herein as "carbocycle," "alicyclic," or "cycloalkyl"), which has 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 1-4 aliphatic carbon atoms. In still other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has 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.

[0013] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon); the quaternized form of any basic nitrogen; or a substitutable nitrogen of a heterocyclic ring, for example, N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl).

[0014] The term "unsaturated," as used herein, means a moiety having one or more units of unsaturation.

[0015] As used herein, the term "saturated or unsaturated, straight-chain or branched, divalent C 1~8 (or C 1~6 ") Hydrocarbon chain" refers to straight or branched, divalent alkylene, alkenylene, and alkynylene chains as defined herein.

[0016] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n -, and 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 group in which one or more methylene hydrogen atoms are replaced by a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0017] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced by a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0018] The term "halogen" means F, Cl, Br or I.

[0019] The term "aryl," used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic or bicyclic ring system having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic, and each ring in the system contains 3 to 7 ring members. The term "aryl" can be used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to an aromatic ring system, including but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Similarly, the term "aryl," as used herein, also includes within its scope groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl.

[0020] The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms, having 6, 10, or 14 pi-electrons shared in the cyclic arrangement, and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroar-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic 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]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which include optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently are optionally substituted.

[0021] As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety, either saturated or partially unsaturated, having, in addition to carbon atoms, one or more heteroatoms, preferably 1 to 4 heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + It may also be NR (as in N-substituted pyrrolidinyl).

[0022] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, These include thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl moieties are independently optionally substituted.

[0023] As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0024] As described herein, the compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety have been replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each of the group's substitutable positions, and if more than one position in any given structure may be substituted with more than one substituent selected from the specified group, these substituents may be the same or different at any position. Combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to a compound that does not substantially change when subjected to conditions that allow for its production, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein.

[0025] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently: halogen; -(CH) 0~4 R°;-(CH2) 0~4 OR°-;-O(CH2) 0~4 R o , -O-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 CH(OR°)2;-(CH2) 0~4 SR°; optionally substituted by R° -(CH2) 0~4 Ph; optionally substituted by R° -(CH2)0~4 O(CH2) 0~1 Ph; optionally substituted by R° -CH=CHPh; optionally substituted by R° -(CH2) 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0~4 N(R°)2;-(CH2) 0~4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0~4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0~4 N(R°)C(O)OR°;-N(R°)N(R°)C(O)R°;-N(R°)N(R°)C(O)NR°2;-N(R°)N(R°)C(O)OR°;-(CH2) 0~4 C(O)R°;-C(S)R°;-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 C(O)SR°;-(CH2) 0~4 C(O)OSiR°3;-(CH2) 0~4 OC(O)R°;-OC(O)(CH2) 0~4 SR-, SC(S)SR°;-(CH2) 0~4 SC(O)R°;-(CH2) 0~4 C(O)NR°2;-C(S)NR°2;-C(S)SR°;-SC(S)SR°, -(CH2) 0~4 OC(O)NR°2;-C(O)N(OR°)R°;-C(O)C(O)R°;-C(O)CH2C(O)R°;-C(NOR°)R°;-(CH2) 0~4 SSR°;-(CH2) 0~4 S(O)2R°;-(CH2) 0~4 S(O)2OR°;-(CH2) 0~4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0~4 S(O)R°;-N(R°)S(O)2NR°2;-N(R°)S(O)2R°;-N(OR°)R°;-C(NH)NR°2;-P(O)2R°;-P(O)R°2;-OP(O)R°2;-OP(O) (OR°)2;-SiR°3;-(C 1~4 Linear or branched alkylene)ON(R°)2; or -(C 1~4linear or branched alkylene)C(O)ON(R°)2, where each R° is optionally substituted as defined below and independently represents hydrogen, C 1~6 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2- (a 5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, as defined above, two independent occurrences of R° taken together with their intervening atom(s) form a 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0026] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with their intervening atoms) are independently halogen, —(CH2), 0~2 R ● ,-(Halo R ● ), -(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 ● and R ● each is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0027] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group are the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2~3 O- or -S(C( R * 2)) 2~3 Contains S-, R * Each independent occurrence of is hydrogen, optionally substituted as defined below C 1~6 The "optionally substituted" group is selected from the group consisting of an aliphatic, unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to a substitutable vicinal carbon of an "optionally substituted" group include the following: -O(CR * 2) 2~3 Contains O- and R * Each independent occurrence of is hydrogen, optionally substituted as defined below C 1~6 It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0028] R *Suitable substituents on the aliphatic group include halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, R ● each is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0029] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2,- C(NH)NR † 2 or -N(R † )S(O)2R † Contains R † each independently represents hydrogen, optionally substituted C as defined below 1~6 an aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R †two independent occurrences of together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0030] R † Suitable substituents on the aliphatic group are independently halogen, —R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2 or -NO2, and R ● each is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0031] As used herein, the term "pharmaceutically acceptable salt" refers to such salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. See, for example, SM Berge describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, Vol. 66, pp. 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts of amino groups formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and 2-hydroxy-ethanesulfonate. Examples of salts that may be used include phosphate, 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, and the like.

[0032] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1~4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0033] Unless otherwise specified, structures depicted herein represent all isomeric forms of the structure. It is also intended to include various isomers (e.g., enantiomers, diastereomeric isomers, and geometric (or conformational) isomers), such as the R and S configurations about each asymmetric center, Z and E double bond isomers, and Z and E stereoisomers. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Unless otherwise stated, all tautomers of the compounds of the invention are within the scope of the invention. Furthermore, unless otherwise stated, structures depicted herein are also intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, replacement of hydrogen by deuterium or tritium, or 13 C or 14 Compounds having the present structures, including the replacement of a carbon with a C-rich carbon, are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the present invention.

[0034] As used herein, the term "leucine mimetic" is defined as a compound that reduces the amount of Sestrin2 bound to GATOR2 by at least about 40% at 25 μM compared to leucine. 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%.

[0035] As used herein, the term "leucine antagonist" is defined as a compound that increases the amount of Sestrin2 bound to GATOR2 by at least about 40% (expressed as -40% of leucine activity) at 25 μM relative to leucine. 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%.

[0036] The terms "measurable affinity" and "measurably inhibit," as used herein, refer to a measurable change in Sestrin2 binding to GATOR2 between a sample containing a compound of the present invention or a composition thereof, as well as Sestrin2, GATOR2 and leucine, and an equivalent sample containing Sestrin2, GATOR2 and leucine in the absence of the compound or composition thereof. 3. Description of Exemplary Embodiments:

[0037] In certain embodiments, the present invention provides a compound of formula I: [ka] or a pharmaceutically acceptable salt thereof, R 1 is H or C 1~6 is alkyl, R 2 is R, -(CH2) n -phenyl, -C(O)R, -SOR or -C(O)N(R), n is 0, 1 or 2; Each R is independently hydrogen, -CN, or a saturated or unsaturated 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 heterocyclic ring independently of nitrogen, oxygen, or sulfur; an optionally substituted group selected from a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms selected from R 3 is ring A, -C(O)R, -C(O)OR, -C(O)N(R), -SOH, -SON(R), -S(O)R, -S(O)R, -S(O)R, -OR, or -B(OR), where two OR groups on the same boron together with their intervening atoms form a 5-8 membered saturated or partially unsaturated monocyclic ring having, in addition to the boron and two oxygens, 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 3 and R 4 together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen or sulfur; L is a covalent bond or a straight or branched C optionally substituted with 1 to 9 fluoro groups. 1~6 is an alkylene chain, Ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R 4 is R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4 together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen or sulfur; R 5 is H or C 1~4 alkyl).

[0038] In some embodiments, compounds of Formula I are provided other than those compounds depicted in Table 2 below.

[0039] As generally defined above, R 1 is H or C 1~6 In some embodiments, R 1 is H. In other embodiments, R 1 is C 1~6 In some embodiments, R 1 is methyl. In some embodiments, R 1 is isobutyl. In some embodiments, R 1 is selected from those depicted in Table 1 below. In some embodiments, R 1 is selected from those illustrated in Table 2 below.

[0040] As generally defined above, R 2 is R, -(CH2) n -phenyl, -C(O)R, -S0R, or -C(O)N(R). In some embodiments, R 2 is R. In some embodiments, R 2 is -(CH2) n In some embodiments, R 2 is —C(O)R. In some embodiments, R 2 is -SO2R. In some embodiments, R 2 is —C(O)N(R). In some embodiments, R 2 is methyl. In some embodiments, R 2 is -(CH)-phenyl. In some embodiments, R 2 is —C(O)CH. In some embodiments, R 2 is selected from those depicted in Table 1 below. In some embodiments, R 2 is selected from those illustrated in Table 2 below.

[0041] 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.

[0042] As generally defined above, R 3 is ring A, -C(O)R, -C(O)OR, -C(O)N(R), -SOH, -SON(R), -S(O)R, -S(O)R, -S(O)R, -OR, or -B(OR), where two -OR groups on the same boron together with their intervening atoms form a 5-8 membered saturated or partially unsaturated monocyclic ring having, in addition to the boron and two oxygens, 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 3 and R 4 taken together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0043] In some embodiments, R 3 is —C(O)OH. In some embodiments, R 3 is —C(O)N(R). In some embodiments, R 3 is —SO3H. In some embodiments, R 3 is —SO 2 N(R) 2 . In some embodiments, R 3 is -B(OR)2, where two -OR groups on the same boron, taken together with their intervening atoms, form a 5-8 membered monocyclic saturated, partially unsaturated, or heterocyclic ring having, in addition to the boron and two oxygens, 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 and R 4 taken together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen, or sulfur.

[0044] In some embodiments, R 3is ring A. As generally defined above, ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5-6 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 1-4 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., maleimide), pyrazolyl, oxazolyl, tetrazolyl, thiazolyl, and triazolyl. In some embodiments, ring A is an optionally substituted 6 membered heteroaryl ring having 1-2 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.

[0045] In some embodiments, R 3 is (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 illustrated in Table 2 below.

[0046] As generally defined above, L is a covalent bond or a linear or branched C group optionally substituted with 1 to 4 fluoro groups. 1~6 In some embodiments, L is an alkylene chain. In some embodiments, L is a covalent bond. In some embodiments, L is a straight or branched C alkylene chain optionally substituted with 1 to 4 fluoro groups. 1~6In some embodiments, L is an alkylene chain. In some embodiments, L is methylene. In some embodiments, L is n-butylenyl. In some embodiments, L is ethylenyl. In some embodiments, L is n-propylenyl. 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.

[0047] In some embodiments, L is a branched C optionally substituted with 1 to 4 fluoro groups. 1~6 is an alkylene chain. In certain embodiments, L is -C(CH3)2-. In other embodiments, L is -C(CH3)(CF3)-.

[0048] As generally defined above, R 4 is R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4 taken together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen, or sulfur. 4 is R. In some embodiments, R 4 is —CF. In some embodiments, R 4 is -OR. In some embodiments, R 4 is -N(R). In some embodiments, R 4 is —Si(R). In some embodiments, R 4 is -SR. In some embodiments, R 4 is isopropyl. In some embodiments, R 4 is tert-butyl. , 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 methoxyl. In some embodiments, R 4is methylthioyl. In some embodiments, R 3 and R 4 are taken together to form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen, or sulfur. 4 is selected from those depicted in Table 1 below. In some embodiments, R 4 is selected from those illustrated in Table 2 below.

[0049] As generally defined above, R 5 is H or C 1~4 In some embodiments, R 5 is H. In some embodiments, R 5 is C 1~4 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 illustrated in Table 2 below.

[0050] In certain embodiments, the present invention provides a compound of formula II: [ka] or a pharmaceutically acceptable salt thereof, wherein each variable, either singly or in combination, is as defined above and as described in the embodiments presented herein.

[0051] In certain embodiments, the present invention provides a compound of formula III: [ka] or a pharmaceutically acceptable salt thereof, Q is —C(R′)2— or —NH—; R x and R y are each hydrogen or R xand R y together form =O, [ka] is a double or single bond, Each R is independently hydrogen, -CN, or C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms, or a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R' is independently hydrogen, halogen, -CN, or C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms, or a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; L is a covalent bond or a straight or branched C optionally substituted with 1 to 9 fluoro groups. 1~6 is an alkylene chain, R 4’ is R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, and R 5’ is H, -OR or C 1~4 alkyl).

[0052] In some embodiments, Q is -NH-. In some embodiments, Q is -CH2-. In some embodiments, Q is -CHF-.

[0053] In some embodiments, L is -CH2-.

[0054] In some embodiments, R x and R y Each is hydrogen. x and Ry together form =O.

[0055] In some embodiments, R 5’ is H. In some embodiments, R 5’ is -OH.

[0056] In some embodiments, [ka] is a single bond. In some embodiments, [ka] is a double bond.

[0057] In certain embodiments, the present invention provides a compound of formula IV-a, IV-b, or IV-c: [ka] [ka] or a pharmaceutically acceptable salt thereof, R 1 is H or C1-6 alkyl, R 2 is R, -(CH2) n -phenyl, -C(O)R, -SOR or -C(O)N(R), R 4’’ are each independently R, halogen, or —CF; Each R is independently hydrogen, -CN, or a saturated or unsaturated C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms, or a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; L 1is a covalent bond or a straight or branched C optionally substituted with 1 to 9 fluoro groups 1~6 alkylene chain) to provide.

[0058] In some embodiments, R 1 is H. In some embodiments, R 1 is C 1~6 It is alkyl.

[0059] In some embodiments, R 1 is selected from those illustrated in Table 1 below.

[0060] In some embodiments, R 2 is R. In some embodiments, R 2 is -(CH2) n In some embodiments, R 2 is -C(O)R.

[0061] In some embodiments, R 2 is selected from those illustrated in Table 1 below.

[0062] In some embodiments, R 4’’ are each independently R, halogen, or —CF. In some embodiments, R 4’’ is R. In some embodiments, R 4’’ is a halogen. In some embodiments, R 4’’ is —CF. In some embodiments, R 4’’ is selected from those illustrated in Table 1 below.

[0063] In some embodiments, L 1 is a covalent bond or a straight or branched C optionally substituted with 1 to 9 fluoro groups 1~6 In some embodiments, the alkylene chain is an alkylene chain. , L 1 is a covalent bond. In some embodiments, L 1is a straight or branched C optionally substituted with 1 to 9 fluoro groups 1~6 In some embodiments, L 1 is selected from those illustrated in Table 1 below.

[0064] Exemplary compounds of the invention are set forth in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Table 1-18] [Table 1-19] [Table 1-20] [Table 1-21] [Table 1-22] [Table 1-23] [Table 1-24]

[0065] Exemplary compounds of the invention are set forth in Table 2 below. [Table 2-1] [Table 2-2]

[0066] In some embodiments, the present invention provides a compound set forth in Table 1 above, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a compound set forth in Table 2 above, or a pharmaceutically acceptable salt thereof. 5. Use, Formulation and Administration Pharmaceutically acceptable compositions

[0067] According to another embodiment, the present invention provides a composition comprising a compound of the present invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of the compound in the composition of the present invention is effective to measurably inhibit or activate Sestrin-GATOR2 interaction in a biological sample or a patient. In certain embodiments, the amount of the compound in the composition of the present invention is effective to measurably inhibit or activate Sestrin-GATOR2 interaction in a biological sample or a patient. In certain embodiments, the composition of the present invention is formulated for administration to a patient in need of such a composition. In some embodiments, the composition of the present invention is formulated for oral administration to a patient.

[0068] The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0069] The term "pharmaceutically acceptable carrier, adjuvant or vehicle" refers to a non-toxic 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 the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphate, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, phosphate These include potassium hydrogen, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0070] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, 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 the present invention can be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations can also be injectable sterile solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0071] For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for the formulation.

[0072] The pharmaceutically acceptable composition of the present invention can be orally administered in any oral acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants such as magnesium stearate are also usually added.For oral administration in the form of capsules, useful diluents include lactose and dry cornstarch.When aqueous suspensions are required for oral use, active ingredient is combined with emulsifiers and suspending agents.If desired, certain sweeteners, flavorings or coloring agents can also be added.

[0073] Alternatively, the pharmaceutically acceptable composition of the present invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, so that it melts in the rectum and releases the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0074] The pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations for each of these areas or organs are readily prepared.

[0075] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical transdermal patches may also be used.

[0076] For topical application, the provided pharmaceutically acceptable composition can be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, the provided pharmaceutically acceptable composition can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0077] For ophthalmic use, the provided pharmaceutically acceptable compositions may be formulated as a micronized suspension in pH-adjusted isotonic sterile saline, or as a solution in pH-adjusted isotonic sterile saline, preferably with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.

[0078] The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers which enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0079] Most preferably, the pharmaceutically acceptable composition of the present invention is formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, the pharmaceutically acceptable composition of the present invention is administered without food. In other embodiments, the pharmaceutically acceptable composition of the present invention is administered with food.

[0080] The amount of the compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending on the host treated and the particular mode of administration. Preferably, the provided compositions should be formulated so that a dosage of between 0.01 and 100 mg / kg body weight / day of the inhibitor can be administered to a patient receiving these compositions.

[0081] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, as well as the judgment of the treating physician and the severity of the specific disease being treated. The amount of the compound of the present invention in the composition will also depend on the specific compound in the composition. Uses of the Compounds and Pharmaceutically Acceptable Compositions

[0082] The compounds and compositions described herein are generally useful for inhibiting or activating Sestrin-GATOR2 interaction. In some embodiments, provided compounds or compositions thereof are activators of Sestrin-GATOR2 interaction.

[0083] The activity of the compounds utilized in this invention as inhibitors or activators of Sestrin-GATOR2 interaction may be determined in vitro, in vivo, or in a cell line. In vitro assays can be performed. In vitro assays include assays that determine inhibition or activation of Sestrin-GATOR2 interaction. An alternative in vitro assay quantifies the ability of an inhibitor or activator to decrease or increase binding of Sestrin to GATOR2. Detailed conditions for assaying compounds utilized in the present invention as inhibitors or activators of Sestrin-GATOR2 interaction are described in the Examples below.

[0084] As used herein, the terms "treatment," "treat," and "treating," as described herein, refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof. In some embodiments, treatment may be administered after the onset of one or more symptoms. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of symptom history and / or genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0085] The provided compounds are inhibitors or activators of Sestrin-GATOR2 interaction and are therefore useful for treating one or more disorders associated with the activity of mTORC1. Accordingly, in certain embodiments, the present invention provides a method for treating an mTORC1-mediated disorder, comprising administering a compound of the present invention or a pharmaceutically acceptable composition thereof to a patient in need of such treatment.

[0086] As used herein, the term "mTORC1-mediated" disorder, disease, and / or condition, as used herein, means any disease or other deleterious condition in which mTORC1 is known to play a role. Accordingly, another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which mTORC1 is known to play a role.

[0087] The methods described herein include methods for treating cancer in a subject. As used in this context, "treating" means improving or ameliorating at least one symptom or clinical parameter of cancer. For example, treatment can result in a decrease in tumor size or growth rate. Treatment does not need to cure cancer or cause 100% remission time in all subjects.

[0088] As described herein, the administration of an agent, such as an inhibitory nucleic acid or small molecule, that activates Sestrin-GATOR2 interaction, thereby reducing mTORC1 activity, reduces cancer cell proliferation, thereby treating cancer in a subject. Thus, in some embodiments, the methods described herein include administering a therapeutically effective dose of one or more agents that activate Sestrin-GATOR2 interaction, thereby indirectly inhibiting the mTORC1 pathway.

[0089] As used herein, the term "cancer" refers to cells capable of autonomous growth, i.e., an abnormal state or condition, characterized by rapid proliferative cell growth. The term includes all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. The term "tumor," as used herein, refers to a mass of cancerous cells, e.g., cancer cells.

[0090] Cancers that can be treated or diagnosed using the methods described herein include those affecting various organ systems, such as those affecting the lung, breast, thyroid, lymphoid, gastrointestinal, and genitourinary tracts. It includes malignant tumors and adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0091] In some embodiments, the methods described herein are used to treat or diagnose carcinoma in a subject. The term "carcinoma" is an art-recognized The term "carcinoma" refers to malignant tumors of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine gland cancer, and melanoma. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include, for example, malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0092] The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal derivation.

[0093] In some embodiments, the cancer treated by the methods described herein is a cancer in which the level of mTORC1 is increased or the expression or activity of mTORC1 is increased compared to normal tissue or other cancers of the same tissue. Methods known in the art and described herein can be used to identify such cancers. In some embodiments, the method includes obtaining a sample containing cancer cells, determining mTORC1 activity in the sample, and administering a treatment agent described herein (e.g., a modulator of Sestrin-GATOR2 interaction). In some embodiments, the cancer is one that has been shown herein to increase the level of mTORC1 activity.

[0094] In some embodiments, the present invention provides methods for treating one or more disorders, diseases and / or conditions, including but not limited to, cell proliferative disorders. Cell proliferative disorders

[0095] The present invention features methods and compositions for the diagnosis and prognosis of cell proliferation disorders (such as cancer), and for the treatment of these disorders by modulating Sestrin-GATOR2 interaction, thereby selectively modulating mTORC1 activity indirectly.Cell proliferation disorders described herein include, for example, cancer, obesity and proliferation-dependent diseases.Such disorders can be diagnosed using methods known in the art. cancer

[0096] Cancers include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous 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 (sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial cell carcinoma, leukemia ... iosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial tumor, mesothelioma, Ewing's tumor, 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 adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, 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 In some embodiments, the cancer is melanoma or breast cancer. Other proliferative disorders

[0097] Other proliferative diseases include, for example, obesity, benign prostatic hyperplasia, psoriasis, dyskeratinization, lymphoproliferative disorders (e.g., disorders in which there is abnormal proliferation of lymphoid cells), rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy. Proliferative diseases, incorporated herein by reference, are described in U.S. Patent Nos. 5,639,600 and 7,087,648. Other disorders

[0098] In some embodiments, the method of activating mTORC1 is used to treat ribosomal diseases (e.g., Diamond-Blackfan anemia, 5q syndrome, Shwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilage-hair hypoplasia, and Treacher Collins syndrome). (Payne et al., (2012) Blood. September 13; 120(11) Issue): 2214-24; Efeyan et al. (2012) Trends Mol Med. September; 18 ( (See, e.g., Vol. 9, pp. 524-533.) Accordingly, in some embodiments, the present invention provides a method of treating a ribosomal disease in a patient in need thereof, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof. In certain embodiments, the present invention provides a method of treating a ribosomal disease selected from Diamond-Blackfan anemia, 5q syndrome, Shwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilage-hair hypoplasia, or Treacher Collins syndrome in a patient in need thereof, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0099] In some embodiments, the method of activating mTORC1 activity is used to treat cohesinopathy (e.g., Roberts syndrome and Cornelia de Lange syndrome). (See Xu et al. (2016) BMC Genomics 17:25.) Thus, in some embodiments, the present invention provides a method of treating a cohesin disease (e.g., Roberts syndrome and Cornelia de Lange syndrome) in a patient in need thereof, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0100] In some embodiments, the method of activating mTORC1 is used to reverse or prevent muscle atrophy caused by lifestyle, disuse caused by orthopedic surgery, immobilization, or the age of the subject, or the disease or condition that the subject has or suffers from. (See Cuthbertson et al. (2005) FASEB J. March; 19 (3): 422-4, Epub 2004 December 13; Rennie, (2009) Appl. Physiol. Nutr. Metab. 34: 377-381; Ham et al. (2014) Clin Nutr. December; 33 (6): 937-45). Thus, in some embodiments, the present invention provides a method of reversing or preventing muscle atrophy in a patient in need thereof due to lifestyle, inactivity caused by orthopedic surgery, immobilization, or the age of the subject, or a disease or condition the subject has or is suffering from, comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0101] In some embodiments, the methods of activating mTORC1 can be used to treat bone fractures, severe burns, Reverse or prevent muscle atrophy due to spinal column injury, amputation, degenerative disease, conditions requiring the subject to recover in bed, stay in an intensive care unit, or prolonged hospitalization. (Gordon et al., (2013) Int J Biochem Cell Biol. October; 45(10):2147-57; Leger et al., (2009) Muscle Nerve. July; 40(1):69-78.) Accordingly, in some embodiments, the present invention provides a method of reversing or preventing muscle atrophy in a patient in need thereof due to fracture, severe burns, spinal column injury, amputation, degenerative disease, conditions requiring the subject to be confined to bed for recovery, stay in an intensive care unit, or prolonged hospitalization, comprising administering to said patient a provided compound or a pharmaceutically acceptable composition thereof.

[0102] In some embodiments, methods of activating mTORC1 are used to treat a disease, condition, or disorder that results in skeletal muscle atrophy, such as sarcopenia, muscle denervation, muscular dystrophy, inflammatory myopathy, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), or myasthenia gravis (Kye et al., (2014) Hum Mol Genet. Dec 1;23(23) ): pp. 6318-6331; Gurpur et al. (2009) Am J Pathol. March; Vol. 174(3): pp. 999-1008; Chauhan et al. (2013) Neurosci Res. September October; 77(1-2): 102-9; Ching et al. (2013) Hum Mol Genet. March 15; 22(6): 1167-79). In some embodiments, the present invention provides a method of treating a disease, condition, or disorder that results in 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 of such treatment, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0103] In some embodiments, methods of activating mTORC1 are used to prevent, maintain, or enhance muscle loss in subjects preparing for, participating in, or recently returned from space travel, respectively. (See Stein et al. (1999) Am J Physiol.; 276:E1014-21.) Thus, in some embodiments, the present invention provides a method for preventing, maintaining, or enhancing muscle loss in subjects in need of preventing, maintaining, or enhancing recovery from space travel, respectively, in subjects preparing for, participating in, or recently returned from space travel, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0104] In some embodiments, methods of activating mTORC1 are used to maintain or enhance recovery from excessive muscle tone and / or fatigue in subjects preparing for, participating in, or recently returning from armed conflict or military training (see Pasiakos et al. (2011) Am J Clin Nutr. September;94(3):809-18). Accordingly, in some embodiments, the present invention provides a method of maintaining or enhancing recovery from excessive muscle tone and / or fatigue in a subject in need thereof, such as a subject preparing for, participating in, or recently returning from armed conflict or military training, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0105] In some embodiments, the method of activating mTORC1 is used to inhibit autophagy in a patient. In some embodiments, the patient has a condition that is dependent on the induction of autophagy. have or are affected by treatment-resistant cancer (Kim and Guan, 2015 (See J Clin Invest. January; 125(1):25-32.) Accordingly, in some embodiments, the present invention provides methods of preventing autophagy in a patient in need thereof, the patient having or suffering from a treatment-resistant cancer dependent on the induction of autophagy, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0106] In some embodiments, methods of activating mTORC are used to treat or prevent depression. (See Ignacio et al. (2015) Br J Clin Pharmacol. November 27.) Accordingly, in some embodiments, the present invention provides a method of treating or preventing depression in a patient in need thereof, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0107] In some embodiments, methods of activating mTORC1 are used to induce rapid onset of antidepressant activity. Accordingly, in some embodiments, the present invention provides a method of inducing rapid onset of antidepressant activity in a patient in need thereof, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0108] In some embodiments, methods of activating mTORC1 are used to treat or prevent jet lag by accelerating the re-entrainment of circadian behavior in response to a shift in daylight / light cycle (see Cao et al. (2013) Neuron. Aug. 21;79(4):712-24, 10.1016). Accordingly, in some embodiments, the present invention provides a method of treating or preventing jet lag by accelerating the re-entrainment of circadian behavior in response to a shift in daylight / light cycle in a patient in need thereof, comprising administering to the patient a provided compound or a pharmaceutically acceptable composition thereof.

[0109] In some embodiments, a method of activating mTORC1 is used to prevent or reverse myocardial atrophy in a subject. In some embodiments, the subject has or has previously had a disease or condition selected from heart attack, congestive heart failure, heart transplant, heart valve repair, atherosclerosis, other major vascular disease, and cardiac bypass surgery (see Song et al. (2010) Am J Physiol Cell Physiol. December;299(6):C1256-C1266). Thus, in some embodiments, the present invention provides a method for preventing or reversing myocardial atrophy in a subject in need thereof, the method comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0110] In some embodiments, methods of activating mTORC1 are used to increase post-exercise strengthening and / or increase muscle mass. In some embodiments, the methods are performed as part of a parenteral total nutrition regimen or in conjunction with physical therapy to promote functional electrical stimulation (Nakamura et al., (2012) Geriatr Gerontol Int. Jan;12( (See, e.g., Vol. 1, pp. 131-9). Thus, in some embodiments, the present invention provides methods for increasing post-exercise strength and / or increasing muscle mass. In some embodiments, the methods provide a method for providing a portion of total parenteral nutrition to a subject in need thereof. The method is performed in conjunction with or in conjunction with physical therapy to promote functional electrical stimulation and includes administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0111] In some embodiments, methods of activating mTORC1 are used to reduce food intake. (Pedroso et al., (2015) Nutrients. May 22;7(5):3914-3 (See page 7.) Thus, in some embodiments, the present invention provides a method of reducing food intake in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0112] In some embodiments, the method of activating mTORC1 is used to treat obesity. Thus, in some embodiments, the present invention provides a method of treating obesity in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0113] In some embodiments, methods of activating mTORC1 are used to improve productivity in the production of therapeutic recombinant proteins from bioreactors (McVey et al., 2016). )Biotechnol Bioeng. February 16, doi:10.1002 / bit.25951 (See, e.g., J. Biol. Chem. Soc. 2009, 103:131-132, 2009). Thus, in some embodiments, the present invention provides methods for increasing productivity in the production of a therapeutic recombinant protein from a bioreactor, comprising adding to said production a provided compound or a pharmaceutically acceptable composition thereof.

[0114] In some embodiments, methods of activating mTORC1 in immune cells are used to promote and / or maintain their anti-tumor activity, including increasing mTORC1 in immune cells in vitro prior to adoptive transfer and increasing mTORC1 in immune cells in vivo upon co-administration with other targeted immunotherapy strategies. In some embodiments, the immune cells include naive T cells, CD4+ or CD8+ T cells, Th1, Th2, T Reg and Th17 cells, dendritic cells, NK cells, and macrophages (Yang et al., (2011) Nat Immunol.; 12:88 pp. 8~897; O'Brien et al. (2011) Eur J Immunol.; vol. 41: 3361~337 0; Delgoffe et al. (2009) Immunity. June 19; Vol. 30 (No. 6): 832~ 44 pages; Chi, (2012) Nat Rev Immunol. April 20; Volume 12 (No. 5): 32 5-338; Pollizzi et al. (2015) J Clin Invest.; Vol. 125(5): 2090-2108; Ali et al. (2015) Front Immunol.; Vol. 6: 355; Katholnig et al. (2013) Biochem Soc Trans. August; Vol. 41(4): 927-33; Wang et al. (2013) Proc Natl Acad Sci USA. December 10; Vol. 110(50): E4894-903; Yang and Chi (2013) J Clin Invest. December 123(12):5165-78). Thus, in some embodiments, the present invention provides methods of activating mTORC1 in immune cells to promote and / or maintain their anti-tumor activity. In some embodiments, the present invention provides methods of increasing mTORC1 in immune cells in vitro prior to adoptive transfer. In some embodiments, the present invention provides methods of increasing mTORC1 in immune cells when co-administered with other targeted immunotherapy strategies in vivo. In certain embodiments, the immune cells include naive T cells, CD4+ or CD8+ T cells, Th1, Th2, T Reg and Th17 cells, dendritic cells, NK cells and macrophages, comprising the step of adding to said immune cells a provided compound or a pharmaceutically acceptable composition thereof.

[0115] In some embodiments, methods of activating mTORC1 in the retina are used to treat retinitis pigmentosa and other forms of ocular neurodegeneration (see Punzo et al. (2009) Nat Neurosci. January;12(1):44-52). Thus, in some embodiments, In one aspect, the present invention provides a method of treating retinitis pigmentosa and other forms of ocular neurodegeneration in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0116] In some embodiments, methods of activating mTORC1 are used to increase central or peripheral axonal regeneration (see Namiko et al. (2010) J Biol Chem. 285:28034-28043). Accordingly, in some embodiments, the present invention provides a method of increasing central or peripheral axonal regeneration in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0117] In some embodiments, methods of activating mTORC1 are used to promote remyelination and neuronal activity after injury or in diseases characterized by demyelination, such as multiple sclerosis and Parkinson's disease (Tyler et al. (2009) J Neurosci. May 13;29 Volume (No. 19): pp. 6367-78; Norrmen et al. (2014) Cell Rep. October 23 (See, e.g., J. Clin. Pathol. Nov. 2006; 9(2):646-60; Love (2006) J. Clin. Pathol. Nov. 2006; 59(11):1151-1159.) Thus, in some embodiments, the present invention provides a method of promoting remyelination and neuronal activity after injury or in diseases characterized by demyelination in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method of treating Parkinson's disease in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0118] In some embodiments, methods of activating mTORC1 are used to treat multiple sclerosis. Accordingly, in some embodiments, the present invention provides methods of treating multiple sclerosis or variants thereof in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides methods of treating Barrow-concentric sclerosis, Schilder's disease, acute (Marburg) multiple sclerosis, inflammatory demyelinating polyneuropathy, or tumorous multiple sclerosis in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. a method for treating multiple sclerosis, the method comprising administering to the subject a compound or compound provided herein. or a pharmaceutically acceptable composition thereof.

[0119] In some embodiments, methods of activating mTORC1 are used to treat Devic's disease, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, and Niemann-Pick disease (see Takikita et al. (2004) J Neuropathol Exp Neurol. June;63(6):660-73). Thus, in some embodiments, the present invention provides methods for treating Devic's disease, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, and Niemann-Pick disease in subjects in need of such treatment. and Niemann-Pick disease, comprising administering to said subject a provided compound or a pharmaceutically acceptable composition thereof.

[0120] In some embodiments, methods of activating mTORC1 are used to treat or prevent forms of autism (Novarino et al., (2012) Science October 19, 338:61 (See, e.g., Issue 05, pages 394-397.) Accordingly, in some embodiments, the present invention provides a method of treating or preventing a form of autism in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0121] In some embodiments, the method of activating mTORC1 is used to treat a neurodegenerative disease. Thus, in some embodiments, the present invention provides a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0122] In some embodiments, the method of activating mTORC1 is used to treat diseases associated with synaptic dysfunction.Thus, in some embodiments, the present invention provides a method for treating diseases associated with synaptic dysfunction in a subject in need thereof, comprising administering to the subject a compound provided herein or a pharmaceutically acceptable composition thereof.

[0123] In some embodiments, methods of activating mTORC1 in the central nervous system are used to increase dendritogenesis and synaptogenesis in neurodegenerative diseases characterized by dendritic spine loss and synapse loss, such as Alzheimer's disease, amyotrophic lateral sclerosis, stroke, and glaucoma (Di Polo et al., (2015) Neural Regen Res. April; 10 (See Vol. (4): pp. 559-561). Thus, in some embodiments, the present invention provides a method of increasing dendrite formation and synaptogenesis in a neurodegenerative disease characterized by a decrease in dendritic spines and loss of synapses in a subject in need thereof, the method comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the present invention provides a method of treating Alzheimer's disease, amyotrophic lateral sclerosis, stroke, or glaucoma in a subject in need thereof, the method comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0124] In some embodiments, methods of 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 (Lin et al., PLoS ONE 8(4):e62572, 2013; Lee et al., (2015) Neuron. January 21; 85(2):303-315; Bowling et al., (2014) Sci Signal. January 14; 7(308):ra4). Thus, in some embodiments, the present invention provides methods of treating diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, schizophrenia, Rett syndrome, Fragile X syndrome, Parkinson's disease, Huntington's disease, stroke, and glaucoma in a subject in need thereof, comprising administering to the subject a provided compound or a pharmaceutically acceptable composition thereof.

[0125] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, such as by oral or nasal spray, enterally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment or drops), depending on the severity of the infection to be treated. In certain embodiments, the compounds of the present invention can be administered buccally. can be administered orally or parenterally, one or more times daily, at a dosage level of about 0.01 mg / kg to about 50 mg / kg, and preferably about 1 mg / kg to about 25 mg / kg of subject body weight per day, to obtain the desired therapeutic effect.

[0126] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain, for example, an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, the oral composition may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and fragrances.

[0127] Injectable preparations, for example, injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants, or wetting agents and suspending agents. Sterile injectable preparations can also be injectable sterile solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution (USP), and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently used as solvents or suspending media. For this purpose, any non-irritating, fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0128] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0129] To prolong the effect of the compounds of the present invention, it is often desirable to delay the absorption of the compound from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends on the dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, the absorption of a parenterally administered compound form can be delayed by dissolving or suspending it in an oil vehicle. Injectable depot formulations are made by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. The release rate of the compound can be controlled depending on the ratio of compound to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0130] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing a compound of the invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity to release the active compound.

[0131] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is present in at least one inert, pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate), and / or a) fillers or extenders (such as starch, lactose, sucrose, glucose, mannitol, and silicic acid), b) binders (such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), c) humectants (such as glycerol), d) disintegrants (such as agar-agar, calcium carbonate, potato starch, and the like). The composition may be mixed with any of the following: starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders (paraffin, etc.), f) absorption accelerators (quaternary ammonium compounds, etc.), g) wetting agents (cetyl alcohol and glycerol monostearate, etc.), h) absorbents (kaolin and bentonite clay, etc.), and i) lubricants (talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, these dosage forms may also contain buffering agents.

[0132] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical compounding art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. It may also be used as a filler.

[0133] The active compound can also be in microencapsulated form with one or more excipients, as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings well known in the pharmaceutical compounding art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. These dosage forms may also contain, as is common practice, additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only or preferentially in a certain portion of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0134] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed with a pharmaceutically acceptable carrier under sterile conditions, and any necessary preservatives or buffers may be required. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of the present invention. Furthermore, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0135] According to one embodiment, the present invention relates to a method for modulating Sestrin-GATOR2 interaction and thereby indirectly and selectively modulating mTORC1 activity in a biological sample, comprising the step of contacting said biological sample with a compound of the present invention or a composition comprising said compound.

[0136] The term "biological sample," as used herein, includes, but is not limited to, a cell culture or extract thereof; a biopsy obtained from a mammal or extract thereof; and blood, saliva, urine, stool, semen, tears, or other bodily fluids, or extracts thereof.

[0137] Another embodiment of the present invention relates to a method of modulating Sestrin-GATOR2 interaction, thereby indirectly and selectively modulating mTORC1 activity in a patient, comprising administering to said patient a compound of the present invention or a composition comprising said compound.

[0138] According to another embodiment, the present invention relates to a method for modulating Sestrin-GATOR2 interaction, thereby indirectly and selectively modulating mTORC1 activity in a patient, the method comprising administering to the patient a compound of the present invention or a composition comprising said compound. In another embodiment, the present invention relates to a method for treating an mTORC1-mediated disorder in a patient in need thereof, the method comprising administering to the patient a compound according to the present invention or a pharmaceutically acceptable composition thereof. Such disorders are described in detail herein.

[0139] Depending on the particular condition, or disease, being treated, additional therapeutic agents, which are normally administered to treat such condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated."

[0140] The compounds of the present invention may also be advantageously used in combination with other antiproliferative compounds, including, but not limited to, aromatase inhibitors, antiestrogens, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule-active compounds, alkylating compounds, histone deacetylase inhibitors, compounds that induce cell differentiation processes, cyclooxygenase inhibitors, MMP inhibitors, mTOR inhibitors, antitumor antimetabolites, platin compounds, compounds that target / inhibit protein kinase activity or lipid kinase activity, and additional antiangiogenic compounds, compounds that target, decrease or inhibit the activity of protein phosphatases or lipid phosphatases, gonadorelin agonists, antiandrogens, methionine agonists, and the like. aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds for use in the treatment of hematological malignancies; compounds that target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors (17-AAG (17-alkylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 (Conforma Therapeutics); temozolomide (Temodal®); kinesin spindle protein inhibitors (SB715992 or SB743921 (GlaxoSmithKline) or pentamidine / chlorpromazine (CombinatoRx); MEK inhibitors (ARRY142886 (Array BioPharma), AZD6244 (AstraZeneca), PD 181461 (Pfizer) and leucovorin. The term "aromatase inhibitor" as used herein refers to compounds that inhibit estrogen production, such as the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. This term includes, but is not limited to, steroids, particularly atamestane, exemestane, and formestane, and nonsteroids, particularly aminoglutethimide, rogletimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole, and letrozole. Exemestane is sold under the trade name Aromasin™. Formestane is sold under the trade name Lentaron™. Fadrozole is sold under the trade name Afema™. Anastrozole is sold under the trade name Arimidex™. Letrozole is sold under the brand names Femara™ or Femar™. Aminoglutethimide is sold under the brand name Orimeten™. Combinations of the present invention that include a chemotherapeutic agent that is an aromatase inhibitor are particularly useful in the treatment of hormone receptor positive tumors, such as breast tumors.

[0141] The term "anti-estrogen" as used herein refers to a compound that antagonizes 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 sold under the trade name Nolvadex™. Raloxifene hydrochloride is sold under the trade name Evista™. Fulvestrant can be administered under the trade name Faslodex™. The combination of the present invention, which includes a chemotherapeutic agent that is an anti-estrogen, is particularly useful for treating estrogen receptor-positive tumors, such as breast tumors.

[0142] The term "antiandrogen," as used herein, refers to any substance capable of inhibiting the biological action of androgen hormones, including, but not limited to, bicalutamide (Casodex™). The term "gonadorelin agonist," as used herein, includes, but is not limited to, abarelix, goserelin, and goserelin acetate. Goserelin may be administered under the trade name Zoladex™.

[0143] The term "topoisomerase I inhibitors" as used herein includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin, and the macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, for example, in the form as it is marketed, for example, under the trademark Camptosar™. Topotecan is marketed under the trademark Hycamptin™.

[0144] The term "topoisomerase II inhibitors," as used herein, includes, but is not limited to, anthracyclines such as doxorubicin (including liposomal formulations such as Caelyx™), daunorubicin, epirubicin, idarubicin, and nemorubicin; the anthraquinones mitoxantrone and losoxantrone; and the podophyllotoxins etoposide and teniposide. Etoposide is sold under the trade name Etopophos™. Teniposide is sold under the trade name VM26-Bristol. Doxorubicin is sold under the trade names Acriblastin™ or Adriamycin™. Epirubicin is sold under the trade name Farmorubicin™. Idarubicin is sold under the trade name Zavedos™. Mitoxantrone is sold under the trade name Novantron™.

[0145] The term "microtubule active agent" includes, but is not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermola The present invention relates to microtubule-stabilizing compounds, microtubule-destabilizing compounds, and microtubule polymerization inhibitors, including steroids; coticin, and epothilones and their derivatives. Paclitaxel is sold under the trade name Taxol™. Docetaxel is sold under the trade name Taxotere™. Vinblastine sulfate is sold under the trade name Vinblastin RP™. Vincristine sulfate is sold under the trade name Farmistin™.

[0146] The term "alkylating agent" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide is sold under the brand name Cyclostin™. Ifosfamide is sold under the brand name Holoxan™.

[0147] The term "histone deacetylase inhibitors" or "HDAC inhibitors" relates to compounds which inhibit histone deacetylase and which have antiproliferative activity, including but not limited to suberoylanilide hydroxamic acid (SAHA).

[0148] The term "antineoplastic 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 folate antagonists (such as pemetrexed). Capecitabine is sold under the trade name Xeloda™. Gemcitabine is sold under the trade name Gemzar™.

[0149] The term "platin compound" as used herein includes, but is not limited to, carboplatin, cisplatin, cisplatinum, and oxaliplatin. Carboplatin can be administered, e.g., in the form as it is marketed, e.g., under the trademark Carboplat™. Oxaliplatin can be administered, e.g., in the form as it is marketed, e.g., under the trademark Eloxatin™.

[0150] The term "compounds which target / increase protein or lipid kinase activity; or protein or lipid phosphatase activity; or further anti-angiogenic compounds", as used herein, includes, but is not limited to, protein tyrosine kinase inhibitors, and / or serine kinase inhibitors, and / or threonine kinase inhibitors, or lipid kinase inhibitors, such as (a) compounds which target, decrease or inhibit the activity of platelet-derived growth factor receptors (PDGFRs) (compounds which target, decrease or inhibit the activity of PDGFRs, especially N-phenyl-2-pyrimidine-amine derivatives such as imatinib, compounds which inhibit PDGF receptors such as SU101, SU6668 and GFB-111); b) compounds which target, decrease or inhibit the activity of fibroblast growth factor receptors (FGFRs); c) compounds which target, decrease or inhibit the activity of insulin-like growth factor receptor I (IGF-IR) ( compounds which target, reduce or inhibit the activity of IGF-IR, in particular compounds which inhibit the kinase activity of the IGF-I receptor), or antibodies which target the extracellular domain of the IGF-I receptor or its growth factors; d) compounds which target, reduce or inhibit the activity of the Trk receptor tyrosine kinase family, or ephrin B4 inhibitors; e) compounds which target, reduce or inhibit the activity of the AxI receptor tyrosine kinase family; f) compounds which target, reduce or inhibit the activity of the Ret receptor tyrosine kinase; g) compounds which target, reduce or inhibit the activity of the Kit / SCFR receptor tyrosine kinase, such as imatinib; h) compounds which target, reduce or inhibit the activity of the C-kit receptor tyrosine kinase, which is part of the PDGFR family, such as compounds which target, reduce or inhibit the activity of the c-Kit receptor tyrosine kinase family, in particular compounds which inhibit the c-Kit receptor, such as imatinib. i) compounds that target, decrease or inhibit the activity of c-Abl family members, their gene fusion products (e.g., BCR-Abl kinase) and mutants, such as compounds that target, decrease or inhibit the activity of c-Abl family members and their gene fusion products, such as N-phenyl-2-pyrimidine-amine derivatives, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC680410; PD173955 from ParkeDavis; or dasatinib (BMS-35 4825);j) compounds that target, decrease or inhibit the activity of members of the cyclin-dependent kinase family (CDK), including members of the protein kinase C (PKC) and the Raf family of serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and TEC families, and / or staurosporine derivatives such as midostaurin; further exemplary compounds are UCN-01, safingo k) compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, such as compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, including compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, including compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, such as mesylate, BAY43-9006, bryostatin 1, perifosine; limofosine; RO318220 and RO320432; GO6976; lsis3521; LY333531 / LY379196; isoquinoline compounds; FTIs; PD184352 or QAN697 (P13K inhibitors) or AT7519 (CDK inhibitors); imatinib nitrate (Gleevec™) or tyrphostins (tyrphostin A23 / RG-50810; AG99; tyrphostin AG213; tyrphostin AG1748; tyrphostin AG490; tyrphostin B44; tyrphostin B44(+) enantiomer; tyrphostin AG555; AG494; tyrphostin AG556, AG957, etc.), and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester; NSC680410, adaphostin);l) Compounds that target, decrease or inhibit the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR1, ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and variants thereof, such as compounds that target, decrease or inhibit the activity of the epidermal growth factor receptor family, in particular compounds that inhibit EGF receptor tyrosine kinase family members such as EGF receptor, ErbB2, ErbB3 and ErbB4, or that bind to EGF or EGF-related ligands. , protein or antibody, CP358774, ZD1839, ZM105180; trastuzumab (Herceptin™), cetuximab (Erbitux™), 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 which target, decrease or inhibit the activity of c-Met, especially c - compounds that target, decrease or inhibit the activity of the c-Met receptor, such as compounds that inhibit the kinase activity of the Met receptor, or antibodies that target the extracellular domain of c-Met or that bind to HGF, n) compounds that inhibit one or more JAK family members (JAK1 / JAK2 / JAK3), including but not limited to, PRT-062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, and ruxolitinib / TYK2 and / or pan-JAK), o) compounds that target, decrease 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, pictorelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765 and idelalisib, and;and q) hedgehog (Hh) or smoothened receptors, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib, and IPI-926 (saridegib); These compounds include compounds that target, decrease, or inhibit the signaling activity of the SMO pathway.

[0151] The term "PI3K inhibitor," as used herein, 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 useful in the present invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictorelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib.

[0152] The term "Bcl-2 inhibitors," as used herein, includes, but is not limited to, compounds having inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including, but not limited to, ABT-199, ABT-731, ABT-737, apogossypol, the pan-Bcl-2 inhibitors of Ascenta, curcumin (and its analogs), dual Bcl-2 / Bcl-xL inhibitors (Infinity Pharmaceuticals / Novartis Pharmaceuticals), genasense (G3139), HA14-1 (and its analogs; see WO2008118802), navitoclax (and its analogs, see US7390799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and its analogs, see WO2004106328), S-001 (Gloria Pharmaceuticals), the TW series of compounds (Univ. of Michigan), and venetoclax. In some embodiments, the Bcl-2 inhibitor is a small molecule therapeutic agent. In some embodiments, the Bcl-2 inhibitor is a peptidomimetic.

[0153] The term "BTK inhibitor," as used herein, 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.

[0154] The term "SYK inhibitor," as used herein, refers to compounds that have inhibitory activity against spleen tyrosine kinase (SYK), including, but not limited to, PRT-062070, R-343, R-333, Excelea, PRT-062607, and fostamatinib.

[0155] Further examples of BTK inhibitory compounds, and conditions treatable by such compounds in combination with the compounds of the present invention, can be found in WO2008039218 and WO2011090760, which are incorporated by reference in their entireties.

[0156] Further examples of SKY inhibitory compounds, and conditions treatable by such compounds in combination with the compounds of the present invention, can be found in WO2003063794, WO2005007623 and WO2006078846, which are incorporated herein by reference in their entireties.

[0157] Further examples of PI3K inhibitory compounds, and conditions treatable by such compounds in combination with the compounds of the present invention, are described in WO2004019973, WO2004089925, WO2007016176, which are incorporated herein by reference in their entireties. , US8138347, WO2002088112, WO2007084786, WO2007129161, WO2006122806, WO2005113554 and WO2007044729.

[0158] Further examples of JAK inhibitory compounds, and conditions treatable by such compounds in combination with the compounds of the present invention, can be found in WO2009114512, WO2008109943, WO2007053452, WO2000142246, and WO2007070514, which are incorporated by reference in their entireties.

[0159] Additional anti-angiogenic compounds include compounds that have another mechanism of activity, such as protein or lipid kinase inhibition, such as a mechanism unrelated to thalidomide (Thalomid™) and TNP-470.

[0160] Examples of proteasome inhibitors useful in combination with the compounds of the invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0161] Compounds which target, decrease or inhibit the activity of protein or lipid phosphatases are eg inhibitors of phosphatase 1, phosphatase 2A or CDC25, such as okadaic acid or a derivative thereof.

[0162] Compounds that induce cell differentiation processes include, but are not limited to, retinoic acid, α-, γ-, or δ-tocopherol, or α-, γ-, or δ-tocotrienol.

[0163] The term cyclooxygenase inhibitors, as used herein, includes, but is not limited to, Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acids and derivatives (such as celecoxib (Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib, or 5-alkyl-2-arylaminophenylacetic acids (such as 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenylacetic acid, i.e., lumiracoxib).

[0164] The term "bisphosphonate," as used herein, includes, but is not limited to, etridonic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid, and zoledronic acid. Etridonic acid is sold under the trade name Didronel™. Clodronic acid is sold under the trade name Bonefos™. Tiludronic acid is sold under the trade name Skelid™. Pamidronic acid is sold under the trade name Aredia™. Alendronic acid is sold under the trade name Fosamax™. Ibandronic acid is sold under the trade name Bondranat™. Risedronic acid is sold under the trade name Actonel™. Zoledronic acid is sold under the trade name Zometa™. The term "mTOR inhibitors" relates to compounds which inhibit the mammalian target of rapamycin (mTOR) and which have antiproliferative activity, such as sirolimus (Rapamune®), everolimus (Certican™), CCI-779 and ABT578.

[0165] The term "heparanase inhibitor," as used herein, refers to a compound that targets, decreases, or inhibits heparin sulfate degradation. This term includes, but is not limited to, PI-88. The term "biological response modifier," as used herein, refers to a lymphokine or interferon.

[0166] The term "inhibitor of Ras oncogenic isoforms" as used herein refers to compounds that target, reduce or inhibit the oncogenic activity of Ras, such as H-Ras, K-Ras or N-Ras; for example, "farnesyltransferase inhibitors" such as L-744832, DK8G557 or R115777 (Zarnestra™).The term "telomerase inhibitor" as used herein refers to compounds that target, reduce or inhibit the activity of telomerase.The compound that targets, reduces or inhibits the activity of telomerase is particularly a compound that inhibits telomerase receptor, such as telomestatin.

[0167] The term "methionine aminopeptidase inhibitor" as used herein refers to a compound that targets, decreases or inhibits the activity of methionine aminopeptidase. Compounds that target, decreases or inhibit the activity of methionine aminopeptidase include, but are not limited to, bengamide or its derivatives.

[0168] The term "proteasome inhibitor," as used herein, refers to a compound that targets, decreases, or inhibits the activity of the proteasome. Compounds that target, decrease, or inhibit the activity of the proteasome include, but are not limited to, bortezomib (Velcade™) and MLN341.

[0169] The term "matrix metalloproteinase inhibitors" or ("MMP" inhibitors), as used herein, includes, but is not limited to, collagen peptidomimetic inhibitors and collagen non-peptidomimetic inhibitors, tetracycline derivatives, such as the hydroxamate peptidomimetic inhibitor batimastat and its orally bioavailable analogs marimastat (BB-2516), prinomastat (AG3340), metastat (NSC683551), BMS-279251, BAY12-9566, TAA211, MMI270B, or AAJ996.

[0170] The term "compounds used in the treatment of hematological malignancies," as used herein, includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, decrease or inhibit the activity of FMS-like tyrosine kinase receptor (Flt-3R); interferon, 1-β-D-arabinofuransylcytosine (ara-c), and bisulfan; and ALK inhibitors, which are compounds that target, decrease or inhibit anaplastic lymphoma kinase.

[0171] Compounds that target, decrease or inhibit the activity of Flt-3R are compounds, proteins or antibodies that inhibit Flt-3R receptor kinase family members, such as PKC412, midostaurin, staurosporine derivatives, SU11248 and MLN518, among others.

[0172] The term "HSP90 inhibitor," as used herein, includes, but is not limited to, compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90; compounds that target, reduce, or inhibit the degradation of HSP90 client proteins via the ubiquitin proteasome pathway. Compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90 include, among others, compounds, proteins, or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino, 17-demethoxygeldanamycin (17AAG), geldanamycin derivatives; other geldanamycin-related compounds; radicicol, and HDAC inhibitors.

[0173] The term "antiproliferative antibody" as used herein includes, but is not limited to, trastuzumab. This includes mab (Herceptin™), trastuzumab-DM1, erbitux, bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40), and 2C4 antibodies. By antibody is meant intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies and antibody fragments, so long as they exhibit the desired biological activity.

[0174] For the treatment of acute myeloid leukemia (AML), the compound of the present invention can be used in combination with standard leukemia therapy, especially in combination with the therapy used for the treatment of AML.In particular, the compound of the present invention can be administered in combination with other drugs useful for the treatment of AML, such as farnesyltransferase inhibitors and / or daunorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatin and PKC412.

[0175] Other anti-leukemia compounds include, for example, Ara-C, a pyrimidine analog, which is a 2'-alpha-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are purine derivatives 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 suberoylanilide hydroxamic 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-propenamide or a pharmaceutically acceptable salt thereof, and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide or a pharmaceutically acceptable salt thereof, especially lactate. Somatostatin receptor antagonists, as used herein, refer to compounds that target, treat, or inhibit somatostatin receptors, such as octreotide and SOM230. Methods of damaging tumor cells refer to methods such as ionizing radiation. The term "ionizing radiation," referred to above and hereinafter, means ionizing radiation that occurs either as electromagnetic waves (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is delivered in, but not limited to, radiation therapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al. (eds.), 4th ed., Vol. 1, pp. 248-275 (1993).

[0176] Similarly, EDG binders and ribonucleotide reductase inhibitors are also included. The term "EDG binders" as used herein refers to a class of immunosuppressants that modulate lymphocyte recirculation, such as FTY720. The term "ribonucleotide reductase inhibitors" includes, but is not limited to, fludarabine, and / or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptoethanol, 5-methyl-2-methyl-2-propanol, 5 ... refers to pyrimidine or purine nucleoside analogs, including putopurine (especially in combination with ara-C for ALL), and / or pentostatin. Ribonucleotide reductase inhibitors are, inter alia, hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0177] Similarly, such compounds, proteins or monoclonal antibodies of VEGF such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate. anthranilamide; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies such as rhuMAb and RHUFab, VEGF aptamers such as makgon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibodies, angiozyme (RPI4610) and bevacizumab (Avastin™).

[0178] Photodynamic therapy, as used herein, refers to a treatment that uses certain chemicals known as photosensitizing compounds to treat or prevent cancer. Examples of photodynamic therapy include treatment with compounds such as Visudyne™ and porfimer sodium.

[0179] Angiostatic steroid, as used herein, refers to compounds that block or inhibit angiogenesis, such as, for example, anecortave, triamcinolone, hydrocortisone, 11-α-epihydrocortisol, cortexolone, 17α-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone, and dextasazone.

[0180] Corticosteroid-containing implants include fluocinolone and dextromethorphan. It refers to compounds such as sazon.

[0181] Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or other compounds or compounds with other or unknown mechanisms of action.

[0182] The structures of the active compounds identified by code numbers, generic names or trade names can be found from the current edition of the standard compendium "The Merck Index" or from databases such as Patents Int. It is available from international sources (e.g., IMS World Publications).

[0183] The compounds of the present invention may also be used in combination with known therapeutic methods, such as hormone administration or radiation therapy. In certain embodiments, provided compounds are used as radiosensitizers, particularly for the treatment of tumors that exhibit poor sensitivity to radiation therapy.

[0184] The compounds of the present invention can be administered alone or in combination with one or more other therapeutic compounds. Possible combination therapies include fixed combinations, or administration of the compounds of the present invention and one or more other therapeutic compounds at different times or independently, or the combined administration of a fixed combination with one or more other therapeutic compounds. The compounds of the present invention can also be administered for tumor treatment in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination thereof, among others. Long-term therapy is equally possible as adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments include therapy to maintain the patient's condition after tumor regression, or chemopreventive therapy, for example, in at-risk patients.

[0185] The additional agents may be administered separately from a composition containing a compound of this invention as part of a multiple dose regimen. Alternatively, the agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition. When administered as part of a multiple dose regimen, the two active agents may be administered simultaneously, sequentially, or usually within 5 hours of each other. The administration of the two drugs can be performed within a period of time of each other.

[0186] As used herein, the terms "combination," "in combination," and related terms refer to simultaneous or sequential administration of therapeutic agents according to the present invention. For example, a compound of the present invention may be administered simultaneously with another therapeutic agent, or sequentially in separate unit dosage forms, or together in a single unit dosage form. Thus, the present invention provides a single unit dosage form comprising a compound of the present invention, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0187] The amounts of both the compounds of the present invention and the additional therapeutic agent (in those compositions containing such additional therapeutic agents) that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated so that the compound of the present invention can be administered at a dosage of between 0.01 and 100 mg / kg body weight / day.

[0188] In such compositions containing an additional therapeutic agent, the additional therapeutic agent and the compound of the present invention may act synergistically. Thus, the amount of the additional therapeutic agent in such compositions is less than the amount required for a monotherapy utilizing only that therapeutic agent. In such compositions, the additional therapeutic agent may be administered at a dosage of between 0.01 and 1,000 μg / kg body weight / day.

[0189] The amount of additional therapeutic agent present in the compositions of the invention is no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the sole active agent. Preferably, the amount of additional therapeutic agent in the disclosed compositions is in the range of about 50% to 100% of the amount that would normally be present in a composition comprising that agent as the sole therapeutic active agent.

[0190] The compounds of the present invention or pharmaceutical compositions thereof may also be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents, and catheters. For example, vascular stents have been used to overcome restenosis (re-narrowing of the blood vessel wall after injury). However, patients who use stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can be prevented or reduced by pre-coating the device with a pharmaceutically acceptable composition containing a kinase inhibitor. An implantable device coated with the compounds of the present invention is another embodiment of the present invention. [Example]

[0191] Example As illustrated in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures: While the general methods illustrate the synthesis of certain compounds of the invention, it will be recognized that the following general methods, and others known to those of skill in the art, are applicable to all compounds described herein, and each subclass and species of these compounds.

[0192] List of abbreviations used in experimental items. 4A MS: 4Å molecular sieve AcOH: acetic acid ACN: acetonitrile Anhyd: Anhydrous Aq: Aqueous, aqueous solution Bn: Benzyl Boc: tert-butoxycarbonyl CbzCl: benzyl chloroformate Cbz-OSU: N-(benzyloxycarbonyloxy)succinimide Cu(OAc)2: Copper(II) acetate d: number of days DAST: Diethylaminosulfur trifluoride DBU: 1,8-diazobicyclo[5.4.0]undec-7-ene DCE: 1,2-dichloroethane DCM: dichloromethane DEA: Diethylamine DIBAL-H: Diisobutylaluminum hydride DIPEA: N,N-diisopropylethylamine DMA: N,N-dimethylacetamide DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMSO - Dimethyl sulfoxide DPPA: Diphenylphosphoryl azide EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ee: enantiomeric excess ESI: electrospray ionization Et3N: Triethylamine Et2O: Diethyl ether EtOAc: ethyl acetate EtOH: ethanol Fmoc: fluorenylmethyloxycarbonyl Fmoc-OSu:N-(9-fluorenylmethoxycarbonyloxy)succinimide h:h HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HCOONH4: Ammonium formate HPLC: High-performance liquid chromatography IBX: 2-iodoxybenzoic acid IPA: Isopropyl alcohol KOAc: Potassium acetate M: Molar concentration Me: Methyl MeOH: Methanol mins: minutes mL: milliliter mM: millimolar concentration mmol: millimolar MTBE: Methyl tert-butyl ether NaBH3CN: sodium cyanoborohydride Na2CO3: Sodium carbonate NaHCO3: Sodium bicarbonate NMP: N-methylpyrrolidine NMR: nuclear magnetic resonance o C: Celsius PBS: phosphate buffered saline Pd / C: Palladium on carbon Pd(OH)2 / C: Pearlman catalyst PE: Petroleum ether PhNH2: Aniline PPh3: Triphenylphosphine Rel: Relative rt: room temperature sat: saturation SFC: Supercritical Fluid Chromatography SOCl2: Thionyl chloride TBAB: tetra-n-butylammonium bromide tBuOK: potassium tert-butoxide TEA: Triethylamine Tf: Trifluoromethanesulfonate TfAA: Trifluoromethanesulfonic anhydride TFA: trifluoroacetic acid TIPS: Triisopropylsilyl THF: tetrahydrofuran TMSCN: Trimethylsilyl cyanide pTSA: para-toluenesulfonic acid TsOH: p-toluenesulfonic acid

[0193] The preparation of representative, non-limiting examples of compounds provided below is described.

[0194] (Example 1) (S)-2-(Dimethylamino)-4-methylpentanoic acid [I-1]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-2-(dimethylamino)-4-methylpentanoic acid:

[0195] To a solution of (S)-2-amino-4-methylpentanoic acid (2.0 g, 15.24 mmol), formaldehyde (38%, 24.0 g) and Pd / C (10%, 500 mg) were added, and the resulting solution was filtered (60 mL). The mixture was hydrogenated at room temperature for 2 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 (400 MHz, MeOD-d4): δ 3.47 (dd, J = 4.4 Hz, 10.0 Hz, 1H), 2.85 (S, 6H), 1.89-1.74 (m, 2H), 1.62-1.55 (m, 1H), 1.00 (dd, J = 2.8 Hz, 6.8Hz, 6H).

[0196] (Examples 2 and 3) (S)-2-Amino-7,7,7-trifluoroheptanoic acid hydrochloride Salt [I-2] and (R)-2-amino-7,7,7-trifluoroheptanoic acid hydrochloride [I-3]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: 1,1,1-trifluoro-5-iodopentane:

[0197] To a solution of 5,5,5-trifluoropentan-1-ol (2.0 g, 14.0 mmol), imidazole (1.48 g, 21.7 mmol), and PPh (5.5 g, 21.0 mmol) in DCM (40 mL) was added I (4.45 g, 17.5 mmol) using an ice bath. The mixture was warmed to room temperature and stirred overnight. EtO (50 mL) was added to the above mixture, which was then stirred for 10 minutes. The mixture was filtered, and the filtrate was evaporated at 65 °C under atmospheric pressure to remove the solvent. The residue was diluted with EtO (30 mL), the mixture was filtered, and the filtrate was used in the next step. Step 2: (S)-tert-butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate and (R)-tert-butyl 2-(diphenylmethylene (amino)-7,7,7-trifluoroheptanoate:

[0198] To a solution of tert-butyl 2-(diphenylmethyleneamino)acetate (2.0 g, 6.78 mmol) and TBAB (109 mg, 0.339 mmol) in toluene (35 mL) and DCM (15 mL) was added KOH (50%, 20 mL) at −10° C. After 5 min, the above solution of 1,1,1-trifluoro-5-iodopentane in EtO (30 mL) was added dropwise over 5 min, and the resulting mixture was stirred at −10° C. to 0° C. for 1 h. 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 (Na 2 SO 4 ), filtered and concentrated in vacuo, and the crude product was purified by chromatography (silica, ethyl acetate / petroleum ether=1 / 10) and then purified by chiral preparative HPLC [column, R,R-whelk-ol 4.6 *250 mm 5 um; solvent, MeOH (0.2% methanolic ammonia)] to give (S)-tert-butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol, 7.1%) and (R)-tert-butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol, 7.1%).

[0199] (S)-tert-Butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol, 7.1%). ESI-MS (EI+, m / z): 243.1 [M+H]+. 1 H-NMR (500 MHz, CDCl3): δ 8.64 (d, J = 8.0 Hz, 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 = 5.5 Hz, 7.5 Hz, 1H), 2.00-2.05 (m, 2H), 1.88-1.92 (m, 2H), 1.31-1.52 (m, 13H).

[0200] (R)-tert-Butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol, 7.1%). ESI-MS (EI+, m / z): 243.1 [M+H]+. 1 H-NMR (500 MHz, CDCl3): δ 8.64 (d, J = 7.0 Hz, 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 = 5.5 Hz, 7.5 Hz, 1H), 2.00-2.05 (m, 2H), 1.88-1.92 (m, 2H), 1.31-1.52 (m, 13H).

[0201] Step 3: (S)-2-Amino-7,7,7-trifluoroheptanoic acid hydrochloride [I-2]:

[0202] A solution of (S)-tert-butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol) in 6 M HCl (10 mL) and dioxane (5 mL) was heated to 100 °C for 17 h. The solution was extracted with EtO (10 mL × 2), and the aqueous phase was concentrated to dryness to give (S)-2-amino-7,7,7-trifluoroheptanoic acid hydrochloride (I-2) as a white solid (82.7 mg, 0.35 mmol, 74%). ESI-MS (EI+, m / z): 200.1 [M+H]+. 1H NMR (500 MHz, DO) 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).

[0203] Step 4: (R)-2-amino-7,7,7-trifluoroheptanoic acid hydrochloride [I-3]:

[0204] A solution of (R)-tert-butyl 2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol) in 6 M HCl (10 mL) and dioxane (5 mL) was heated to 100 °C for 17 h. The solution was extracted with EtO (10 mL × 2) and the aqueous phase was concentrated to dryness to give (R)-2-amino-7,7,7-trifluoroheptanoate (200 mg, 0.48 mmol). Trifluoroheptanoic acid hydrochloride (I-3) was obtained as a white solid (91.6 mg, 0.39 mmol, 82%). ESI-MS (EI+, m / z): 200.1 [M+H]+. 1H NMR (500 MHz, DO) 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).

[0205] (Examples 4 and 5) (S)-2-amino-4,4,4-trifluorobutanoic acid [I-4] and (R)-2-amino-4,4,4-trifluorobutanoic acid [I-5]. [ka] Synthesis scheme: [ka] Procedures and characterization:

[0206] Step 1: (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid

[0207] To a solution of 2-amino-4,4,4-trifluorobutanoic acid (1.0 g, 6.36 mmol) and NaHCO3 (589 mg, 7.01 mmol) in acetone (60 mL), N-(benzyloxycarbonyloxy)succinimide (1.75 g, 7.01 mmol) was slowly added, and the resulting solution (60 mL) was filtered at 0 °C. The mixture was stirred at room temperature for 16 h. The reaction mixture was extracted with CHCl2 (2 × 100 mL), and the aqueous layer was acidified with HCl (3 M) to approximately pH 4, followed by extraction with EtOAc (3 × 150 mL). The organic phase was dried over Na2SO4, and the solvent was evaporated under vacuum. The resulting crude product was purified by chiral preparative HPLC (column, AY-H 4.6 * Purification by HPLC (250 mm 5 μm; solvent, EtOH) gave (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid (700 mg, 2.40 mmol, 37.8%) and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid (700 mg, 2.40 mmol, 37.8%) as a white solid. ESI-MS (EI+, m / z): 314.0 [M+Na]+.

[0208] (S)-2-(Benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid.1 H-NMR (500 MHz, DMSO-d6): δ 13.20 (s, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.40-7.30 (m, 5H), 5.06 (s, 2H), 4.31-4.27 (m, 1H), 2.85-2.58 (m, 2H).

[0209] (R)-2-(Benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid. 1 H-NMR (500 MHz, DMSO-d6): δ 13.21 (s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.38-7.30 (m, 5H), 5.06 (s, 2H), 4.31-4.27 (m, 1H), 2.83-2.59 (m, 2H).

[0210] Step 2: (S)-2-Amino-4,4,4-trifluorobutanoic acid [I-4].

[0211] A mixture of (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid (700 mg, 2.40 mmol) and 10% Pd / C (200 mg) in MeOH (50 mL) was stirred under a hydrogen atmosphere at room temperature for 2 h. 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-trifluorobutanoic 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 DO): δ 4.32 (t, J = 6.0 Hz, 1H), 3.03-2.82 (m, 2H).

[0212] Step 3: (R)-2-Amino-4,4,4-trifluorobutanoic acid [I-5].

[0213] A mixture of (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutanoic acid (700 mg, 2.40 mmol) and 10% Pd / C (200 mg) in MeOH (50 mL) was stirred under a hydrogen atmosphere at room temperature 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-4,4,4-trifluorobutanoic acid (I-5) (250 mg, 1.59 mmol, 66.3%) as a white solid. ESI-MS (EI + , m / z): 158.1 [M+H] + . 1 H-NMR (500 MHz, DMSO-d + 1 drop TFA + 1 drop DO): δ 4.31 (t, J = 6.0 Hz, 1H), 3.03-2.83 (m, 2H).

[0214] (Examples 6 and 7) (S)-2-amino-5,5,5-trifluoropentanoic acid [I-6] and (R)-2-amino-5,5,5-trifluoropentanoic acid [I-7]. [ka] Synthesis scheme: [ka] Procedures and characterization:

[0215] Step 1: 4,4,4-trifluorobutanal:

[0216] To a solution of 4,4,4-trifluorobutan-1-ol (4.0 g, 31.3 mmol) in DMSO (80 mL) was added IBX (13.0 g, 46.9 mmol) under ice bath. The mixture was warmed to room temperature and stirred overnight. The reaction mixture was poured into water (200 mL) and extracted with EtO (100 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (NaSO), and used in the next step.

[0217] Step 2: 2-(benzylamino)-5,5,5-trifluoropentanenitrile:

[0218] To a solution of the above 4,4,4-trifluorobutanal in EtO (200 mL) was added benzylamine (4 mL), AcOH (3.0 mL), and then TMSCN (3.5 mL) using an ice bath. The mixture was warmed 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 (NaSO), filtered, and concentrated in vacuo to give 2-(benzylamino)-5,5,5-trifluoropentanenitrile (6.7 g, crude) as a brown solid, which was used in the next step. ESI-MS (EI+, m / z): 243.1 [M+H]+.

[0219] Step 3: 2-(benzylamino)-5,5,5-trifluoropentanoic acid:

[0220] 2-(benzylamino)-5, in concentrated HCl (80 mL) and AcOH (30 mL) A solution of 5,5-trifluoropentanenitrile (6.7 g, crude) was heated to 95 °C for 17 h. The solution was concentrated to dryness and diluted with ACN (50 mL). The resulting solution was filtered (100 mL). The pH was adjusted to 3-4 with saturated NaHCO solution. The mixture was filtered and dried to give 2-(benzylamino)-5,5,5-trifluoropentanoic acid (3.5 g, 13.4 mmol, 43% over 3 steps) as a white solid. ESI-MS (EI + , m / z): 262.1 [M+H] + .

[0221] Step 4: 2-Amino-5,5,5-trifluoropentanoic acid:

[0222] A mixture of 2-(benzylamino)-5,5,5-trifluoropentanoic 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 h. The mixture was filtered, and the filtrate was concentrated to dryness to give 2-amino-5,5,5-trifluoropentanoic acid (3.0 g, crude) as a brown solid. ESI-MS (EI+, m / z): 172.2 [M+H]+.

[0223] Step 5: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid:

[0224] To a solution of 2-amino-5,5,5-trifluoropentanoic acid (3.0 g, crude) in saturated NaHCO3 (100 mL) and acetone (100 mL) was added Cbz-OSu (3.45 g, 13.9 mmol) using an ice bath. After 2 h, the mixture was adjusted to pH 3 with 6 M HCl and extracted with EtOAc (50 mL × 2). The organic phase was washed with water (50 mL) and brine (100 mL), dried (Na2SO4), concentrated in vacuo, and the crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 2) and then purified by chiral preparative HPLC [column, AY-H 4.6 * Purification by HPLC using a 250 mm column (5 μm column); solvent, MeOH (0.5% NHOH)] afforded (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid (1.50 g, 4.92 mmol, 28%, two steps) and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid (1.50 g, 4.92 mmol, 28% overall for two steps) as white solids.

[0225] (S)-2-(Benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid (1.50 g, 4.92 mmol, 28% over two 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).

[0226] (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic 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).

[0227] Step 6: (S)-2-Amino-5,5,5-trifluoropentanoic acid [I-6]:

[0228] (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid (500 mg, 1.64 mmol) and MeO on Pd / C (10%) (50 mg) The mixture in H (20 mL) was stirred under hydrogen at room temperature for 2 h. 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-trifluoropentanoic 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 (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).

[0229] Step 7: (R)-2-amino-5,5,5-trifluoropentanoic acid [I-7]:

[0230] A mixture of (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoropentanoic acid (500 mg, 1.64 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred under hydrogen for 2 h at room temperature. 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-trifluoropentanoic 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).

[0231] (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]. [ka] Synthesis scheme: [ka] Procedures and characterization:

[0232] Step 1: (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid and (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid

[0233] 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) was slowly added benzyl chloroformate (554 mg, 3.25 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 h. The reaction mixture was extracted with DCM (2 × 100 mL), and the aqueous layer was acidified with HCl (3 M) to approximately pH 4, followed by extraction with EtOAc (3 × 50 mL). The organic phase was dried over NaSO, and the solvent was evaporated under vacuum. The resulting crude product was purified by chiral preparative HPLC (column: AY-H (250 * 4.6mm 5um); Purification with a mobile phase of n-hexane (0.1% DEA):EtOH (0.1% DEA) = 90:10 gave (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]+.

[0234] (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid, 1H-NMR (500 MHz, DMSO-d6): δ 12.68 (s, 1H), 7.66 (d, J = 7.5 Hz, 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).

[0235] (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid, 1H-NMR (500 MHz, DMSO-d6): δ 12.68 (s, 1H), 7.67 (d, J = 8.5 Hz, 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).

[0236] Step 2: (S)-2-Amino-6,6,6-trifluorohexanoic acid [I-8].

[0237] A mixture of (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (200 mg, 0.63 mmol) and 10% Pd / C (50 mg) in MeOH (20 mL) was stirred under a hydrogen atmosphere at room temperature 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 H-NMR (500 MHz, DMSO-d + 1 drop TFA + 1 drop DO): 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).

[0238] Step 3: (R)-2-Amino-6,6,6-trifluorohexanoic acid [I-9].

[0239] A mixture of (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (250 mg, 0.78 mmol) and 10% Pd / C (50 mg) in MeOH (20 mL) was stirred under a hydrogen atmosphere at room temperature 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] + . 1 H-NMR (500 MHz, DMSO-d + 1 drop TFA + 1 drop DO): 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).

[0240] (Example 11) (S)-2-(benzylamino)-4-methylpentanoic acid [I-11]. [ka] Synthesis scheme: [ka] Procedures and characterization:

[0241] Step 1: (S)-Benzyl 2-(benzylamino)-4-methylpentanoate:

[0242] To a stirred solution of p-toluenesulfonic acid L-leucine benzyl ester (800 mg, 2.0 mmol) in MeOH (30 mL) was added benzaldehyde (0.26 g, 2.4 mmol) and potassium acetate (0.4 g, 4.1 mmol), and the mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (0.2 g, 3.0 mmol) was then added, and the mixture was stirred at room temperature for an additional 5 hours. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL x 2), washed with brine (50 mL), and the resulting solution was filtered (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21 * Purification by 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-(benzylamino)-4-methylpentanoate (200 mg, 0.64 mmol, 32%) as a colorless oil. MS (EI+, m / z): 312.3 [M+H]+. 1H-NMR (500 MHz, , MeOD): δ 7.41~7.49 (m, 10H), 5.34 (dd, J = 12.0 Hz, 45.0 Hz, 2H), 4.23 (q, J = 12.0 Hz, 2H), 4.07~4.09 (m, 3H), 1.68~1.85 (m, 3H), 0.94 (dd, J = 8.5 Hz, 20.5 Hz, 6H).

[0243] Step 2: (S)-2-(benzylamino)-4-methylpentanoic acid [I-11]:

[0244] To a stirred solution of (S)-benzyl 2-(benzylamino)-4-methylpentanoate (50 mg, 0.16 mmol) in MeOH (5 mL) was added 1 M NaOH (0.5 mL). The reaction was stirred at room temperature for 4 h. The resulting solution was concentrated and the residue was purified by preparative HPLC (Boston C18 21 *Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) afforded (S)-2-(benzylamino)-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 (500 MHz, DMSO-d6): δ 9.32 (s, 1H), 7.43–7.50 (m, 5H), 4.17 (dd, J = 13.0 Hz, 44.0 Hz, 2H), 3.82 (t, J = 6.5 Hz, 1H), 1.68–1.76 (m, 3H), 0.85~0.90 (m, 6H).

[0245] (Example 12) (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid [ I-12]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0246] Step 1: (S)-Benzyl 4-methyl-2-(2-phenylacetamido)pentanoate:

[0247] To a solution of p-toluenesulfonic acid L-leucine benzyl ester (500 mg, 1.27 mmol), 2-phenylacetic acid (260 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL) was added DIPEA (410 mg, 3.18 mmol), and the solution was stirred at room temperature for 2 hours. This solution was purified by preparative HPLC (Boston C18 21 *Purification by 250 mm 10 μm (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 4-methyl-2-(2-phenylacetamido)pentanoate (300 mg, 0.88 mmol, 70%) as a white solid. MS (EI+, m / z): 340.2 [M+H]+.

[0248] Step 2: (S)-4-Methyl-2-(2-phenylacetamido)pentanoic acid [I-12]:

[0249] To a stirred solution of (S)-benzyl 4-methyl-2-(2-phenylacetamido)pentanoate (250 mg, 0.74 mmol) in EtOH (10 mL) was added catalytic Pd / C (10%, 20 mg). The reaction was stirred under a hydrogen atmosphere at 50 °C for 3 h. 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 (500 MHz, MeOD): δ 7.24-7.32 (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).

[0250] (Example 13) (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0251] Step 1: (S)-Benzyl 2-(isopropylamino)-4-methylpentanoate:

[0252] To a stirred solution of p-toluenesulfonic acid L-leucine benzyl ester (1.0 g, 2.53 mmol) in MeOH (30 mL) was added acetone (177 mg, 3.05 mmol) and potassium acetate (0.5 g, 5.08 mmol), and the mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (0.24 g, 3.81 mmol) was then added, and the mixture was stirred at room temperature for an additional 3 hours. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL x 2), washed with brine (50 mL), and the resulting solution was filtered (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21 * Purification by 250 mm 10 μm (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-(isopropylamino)-4-methylpentanoate (200 mg, 0.76 mmol, 30%) as a colorless oil. MS (EI+, m / z): 264.3 [M+H]+. 1H-NMR (500 MHz, , MeOD): δ 7.22~7.29 (m, 5H), 5.07 (dd, J = 11.5 Hz, 17.0 Hz, 2H), 3.33(dd, J = 6.5 Hz, 8.5 Hz, 1H), 2.54~2.59 (m, 1H), 1.30~1.48 (m, 3H), 0.72~0.94 (m, 12H).

[0253] Step 2: (S)-2-(Isopropylamino)-4-methylpentanoic acid [I-13]:

[0254] To a stirred solution of (S)-benzyl 2-(isopropylamino)-4-methylpentanoate (200 mg, 0.76 mmol) in MeOH (10 mL) was added a catalytic amount of Pd / C (10%, 50 mg). The reaction was stirred under a hydrogen atmosphere at room temperature for 24 h. The resulting solution was filtered, and the filtration was 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 (500 MHz, MeOD): δ 3.56 (dd, J = 6.0 Hz, 8.5 Hz, 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, 6H). 3.85 (dd, J = 5.5 Hz, 8.5 Hz, 1H), 2.87 (q, J = 6.0 Hz, 1H), 2.68 (dd, J = 7.5 Hz, 12.0 Hz, 1H), 1.92~1.99 (m, 1H), 1.65~1.78 (m, 3H), 0.88~0.96 (m, 12H).

[0255] (Example 14) (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14 ]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0256] Step 1: (S)-Benzyl 2-(isobutylamino)-4-methylpentanoate:

[0257] To a stirred solution of p-toluenesulfonic acid L-leucine benzyl ester (1.0 g, 2.53 mmol) in MeOH (30 mL) was added isobutyraldehyde (0.22 g, 3.05 mmol) and potassium acetate (0.5 g, 5.08 mmol). The mixture was stirred at room temperature for 30 minutes, and then sodium cyanoborohydride (0.24 g, 3.81 mmol) was added. The mixture was stirred at room temperature for an additional 5 hours. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL x 2), washed with brine (50 mL), and the resulting solution was filtered (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21 * Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-(isobutylamino)-4-methylpentanoate (300 mg, 1.08 mmol, 50%) as a colorless oil. MS (EI+, m / z): 278.2 [M+H]+. 1H-NMR (500 MHz, DMSO-d6): δ 9.16 (s, 1H), 9.14 (d, J = 17.5 Hz, 2H), 7.42-7.43 (m, 5H), 5.28 (q, J = 12.0 Hz, 2H). 4.08-4.09 (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).

[0258] Step 2: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0259] To a stirred solution of (S)-benzyl 2-(isobutylamino)-4-methylpentanoate (300 mg, 1.08 mmol) in MeOH (10 mL) was added catalytic Pd / C (10%, 50 mg). The reaction was stirred under a hydrogen atmosphere at room temperature for 24 h. 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 (500 MHz, DMSO-d6): δ 8.82 (s, 2H), 3.85 (dd, J = 5.5 Hz, 8.5 Hz, 1H), 2.87 (q, J = 6.0 Hz, 1H), 2.68 (dd, J = 7.5 Hz, 12.0 Hz, 1H), 1.92~1.99 (m, 1H), 1.65~1.78 (m, 3H), 0.88~0.96 (m, 12H).

[0260] (Example 15) (S)-2-benzamido-4-methylpentanoic acid [I-15]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0261] Step 1: (S)-Benzyl 2-benzamido-4-methylpentanoate:

[0262] To a solution of p-toluenesulfonic acid L-leucine benzyl ester (500 mg, 1.27 mmol), benzoic acid (223 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL) was added DIPEA (410 mg, 3.18 mmol), and the solution was stirred at room temperature for 2 hours. This solution was purified by preparative HPLC (Boston C18 21 *Purification by 250 mm 10 μm (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-benzamido-4-methylpentanoate (300 mg, 0.92 mmol, 73%) as a white solid. MS (EI+, m / z): 326.2 [M+H]+.

[0263] Step 2: (S)-2-Benzamido-4-methylpentanoic acid [I-15]:

[0264] To a stirred solution of (S)-benzyl 2-benzamido-4-methylpentanoate (100 mg, 0.46 mmol) in EtOH (10 mL) was added catalytic amount of Pd / C (10%, 20 mg). The reaction was stirred under hydrogen atmosphere at 50 °C for 3 h. 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 (400 MHz, MeOD): δ 7.87 (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).

[0265] (Example 16) (S)-2-Isobutyramido-4-methylpentanoic acid [I-16]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0266] Step 1: (S)-Benzyl 2-isobutyramido-4-methylpentanoate:

[0267] To a solution of p-toluenesulfonic acid L-leucine benzyl ester (500 mg, 1.27 mmol), isobutyric acid (168 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL) was added DIPEA (410 mg, 3.18 mmol), and the solution was stirred at room temperature for 2 hours. This solution was purified by preparative HPLC (Boston C18 21 * Purification by 250 mm 10 μm (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-isobutyramido-4-methylpentanoate (300 mg, 1.03 mmol, 81%) as a white solid. MS (EI+, m / z): 292.2 [M+H]+.

[0268] Step 2: (S)-2-Isobutyramido-4-methylpentanoic acid [I-16]:

[0269] To a stirred solution of (S)-benzyl 2-(cyclohexanecarboxamido)-4-methylpentanoate (200 mg, 0.69 mmol) in EtOH (10 mL) was added a catalytic amount of Pd / C (10%, 20 mg). The reaction was stirred under a hydrogen atmosphere at 50 °C for 3 h. The resulting solution was filtered and concentrated to give (S)-2-isobutyramido-4-methylpentanoic acid (100 mg, 0.50 mmol, 73%) as a white solid. MS (EI+, m / z): 202.2 [M+H]+. 1H-NMR (400 MHz, MeOD): δ 4.43 (t, J = 6.4 Hz, 1H), 2.49-2.56 (m, 1H), 1.60-1.74 (m, 3H), 1.12 (dd, J = 2.4 Hz, 6.8 Hz, 6H), 0.96 (dd, J = 6.4 Hz, 16.0 Hz, 6H).

[0270] (Example 17) (S)-2-(cyclohexanesulfonamido)-4-methylpentanoic acid [I-17]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0271] Step 1: (S)-Benzyl 2-(cyclohexanesulfonamido)-4-methylpentanoate:

[0272] To a solution of (S)-benzyl 2-amino-4-methylpentanoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and EtN (642.89 mg, 6.35 mmol) in DMF (3 mL) cooled in an ice bath, cyclohexanesulfonyl chloride (278.53 mg, 1.52 mmol) was added. The mixture was stirred at 25° C. for 2 hours. The solution was diluted with ethyl acetate (10 mL) and washed with brine (10 mL). The resulting solution was filtered (10 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by preparative HPLC (Boston C18 21 * Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-(cyclohexanesulfonamido)-4-methylpentanoate (200 mg, 0.544 mmol, 98%) as a white solid. ESI-MS (EI+, m / z): 368.3 [M+H]+. 1H-NMR (500 MHz, DMSO-d6) δ 7.70 (d, J = 9.0 Hz, 1H), 7.38 (t, J = 6.5 Hz, 4H), 7.37 - 7.32 (m, 1H), 5.14 (q, J = 12.5 Hz, 2H), 3.91 (td, J = 5.0 Hz, 9.5 Hz, 1H), 2.69-2.74 (m, 1H), 2.05 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.5 Hz, 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 = 10.5 Hz, J = 20.5 Hz, 6H).

[0273] Step 2: (S)-2-(Cyclohexanesulfonamido)-4-methylpentanoic acid [I-17]:

[0274] To a solution of (S)-benzyl 2-(cyclohexanesulfonamido)-4-methylpentanoate (192 mg, 0.552 mmol) in EtOH (3 mL) was added Pd / C (20 mg, 10%). The reaction mixture was stirred under hydrogen at 50 °C for 4 h. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-(cyclohexanesulfonamido)-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 (500 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.47 (d, J = 9.0 Hz, 1H), 3.76 (td, J = 5.0 Hz, 9.5 Hz, 1H), 2.82 - 2.69 (m, 1H), 2.18 - 1.97 (m, 2H), 1.82 - 1.69 (m, 3H), 1.61 (d, J = 12.5 Hz, 1H), 1.54 - 1.40 (m, 2H), 1.39 - 1.07 (m, 5H), 0.95 - 0.80 (m, 6H).

[0275] (Example 18) (S)-4-methyl-2-(phenylmethylsulfonamido)pentanoic acid [I-18]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0276] Step 1: (S)-Benzyl 4-methyl-2-(phenylmethylsulfonamido)pentanoate:

[0277] To a solution of (S)-benzyl 2-amino-4-methylpentanoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and EtN (642.89 mg, 6.35 mmol) in DMF (3 mL) cooled in an ice bath, phenylmethanesulfonyl chloride (290.71 mg, 1.52 mmol) was added. The mixture was stirred at 25° C. for 2 hours. The solution was diluted with ethyl acetate (10 mL) and washed with brine (10 mL). The resulting solution was filtered (10 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by preparative HPLC (Boston C18 21 * Purification by 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 4-methyl-2-(phenylmethylsulfonamido)pentanoate (149 mg, 0.396 mmol, 90%) as a white solid. ESI-MS (EI+, m / z): 398.0 [M+Na]+. 1H -NMR (500 MHz, DMSO-d6) δ 7.81 (d, J = 8.5 Hz, 1H), 7.52 - 7.18 (m, 9H), 5.15 (s, 2H), 4.28 (dd, J = 13.5 Hz, 44.5 Hz, 2H), 3.87 (dd, J = 8.0 Hz, 15.0 Hz, 1H), 1.57 - 1.15 (m, 4H), 0.82 (dd, J = 4.5 Hz, 6.0 Hz, 6H).

[0278] Step 2: (S)-4-Methyl-2-(phenylmethylsulfonamido)pentanoic acid [I-18]:

[0279] To a solution of (S)-benzyl 4-methyl-2-(phenylmethylsulfonamido)pentanoate (121 mg, 0.322 mmol) in EtOH (3 mL) was added Pd / C (20 mg, 10%). The reaction mixture was stirred under hydrogen at 50 °C for 4 h. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(phenylmethylsulfonamido)pentanoic acid (I-18) (41.2 mg, 0.144 mmol, 100%) as a white solid. ESI-MS (EI+, m / z): 308.0 [M+Na]+. 1H NMR (500 MHz, DMSO-d6) δ 12.77 (s, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.47 - 7.25 (m, 5H), 4.30 (dd, J = 13.5 Hz, 37.0 Hz, 2H), 3.75 (dd, J = 7.5 Hz, 15.5 Hz, 1H), 1.65 (dt, J = 6.5 Hz, 13.5 Hz, 1H), 1.45 (t, J = 7.2 Hz, 2H), 0.85 (dd, J = 1.5 Hz, 6.5 Hz, 6H).

[0280] (Example 19) (S)-4-methyl-2-(methylsulfonamido)pentanoic acid [I-19]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0281] Step 1: (S)-Benzyl 4-methyl-2-(methylsulfonamido)pentanoate:

[0282] To a solution of (S)-benzyl 2-amino-4-methylpentanoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and EtN (642.89 mg, 6.35 mmol) in DMF (3 mL) cooled in an ice bath, methanesulfonyl chloride (290.71 mg, 1.52 mmol) was added, and the mixture was stirred at 25° C. for 2 hours. The solution was diluted with ethyl acetate (10 mL) and washed with brine (10 mL). The resulting solution was filtered (10 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by preparative HPLC (Boston C18 21 * Purification by 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 4-methyl-2-(methylsulfonamido)pentanoate (192 mg, 0.641 mmol, 98%) as a white solid. + , m / z): 323.0 [M + Na] + . 1 H -NMR (500 MHz, DMSO-d6) δ 7.79 (d, J = 8.8 Hz, 1H), 7.42 - 7.36 (m, 4H), 7.37 - 7.32 (m, 1H), 5.16 (s, 2H), 3.97 (td, J = 6.0 Hz, 9.0 Hz, 1H), 2.85 (s, 3H), 1.68 (dq, J = 6.5 Hz, 13.0 Hz, 1H), 1.54 - 1.46 (m, 2H), 0.91 - 0.82 (m, 6H).

[0283] Step 2: (S)-4-Methyl-2-(methylsulfonamido)pentanoic acid [I-19]:

[0284] To a solution of (S)-benzyl 4-methyl-2-(methylsulfonamido)pentanoate (149 mg, 0.497 mmol) in EtOH (3 mL) was added Pd / C (20 mg, 10%). The reaction mixture was stirred under a hydrogen atmosphere at 50 °C for 4 h. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(methylsulfonamido)pentanoic acid (I-19) (31.4 mg, 0.150 mmol, 100%) as a white solid. ESI-MS (EI + , m / z): 232.1 [M + Na] + . 1 H NMR (500 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.56 (d, J = 9.0 Hz, 1H), 3.82 (dd, J = 8.0 Hz, 15.5 Hz, 1H), 2.88 (s, 3H), 1.72 (dt, J = 6.5 Hz, 13.0 Hz, 1H), 1.48 (t, J = 7.0 Hz, 2H), 0.89 (t, J = 7.0 Hz, 6H).

[0285] (Example 20) (S)-2-amino-4-methyl-N-phenylpentanamide [I- 20]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0286] Step 1: (S)-Benzyl 4-methyl-1-oxo-1-(phenylamino)pentan-2-ylcarbamate:

[0287] To a solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 3.77 mmol) in DMF (20 mL) was added aniline (702 mg, 7.55 mmol), HATU (1.72 g, 4.52 mmol), and EtN (1.14 g, 11.31 mmol) at room temperature. After 2 h, the solution was diluted with EtOAc (80 mL), washed with brine (80 mL), and the resulting solution was filtered (80 mL × 3), dried (NaSO), and concentrated in vacuo. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 3) to give (S)-benzyl 4-methyl-1-oxo-1-(phenylamino)pentan-2-ylcarbamate (350 mg, 1.03 mmol, 27%) as a white solid. ESI-MS (EI+, m / z): 341.1[M+H]+.

[0288] Step 2: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0289] A mixture of (S)-benzyl 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 for 2 hours at room temperature. 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 (500 MHz, DMSO-d6): δ 9.86 (s, 1H), 7.63 (dd, J = 1.0 Hz, 8.5 Hz, 2H), 7.31-7.27 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H), 3.31 (dd, J = 5.0 Hz, 8.5 Hz, 1H), 1.80-1.71 (m, 1H), 1.50-1.44 (m, 1H), 1.35-1.29 (m, 1H), 0.90 (dd, J = 6.5 Hz, 14.0 Hz, 6H).

[0290] (Example 21) (S)-2-amino-N,4-dimethylpentanamide [I-21]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0291] Step 1: (S)-Benzyl 4-methyl-1-(methylamino)-1-oxopentan-2-ylcarbamate:

[0292] To a solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 3.77 mmol) in DMF (20 mL) was added MeNH₂·HCl (509 mg, 7.54 mmol), HATU (1.72 g, 4.52 mmol), and Et₃N (1.14 g, 11.31 mmol) at 25 °C. After 2 h, the solution was diluted with EtOAc (80 mL), washed with brine (80 mL), filtered (80 mL × 3), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 3) to afford (S)-benzyl 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]+.

[0293] Step 2: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0294] A mixture of (S)-benzyl 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 under hydrogen for 2 h at room temperature. 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 (500 MHz, DMSO-d6): δ 7.80 (s, 1H), 3.10 (dd, J = 5.0 Hz, 9.0 Hz, 1H), 2.57 (dd, J = 3.0 Hz, 5.0 Hz, 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).

[0295] (Example 22) (S)-4-methyl-2-(phenylamino)pentanoic acid [I-22]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0296] Step 1: (S)-Benzyl 2-(cyclohexanecarboxamido)-4-methylpentanoate:

[0297] To a solution of p-toluenesulfonic acid L-leucine benzyl ester (500 mg, 1.27 mmol), cyclohexanecarboxylic acid (244 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL) was added DIPEA (410 mg, 3.18 mmol), and the solution was stirred at room temperature for 2 hours. This solution was purified by preparative HPLC (Boston C18 21 *Purification on a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-(cyclohexanecarboxamido)-4-methylpentanoate (300 mg, 0.91 mmol, 71%) as a white solid. MS (EI+, m / z): 332.3 [M+H]+.

[0298] Step 2: (S)-2-(cyclohexanecarboxamido)-4-methylpentanoic acid [I-22]:

[0299] To a stirred solution of (S)-benzyl 2-(cyclohexanecarboxamido)-4-methylpentanoate (200 mg, 0.60 mmol) in EtOH (10 mL) was added a catalytic amount of Pd / C (10%, 20 mg). The reaction was stirred under a hydrogen atmosphere at 50 °C for 3 h. The resulting solution was filtered and concentrated to give (S)-2-(cyclohexanecarboxamido)-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 (500 MHz, CD3OD): δ 4.43 (t, J = 7.5 Hz, 1H), 2.29 (td, J = 8.0 Hz, 11.0 Hz, 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 = 6.0 Hz, 20.5 Hz, 6H).

[0300] (Example 25) (S)-4-methyl-2-(phenylsulfonamido)pentanoic acid [I-25]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0301] Step 1: (S)-Benzyl 4-methyl-2-(phenylsulfonamido)pentanoate:

[0302] To an ice-bath cooled solution of (S)-benzyl 2-amino-4-methylpentanoate 4-methylbenzenesulfonate (300 mg, 0.762 mmol) and EtN (385.73 mg, 3.81 mmol) in DMF (3 mL) was added benzenesulfonyl chloride (148.12 mg, 0.838 mmol). The mixture was stirred at 25 °C for 2 h. The solution was diluted with ethyl acetate (10 mL) and washed with brine (10 mL). The resulting solution was filtered (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product (280 mg, purity: 85%, yield: 74%) was used directly in the next step. ESI-MS (EI+, m / z): 384.1 [M+Na]+.

[0303] Step 2: (S)-4-Methyl-2-(phenylsulfonamido)pentanoic acid [I-25]:

[0304] To a solution of (S)-benzyl 4-methyl-2-(phenylsulfonamido)pentanoate (200 mg, 0.553 mmol) in EtOH (3 mL) was added Pd / C (20 mg, 10%). The reaction mixture was stirred under a hydrogen atmosphere at 50 °C for 4 h. 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 (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.79 - 7.73 (m, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.56 (t, J = 7.4 Hz, 2H), 3.63 (dd, J = 8.5 Hz, 14.5 Hz, 1H), 1.53 (td, J = 6.5 Hz, 13.5Hz, 1H), 1.41 - 1.31 (m, 2H), 0.79 (d, J = 6.6 Hz, 3H), 0.66 (d, J = 6.5 Hz, 3H).

[0305] (Example 26) (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0306] Step 1: (S)-Benzyl 4-methyl-2-(phenylamino)pentanoate:

[0307] To a mixture of p-toluenesulfonic acid L-leucine benzyl ester (200 mg, 0.51 mmol), phenylboronic acid (186 mg, 1.52 mmol), and Cu(OAc) (462 mg, 2.54 mmol) in DCM (10 mL) was added 4A MS (1.0 g) and EtN (155 mg, 1.52 mmol), and the mixture was stirred at room temperature for 18 h. The mixture was quenched, and the resulting solution was filtered (50 mL), extracted with EtOAc (50 mL × 2), washed with brine (50 mL), and filtered (50 mL). The organic phase was concentrated and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 20) to give (S)-benzyl 4-methyl-2-(phenylamino)pentanoate (100 mg, 0.34 mmol, 66%) as a colorless oil. MS (EI+, m / z): 298.2 [M+H]+.

[0308] Step 2: (S)-4-Methyl-2-(phenylamino)pentanoic acid [I-26]:

[0309] To a stirred solution of (S)-benzyl 4-methyl-2-(phenylamino)pentanoate (100 mg, 0.34 mmol) in EtOH (10 mL) was added catalytic Pd / C (10%, 20 mg). The reaction was stirred under a hydrogen atmosphere at 50 °C for 2 h. 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 (400 MHz, CDCl3): δ 7.23 (t, J = 8.0 Hz, 2H), 6.83 (t, J = 7.6 Hz, 1H), 6.66 (d, J = 8.0 Hz, 2H), 3.99 (d, J = 8.4 Hz, 1H), 2.87 (q, J = 6.0 Hz, 1H), 1.72~1.86 (m, 2H), 1.62~1.68 (m, 1H), 0.85~1.03 (m, 6H).

[0310] (Example 36) (S)-2-acetamido-4-methylpentanoic acid [I-36]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0311] Step 1: (S)-Benzyl 2-acetamido-4-methylpentanoate:

[0312] To a solution of p-toluenesulfonic acid L-leucine benzyl ester (500 mg, 1.27 mmol), acetic acid (114 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL) was added DIPEA (410 mg, 3.18 mmol), and the solution was stirred at room temperature for 2 hours. This solution was purified by preparative HPLC (Boston C18 21 *Purification by 250 mm 10 μm (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-benzyl 2-acetamido-4-methylpentanoate (300 mg, 1.14 mmol, 89%) as a white solid. MS (EI+, m / z): 264.2 [M+H]+.

[0313] Step 2: (S)-2-Acetamido-4-methylpentanoic acid [I-36]:

[0314] To a stirred solution of (S)-benzyl 2-acetamido-4-methylpentanoate (250 mg, 0.74 mmol) in EtOH (10 mL) was added a catalytic amount of Pd / C (10%, 20 mg). The reaction was stirred under a hydrogen atmosphere at 50 °C for 3 h. 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 (500 MHz, MeOD): δ 4.43 (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).

[0315] (Example 45) (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid [I-45]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0316] Step 1: (S,E)-2-(4-methoxy-4-oxobut-2-enamido)-4-methylpentanoic acid [I-45]:

[0317] To a solution of (E)-4-methoxy-4-oxobut-2-enoic acid (1.0 g, 7.69 mmol) in DCM (30 mL) was added SOCl (1.83 g, 15.38 mmol) followed by DMF (0.1 mL). The solution was heated to 40° C. for 4 hours. The solution was concentrated to dryness to give an oil. The oil was diluted with DCM (10 mL). The ice bath A solution of (S)-2-amino-4-methylpentanoic acid (1.0 g, 7.62 mmol) in acetone (20 mL) and saturated Na2CO3 (20 mL) was added dropwise to a cooled aliquot. After 1 h, the solution was adjusted to pH 2 with 6 M HCl solution, extracted with EtOAc (40 mL x 2), washed with brine (80 mL), and the resulting solution was filtered (80 mL x 3), dried (Na2SO4), filtered, and concentrated in vacuo. The crude product was purified by chromatography (silica, 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 (400 MHz, CDCl3): δ 7.32 (d, J = 15.2 Hz, 1H), 7.05 (d, J = 15.2 Hz, 1H), 6.85-6.89 (m, 2H), 7.30-7.46 (m, 1H), 3.82 (s, 1H), 1.63-1.78 (m, 3H), 0.97 (d, J = 4.8 Hz, 6H).

[0318] (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]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0319] Step 1: Ethyl 2,2-difluoro-3-methylbutanoate:

[0320] A mixture of ethyl 3-methyl-2-oxobutanoate (10 g, 0.069 mol) and DAST (16.8 g, 0.10 mol) was stirred at room temperature for 12 hours. After checking by TLC, the reaction mixture was slowly added dropwise to cold saturated aqueous sodium bicarbonate solution. The mixture was extracted with EtO (300 mL × 2), and the organic layer was washed with brine, dried, and concentrated to give crude ethyl 2,2-difluoro-3-methylbutanoate (8.3 g), which was used directly in the next step.

[0321] Step 2: 2,2-Difluoro-3-methylbutanal:

[0322] A solution of crude ethyl 2,2-difluoro-3-methylbutanoate (8.3 g) in CH2Cl2 (200 mL) was added to a hexane solution of DIBAL-H (1. 0M, 69 mL, 69.0 mmol) was added dropwise, and the mixture was stirred at −78° C. for 30 minutes. After checking by TLC, the reaction was quenched with saturated citric acid and extracted with EtO. The extract was washed with saturated citric acid, brine, dried over NaSO, and concentrated under reduced pressure to give the oily aldehyde 2,2-difluoro-3-methylbutanal (4.2 g), which was used immediately in the next step without purification.

[0323] Step 3: 2-(benzylamino)-3,3-difluoro-4-methylpentanenitrile:

[0324] A solution of crude 2,2-difluoro-3-methylbutanal (4.2 g) in MeOH (50 mL) was cooled to 0° C. Acetic acid (glacial, 2.1 mL) was added, followed by the dropwise addition of trimethylsilyl cyanide (4.2 mL) over 15 minutes, while maintaining a temperature of approximately 0° C. The reaction mixture was allowed to warm to 25° C. and stirred overnight. The resulting cold solution was filtered (200 mL) and poured into the reaction mixture, which was then diluted with dichloromethane (2 * The dichloromethane layer was then washed with brine (2*50 mL) and the resulting solution was filtered (2*100 mL). The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude 2-(benzylamino)-3,3-difluoro-4-methylpentanenitrile (2.8 g), which was used immediately in the next step without purification. ESI-MS (EI+, m / z): 238.2 [M+H]+.

[0325] Step 4: 2-(benzylamino)-3,3-difluoro-4-methylpentanoic acid:

[0326] A solution of crude 2-(benzylamino)-3,3-difluoro-4-methylpentanenitrile (2.8 g) in concentrated hydrochloric acid (50 mL) and HOAc (10 mL) was stirred at 90 °C for 24 h and concentrated. The residue was purified by preparative HPLC to give 2-(benzylamino)-3,3-difluoro-4-methylpentanoic acid (513 mg) as a white solid. The pure product was purified by chiral HPLC to give (R)-2-(benzylamino)-3,3-difluoro-4-methylpentanoic acid (80 mg) and (S)-2-(benzylamino)-3,3-difluoro-4-methylpentanoic acid (63 mg), both of which were white solids. ESI-MS (EI+, m / z): 258.2 [M+H]+.

[0327] Step 5-A: (R)-2-Amino-3,3-difluoro-4-methylpentanoic acid [I-46]:

[0328] To a solution of (R)-2-(benzylamino)-3,3-difluoro-4-methylpentanoic acid (80 mg, 0.31 mmol) in MeOH (20 mL) was added HCOONH4 (98 mg, 1.56 mmol) and Pd / C (100 mg) at room temperature. The mixture was stirred at 60 °C for 2 h. The reaction mixture was filtered and concentrated to give the crude product, which was purified by reverse-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, 1 H), 2.55-2.42 (m, 1 H), 1.04 (d, J = 7.0 Hz, 3 H), 0.993 (d, J = 6.5 Hz, 3 H).

[0329] Step 5-B: (S)-2-Amino-3,3-difluoro-4-methylpentanoic acid [I-47]:

[0330] (S)-2-(benzylamino)-3,3-difluoro-4-methylpentanoic acid (63 To a solution of (S)-2-amino-3,3-difluoro-4-methylpentanoic acid (I-47) (14 mg, 34%) as a white solid, was added HCOONH4 (77 mg, 1.22 mmol) and Pd / C (100 mg) at room temperature. The mixture was stirred at 60 °C for 2 h. The reaction mixture was filtered and concentrated to give the crude product, which was purified by reverse-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, DO): δ 4.27 (dd, J = 24.0, 3.5 Hz, 1 H), 2.55-2.42 (m, 1 H), 1.04 (d, J = 7.0 Hz, 3 H), 0.993 (d, J = 6.5 Hz, 3 H).

[0331] (Example 147) (S)-2-Amino-4-methyl-N-(methylsulfonyl)pentanamide hydrochloride [I-147]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-tert-butyl 4-methyl-1-(methylsulfonamido)-1-oxopentan-2-ylcarbamate:

[0332] 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) was added TEA (1.3 g, 12.9 mmol), and the solution was stirred at room temperature for 17 hours. This solution was purified by preparative HPLC (Boston C18 21 * Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-tert-butyl 4-methyl-1-(methylsulfonamido)-1-oxopentan-2-ylcarbamate (130 mg, 0.42 mmol, 8.9%) as a white solid. MS (EI-, m / z): 307.0 [MH] - . Step 2: (S)-2-Amino-4-methyl-N-(methylsulfonyl)pentanamide hydrochloride [I-147]:

[0333] To a solution of (S)-tert-butyl 4-methyl-1-(methylsulfonamido)-1-oxopentan-2-ylcarbamate (130 mg, 0.42 mmol) in EtO (15 mL), 4 M HCl / dioxane (5 mL) was added and stirred at room temperature for 3 h. The solid was filtered to give (S)-2-amino-4-methyl-N-(methylsulfonyl)pentanamide hydrochloride [I-147] as a white solid (32 mg, 0.13 mmol, 31%). ESI-MS (EI+, m / z): 209.1 [M+H] + . 1H NMR (500 MHz, CD3OD) δ 3.96 (t, J = 3.0 Hz, 1H), 3.32 (s, 3H), 1.74-1.79 (m, 3H), 1.02-1.05 (m, 6H).

[0334] (Example 193) (S)-2-Amino-N,4,4-trimethyl-N-(methylsulfonyl)pentanamide hydrochloride [I-193]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-tert-butyl 4,4-dimethyl-1-(N-methylmethylsulfonamido)-1-oxopentan-2-ylcarbamate:

[0335] To a solution of (S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanoic acid (500 mg, 1.97 mmol) in DCM (60 mL), HATU (900 mg, 2.36 mmol) was added and stirred at room temperature for 2 hours. Next, CsCO (1.92 g, 5.91 mmol) and N-methylmethanesulfonamide (322 mg, 2.95 mmol) were added to the mixture and stirred at room temperature overnight. 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 (NaSO), filtered, concentrated in vacuo, and the crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 5) to give (S)-tert-butyl 4,4-dimethyl-1-(N-methylmethylsulfonamido)-1-oxopentan-2-ylcarbamate (420 mg, 1.25 mmol, 63%) as a yellow oil. ESI-MS (EI+, m / z): 359.1 [M+Na] + . Step 2: (S)-2-Amino-N,4,4-trimethyl-N-(methylsulfonyl)pentanamide hydrochloride [I-193]:

[0336] To a solution of (S)-tert-butyl 4,4-dimethyl-1-(N-methylmethylsulfonamido)-1-oxopentan-2-ylcarbamate (420 mg, 1.25 mmol) in EtO (20 mL), 4 M HCl / dioxane (10 mL) was added and stirred at room temperature for 17 h. The solid was filtered to give (S)-2-amino-N,4,4-trimethyl-N-(methylsulfonyl)pentanamide hydrochloride [I-193] as a white solid (250 mg, 0.13 mmol, 71%). ESI-MS (EI+, m / z): 237.1 [M+H] + . 1H NMR (500 MHz, DMSO) δ 8.55 (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).

[0337] (Example 192) 2-amino-4-fluoro-4-methyl-N-(methylsulfonyl)pentanamide hydrochloride [I-192]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: tert-butyl 4-fluoro-4-methyl-1-(methylsulfonamido)-1-oxopentan-2-ylcarbamate:

[0338] To a solution of tert-butyl 4-fluoro-4-methyl-1-(methylsulfonamido)-1-oxopentan-2-ylcarbamate (270 mg, 1.08 mmol) in DCM (50 mL), HATU (451 mg, 1.19 mmol) was added and stirred at room temperature for 2 hours. Next, CsCO (1.06 g, 3.24 mmol) and methanesulfonamide (206 mg, 2.17 mmol) were added to this mixture and stirred at room temperature overnight. 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 (NaSO), filtered, concentrated in vacuo, and the crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 5) to give (S)-tert-butyl 4,4-dimethyl-1-(N-methylmethylsulfonamido)-1-oxopentan-2-ylcarbamate (200 mg, 0.6 mmol, 55%) as a yellow oil. ESI-MS (EI+, m / z): 344.1 [M+NH4] + . Step 2: 2-amino-4-fluoro-4-methyl-N-(methylsulfonyl)pentanamide hydrochloride [I-192].

[0339] To a solution of (S)-tert-butyl 4,4-dimethyl-1-(N-methylmethylsulfonamido)-1-oxopentan-2-ylcarbamate (200 mg, 0.6 mmol) in EtO (20 mL), 4 M HCl / dioxane (10 mL) was added and stirred at room temperature for 17 h. The solid was filtered to give 2-amino-4-fluoro-4-methyl-N-(methylsulfonyl)pentanamide hydrochloride [I-192] as a white solid (89.8 mg, 0.34 mmol, 57%). ESI-MS (EI+, m / z): 227.1 [M+H] + . 1H NMR (500 MHz, DMSO) δ 8.44 (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).

[0340] (Example 190) (S)-Methyl 2-((S)-2-amino-4,4-dimethylpentanamido)-4-methylpentanoate hydrochloride [I-190]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-Methyl 2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanamido)-4-methylpentanoate:

[0341] To a solution of (S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanoic acid (500 mg, 2.0 mmol) in DCM (80 mL), HATU (900 mg, 2.3 mmol) was added and stirred at room temperature for 2 hours. Next, CsCO (1.95 g, 6.0 mmol) and (S)-methyl 2-amino-4-methylpentanoate hydrochloride (555 mg, 3.0 mmol) were added to the mixture and stirred at room temperature overnight. 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 (NaSO), filtered, and concentrated in vacuo, and the crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 5) to give (S)-methyl 2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanamido)-4-methylpentanoate (500 mg, 1.34 mmol, 67%) as a white solid. ESI-MS (EI+, m / z): 317.2 [M-56] + . Step 2: (S)-Methyl 2-((S)-2-amino-4,4-dimethylpentanamido)-4-methylpentanoate hydrochloride [I-190].

[0342] To a solution of (S)-methyl 2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanamido)-4-methylpentanoate (500 mg, 1.34 mmol) in EtO (20 mL) was added 4 M HCl / dioxane (10 mL) and stirred at room temperature for 17 hours. The solid was filtered to give (S)-methyl 2-(S)-2-amino-4,4-dimethylpentanamido)-4-methylpentanoate hydrochloride [I-190] as a white solid (300 mg, 0.97 mmol, 73%). ESI-MS (EI+, m / z): 273.2 [M+H] +. 1H NMR (500 MHz, DMSO) δ 9.07-9.09 (d, J = 7.5 Hz, 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).

[0343] (Example 122) (S)-Methyl 2-amino-4,4-dimethylpentanoate Hydrochloride salt [I-122]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-Methyl 2-amino-4,4-dimethylpentanoate hydrochloride [I-122]:

[0344] A solution of (S)-2-amino-4,4-dimethylpentanoic acid (100 mg, 0.69 mmol) in MeOH (10 mL) was added to 4 M HCl / dioxane (10 mL) and stirred at 80 °C for 24 h. The mixture was concentrated, and the residue was vigorously stirred with EtO to give (S)-methyl 2-amino-4,4-dimethylpentanoate hydrochloride [I-122] as a white solid (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),.

[0345] (Example 123) (R)-Methyl 2-amino-4,4-dimethylpentanoate hydrochloride [I-123]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (R)-Methyl 2-amino-4,4-dimethylpentanoate hydrochloride [I-123]:

[0346] To a mixture of (R)-2-amino-4,4-dimethylpentanoic acid (50 mg, 0.34 mmol) in dry MeOH (10 mL) was added SOCl (0.5 mL) and stirred at room temperature for 17 h. The mixture was concentrated, and the residue was vigorously stirred with EtO to give (R)-methyl 2-amino-4,4-dimethylpentanoate hydrochloride [I-123] as a white solid. (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).

[0347] (Example 205) 2-amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid:

[0348] A mixture of 2-amino-5,5,5-trifluoro-4-methylpentanoic acid (250 mg, 1.35 mmol), BocO (353 mg, 1.62 mmol), and NaOH (80 mg, 2.0 mmol) was dissolved in dioxane (10 mL) and HO (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 (NaSO), 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] + . Step 2: 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoate:

[0349] 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. After filtration, the filtrate was washed with brine (20 mL), dried (NaSO), filtered, and concentrated to give crude 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (385 mg, 1.35 mmol). (400 mg) of 5,5,5-trifluoro-4-methylpentanoate was obtained as a white solid. ESI-MS (EI + , m / z): 282.9 [M-100] + . Step 3: tert-butyl 1-cyanamid-5,5,5-trifluoro-4-methyl-1-oxopentan-2-ylcarbamate:

[0350] A mixture of 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoate (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 hours and at room temperature for 17 hours. The solution was purified by preparative HPLC (Boston C18 21 * Purification on a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave tert-butyl 1-cyanamido-5,5,5-trifluoro-4-methyl-1-oxopentan-2-ylcarbamate (45 mg, 0.14 mmol) as a white solid. MS (EI+, m / z): 310.3 [M+H] + . Step 4: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205]:

[0351] To a solution of tert-butyl 1-cyanamido-5,5,5-trifluoro-4-methyl-1-oxopentan-2-ylcarbamate (45 mg, 0.14 mmol) in EtO (20 mL), 4 M HCl / dioxane (10 mL) was added and stirred at room temperature for 24 hours. This solution was purified by preparative HPLC (Boston C18 21 * Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave 2-amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205] as a white solid (12.3 mg, 0.05 mmol, 27%). MS (EI+, m / z): 210.1 [M+H] + . 1H NMR (500 MHz, CD3OD) δ 4.06-4.09 (m, 1H), 2.43-2.65 (m, 1H), 1.67-1.85 (m, 2H), 1.18-1.22 (m, 3H).

[0352] (Example 206) 2-amino-3-(1-methylcyclobutyl)propanoic acid [I-206]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: N-Methoxy-N,1-dimethylcyclobutanecarboxamide:

[0353] 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) was added TEA (30.3 g, 0.3 mol), 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 x 2). The organic phase was washed with 1N HCl, saturated NaHCO3, and brine (100 mL), dried (Na2SO4), filtered, and concentrated in vacuo 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] + . Step 2: 1-Methylcyclobutanecarbaldehyde:

[0354] To a solution of N-methoxy-N,1-dimethylcyclobutanecarboxamide (2.0 g, 12.7 mmol) in dry THF (20 mL) was added 1 M LiAlH (19 mL, 19 mmol) dropwise at 0 °C under N. The mixture was allowed to warm to room temperature and stirred for 2 h. The solution was slowly quenched with saturated Saigneit salt and extracted with EtO (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (NaSO), filtered, and used in the next step. Step 3: (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)acrylate:

[0355] To a solution of Wittig reagent (2.15 g, 5.86 mmol) in dry THF (80 mL) was added t-BuONa (844 mg, 8.79 mmol) at 0 °C and stirred for 1 h. Next, a solution of 1-methylcyclobutanecarbaldehyde was added and stirred at room temperature for 17 h. The solution was extracted with EtOAc (100 mL × 2). The organic phase was washed with brine (100 mL), dried (NaSO), filtered, and concentrated in vacuo. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 30) to give (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)acrylate (700 mg, 2.2 mmol) as a colorless oil. ESI-MS (EI + , m / z): 200.2 [M-56 * 2] + . Step 4: tert-Butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propanoate:

[0356] A mixture of (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)acrylate (700 mg, 2.2 mmol) and Pd / C (10%, 100 mg) in MeOH (100 mL) was stirred at 30° C. for 17 h. The mixture was filtered, and the filtrate was concentrated to dryness to give tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propanoate (600 mg, crude) as a colorless oil. ESI-MS (EI + , m / z): 158.2 [M-156] + . Step 5: 2-Amino-3-(1-methylcyclobutyl)propanoic acid [I-206]:

[0357] To a solution of tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propanoate (600 mg, crude) in EtO (20 mL) was added 4 M HCl / dioxane (10 mL) and stirred at room temperature for 17 h. The solution was concentrated to give 2-amino-3-(1-methylcyclobutyl)propanoic acid. MS (EI + , m / z): 158.0 [M+H] + . 1 H NMR (500 MHz, D2O) δ 3.91 (t, J = 7.5 Hz, 1H), 2.06-2.02 (m, 1H), 1.88-1.64 (m, 7H), 1.15 (s, 3H).

[0358] (Example 93) S-2-amino-3-(1-methylcyclobutyl)propanoic acid [I-93]. [ka] Synthesis scheme: [ka] Procedures and characterization: The procedure for 2-amino-3-(1-methylcyclobutyl)propanoic acid was the same as in Example 8. Step 6: 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propanoic acid:

[0359] 2-Amino-3-(1-methylcyclobutyl)propanoic acid (300 mg, crude), CbzOSu (714 mg, 2.8 mmol) in acetone (10 mL) and saturated NaHCO The mixture in 3 mL of 3 was stirred at room temperature for 5 hours. This solution was purified by preparative HPLC (Boston C18 21 *Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propanoic acid (160 mg, 0.54 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + . Step 7: (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propanoic acid:

[0360] 2-(Benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propanoic acid (160 mg, 0.54 mmol) was purified by chiral HPLC to give (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propanoic acid (50 mg, 0.17 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + . Step 8: (S)-2-Amino-3-(1-methylcyclobutyl)propanoic acid [I-93]:

[0361] A mixture of (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propanoic acid (50 mg, 0.17 mmol) and Pd / C (10%, 10 mg) in MeOH (10 mL) was stirred at room temperature for 1 h. This solution was purified by preparative HPLC (Boston C18 21 * Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) afforded (S)-2-amino-3-(1-methylcyclobutyl)propanoic acid [I-93] (2 mg, 0.01 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + 1H NMR (500 MHz, DO) δ 3.76-3.79 (t, 1H), 1.96-2.00 (m, 1H), 1.61-1.86 (m, 7H), 1.11 (s, 3H).

[0362] (Example 204) 2-Amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-204] [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propanoate:

[0363] A solution of tert-butyl 2-(diphenylmethyleneamino)acetate (2.5 g, 8.47 mmol) in THF (20 mL) was cooled to -78 °C and then heated under N2 with LiHMD S (8.47 mL, 8.47 mmol) was added dropwise. 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 at -78 °C to room temperature overnight. The solution was diluted with brine (25 mL * 2), dried (NaSO), concentrated, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 30) to give tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propanoate (2.3 g, 6.04 mmol, 71%) as a yellow solid. ESI-MS (EI+, m / z): 382.3 [M+H] + . Step 2: 2-Amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-204]:

[0364] A solution of tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propanoate (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 to give 2-amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-204] as a white solid (113 mg, 0.57 mmol, 44%). ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500 MHz, CD3OD) δ 13.78 (br, 1H), 8.33 (br, 1H), 3.75 (m, 1H), 1.00-1.14 (m, 2H), 0.06 (s, 9H).

[0365] (Example 201) (S)-2-Amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-201]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-2-Amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-201]:

[0366] A solution of (S)-tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propanoate (300 mg, 0.79 mmol) in 4 M HCl / dioxane (3 mL) was stirred at room temperature for 17 h. DCM (40 mL) was added. The solid was filtered to give (S)-2-amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-201] as a white solid (92 mg, 0.47 mmol, 62%). ESI-MS (EI+, m / z): 162.2 [M+H] +. 1H NMR (500 MHz, CD3OD) δ 13.76 (br, 1H), 8.38 (br, 1H), 3.76 (m, 1H), 1.02-1.16 (m, 2H), 0.06 (s, 9H).

[0367] (Example 200) (R)-2-amino-3-(trimethylsilyl)propanoic acid hydrochloride [ I-200]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (R)-2-Amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-200]:

[0368] A solution of (R)-tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propanoate (300 mg, 0.79 mmol) in 4 M HCl / dioxane (3 mL) was stirred at room temperature for 17 h. DCM (40 mL) was added. The solid was filtered to give (R)-2-amino-3-(trimethylsilyl)propanoic acid hydrochloride [I-200] as a white solid (80 mg, 0.41 mmol, 52%). ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500 MHz, CD3OD) δ 13.77 (br, 1H), 8.33 (br, 1H), 3.76 (m, 1H), 1.02-1.14 (m, 2H), 0.06 (s, 9H).

[0369] (Example 194) (S)-2-Amino-4-fluoro-4-methylpentanoic acid [I-194]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-2-Amino-4-fluoro-4-methylpentanoic acid [I-194]:

[0370] A mixture of (S)-ethyl 2-amino-4-fluoro-4-methylpentanoate hydrochloride (65 mg, 0.31 mmol), LiOH.HO (29 mg, 0.69 mmol) in HO (2 mL) was stirred at room temperature for 2.5 hours. 1N HCl was then added to adjust the pH to 3. This mixture was analyzed by reverse HPLC (Boston C18 21 * Direct purification by 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) afforded (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 (500 MHz, 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=3.5 Hz, 3H), 1.37 (d, Jz=4.0 Hz, 3H).

[0371] (Example 94) (S)-3,3-Dimethyl-1-(2H-tetrazol-5-yl)butan-1-amine [I-94]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-tert-butyl 1-cyano-3,3-dimethylbutylcarbamate:

[0372] To a solution of (S)-tert-butyl 1-amino-4,4-dimethyl-1-oxopentan-2-ylcarbamate (500 mg, 2.1 mmol) in DMF (10 mL) was added cyanuric chloride (450 mg, 2.5 mmol) and stirred at room temperature for 2 h. The mixture was then diluted with brine (100 mL), extracted with ethyl acetate (50 mL), dried (NaSO), and concentrated to give crude (S)-tert-butyl 1-cyano-3,3-dimethylbutylcarbamate (500 mg) as a yellow dope. ESI-MS (EI+, m / z): 249.2 [M+Na] + . Step 2: (S)-tert-butyl 3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate:

[0373] A mixture of (S)-tert-butyl 1-cyano-3,3-dimethylbutylcarbamate (crude 500 mg), ZnBr (900 mg, 4.0 mmol), and NaN (260 mg, 4.0 mmol) in DMF (20 mL) was stirred at 100 °C for 17 h. The mixture was then diluted with brine (200 mL), extracted with ethyl acetate (60 mL), dried (NaSO), and concentrated to give crude (S)-tert-butyl 3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate (400 mg) as a yellow solid. ESI-MS (EI+, m / z): 214.3 [M+H-56] + . Step 3: ((S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butan-1-amine [I-94]:

[0374] A solution of (S)-tert-butyl 3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate (crude 300 mg) in 4 M HCl / dioxane (3.5 mL) was stirred at room temperature for 17 h. The solution was then concentrated and purified by reverse-phase HPLC (Boston C18 21 *Direct purification on a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butan-1-amine 2,2,2-trifluoroacetate [I-94] as a white solid (30 mg, 0.11 mmol, 9%, 3 steps). MS (EI+, m / z): 170.2 [M+H] + . 1H NMR (500 MHz, CD3OD) δ 8.18 (br, 3H), 4.48 (m, 1H), 2.14 (m, 1H), 1.73 (dd, Jz=3.5, 16.5 Hz 1H), 0.72 (s, 9H).

[0375] (Example 175) Synthesis of 2-amino-5,5,5-trifluoro-4-methoxypentanoic acid [I-175]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0376] Step 1: (S)-Benzyl 4-methyl-2-(phenylmethylsulfonamido)pentanoate:

[0377] To a solution of 3-(benzyloxy)propan-1-ol (10.0 g, 60.24 mmol) in DMSO (100 mL) was added IBX (20.2 g, 72.29 mmol) under ice bath. was added. The mixture was warmed to room temperature and stirred at this temperature for 17 h. 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) and dried (NaSO). The solution was concentrated and the crude was purified by SGC to give a light yellow liquid (8.0 g, 81%). 1H NMR (500 MHz, CDCl3) δ 9.77 (s, 1H), 7.36-7.26 (m, 5H), 4.53 (s, 2H), 3.8-3.83 (m, 2H), 2.71-2.68 (m, 2H).

[0378] Step 2: (4-(benzyloxy)-1,1,1-trifluorobutan-2-yloxy)trimethylsilane:

[0379] To a solution of 3-(benzyloxy)propanal (4.0 g, 24.4 mmol) in THF (50 mL) was added trimethyl(trifluoromethyl)silane (10.4 g, 73.2 mmol) at room temperature, followed by CsF (0.37 g, 2.44 mmol). The resulting solution was stirred at room temperature for 2 hours. It 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 give (4-(benzyloxy)-1,1,1-trifluorobutan-2-yloxy)trimethylsilane as a colorless liquid (4.5 g, 60%). 1H NMR (500 MHz, CDCl3) δ 7.38-7.29 (m, 5H), 4.51 (t, J= 12 Hz, 2 H), 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).

[0380] Step 3: 4-(benzyloxy)-1,1,1-trifluorobutan-2-ol:

[0381] A solution of 4-(benzyloxy)-1,1,1-trifluorobutan-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, then concentrated and purified by ISCO Biotage to give 4-(benzyloxy)-1,1,1-trifluorobutan-2-ol (2.75 g, 80%) as a colorless liquid.

[0382] Step 4: ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene:

[0383] To a solution of 4-(benzyloxy)-1,1,1-trifluorobutan-2-ol (2.75 g, 11.75 mmol) in THF (100 mL) was added t-BuOK (1.58 g, 14.1 mmol) at 0 °C and stirred at this temperature for 30 min. Next, MeI (2.17 g, 15.28 mmol) was added and stirred at room temperature for another 1 h. The reaction 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 (Na SO ), filtered, and concentrated. The crude product was purified by ISCO Biotage to give ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene (2.04 g, 70%) as a colorless liquid. 1H NMR (500 MHz, CDCl3) δ 7.38-7.29 (m, 5H), 4.53 (t, J= 12 Hz, 2 H), 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).

[0384] Step 5: 4,4,4-trifluoro-3-methoxybutan-1-ol:

[0385] 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 h, then filtered and concentrated to give 4,4,4-trifluoro-3-methoxybutan-1-ol as a colorless liquid. This crude material was used directly in the next step.

[0386] Step 6: 4,4,4-trifluoro-3-methoxybutanal:

[0387] To a solution of 4,4,4-trifluoro-3-methoxybutan-1-ol (1.3 g, crude from the last step) in DMSO (20 mL) was added IBX (2.76 g, 9.88 mmol) under an ice bath. The mixture was warmed to room temperature and stirred at this temperature for 17 hours. The reaction mixture was poured into water (80 mL) and extracted with EtO (80 mL × 2). The organic phase was washed with water (80 mL × 3) and brine (80 mL), and the solution was used directly in the next step.

[0388] Step 7: 2-(benzylamino)-5,5,5-trifluoro-4-methoxypentanenitrile:

[0389] To a solution of the above 4,4,4-trifluoro-3-methoxybutanal in EtO (160 mL) was added benzylamine (2 mL), AcOH (2.0 mL), and then TMSCN (3 mL) using an ice bath. The mixture was warmed 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 (NaSO), filtered, and concentrated in vacuo to give 2-(benzylamino)-5,5,5-trifluoro-4-methoxypentanenitrile (2.0 g, crude) as a brown, thick oil, which was used in the next step. ESI-MS (EI + , m / z):

[0390] Step 8: 2-(benzylamino)-5,5,5-trifluoro-4-methoxypentanoic acid:

[0391] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-methoxypentanenitrile (2.0 g, crude) in concentrated HCl (30 mL) and AcOH (10 mL) was heated to 100 °C for 17 h. The solution was concentrated to dryness, diluted with HO (100 mL) and ACN (50 mL), the pH was adjusted to 3-4 with saturated NaHCO solution, and the mixture was filtered and dried to give 2-(benzylamino)-5,5,5-trifluoro-4-methoxypentanoic acid (0.8 g, 35% over 4 steps) as a brown solid. ESI-MS (EI + , m / z):[M+H] + .

[0392] Step 9: 2-Amino-5,5,5-trifluoro-4-methoxypentanoic acid [I-175]:

[0393] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-methoxypentanoic acid (300 mg, 1.03 mmol) and HCOONH4 (650 mg, 10.3 mmol) in MeOH (10 mL) was stirred at 60 °C for 2 h, then filtered and concentrated. The crude was purified by reverse-phase Biotage to give 2-amino-5,5,5-trifluoro-4-methoxypentanoic acid [I-175] as a white solid. 1H NMR (500 MHz, 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).

[0394] (Example 176) 2-amino-4,4,5-trimethylhexanoic acid [I-176]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0395] Step 1: Diethyl 2-(2,3-dimethylbutan-2-yl)malonate:

[0396] A solution of diethyl 2-(propan-2-ylidene)malonate (2 g, 10.0 mmol) in THF (60 mL) was cooled to 0° C., and then copper(I) iodide (2.9 g, 15.0 mmol) was added. The mixture was stirred at 0° C. for 0.5 hours. Next, isopropylmagnesium bromide (1 mol / L, 30.0 mL, 30.0 mmol) was added dropwise to the above mixture at 0° C. The mixture was stirred at 0° C. for 2 hours. The mixture was quenched with HCl (1 mol / L) and EtOAc (60 mL) was added. * The organic phase was separated, washed with water (100 mL x 2) and brine (130 mL), dried (NaSO), filtered, and concentrated in vacuo to give diethyl 2-(2,3-dimethylbutan-2-yl)malonate (2.4 g, 10.0 mmol, 98%) as a yellow solid. ESI-MS (EI + , m / z): 245.3 [M+H] + .

[0397] Step 2: 2-(2,3-dimethylbutan-2-yl)malonic acid:

[0398] A mixture of diethyl 2-(2,3-dimethylbutan-2-yl)malonate acetamide (2.4 g, 10.0 mmol) and lithium hydroxide hydrate (2.1 g, 50.0 mmol) in DMSO (50 mL) and water (10 mL) was heated to 98 °C and maintained for 20 h. 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 (NaSO), filtered, and concentrated in vacuo to give 2-(2,3-dimethylbutan-2-yl)malonic acid (1.8 g, 10.0 mmol, 95%) as a yellow oil. ESI-MS (EI + , m / z): 212.2 [M+H] + .

[0399] Step 3: 3,3,4-trimethylpentanoic acid:

[0400] A solution of 2-(2,3-dimethylbutan-2-yl)malonic acid (1.8 g, 10.0 mmol) in DMSO (30 mL) was heated to 120 °C and maintained for 12 h. 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 in vacuo to give 3,3,4-trimethylpentanoic acid (1.4 g, 10.0 mmol, 95%) as a yellow oil. ESI-MS (EI-, m / z): 143.2 [M−H] + .

[0401] Step 4: N-Methoxy-N,3,3,4-tetramethylpentanamide:

[0402] To a solution of 3,3,4-trimethylpentanoic acid (1.4 g, 10.0 mmol) in DMF (30 mL) at 20° C. was added N,O-dimethylhydroxylamine hydrochloride (1.2 g, 12.0 mmol) followed by DIEA (3.8 g, 30.0 mmol). HATU (5.8 g, 15.0 mmol) was then added. The mixture was heated to 25° C. with stirring and maintained for 18 hours. The reaction mixture was then diluted with water followed by methyl tert-butyl ether (50 mL). * The mixture was quenched with 2). The phases were separated and the organic layer was washed with brine (80 mL * 3), dried over NaSO, filtered, and concentrated in vacuo to give N-methoxy-N,3,3,4-tetramethylpentanamide (1.5 g, 90%) as a brown oil. ESI-MS (EI + , m / z): 188.2 [M+H] + .

[0403] Step 4: 3,3,4-trimethylpentanal:

[0404] To a solution of N-methoxy-N,3,3,4-tetramethylpentanamide (1.9 g, 0.01 mol) in THF (30 mL) was added LiAlH (1 g, 0.03 mol) at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was diluted with water, followed by methyl tert-butyl ether (50 mL). * The layers were separated and the organic layer was washed with brine (80 mL * The extract was washed with 3), dried over NaSO, 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.

[0405] Step 5: 2-(benzylamino)-4,4,5-trimethylhexanenitrile:

[0406] To a solution of the above 3,3,4-trimethylpentanal in methyl tert-butyl ether (120 mL) was added benzylamine (1.6 mL), AcOH (1.0 mL), and then TMSCN (1.8 mL) using an ice bath. The mixture was warmed 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 (Na SO ), filtered, and concentrated in vacuo to give 2-(benzylamino)-4,4,5-trimethylhexanenitrile (2 g, crude) as a brown oil, which was used in the next step. ESI-MS (EI+, m / z): 245.4 [M+H] + .

[0407] Step 6: 2-(benzylamino)-4,4,5-trimethylhexanoic acid:

[0408] A solution of 2-(benzylamino)-4,4,5-trimethylhexanenitrile (2 g, crude) in concentrated HCl (60 mL) and AcOH (10 mL) was heated to 95 °C for 18 h. The solution was cooled to 15 °C, the pH was adjusted to 3-4 with saturated NaHCO solution, and the mixture was filtered and dried to give 2-(benzylamino)-4,4,5-trimethylhexanenitrile. The carboxylic acid (0.6 g, 2.3 mmol, 30% over three steps) was obtained as a white solid. ESI-MS (EI + , m / z): 264.4 [M+H] + .

[0409] 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0410] To a solution of 2-(benzylamino)-4,4,5-trimethylhexanoic acid (78 mg, 0.3 mmol) in MeOH (8 mL) was added HCOONH4 (0.13 g, 2.0 mmol) and Pd / C (30 mg) at room temperature. The mixture was stirred at 60 °C for 2 h. The reaction mixture was filtered and concentrated to give the crude product, which was purified by reverse-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 (500 MHz, MeOD) δ 3.56 (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).

[0411] (Example 178) 2-amino-4,4-dimethylheptanoic acid [I-178] [ka] Synthesis scheme: [ka] Procedures and characterization:

[0412] The procedure was the same as that used in Example 176.

[0413] 2-Amino-4,4-dimethylheptanoic acid [I-178]: 1 H NMR (500 MHz, MeOD-d4) δ 3.77 (t, J = 6 Hz, 1H), 2.09-2.05 (m, 1H), 1.6-1.56 (m, 1H), 1.37-1.26 (m, 4H), 1.01-0.92 (m, 9 H).

[0414] (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]. [ka] Synthesis scheme: [ka] Procedures and characterization:

[0415] The procedure was the same as that used in Example 176.

[0416] 2-Amino-4,4-dimethylheptanoic acid [I-195]: 1 H NMR (500 MHz, D2O) δ 3.87 (t, J = 6.0 Hz, 1H), 1.93 (dd, J = 15.0 Hz, J = 5.5 Hz, 1H), 1.57 (dd, J = 15.0 Hz, J = 6.5 Hz, 1H), 1.22-1.26 (m, 2H), 0.86 (d, (dd, J = 2.0 Hz, 6H), 0.76 (t, J = 7.5 Hz, 3H).

[0417] (S)-2-Amino-4,4-dimethylhexanoic acid [I-120]: 1 H NMR (500 MHz, MeOD-d4) δ 3.43 (dd, J = 7.0 Hz, J = 5.0 Hz, 1H), 1.95 (dd, J = 15.0 Hz, J = 5.0 Hz, 1H), 1.42 (dd, J = 15.0 Hz, J = 7.0 Hz, 1H), 1.23-1.28 (m, 2H), 0.87 (d, (dd, J = 4.5 Hz, 6H), 0.80 (t, J = 7.5 Hz, 3H).

[0418] (R)-2-Amino-4,4-dimethylhexanoic acid [I-191]: 1 H NMR (500 MHz, MeOD-d4) δ 3.43 (dd, J = 7.0 Hz, J = 5.0 Hz, 1H), 1.95 (dd, J = 15.0 Hz, J = 5.0 Hz, 1H), 1.42 (dd, J = 15.0 Hz, J = 7.0 Hz, 1H), 1.23-1.28 (m, 2H), 0.87 (d, (dd, J = 4.5 Hz, 6H), 0.80 (t, J = 7.5 Hz, 3H).

[0419] (Example 177) 2-amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0420] Step 1: N-Methoxy-N-methyl-2-(triphenyl-15-phosphanylidene)acetamide:

[0421] A mixture of 2-chloro-N-methoxy-N-methylacetamide (13.7 g, 0.1 mol) and triphenylphosphane (26.2 g, 0.1 mol) in acetonitrile (200 mL) was heated to 80° C. and held for 20 hours. The mixture was cooled and concentrated below 40° C. to remove the solvent. The residue was dissolved in dichloromethane (200 mL) and then 2N KOH (100 mL). The resulting mixture was stirred at 20° C. for 1 hour. The layers were separated and the organic layer was diluted with brine (200 mL). * The mixture was washed with 3), dried over NaSO, and filtered. The filtrate was concentrated in vacuo to give N-methoxy-N-methyl-2-(triphenyl-15-phosphanylidene)acetamide (36 g, 0.1 mol, 98%) as a yellow solid. ESI-MS (EI+ , m / z): 364.4 [M+H] + .

[0422] Step 2: (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide:

[0423] A mixture of N-methoxy-N-methyl-2-(triphenyl-15-phosphanylidene)acetamide (36.3 g, 0.1 mol) and 4,4,4-trifluorobutan-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 and concentrated in vacuo below 40 °C to remove the solvent. The residue was purified on a silica gel column (200 g, 200-300 mesh, UV 254 nm) eluted with 0-35% ethyl acetate in petroleum ether to give (£)-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] + .

[0424] Step 3: 5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanamide:

[0425] A mixture of (£)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide (6 g, 0.03 mol) and Pd / C (10%, 400 mg) in THF (100 mL) was stirred at 30° C. for 18 h. The mixture was filtered, and the filtrate was concentrated to dryness in vacuo to give 5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanamide (6 g, 0.03 mol, 98%) as a yellow oil. ESI-MS (EI+, m / z): 214.2 [M+H] + .

[0426] Step 4: 5,5,5-trifluoro-3-methylpentanal:

[0427] To a solution of 5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanamide (6 g, 0.03 mol) in THF (100 mL) was added LiAlH (1 g, 0.03 mol) at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was diluted with water and then with methyl tert-butyl ether (60 mL). * The layers were separated and the organic layer was washed with brine (80 mL * The extract was washed with 3), dried over NaSO, and filtered. The filtrate was concentrated to give 5,5,5-trifluoro-3-methylpentanal (4.5 g, 95%) as a colorless solution, which was used directly in the next step.

[0428] Step 5: 2-(benzylamino)-6,6,6-trifluoro-4-methylhexanenitrile:

[0429] To a solution of the above 5,5,5-trifluoro-3-methylpentanal in methyl tert-butyl ether (200 mL) was added benzylamine (5 mL), AcOH (4.0 mL), and then TMSCN (5 mL) using an ice bath. The mixture was warmed 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 (Na SO ), filtered, and concentrated in vacuo to give 2-(benzylamino)-6,6,6-trifluoro-4-methylhexanenitrile (6 g, crude) as a brown oil, which was used in the next step. ESI-MS (EI+, m / z): 271.3 [M+H] + .

[0430] Step 6: 2-(benzylamino)-6,6,6-trifluoro-4-methylhexanoic acid:

[0431] A solution of 2-(benzylamino)-6,6,6-trifluoro-4-methylhexanenitrile (3 g, crude) in concentrated HCl (100 mL) and AcOH (20 mL) was heated to 100 °C for 17 h. The solution was cooled to 15 °C, the pH was adjusted to 3-4 with saturated NaHCO solution, and the mixture was filtered and dried to give 2-(benzylamino)-6,6,6-trifluoro-4-methylhexanoic acid (1 g, 13.4 mmol, 33% over 3 steps) as a white solid. ESI-MS (EI + , m / z): 290.3 [M+H] + .

[0432] 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0433] To a solution of 2-(benzylamino)-6,6,6-trifluoro-4-methylhexanoic acid (88 mg, 0.31 mmol) in MeOH (8 mL) was added HCOONH4 (0.13 g, 2.0 mmol) and Pd / C (30 mg) at room temperature. The mixture was stirred at 60 °C for 2 h. The reaction mixture was filtered and concentrated to give the crude product, which was purified by reverse-phase silica gel chromatography. Purification by column chromatography gave 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 (500 MHz, DMSO) δ 3.15 (d, J = 5.7 Hz, 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, 6.2 Hz, 3H).

[0434] (Example 179) (S)-2-Amino-5-fluoro-4-(fluoromethyl)pentanoic acid [I-179] [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane:

[0435] To a solution of (2,2-dimethyl-1,3-dioxan-5-yl)methanol (0.29 g, 2.0 mmol) in DMF (10 mL) at 0° C. was added NaH (60% in oil, 0.12 g, 3.0 mmol). 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 warmed to 10° C. over 3 h and held for 18 h. The reaction mixture was quenched with ice water followed by EtOAc (60 mL). The layers were separated and the organic layer was washed with brine (60 mL). * The mixture was washed with 3), dried over NaSO, and filtered. The filtrate was concentrated, and the residue was purified on a silica gel column (20 g, UV 254 nm, eluted with 10% to 50% EtOAc in 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] + . Step 2: 2-(benzyloxymethyl)propane-1,3-diol:

[0436] To a solution of 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane (930 mg, 3.94 mmol) in MeOH (20 mL) was added 3N aqueous HCl (2 mL). The mixture was stirred at 50° C. for 2 hours. The reaction mixture was concentrated to give DCM ( The mixture was diluted with 20 mL of HCl, washed with brine (15 mL), dried and evaporated to give a crude colorless oil (780 mg, 100%). + , m / z): 197 [M+H] + . Step 3: ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene:

[0437] To a pre-cooled solution of 2-(benzyloxymethyl)propane-1,3-diol (780 mg, 3.94 mmol) in DCM (20 mL) was added DAST (1.9 g, 11.8 mmol) dropwise at -78 °C. The mixture was stirred at 20 °C for 24 h. The reaction mixture was quenched with saturated aqueous NaHCO (10 mL) at -78 °C. The DCM phase was separated, washed with brine, dried over MgSO, 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 (500 MHz, CDCl3) δ 7.37 - 7.28 (m, 5H), 4.65 - 4.57 (m, 2H), 4.55 - 4.48 (m, 4H), 3.57 (d, J = 6.2 Hz, 2H), 2.50 - 2.34 (m, 1H). Step 4: 3-Fluoro-2-(fluoromethyl)propan-1-ol:

[0438] To a pre-cooled solution of ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene (800 mg, 3.94 mmol) in DCM (20 mL) at −78 °C, BCl / toluene (1 M, 6 mL, 6.0 mmol) was added dropwise. The mixture was stirred at −78 to 0 °C for 2 h. The reaction mixture was quenched with HO (0.5 mL) at −78 °C. The DCM phase was dried over MgSO and filtered, and the solution (approximately 20 mL) was used directly in the next step. Step 5: 3-Fluoro-2-(fluoromethyl)propyl trifluoromethanesulfonate:

[0439] To a pre-cooled solution of 3-fluoro-2-(fluoromethyl)propan-1-ol (8 mL of the solution from Step 4, 1.6 mmol) at −40° C. was added pyridine (380 mg, 4.8 mmol) and then TfO (1.36 g, 4.8 mmol) dropwise. The mixture was stirred at −30° C. for 1 h. The reaction mixture was quenched with brine (20 mL) at −40° C. The DCM phase was separated, dried over MgSO, filtered, and concentrated to give a crude tan oil (200 mg, 51%), which was used directly in the next step. Step 6: tert-butyl 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)pentanoate:

[0440] To a pre-cooled solution of tert-butyl 2-(diphenylmethyleneamino)acetate (944 mg, 3.2 mmol) in THF (20 mL) was added LDA (2.5 M in THF / toluene / hexane, 1.28 mL, 3.2 mmol) at −78° C. over 25 min. The mixture was stirred at this temperature for 10 min. A solution of 3-fluoro-2-(fluoromethyl)propyl trifluoromethanesulfonate (200 mg, 0.82 mmol) in THF (2 mL) was added dropwise at −78° C. The reaction mixture was placed in a fresh cooling bath and stirred for an additional 1 h. The reaction mixture was quenched with saturated aqueous NH4Cl (20 mL) and MTBE (30 mL). * Extraction with 2), washing with HO, brine (50 mL each), drying and concentration gave the crude product, which was purified twice by chromatography (silica gel, PE to 5% EA / PE) to give the desired product. The product (22 mg, 6.9%) was obtained as a white solid. + , m / z): 388 [M+H] + . 1 H NMR (500 MHz, DMSO) δ 7.56 - 7.45 (m, 6H), 7.41 (t, J = 7.4 Hz, 2H), 7.18 (d, J = 6.3 Hz, 2H), 4.50 - 4.17 (m, 4H), 3.91 (dd, J = 7.7, 5.5 Hz, 1H), 2.11 - 1.97 (m, 1H), 1 .87 (dd, J = 12.7, 5.5 Hz, 2H), 1.38 (s, 9H). Step 7: (S)-2-amino-5-fluoro-4-(fluoromethyl)pentanoic acid hydrochloride:

[0441] A solution of tert-butyl 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)pentanoate (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 EtO 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 EtO 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 the 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 (500 MHz, DMSO) δ 7.85 (bs, 3H), 4.48 (dd, J = 48.3, 14.2 Hz, 4H), 3.46 - 3.36 (m, 1H), 2.47 - 2.26 (m, 1H), 1.78 (dt, J = 14.3, 7.3 Hz, 1H), 1.63 - 1.53 (m, 1H).

[0442] (Example 187) (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]: [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: (S)-3-Amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0443] To a round-bottom flask containing (S)-2-amino-4-methylpent-4-enoic acid (100 mg) was added concentrated HCl (1 mL) and SOCl (0.2 mL). The mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated and washed with EtO 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] + . 1 H NMR (500 MHz, DMSO) δ 8.80 (bs, 3H), 4.58 (dd, J = 11.2, 9.3 Hz, 1H), 2.53 - 2.48 (m, 1H), 2.13 (t, J = 11.7 Hz, 1H), 1.45 (s, 3H), 1.40 (s, 3H).

[0444] (Example 90) Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-90]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0445] Step 1: Diethyl 2-(1,1,1-trifluoropropan-2-ylidene)malonate:

[0446] To THF (1 L) was added TiCl4 (65.8 mL, 600 mmol) dropwise over 20 minutes using an ice bath, followed by CCl4 (30 mL). To this mixture was added diethyl malonate (48.0 g, 300 mmol) and 1,1-difluoropropan-2-one (56.4 g, 600 mmol). The mixture was warmed to room temperature and stirred overnight. Pyridine (200 mL) was added dropwise over 20 minutes using an ice bath, and the reaction mixture was poured into water (2 L) and filtered. The filtrate was extracted with EtOAc (500 mL x 2). The organic phase was washed with water (600 mL), 1 M HCl (600 mL x 2), water (600 mL), saturated NaHCO3 (600 mL), and brine (600 mL), and dried (Na2SO4). 4), filtered, concentrated in vacuo, and purified by chromatography (silica, ethyl acetate / petroleum ether 0% to 5%) to give diethyl 2-(1,1-difluoropropan-2-ylidene)malonate (60.9 g, 258 mmol, 86%) as a colorless liquid. ESI-MS (EI + , m / z): 237.0 [M+H] + . 1 H-NMR (500 MHz, CDCl3): δ 6.97 (t, J = 55.5 Hz, 1H), 4.25-4.33 (m, 4H), 2.03 (s, 3H), 1.29-1.34 (m, 6H).

[0447] Step 2: Diethyl 2-(1,1-difluoro-2-methylpropan-2-yl)malonate:

[0448] To a mixture of diethyl 2-(1,1-difluoropropan-2-ylidene)malonate (10.0 g, 42.3 mmol) and CuI (12.1 g, 63.5 mmol) in DCM (100 mL) and THF (25 mL) was added MeMgI (42.3 mL, 130.5 mmol) at −20° C. over 1 h, and the solution was poured into ice water (200 mL) and treated with saturated NH4Cl solution (100 mL). The mixture was stirred for 30 min, filtered, the filtrate was extracted with DCM (100 mL), the organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (NaSO), filtered and concentrated in vacuo to give diethyl 2-(1,1-difluoro-2-methylpropan-2-yl)malonate (10.1 g, 40.2 mmol, 95%) as a brown liquid, which was used in the next step. ESI-MS (EI + , m / z): 253.1 [M+H] + . 1 H-NMR (500 MHz, CDCl3): δ 6.05 (t, J = 57.5 Hz, 1H), 4.17-4.23 (m, 4H), 3.49 (s, 1H), 1.22-1.28 (m, 6H), 1.20 (s, 6H).

[0449] Step 3: 4,4-Difluoro-3,3-dimethylbutanoic acid:

[0450] A mixture of diethyl 2-(1,1-difluoro-2-methylpropan-2-yl)malonate (6.1 g, 24.2 mmol) and LiOH.HO (5.1 g, 121 mmol) in DMSO (50 mL) and HO (0.5 mL) was heated to 90 °C for 17 h. The mixture was diluted with water (200 mL) and extracted with DCM (100 mL). The pH of the aqueous phase was adjusted to 3-4 with 6 M HCl solution, extracted with DCM (100 mL x 2), dried (NaSO), filtered, and concentrated in vacuo to give 4,4-difluoro-3,3-dimethylbutanoic acid (3.6 g, crude) as a brown liquid. ESI-MS (EI + , m / z):151.1[MH] - .

[0451] Step 4: 4,4-Difluoro-N-methoxy-N,3,3-trimethylbutanamide:

[0452] To a solution of 4,4-difluoro-3,3-dimethylbutanoic 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) was added EtN (7.18 g, 71.1 mmol), followed by stirring at room temperature for 17 h. The mixture was filtered, and the filtrate was diluted with water (200 mL), extracted with EtO (100 mL x 2), washed with water (100 mL), 1 M HCl (100 mL), and brine (100 mL), dried (NaSO), filtered, and concentrated in vacuo to give 4,4-difluoro-N-methoxy-N,3,3-trimethylbutanamide (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 (500 MHz, CDCl3): δ 5.95 (t, J = 57.5 Hz, 1H), 3.69 (s, 3H), 3.17 (s, 3H), 2.51 (s, 2H), 1.12 (s, 6H).

[0453] Step 5: 4,4-Difluoro-3,3-dimethylbutanal:

[0454] To a solution of 4,4-difluoro-N-methoxy-N,3,3-trimethylbutanamide (3.1 g, 15.9 mmol) in THF (80 mL) was added LiAlH (24 mL, 24 mmol) dropwise using an ice bath. After 1 h, the mixture was quenched with citric acid solution (100 mL), the solution was extracted with EtO (100 mL × 2), the organic phase was washed with brine (100 mL), dried (NaSO), and the solution was used in the next step.

[0455] Step 6: 2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanenitrile:

[0456] To a solution of the above 4,4-difluoro-3,3-dimethylbutanal in EtO (200 mL) was added benzylamine (3 mL), AcOH (3 mL), and then TMSCN (3 mL) using an ice bath. The solution was stirred from 0 to RT for 17 h and then diluted with EtOAc (100 mL). The solution was washed with HO (100 mL × 2) and then concentrated to give 2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanenitrile (3.2 g, crude) as a brown liquid. ESI-MS (EI + , m / z): 253.0 [M+H] + .

[0457] Step 7: 2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanoic acid:

[0458] A solution of 2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanenitrile (1.8 g, crude) in concentrated HCl (50 mL) and AcOH (10 mL) was heated to 100 °C for 64 h. The mixture was concentrated to remove the solvent, the pH adjusted to 12 with 1 M NaOH solution, extracted with PE (100 mL), and the aqueous phase adjusted to pH 5–6 with 6 M HCl. A white solid formed, which was filtered, and the filter cake was washed with water (50 mL) and dried in vacuo to give 2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (1.3 g, 4.80 mmol, 54%, 3 steps) as a white solid. ESI-MS (EI + , m / z):272.0.

[0459] Step 8: 2-Amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0460] A mixture of 2-(benzylamino)-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 h. The mixture was filtered, and the filtrate was concentrated to give 2-amino-5,5-difluoro-4,4-dimethylpentanoic acid (1.0 g, crude) as a white solid. ESI-MS (EI + , m / z):182.0.

[0461] Step 9: 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid

[0462] To a solution of 2-amino-5,5-difluoro-4,4-dimethylpentanoic acid (1.0 g, crude) and NaHCO3 (1.27 g, 14.4 mmol) in acetone (30 mL) and HO (30 mL) was added CbzOSu (2.39 g, 9.6 mmol) using an ice bath. After stirring for 17 h, the pH of the mixture was adjusted to 3-4 with 1 M HCl solution, and the solution was extracted with EtOAc (50 mL × 2), washed with brine (50 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The crude product was purified by reverse-phase silica gel chromatography followed by chiral preparative HPLC [column, CC4 4.6]. * 250m Purification by HPLC using a 5 μm column (solvent: MeOH (0.2% methanolic ammonia) [molecular weight: 5 μm; solvent: MeOH (0.2% methanolic ammonia)] afforded (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (400 mg, 1.27 mmol, 26%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (380 mg, 1.21 mmol, 25%, 2 steps) as two colorless oils. + , m / z):316.0.

[0463] Step 10: (S)-2-Amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0464] A solution of (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic 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 in vacuo, and purified by reverse-phase silica gel chromatography to give (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid (115.7 mg, 0.64 mmol, 50%). ESI-MS (EI + , m / z):182.0 1 H-NMR (500 MHz, MeOD-d4): δ 5.60 (t, J = 56.5 Hz, 1H), 3.97 (t, J = 6.0 Hz, 1H), 2.07 (dd, J = 15.5 Hz, J = 5.5 Hz, 1H), 1.77 (dd, J = 15.5 Hz, J = 6.5 Hz, 1H), 0.96 (d, J = 9.5 Hz, 6H).

[0465] (Example 88) Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-88]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0466] Step 1: (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide:

[0467] To a solution of 2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanenitrile (1.2 g, 4.76 mmol) in DCM (20 mL) was added concentrated HSO (10 mL) dropwise over 5 min using an ice bath, and the mixture was warmed to room temperature and stirred for 6 h. The mixture was poured into ice water (100 mL) and the pH of the solution was adjusted to 8-9 with 10% NaOH solution. It was then extracted with EtOAc (100 mL x 2). The organic phase was washed with water (100 mL) and brine (100 mL), dried (NaSO), filtered, concentrated in vacuo, and purified by chiral preparative HPLC (MeOH / DCM 0% to 5%) [column, CC4 4.6]. * 250mm 5um; Solvent, M Purification with HCl (0.2% methanolic ammonia)] afforded (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide (400 mg, 1.48 mmol, 31%) and (R)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide (380 mg, 1.41 mmol, 30%) as two colorless liquids. + , m / z): 253.0 [M+H] + .

[0468] Step 2: (S)-2-amino-5,5-difluoro-4,4-dimethylpentanamide:

[0469] 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 h. The mixture was filtered, and the filtrate was concentrated and purified by reverse-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 (500 MHz, MeOD-d4): δ 5.66 (t, J = 56.5 Hz, 1H), 3.95 (dd, J = 8.0 Hz, J = 5.0 Hz, 1H), 2.14 (dd, J = 10.0 Hz, J = 8.0 Hz, 1H), 1.83 (dd, J = 14.5 Hz, J = 5.5 Hz, 1H), 1.12 (d, J = 15.0 Hz, 6H).

[0470] (Example 185) Synthesis of (S)-methyl 2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate [I-185]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0471] The procedure for 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid was the same as in Example 90.

[0472] Step 1: (S)-Methyl 2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate:

[0473] 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)-methyl 2-amino-4-methylpentanoate 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 Biotage reverse-phase silica gel chromatography to give (S)-methyl 2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate (95 mg, 45%) as a white solid. ESI-MS (EI + , m / z):443.0.

[0474] Step 2: (S)-Methyl 2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate:

[0475] A solution of (S)-methyl 2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanamido)-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 reverse-phase silica gel chromatography. Purification by Biotage afforded (S)-methyl 2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate (45 mg, 69%) as a white solid. ESI-MS (EI + , m / z):309.0. 1H-NMR (500 MHz, DMSO-d6): 9.11 (d, J = 7 Hz, 1H), 8.41 (s, 3H), 5.81 (t, J = 56.5 Hz, 1H), 4.34-4.31 (m, 1H), 3.89-3.81 (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).

[0476] (Example 184) Synthesis of (S)-methyl 2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate [I-184] [ka] The procedure was the same as in Examples 90 and 185.

[0477] (S)-Methyl 2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate: ESI-MS (EI + , m / z):309.0. 1 H-NMR (500 MHz, DMSO-d6): 9.18 (d, J = 7 Hz, 1H), 8.38 (s, 3H), 5.79 (t, J = 56.5 Hz, 1H), 4.37-4.32 (m, 1H), 3.85-3.78 (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).

[0478] Example 145 Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid, (2R,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid, (2R,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid, and (2S,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid: [3d; I-145]; [3c; I-146]; [3a; I-167]; [3b; I-250] [ka] Synthesis scheme: [ka] Procedures and characterization:

[0479] Step 1: Synthesis of (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid, (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid, (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid and (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid:

[0480] 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 NaHCO (10 mL) was added CbzOSu (970 mg, 3.9 mmol). The mixture was stirred at room temperature for 3 hours. Then, EtOAc (20 mL) and HO (20 mL) were added, and the water was separated and EtOAc (2 *The combined extracts were washed with brine (20 mL), dried over anhydrous NaSO, 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 pure product was purified by chiral HPLC to give four isomers: (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (150 mg, 15%), (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (40 mg, 3.9%), (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (50 mg, 4.9%), and (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (80 mg, 7.8%), all of which were white solids. ESI-MS (EI+, m / z): 342.0 [M+Na]+.

[0481] Step 2-A: Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0482] (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro A solution of 2S,4R-4-methylpentanoic acid (150 mg, 0.47 mmol) and Pd / C (75 mg) in MeOH (15 mL) was stirred at room temperature for 3 h. The reaction mixture was filtered and concentrated to give (2S,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid (51.7 mg, 59%) as a white solid. ESI-MS (EI + , m / z): 186.2 [M+H] + . 1 H-NMR (500 MHz, 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 = 7.0 Hz, 3 H).

[0483] Step 2-B: Synthesis of (2R,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0484] A solution of (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic 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-methylpentanoic acid (13.3 mg, 60%) as a white solid. ESI-MS (EI + , m / z): 186.2 [M+H] + . 1 H-NMR (500 MHz, MeOD): δ 3.51-3.47 (m, 1H), 2.64-2.58 (br, 1H), 2.12-2.06 (m, 1H),1.63-1.57 (m, 1H),1.13 (d, J = 7.0 Hz, 3 H).

[0485] Step 2-C: Synthesis of (2R,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0486] A solution of (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic 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-methylpentanoic acid (18.0 mg, 61%) as a white solid. ESI-MS (EI + , m / z): 186.1 [M+H] + . 1H-NMR (500 MHz, MeOD): δ 3.51-3.47 (m, 1H), 2.46-2.44 (br, 1H), 1.95-1.87 (m, 2H), 1.11 (d, J = 7.0 Hz, 3H).

[0487] Step 2-D: Synthesis of (2S,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0488] A solution of (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic 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-methylpentanoic acid (38.1 mg, 82%) as a white solid. ESI-MS (EI + , m / z): 186.2 [M+H] + . 1 H-NMR (500 MHz, MeOD): δ 3.51-3.47 (m, 1H), 2.46-2.44 (br, 1H), 1.95-1.87 (m, 2H), 1.11 (d, J = 7.0 Hz, 3H).

[0489] (Example 128) (S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanoic acid (I-128): [ka] Synthesis scheme: [ka] Procedures and characterization: The procedure used was the same as that used in Example 187.

[0490] (S)-2-Amino-5,5,5-trifluoro-4,4-dimethylpentanoic acid: ESI-MS (EI + , m / z):200.11 H-NMR (500 MHz, D2O): δ 3.94 (t, J = 5.5 Hz, 1H), 2.23 (dd, J = 15.5 Hz, J = 5.5 Hz, 1H), 1.90 (dd, J = 15.5 Hz, J = 6.0 Hz, 1H), 1.13 (d, J = 8.5 Hz, 6H).

[0491] (Example 188) (S)-methyl 2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanamido)-4-methylpentanoate [I-188] [ka] Synthesis scheme: [ka] Procedures and characterization:

[0492] The procedure for 2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylpentanoic acid was the same as in Example 90.

[0493] Step 1: (S)-Methyl 2-((R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylpentanamido)-4-methylpentanoate:

[0494] 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)-methyl 2-amino-4-methylpentanoate 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 reverse-phase silica gel chromatography using Biotage to give (S)-methyl 2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanamido)-4-methylpentanoate (120 mg, 58%) as a white solid. ESI-MS (EI + , m / z):461.0.

[0495] Step 2: (S)-Methyl 2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanamido)-4-methylpentanoate:

[0496] A solution of (S)-methyl 2-((S)-2-(benzyloxycarbonylamino)-5,5,5-difluoro-4,4-dimethylpentanamido)-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 material was purified by Biotage reverse-phase silica gel chromatography to give (S)-methyl 2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanamido)-4-methylpentanoate (49 mg, 57%) as a white solid. ESI-MS (EI + , m / z):326.0. 1H-NMR (500 MHz, MeOD-d4): 4.47 (t, J = 7.5 Hz, 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).

[0497] (Example 189) (S)-methyl 2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanamido)-4-methylpentanoate [I-189]: [ka] Synthesis scheme: The procedure used was the same as that used in Example 188. Procedures and characterization: (Example 189) (S)-methyl 2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanamido)-4-methylpentanoate [I-189]: 1 H-NMR (500 MHz, MeOD-d4): 4.52 (t, J = 7.5 Hz, 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).

[0498] (Example 108) (S)-2-Amino-6-fluorohexanoic acid [I-108]. [ka]

[0499] (Example 109) (R)-2-Amino-6-fluorohexanoic acid [I-109]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: tert-butyl 2-(diphenylmethyleneamino)-6-fluorohexanoate:

[0500] A mixture of 1-fluoro-4-iodobutane (2.0 g, 9.90 mmol), tert-butyl 2-(diphenylmethyleneamino)acetate (2.43 g, 8.25 mmol), TBAB (266 mg, 0.83 mmol), and KOH (aqueous 50%) (10 mL) in DCM (10 mL) and toluene (25 mL) was stirred at 50 °C for 16 h. The solution was purified by SGC (silica, ethyl acetate / petroleum ether = 1 / 5) to give tert-butyl 2-(diphenylmethyleneamino)-6-fluorohexanoate (0.91 g, 2.47 mmol, 30%) as a colorless oil. MS (EI+, m / z): 370.2 [M+H] + . Step 2: (S)-2-Amino-6-fluorohexanoic acid [I-108]:

[0501] A solution of (S)-tert-butyl 2-(diphenylmethyleneamino)-6-fluorohexanoate (360 mg, 0.97 mmol) in dioxane (10 mL) and HCl (aqueous 6 M) was stirred at room temperature for 16 h. 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] as a white solid (125 mg, 0.84 mmol, 86%). ESI-MS (EI+, m / z): 150.3 [M+H] +. 1H NMR (500 MHz, D2O) δ 4.469 (t, J = 6.0 Hz, 1H), 4.351 (t, J = 6.0 Hz, 1H), 3.950 (t, J = 6.0 Hz, 1H), 1.904-1.820 (m, 2H), 1.690-1.588 (m, 2H), 1.456-1.388 (m, 2H). Step 2: (R)-2-Amino-6-fluorohexanoic acid [I-109]:

[0502] A solution of (R)-tert-butyl 2-(diphenylmethyleneamino)-6-fluorohexanoate (300 mg, 0.81 mmol) in dioxane (10 mL) and HCl (aqueous 6 M) was stirred at room temperature for 16 h. 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] as a white solid (35 mg, 0.23 mmol, 29%). ESI-MS (EI+, m / z): 150.2 [M+H] + . 1H NMR (500 MHz, D2O) δ 4.505 (t, J = 6.0 Hz, 1H), 4.410 (t, J = 6.0 Hz, 1H), 3.823 (t, J = 6.0 Hz, 1H), 1.906-1.827 (m, 2H), 1.722-1.639 (m, 2H), 1.485-1.399 (m, 2H).

[0503] (Example 198) Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoate (I-198): [ka] Synthesis scheme: [ka] Procedures and characterization:

[0504] Step 1: 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide:

[0505] To a stirred solution of hexafluoroacetone trihydrate (30 g, 136 mmol), H2SO4 (100 mL, concentrated) was slowly added dropwise over 1 h, and gaseous hexafluoroacetone was introduced into a solution of N-methoxy-N-methyl-2-(triphenylphosphoranylidene)-acetamide (10 g, 27.5 mmol) in THF (200 mL). The mixture was stirred at room temperature for 16 h. Petroleum ether (200 mL) was then added, and the 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 small oil. ESI-MS (EI + , m / z): 252.1 [M+H] + . 1 H-NMR (500 MHz, CDCl3): δ 7.15 (s, 1H), 3.67 (s, 3H), 3.26 (s, 3H).

[0506] Step 2: 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butanamide:

[0507] A mixture of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide (4.5 g, 17.9 mmol), Pd(OH) / C (620 mg) in MeOH (100 mL) was stirred under a hydrogen atmosphere at room temperature for 16 h. It was then filtered and concentrated to give 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butanamide (1.8 g, 7.1 mmol, 40%) as a small oil. The product was obtained as a solid. + , m / z): 254.1 [M+H] + .

[0508] Step 3: 4,4,4-trifluoro-3-(trifluoromethyl)butanal:

[0509] To a solution of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butanamide (1.8 g, 7.1 mmol) in THF (50 mL) was added LiAlH (8.5 mL, 8.5 mmol) dropwise using an ice bath. After 1 h, the mixture was quenched with citric acid solution (100 mL), the solution was extracted with EtO (100 mL × 2), the organic phase was washed with brine (100 mL) and dried (NaSO), and this solution was used in the next step.

[0510] Step 4: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanenitrile:

[0511] To a solution of the above 4,4,4-trifluoro-3-(trifluoromethyl)butanal in EtO (200 mL) was added benzylamine (2 mL), AcOH (2 mL), and then TMSCN (2 mL) using an ice bath. The solution was stirred from 0 to RT for 17 h and then diluted with EtOAc (100 mL). The solution was washed with HO (100 mL × 2) and then concentrated to give 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanenitrile (2.1 g, crude) as a brown liquid. ESI-MS (EI + , m / z): 311.2 [M+H] + .

[0512] Step 5: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid:

[0513] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanenitrile (2.1 g, crude) in concentrated HCl (50 mL) and AcOH (10 mL) was heated to 100 °C for 40 h. The mixture was concentrated to remove the solvent, the pH adjusted to 12 with 1 M NaOH solution, extracted with PE (100 mL), and the pH of the aqueous phase adjusted to 5-6 with 6 M HCl to form a white solid. The filter cake was filtered, washed with water (50 mL), and dried in vacuo to give 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (1.0 g, 3.0 mmol, 42%, 3 steps) as a white solid. ESI-MS (EI + , m / z):272.0.

[0514] Step 6: Methyl 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoate:

[0515] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (800 mg, 2.4 mmol) in HCl / MeOH (50 mL, 2 M) was heated to 75° C. for 17 h. The solution was concentrated and purified by preparative HPLC (Boston C18 21 * Purification by 250 mm H2O (mobile phase: A: 0.1% TFA; B: ACN) gave methyl 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoate (120 mg, 0.35 mmol, 15%) as a colorless oil. + , m / z): 344.1 [M+H] + .

[0516] Step 7: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoate trifluoroacetate:

[0517] Methyl 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl) A mixture of methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoate (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 h. The mixture was filtered, and the filtrate was concentrated and purified by reverse-phase silica gel chromatography to give methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoate trifluoroacetate (76 mg, 0.21 mmol, 70%) as a white solid. ESI-MS (EI + , m / z): 254.1 [M+H] + . 1 H NMR (500 MHz, MeOD-d4) δ 4.26 (dd, J = 7.5 Hz, J = 6.0 Hz, 1H), 3.91 (m, 4H), 2.49 (dd, J = 8.5 Hz, J = 5.0 Hz, 1H), 2.33-2.37 (m, 1H).

[0518] (Example 164) (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (I-164). [ka] Synthesis scheme: [ka] Procedures and characterization:

[0519] Step 1: 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid:

[0520] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (480 mg, 1.46 mmol) and Pd(OH) / C (20%, 100 mg) in AcOH (15 mL) was stirred under hydrogen at 35° C. for 17 h. The mixture was filtered, and the filtrate was concentrated in vacuo to give 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (460 mg, crude) as a white solid. ESI-MS (EI + , m / z): 240.2 [M+H] + .

[0521] Step 2: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid:

[0522] To a solution of 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (460 mg, crude) and NaHCO (368 mg, 4.38 mmol) in acetone (30 mL) and HO (30 mL) was added CbzOSu (727 mg, 2.92 mmol) using an ice bath. After 17 h, the pH of the reaction mixture was adjusted to 1 M. The mixture was adjusted to 3-4 with HCl solution, and the solution was extracted with EtOAc (50 mL x 2), washed with brine (50 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The crude product was purified by reverse-phase silica gel chromatography and then by chiral preparative HPLC [column, CC4 4.6 * Purification by HPLC using a 500 rpm column (250 mm, 5 μm; solvent, MeOH (0.2% methanolic ammonia)] afforded (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (27 mg, 0.072 mmol, 5%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (22 mg, 0.059 mmol, 4%, 2 steps) as two colorless oils. I+ , m / z): 396.0 [M + Na] + .

[0523] Step 3: (S)-2-Amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid:

[0524] A mixture of (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid (27 mg, 0.072 mmol) and Pd / C (10%, 5 mg) in MeOH (10 mL) was stirred at room temperature for 1 h. The solution was filtered and purified by reverse-phase silica gel chromatography to give (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)pentanoic acid [I-164] (8.5 mg, 0.036 mmol, 49%) as a white solid. MS (EI) + , m / z): 240.2 [M+H] + . 1 H NMR (500 MHz, D2O) δ 3.74-3.80 (m, 2H), 2.88-2.31 (m, 1H), 1.91-2.20 (m, 1H).

[0525] (Example 203) 2-amino-4-cyclopentylbutanoic acid [I-203]: [ka] Synthesis scheme: [ka]

[0526] Procedures and characterization:

[0527] Step 1: 2-Cyclopentylacetaldehyde:

[0528] 3-Cyclopentylpropan-1-ol (2.0 g, 17.5 mmol) in DMSO To a solution of 1H 2 O (40 mL) was added IBX (7.35 g, 26.3 mmol) under an ice bath. The mixture was warmed to room temperature and stirred overnight. The reaction mixture was poured into water (200 mL) and extracted with EtO (100 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (NaSO), and used in the next step.

[0529] Step 2: (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoate:

[0530] To a solution of Wittig reagent (2.5 g, 6.8 mmol) in THF (50 mL) was added NaOt-Bu (785 mg, 8.2 mmol) using an ice bath. After 1 h, the above solution of 2-cyclopentylacetaldehyde in EtO (200 mL) was added. The mixture was warmed 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 (NaSO), filtered, concentrated in vacuo, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 20) to give (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoate (1.0 g, 3.1 mmol, 45%, 2 steps) as a colorless liquid. ESI-MS(EI + , m / z): 326.2 [M+H] + .

[0531] Step 3: tert-Butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbutanoate:

[0532] A mixture of (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoate (240 mg, 0.74 mmol), HCOONH (233 mg, 3.7 mmol), and Pd / C (10%, 30 mg) in MeOH (15 mL) was heated to reflux for 4 h. The mixture was filtered, concentrated, diluted with EtO (50 mL), washed with water (50 mL) and brine (50 mL), dried (NaSO), filtered, and concentrated in vacuo to give tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbutanoate (224 mg, 0.69 mmol, 93%) as a colorless liquid. ESI-MS (EI + , m / z): 328.2 [M+H] + .

[0533] Step 4: 2-amino-4-cyclopentylbutanoic acid:

[0534] A solution of tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbutanoate (224 mg, 0.69 mmol) in 6 M HCl (20 mL) and dioxane (10 mL) was heated to 70 °C for 2 h. The mixture was concentrated in vacuo, diluted with water (30 mL), extracted with EtO (20 mL × 2), and the filtrate was concentrated to dryness to give 2-amino-4-cyclopentylbutanoic acid (114.9 mg, 0.52 mmol, 81%) as a white solid. ESI-MS (EI + , m / z): 172.3 [M+H] + . 1 H-NMR (500 MHz, D2O): δ 3.91 (t, J = 6.0 Hz, 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).

[0535] (Example 202) 2-amino-5-cyclopentylpentanoic acid [I-202]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0536] Step 1: 3-Cyclopentylpropanal:

[0537] To a solution of 3-cyclopentylpropan-1-ol (1.0 g, 7.8 mmol) in DMSO (20 mL) was added IBX (3.28 g, 11.7 mmol) under ice bath. The mixture was warmed to room temperature and stirred overnight. The reaction mixture was poured into water (100 mL) and extracted with EtO (60 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (NaSO), and used in the next step.

[0538] Step 2: (E)-tert-butyl 2-(tert-butoxycarbonylamino)-5-cyclopentylpent-2-enoate:

[0539] To a solution of Wittig reagent (500 mg, 1.36 mmol) in THF (15 mL) was added NaOt-Bu (157 mg, 1.63 mmol) using an ice bath. After 1 h, a solution of the above 3-cyclopentylpropanal in EtO (100 mL) was added. The mixture was warmed 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 (NaSO), filtered, concentrated in vacuo, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 20) to give (E)-tert-butyl 2-(tert-butoxycarbonylamino)-5-cyclopentylpent-2-enoate (250 mg, 0.74 mmol, 9.5%, 2 steps) as a colorless liquid. ESI-MS(EI + , m / z): 340.2 [M+H] + .

[0540] Step 3: tert-Butyl 2-(tert-butoxycarbonylamino)-5-cyclopentylpentanoate:

[0541] 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 h. 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] + .

[0542] Step 4: 2-amino-5-cyclopentylpentanoic acid:

[0543] A solution of 2-(tert-butoxycarbonylamino)-5-cyclopentylpentanoate (250 mg, 0.73 mmol) in 6 M HCl (20 mL) and dioxane (10 mL) was heated to 80 °C for 5 h. The mixture was concentrated in vacuo, diluted with water (30 mL), extracted with EtO (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 (400 MHz, D2O): δ 3.84 (t, J = 6.0 Hz, 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.

[0544] (Example 197) Synthesis of 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide [I-197]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0545] Step 1: 2-(benzylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0546] A mixture of 2-(benzylamino)-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 EtN (94 mg, 0.93 mmol) in DMF (2 mL) was stirred at room temperature for 3 hours. This mixture was purified by preparative HPLC (Boston C18 21 * Purification by 250 mm H NMR (mobile phase: A: 0.1% TFA; B: ACN) gave 2-(benzylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (45 mg, 0.13 mmol, 43%) as a white solid. + , m / z):339.0.

[0547] Step 2: 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0548] 2-(benzylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (45 mg, 0.13 mmol), HCOONH4 (41 mg, 0.6 A mixture of 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (16.3 mg, 0.066 mmol, 49%) and Pd / C (10%, 10 mg) in MeOH (5 mL) was heated to 60° C. for 1 h. The mixture was filtered, and the filtrate was concentrated and purified by reverse-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):249.2 1H NMR (500 MHz, MeOD-d4) δ 5.26 (dd, J = 15.5 Hz, J = 6.0 Hz, 0.5H), 5.08 (dd, J = 16.5 Hz, J = 5.0 Hz, 1H), 4.28-4.31 (m, 0.5H), 2.97 (d, J = 48.5 Hz, 3H), 2.38 (m, 1H), 1.65-1.99 (m, 8H), 11.16 (dt, J = 6.5 Hz, J = 3.0 Hz, 6H).

[0549] (Example 196) 2-amino-5-fluoro-4,4-dimethylpentanoic acid [I-196]. [ka] Synthesis scheme: [ka] Procedures and characterization: Step 1: 3-hydroxy-N-methoxy-N,2,2-trimethylpropanamide:

[0550] A mixture of 3-hydroxy-2,2-dimethylpropanoic 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), and the combined organic layers were diluted with 1 N HCl (30 mL). * 2), washed with 1N NaHCO3 (30 mL x 2) and brine (50 mL), dried and concentrated to give a residue which was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 2) to give 3-hydroxy-N-methoxy-N,2,2-trimethylpropanamide (6.9 g, 50%) as a colorless oil. ESI-MS (EI+ , m / z): 162.2 [M+H] + . Step 2: 3-Fluoro-N-methoxy-N,2,2-trimethylpropanamide:

[0551] To a mixture of 3-hydroxy-N-methoxy-N,2,2-trimethylpropanamide (4.5 g, 27.9 mmol) in DCM (40 mL) cooled to −78 °C, DAST (7.4 mL, 55.9 mmol) was added dropwise. The mixture was then stirred at room temperature for 1–2 h, cooled again to −78 °C, and DAST (4 mL, 27.9 mmol) was added dropwise. The reaction mixture was stirred at room temperature for an additional 1 h. The reaction mixture was cooled to −78 °C, saturated NH4Cl (15 mL) was added slowly, and DCM (50 mL) was added. The organic layer was separated, washed with saturated NH4Cl (30 mL), brine (30 mL × 2), dried, and concentrated to give a residue that was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 4) to give 3-fluoro-N-methoxy-N,2,2-trimethylpropanamide (1.9 g, 28%) as a colorless oil. ESI-MS(EI + , m / z): 164.2 [M+H] + . Step 3: 3-Fluoro-2,2-dimethylpropanal:

[0552] To a mixture of 3-fluoro-N-methoxy-N,2,2-trimethylpropanamide (1.0 g, 61.3 mmol) in THF (10 mL) cooled to 0 °C, LiAlH (6.1 mL, 61.3 mmol, 1 M in THF) was added dropwise. The mixture was then stirred at this temperature for 0.5–1 h. NH Cl (10 mL) was added slowly, and the mixture was extracted with Et O (20 mL × 3), washed with water (15 mL × 2) and brine (15 mL), dried, and used directly in the next step. ESI-MS (EI + , m / z): No MS. Step 4: (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoate:

[0553] A mixture of 3-fluoro-2,2-dimethylpropanal (approximately 630 mg, 6.1 mmol, EtO solution from the above step), tert-butyl 2-(tert-butoxycarbonylamino)-2-diethoxyphosphoryl-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 h. Saturated NH4Cl (15 mL) was added, extracted with EA (30 mL x 3), the organic phases were combined, washed with water (15 mL) and brine (15 mL), dried and concentrated to give a residue which was purified by chromatography (silica, petroleum ether to DCM) to give (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoate (190 mg, 0.60 mmol, 8%) as a white solid. ESI-MS (EI+, m / z): 206 [M-111] + . Step 5: tert-Butyl 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoate: A mixture of (Z)-tert-butyl 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoate (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 tert-butyl 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoate (200 mg, crude) as a colorless liquid. ESI-MS (EI + , m / z): 342.2 [M + Na] + . Step 6: 2-amino-5-fluoro-4,4-dimethylpentanoic acid trifluoroacetic acid: A solution of tert-butyl 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoate (200 mg, crude) in 6 M HCl (20 mL) and dioxane (10 mL) was heated to 50° C. for 17 h. The mixture was concentrated in vacuo, diluted with water (30 mL), extracted with EtO (20 mL×2), and the filtrate was evaporated in vacuo. The extract was concentrated with HCl and purified by reverse-phase silica gel chromatography to give 2-amino-5-cyclopentylpentanoic acid trifluoroacetate (31.7 mg, 0.11 mmol, 19%) as a white solid. + , m / z): 164.2 [M+H] + . 1 H-NMR (500 MHz, D2O): δ 4.16 (d, J = 47.5 Hz, 1H), 3.97 (t, J = 5.5 Hz, 1H), 2.03 (dd, J = 15.5 Hz, J = 5.5 Hz, 1H), 1.71 (dd, J = 15.5 Hz, J = 6.0 Hz, 1H), 0.91 (dd, J = 15.0 Hz, J = 2.0 Hz, 6H).

[0554] (Example 186) Synthesis of 2,4-diamino-4-methylpentanoic acid [I-186]: [ka] Synthesis scheme: [ka]

[0555] Procedures and characterization:

[0556] Step 1: tert-butyl 4-(methoxy(methyl)amino)-2-methyl-4-oxobutan-2-ylcarbamate:

[0557] To a solution of 3-(tert-butoxycarbonylamino)-3-methylbutanoic 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) was added DIPEA (1.49 g, 11.53 mmol). The solution was stirred at room temperature for 2 hours, and then the mixture was diluted with brine (100 mL) and extracted with EtOAc (50 mL × 2). The combined organic layer was concentrated and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 3) to give tert-butyl 4-(methoxy(methyl)amino)-2-methyl-4-oxobutan-2-ylcarbamate (1.0 g, 3.8 mmol, 82%) as a colorless oil. ESI-MS (EI + , m / z): 261.2 [M+H] + .

[0558] Step 2: tert-Butyl 2-methyl-4-oxobutan-2-ylcarbamate:

[0559] To a solution of tert-butyl 4-(methoxy(methyl)amino)-2-methyl-4-oxobutan-2-ylcarbamate (3.8 g, 14.6 mmol) in THF (50 mL) was added LiAlH (16 mL, 1 M in THF) at room temperature. The solution was stirred at room temperature for 2 hours, quenched with NaSO.10H O, filtered, and washed with THF to give tert-butyl 2-methyl-4-oxobutan-2-ylcarbamate as a yellow solution (approximately 14 mmol in THF (110 mL)). MS (EI + , m / z): 146.3 [M + H-56] + .

[0560] Step 3: tert-butyl 4-(benzylamino)-4-cyano-2-methylbutan-2-ylcarbamate:

[0561] To a solution of tert-butyl 2-methyl-4-oxobutan-2-ylcarbamate (crude, ca. 14 mmol in 110 mL of THF) was added BnNH (2.2 mL) and AcOH (2.2 mL). 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 purified by chromatography (silica, ethyl acetate / petroleum ether=1 / 4) to give tert-butyl 4-(benzylamino)-4-cyano-2-methylbutan-2-ylcarbamate (670 mg, 2.11 mmol, 15%) as a yellow dope. MS (EI + , m / z): 318.3 [M+H] + .

[0562] Step 4: tert-butyl 5-amino-4-(benzylamino)-2-methyl-5-oxopentan-2-ylcarbamate:

[0563] To a mixture of tert-butyl 4-(benzylamino)-4-cyano-2-methylbutan-2-ylcarbamate (640 mg, 2.00 mmol) and KCO (550 mg, 3.98 mmol) in DMSO (16 mL) was added 30% HO (0.64 mL, 5.67 mmol) and stirred at room temperature for 17 hours. The reaction mixture was then diluted with HO (200 mL) and extracted with EtOAc (100 mL × 2). The combined organic layers were concentrated to give 2-(benzylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (crude, 890 mg) as a yellow dope. MS (EI+, m / z): 336.0 [M+H] + .

[0564] Step 5: 2-(benzylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid:

[0565] A mixture of tert-butyl 5-amino-4-(benzylamino)-2-methyl-5-oxopentan-2-ylcarbamate (crude 890 mg, ca. 2.0 mmol), KOH (406 mg, 7.25 mmol) in ethane-1,2-diol (9 mL) and HO (9 mL) was stirred at 100° C. for 5 h. The reaction mixture was then diluted with brine (200 mL) and extracted with THF / EA = 2:1 (90 mL × 5). The organic layers were combined, concentrated and purified by reverse HPLC (Boston C18 21 * Purification using a 250 mm 10 μm column (mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) gave 2-(benzylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (120 mg, 0.36 mmol, 18%) as a white solid. MS (EI+, m / z): 337.3 [M+H] + .

[0566] Step 6: 2-amino-4-(tert-butoxycarbonylamino)-4-methyl Pentanoic acid:

[0567] A mixture of 2-(benzylamino)-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 h. The mixture was filtered, and the filtrate was concentrated and purified by reverse-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.

[0568] Step 7: 2,4-Diamino-4-methylpentanoic acid:

[0569] 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 h. The solution was concentrated in vacuo 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 (500 MHz, D2O) δ 4.04 (dd, J = 9.5 Hz, J = 3.5 Hz, 1H), 2.32 (dd, J = 15.0 Hz, J = 9.5 Hz, 1H),1.94 (dd, J = 15.0 Hz, J = 3.0 Hz, 1H), 1.38 (dd, J = 9.5 Hz, J = 5.0 Hz, 6H).

[0570] (Example 199) Synthesis of 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)butan-1-amine [I-199]: [ka] Synthesis scheme: [ka] Procedures and characterization:

[0571] Step 1: N-Methoxy-N-methyl-2-(triphenyl-15-phosphanylidene)acetamide:

[0572] A mixture of 2-chloro-N-methoxy-N-methylacetamide (13.7 g, 0.1 mol) and triphenylphosphane (26.2 g, 0.1 mol) in acetonitrile (200 mL) was heated to 80 °C and maintained for 20 h. The mixture was cooled and concentrated below 40 °C to remove the solvent. The residue was dissolved in dichloromethane (200 mL) and then 2N KOH (100 mL). The resulting mixture was stirred at 20 °C for 1 h. The organic layer was washed with brine (200 mL × 3), dried over NaSO, and filtered. The filtrate was concentrated in vacuo to give N-methoxy-N-methyl-2-(triphenyl-15-phosphanylidene)acetamide (36 g, 0.1 mol, 98%) as a yellow solid. ESI-MS (EI + , m / z): 364.4 [M+H] + .

[0573] Step 2: (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide:

[0574] A mixture of N-methoxy-N-methyl-2-(triphenyl-15-phosphanylidene)acetamide (36.3 g, 0.1 mol) and 1,1,1-trifluoropropan-2-one (22.4 g, 0.2 mol) in tetrahydrofuran (500 mL) was heated to 20 °C and maintained for 20 h. The mixture was cooled and concentrated in vacuo below 40 °C to remove the solvent. The residue was purified on a silica gel column (200 g, 200-300 mesh, UV 254 nm) eluted with 0 to 25% ethyl acetate in petroleum ether to give (£)-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] + .

[0575] Step 3: 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutanamide:

[0576] A mixture of (£)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide (2 g, 0.01 mol) and Pd / C (10%, 200 mg) in THF (50 mL) was stirred at 26 °C for 18 h. The mixture was filtered, and the filtrate was concentrated to dryness in vacuo to give 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutanamide (2 g, 0.01 mol, 98%) as a yellow oil. ESI-MS (EI + , m / z): 200.2 [M+H] + .

[0577] Step 4: 4,4,4-trifluoro-3-methylbutanal:

[0578] To a solution of 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutanamide (2 g, 0.01 mol) in THF (40 mL) was added LiAlH (0.4 g, 0.01 mol) at 0 °C. The mixture was stirred at 0 °C for 1 hour. The reaction mixture was quenched with water and then methyl tert-butyl ether (30 mL × 2). The organic layer was washed with brine (50 mL × 3), dried over Na SO and filtered. The filtrate contained 4,4,4-trifluoro-3-methylbutanal (1.4 g, crude) as a colorless solution, which was used directly in the next step.

[0579] Step 5: 2-(benzylamino)-5,5,5-trifluoro-4-methylpentanenitrile:

[0580] To a solution of the above 4,4,4-trifluoro-3-methylbutanal in methyl tert-butyl ether (100 mL), benzylamine (1.5 mL), AcOH (1.0 mL), and then TMSCN (1.5 mL) were added using an ice bath. The mixture was warmed to 20° C. and stirred overnight. The solution was diluted with water (30 mL) and dissolved in EtOAc (30 mL). The organic phase was washed with water (30 mL x 2) and brine (50 mL), dried (NaSO), filtered, and concentrated in vacuo to give 2-(benzylamino)-5,5,5-trifluoro-4-methylpentanenitrile (2.6 g, crude) as a brown oil, which was used in the next step. ESI-MS (EI + , m / z): 257.3 [M+H] + .

[0581] Step 6: N-benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)butan-1-amine:

[0582] A solution of 2-(benzylamino)-5,5,5-trifluoro-4-methylpentanenitrile (0.3 g, crude) in DMF (10 mL) was added with NH4Cl (0.15 g, 0.003 mol) and NaN3 (0.21 g, 0.003 mol) and heated to 95 °C for 18 h. 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 in vacuo to give N-benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)butan-1-amine (0.1 g, 0.5 mmol, 33% over 3 steps) as a white solid. ESI-MS (EI + , m / z): 300.3 [M+H] + .

[0583] 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)butan-1-amine trifluoroacetic acid:

[0584] To a solution of N-benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)butan-1-amine (160 mg, 0.54 mmol) in MeOH (15 mL) at room temperature, HCOONH4 (0.17 g, 2.7 mmol) and Pd / C (30 mg) were added. The mixture was stirred at 60 °C for 2 h. The reaction mixture was filtered and concentrated to give the crude product, which was purified by reverse-phase silica gel chromatography to give 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)butan-1-amine trifluoroacetate (72.8 mg, 0.23 mmol, 42%) as a white solid. ESI-MS (EI + , m / z): 210.2 [M+H] + ;1H NMR (500 MHz, DMSO-d6) δ 4.67-4.93 (m, 1H), 2.31 - 2.41 (m, 1H), 2.00-2.12 (m, 2H), 0.99 (dd, J = 16.8, J = 6.4 Hz, 6H).

[0585] (Example 210) Western blot assay

[0586] This screening assay measured the in vitro activity of test compounds against the GATOR2 / Sestrin2 complex purified by immunoprecipitation of stably expressed FLAG-WDR24 from HEK293T cells. HEK293T cells (293T) were genetically engineered to stably express N-terminally tagged FLAG-WDR24 by lentiviral transduction. Lentivirus was generated by co-transfecting HEK-293T cells with the lentiviral transfer vector pLJM60, along with the ΔVPR envelope and CMV VSV-G packaging plasmids, using XTremeGene9 transfection reagent (Roche Diagnostics). The medium was changed to Dulbecco's modified Eagle's medium (DMEM) supplemented with 30% inactivated bovine serum 24 hours after transfection. Virus-containing supernatants were collected 48 and 72 hours after transfection and passed through a 0.45 μm filter to remove cells. Target cells in 6-well tissue culture plates were infected in medium containing 8 μg / mL polybrene, and spin-infection was performed by centrifugation at 2,200 rpm for 1 hour. 24 hours after infection, the virus was removed and cells were selected with the appropriate antibiotics. Cells were then cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. The cells were grown.

[0587] To screen for leucine mimetic 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 without amino acids and supplemented with 5 mM glucose (-AA RPMI, US Biological Life Sciences) for 1 hour, and then subsequently lysed in lysis buffer (40 mM HEPES, 1% Triton, 10 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 2.5 mM MgCl2, and protease inhibitors). To isolate the FLAG-WDR24 / endogenous Sestrin2 complex, 1 ml aliquots of crude lysate (equivalent to 2-4 mg of total protein) were subjected to immunoprecipitation with 30 μl of anti-FLAG resin (SIGMA) for 2 h at 4°C, washed twice in cold lysis buffer and 0.5 M NaCl, 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). Test compounds or controls (the resulting solutions were filtered or leucine) were then added at various concentrations to each immunoprecipitation sample and incubated for 60 min at 4°C with rotation. After the incubation period, the samples were centrifuged to pellet the FLAG-WDR24 / endogenous Sestrin 2 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 subjected to SDS-PAGE and Western blot analysis using anti-FLAG (SIGMA) and anti-Sestrin 2 (Cell Signaling Technology) antibodies as described in L. Chantranupong et al., Cell Reports 9:1-8 (2014).

[0588] The resulting Western blots were scanned, and the band intensities corresponding to Sestrin2 and FLAG-WDR24 were quantified using the LI-COR® imaging platform. To determine the amount of Sestrin2 bound to GATOR2 for each condition, the Sestrin2 band intensity was normalized to that of FLAG-WDR24. A negative control (filtered solution) and a positive control (leucine, 25 μM, SIGMA) were also run for each dose of test compound. Absence of endogenous Sestrin2 bound to FLAG-WDR24 by leucine was normalized to represent 100% activity. Compounds were assayed in duplicate, and the activity of each compound was quantified as a percentage of leucine activity and averaged. Repeat assays yielded an average leucine activity with a standard deviation of 20% compared to water. Therefore, a reduction of at least 40% in the amount of Sestrin2 bound to GATOR2 at 25 μM in duplicate was considered statistically significant and characterized as a leucine mimetic. Some compounds increased the amount of Sestrin 2 bound to FLAG-WDR24. Compounds that increased the amount of Sestrin 2 bound to GATOR2 by more than 40% (expressed as less than -40% of leucine activity) were characterized as leucine antagonists.

[0589] Example 211: Methods for identifying compounds that mimic or antagonize the activity of leucine during Sestrin2 and Sestrin2 / GATOR2 interaction

[0590] introduction

[0591] Sestrin1 and Sestrin2 interact with GATOR2 through the GATOR2 components WDR24 and Seh1L under insufficient leucine levels. Under leucine-sufficient conditions, leucine directly binds to Sestrin2, causing its dissociation from GATOR2. The goal of the following method is to identify compounds that mimic the action of leucine in binding to Sestrin2 and disrupting Sestrin2 / GATOR2. Additionally, the method identifies compounds that antagonize the binding of leucine to Sestrin2 and compounds that block the dissociation of Sestrin2 from GATOR2 in response to leucine.

[0592] Method 1 (in vitro PPI assay)

[0593] This screening assay measured the in vitro activity of compounds against the GATOR2 / Sestrin2 complex purified by immunoprecipitation of stably expressed Flag-WDR24 from HEK293T cells. HEK293T cells (293T) were genetically engineered to stably express N-terminally tagged Flag-WDR24 by lentiviral transduction. Lentivirus was generated by co-transfecting HEK-293T cells with the lentiviral transfer vector pLJM60, along with the ΔVPR envelope and CMV VSV-G packaging plasmids, using XTremeGene9 transfection reagent. 24 hours after transfection, the medium was changed to Dulbecco's modified Eagle's medium (DMEM) supplemented with 30% inactivated bovine serum. Virus-containing supernatants were collected 48 and 72 hours after transfection and passed through a 0.45 μm filter to remove cells. Target cells in 6-well tissue culture plates were infected in medium containing 8 μg / mL polybrene, and spin-infection was performed by centrifugation at 2,200 rpm for 1 hour. 24 hours after infection, the virus was removed and cells were selected with the appropriate antibiotic. Cells were then grown in DMEM supplemented with 10% fetal bovine serum and antibiotics.

[0594] To screen for leucine mimetic 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 without amino acids and supplemented with 5 mM glucose (-AA RPMI, USBiological Life Sciences) for 1 hour, and then subsequently lysed in lysis buffer (40 mM HEPES, 1% Triton, 10 mM sodium beta-glycerophosphate, 10 mM sodium pyrophosphate, 2.5 mM MgCl and protease inhibitors). Flag-WDR24 / endogenous Sestrin2 complexes were isolated as follows: 1 ml aliquots of crude lysate (equivalent to 2–4 mg total protein) were subjected to immunoprecipitation (IP) with 30 μl of anti-Flag resin (SIGMA) for 2 h at 4°C, washed twice in cold lysis buffer and 0.5 M NaCl, and resuspended in 1 ml of cold cytoplasmic buffer (40 mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl, 0.1% Triton X-100). Compounds were then added to each sample at a given concentration of 25 μM and incubated for 30 min at 4°C with rotation. After the incubation period, the samples were centrifuged to pellet the Flag-WDR24 / endogenous Sestrin2 complex bound to the anti-Flag resin. The supernatant was completely removed, and the resin was resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 5 minutes. The samples were then subjected to SDS-PAGE and Western blot analysis using anti-Flag (SIGMA) and anti-Sestrin2 (Cell Signaling Technology) antibodies as described in L. Chantranupong et al., Cell Reports 9:1-8 (2014).

[0595] The resulting Western blots were scanned, and the band intensities corresponding to Sestrin2 and Flag-WDR24 were quantified using the LI-COR® imaging platform. To determine the amount of Sestrin2 bound to GATOR2 for each condition, the band intensity of Sestrin2 was normalized to that of Flag-WDR24. A negative control (water) and a positive control (leucine, 25 μM, SIGMA) were also run for each dose of test compound. Endogenous sestrin2 bound to Flag-WDR24 by leucine were also measured. The absence of Sestrin 2 is normalized to represent 100% activity. Compounds were assayed in duplicate, and the activity of each compound was quantified as a percentage of leucine activity and averaged. A summary of the quantified data from the test compounds is shown in Table 3. Replicate assays yielded an average activity of leucine with a standard deviation of 20% compared to water. Therefore, compounds that reduced the amount of Sestrin 2 bound to GATOR2 by at least 40% in both duplicates at 25 μM were considered statistically significant and designated leucine mimetics. Some compounds also increased the amount of Sestrin 2 bound to Flag-WDR24 (shown as negative leucine activity percentages in Table 3). Compounds that showed less than -40% leucine activity were also considered hits and designated leucine antagonists.

[0596] Method 2 (cell-based mTORC1 activation)

[0597] To demonstrate the efficacy of compounds identified as leucine mimetics in intact cells, mTORC1 signaling in response to compound treatment after leucine starvation was measured by Western blotting. The addition of exogenous leucine during leucine starvation significantly increased mTORC1 signaling in intact cells (Wang, S., Tsun, Z., et al., Science 347(6218):188-194 (2015)). As described previously, mTORC1 activation is measured 10–90 min after the addition of leucine. Therefore, we designed a similar assay to test whether compounds identified as leucine mimetics activate mTORC1 in a similar manner. Briefly, 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 following day, cells were placed in modified DMEM without leucine (Thermo Scientific) or modified DMEM without serum for 1 h, and then leucine mimetics were added at a given concentration for a period longer than 10 min (n=3). Cells were then lysed, subjected to SDS-PAGE, and Western blot analysis was performed using antibodies directed against mTORC1 substrates phosphorylated S6 kinase (Thr389) and phosphorylated 4EBP1 (Thr37 / 46) (Cell Signaling Technology), as well as a loading control (beta-actin, Santa Cruz Biotechnology), as described in Kang, SA et al., Science 341(6144):364-374 (2013). The intensity of the bands corresponding to the phosphorylated substrates was then normalized to the actin band using the LI-COR® imaging platform. Compounds that significantly increased mTORC1 signaling relative to untreated leucine-starved cells (Student's t-test, p<0.05) were considered active in the cells. As a positive control, leucine was added to leucine-starved cells at 100 μM for 60 minutes.

[0598] Method 3 (cell-based mTORC1 activation)

[0599] To demonstrate the efficacy of compounds identified as leucine antagonists or to determine whether weak leucine mimetics enhance the activity of leucine in intact cells, the same procedure as above was repeated with the following modification: cells were placed in DMEM medium without leucine for 60 minutes (as described in Method 3), and then in compound for a period greater than or equal to 60 minutes (n=3). 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 statistically significantly reduced the levels of actin-normalized phosphorylated substrates of mTORC1 in response to leucine at either 30 μM or 100 μM (Student's t-test, p<0.05) were considered active in cells. Compounds that increased actin-normalized phosphorylated substrate levels of mTORC1 in response to leucine in a statistically significant manner (Student's t-test, p<0.05) at either 30 μM or 100 μM Compounds were considered leucine enhancers in cells. As a control, leucine-starved cells were pretreated with water before adding leucine. Alternatively, potential leucine antagonists were assayed in HEK293T cells using the same method as above, but without leucine starvation and stimulation. Western blots were performed to determine whether compound treatment attenuated baseline mTORC1 signaling under sufficient culture conditions.

[0600] Method 4.

[0601] The ability of compounds to modulate the interaction between Sestrin2 and GATOR2 in cells was measured by repeating the assay described in Methods 2 and 3 in HEK293T cells stably engineered to 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 after treatment with the compounds described in Method 1 (n = 3). Briefly, after cell treatment, immunoprecipitation was performed using anti-Flag resin to measure the amount of endogenous Sestrin2 bound to Flag-WDR24, and the resulting samples were subjected to SDS-PAGE and Western blotting to measure the amount of endogenous Sestrin2 bound to Flag-WDR24. Compounds that modulated the amount of Sestrin2 bound to GATOR2 in a statistically significant manner (Student's t-test, p < 0.05) were considered suitable.

[0602] Method 5 (ALPHALisa cell-based assay)

[0603] To demonstrate the efficacy of compounds identified as leucine mimetics in intact cells in a plate-based format, mTORC1 signaling in response to compound treatment after leucine starvation was measured by AlphaLISA. Briefly, 1,000,000 HEK293T cells were plated in T-75 cell culture flasks with DMEM supplemented with 10% fetal bovine serum. After the cells reached confluence, they were placed in modified DMEM without leucine (Thermo Scientific) and containing 10% dialyzed fetal bovine serum for 1 hour. The cells were then trypsinized and replated in 96-well black clear-bottom plates at 50,000 cells / well in DMEM without leucine and containing 10% dialyzed fetal bovine serum. The cells were allowed to adhere to the plate for 2 hours, and then compounds were added at the given concentrations (n=4) for a period of more than 1 hour. After reaching the time points, cells were lysed and analyzed by p-p70 S6K (Thr389) SureFire Ultra AlphaLISA kit according to the manufacturer's instructions (http: / / www.perkinelmer.com / CMSRes The compounds were treated with 100 mg TGR70S-p70P / T389 / TGR70S_p70_pT389.pdf. Compounds that significantly increased mTORC1 signaling relative to leucine-starved cells without treatment (Student's t-test, p<0.05) were considered mTORC1 activators. Compounds that significantly decreased mTORC1 signaling relative to leucine-starved cells without treatment (Student's t-test, p<0.05) were considered inhibitors. As a positive control, leucine was added at 100 μM to leucine-starved cells for a period equivalent to compound treatment.

[0604] Method 6, Thermal Shift Protocol (Tm Shift):

[0605] Full-length, codon-optimized human Sestrin2 was N-terminally fused with a His-MBP tag and cloned into the pMAL6H-C5XT bacterial expression vector. This vector was transformed into Escherichia coli LOBSTR(DE3) cells (Kerafast). The cells were transformed with sestrin 2. Cells were grown at 37°C to 0.6 OD, and then protein production was induced with 0.2 mM IPTG at 18°C ​​for 12–14 hours. Cells were harvested 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 benzonase, and 1 mM PMSF), and lysed by sonication. The lysate was clarified by centrifugation at 10,000 g for 20 minutes. Sestrin 2 protein was isolated from the soluble fraction and purified to nearly 100% purity by His-tag affinity capture followed by ion-exchange and size-exclusion chromatography. For the thermal shift assay, Sestrin 2 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 the thermal shift assay, 2 μl of Sestrin 2 protein was combined with 8 μl of ROX dye (Thermo Fisher), 1 μL of vehicle or compound, and 14 μL of dilution buffer per well of a 96-well plate and incubated on ice for 1 hour to allow compound binding. Thermal shift assays were then performed on an Agilent MX3005p, with each compound assayed in triplicate at 10 μM, 100 μM, and 1000 μM. Incubation with leucine shifted the melting temperature of Sestrin 2 from 2.16 to 11.61 degrees Celsius in a dose-dependent manner. Based on the CV% variation of replicate measurements of thermal shifts of Sestrin 2 incubated with vehicle, a positive shift of 2 degrees or more is considered statistically significant.

[0606] Method 7, Indirect Ligand Binding Assay (ILBA)

[0607] Binding of Sestrin 2 to leucine or other ligands was detected either in vitro in intact cells or in purified proteins by immunodetection using a rabbit monoclonal anti-Sestrin 2 antibody from Cell Signaling Technology (CST, Catalog No. 8487). The binding of the CST antibody to native (non-denatured) Sestrin 2 was modulated by leucine binding, such that the affinity of this antibody decreased upon leucine binding. Similarly, the affinity of the CST antibody for native Sestrin 2 decreased upon binding of compounds to native Sestrin 2, as did leucine. Conversely, compounds that destabilize Sestrin 2 increased the affinity of the CST antibody for non-denatured Sestrin 2, as measured by thermal shift assay. Consequently, we developed a multiplexed format, an indirect ligand binding assay (ILBA), to measure the affinity of the CST anti-Sestrin 2 antibody after leucine or compound binding. In one version, this assay was performed using crude lysates produced from human cell lines after a 1-hour amino acid starvation period (cells were lysed in 1% Triton, 10 mM beta-glycerol phosphate, 10 mM sodium pyrophosphate, 40 mM HEPES [pH 7.4], 150 mM NaCl, and 2.5 mM MgCl). The lysates were then incubated with leucine or other compounds for 1 hour on ice or at room temperature. L. Chantranupong et al. As described in Cell Reports, Vol. 9, pp. 1-8 (2014), After incubation, samples were subjected to immunoprecipitation with CST anti-Sestrin 2 antibody for 1.5 hours and then incubated with Protein A-Sepharose for 30 minutes. Sepharose-conjugated antibody-protein complexes were precipitated by centrifugation, and the flow-through was subjected to two immunoprecipitations using a rabbit polyclonal anti-Sestrin 2 antibody (Protein Tech, #10795-1-AP) to determine equal total Sestrin 2 protein levels between samples. Immunoprecipitated samples were subjected to SDS-PAGE followed by Western blotting using a mouse monoclonal anti-Sestrin 2 antibody from SIGMA (catalog no. WH0083667M3). In immunoblots using samples immunoprecipitated with the anti-Sestrin 2 antibody from CST, leucine binding induced a significant decrease in the intensity of the band corresponding to Sestrin 2, by 50% or more. However, immunoblotting using samples immunoprecipitated with Protein Tech antibodies did not result in changes in Sestrin 2. This type of assay also measured increased instability of Sestrin 2 induced by incubation with compounds. This assay was performed in the same way, but compounds that destabilized Sestrin 2 (as measured by thermal shift assay) resulted in an increase in the intensity of the immunoblot band corresponding to Sestrin 2 immunoprecipitated using CST antibodies.

[0608] This assay was also performed in cultured human cells overexpressing Sestrin2 N-terminally fused to a Flag tag. In this type of assay, the procedure remained the same, except that immunoblotting was performed using a mouse anti-Flag antibody (#F3165, SIGMA). When ILBA was performed using a point mutant form of Sestrin2 that is unable to bind leucine, no decrease in the affinity of the CST antibody for leucine or γ-methylleucine was observed.

[0609] In another version of the assay, cultured human cells were subjected to several combinations of amino acid starvation for 1 hour and then stimulated with leucine or compound. After 1 hour of stimulation, cells were lysed and treated as described above, except for the 1 hour ligand binding step.

[0610] Indirect ligand binding assays were also performed in a multiwell format using ALPHAlisa technology (Perkin Elmer). This type of assay required biotinylated anti-Sestrin 2 antibody, streptadipin donor beads (Perkin Elmer) conjugated to either anti-Flag receptor beads (Perkin Elmer) or mouse anti-Sestrin 2 antibody (SIGMA) for detection of overexpressed Flag-Sestrin 2, and anti-mouse receptor beads (Perkin Elmer) for detection of endogenous Sestrin 2.

[0611] The assay was performed as described above with the following modifications: For the leucine or compound binding portion of the assay, crude lysates produced from cells transiently or stably overexpressing human Flag-Sestrin 2 after 1 hour of amino acid starvation were diluted in lysis buffer to 0.8 mg / ml total protein and arrayed in a multiwell plate, such as a 96-well plate. For detection of endogenous Sestrin 2, crude lysates were diluted in lysis buffer to 4 mg / ml total protein. Leucine or compound was added to each well, and the plate was incubated on ice or at room temperature for 1 hour with gentle agitation. During the ligand binding step, biotinylated anti-Sestrin 2 antibody (CST) was diluted to 5 nM in ALPHAlisa immunoassay buffer (Perkin Elmer) and 5 nM mouse anti-Sestrin 2 antibody (from SIGMA, combined with a 4X stock solution of anti-mouse receptor beads (40 μg / ml) for the assay detecting endogenous Sestrin 2). For detection of Flag-Sestrin2, a 4X stock solution of anti-Flag receptor beads (40 μg / ml) was prepared in immunoassay buffer. After the ligand binding step, 5 μL of lysate was combined with 10 μL of biotinylated anti-Sestrin2 antibody, 12.5 μL of mouse Sestrin2 antibody / anti-mouse receptor bead mix or anti-Flag receptor beads, and 10 μL of ALPHAlisa immunoassay buffer and incubated for 1 hour at room temperature. Finally, 12.5 μL of streptavidin donor beads (160 μg / ml in immunoassay buffer) were added and incubated for an additional hour in the dark before reading the plate on the Envision plate reader.

[0612] ALPHAlisa assays were also performed as described, but with purified Sestrin 2 protein diluted in immunoassay buffer at a final reaction concentration of 3 ng / ml.

[0613] Finally, cells treated with leucine or compounds under amino acid starvation conditions before lysis were ALPHAlisa was performed using lysates derived from cells. Cell-based procedures were performed in multiwell plates, using 15 μL of lysate (1 mg / ml total protein) in combination with 10 μL of biotinylated antibody, 12.5 μL of antibody / receptor bead mix, and 12.5 μL of streptadipin donor bead mix per ALPHAlisa reaction.

[0614] Indirect ligand binding assays were also performed using capture-based methods, such as sandwich ELISAs, as performed in the art. In one type of assay, ILBA was performed using the MULTI-ARRAY® technology developed by Meso-Scale Discovery (MSD). The MSD system was based on electrochemiluminescence detection of antibody binding to the analyte. ILBA was performed using crude lysates expressing endogenous Sestrin 2 or overexpressing Flag-Sestrin 2, and leucine treatment was performed either in vitro or in cells prior to lysis. For in vitro ILBA with endogenous Sestrin 2, crude lysates (0.8 mg / ml total protein) were prepared, and leucine binding was performed in the same manner as described for ALPHAlisa ILBA. After ligand binding was complete, biotinylated anti-Sestrin 2 antibody from CST was added to each well to a final concentration of 0.25 μg / ml and incubated for 1 hour at 4°C with gentle agitation. Capture of each sample into wells of a 96-well plate was performed in one of the following ways: on streptavidin-coated MSD plates or bare MSD plates coated with mouse anti-Sestrin 2 antibody from SIGMA. Capture required 25 μL of sample per well, followed by incubation for 1 hour with shaking at 350 rpm. After sample capture, wells were washed three times with Tris-buffered saline containing 0.1% Tween (TBS-T). If samples were captured on streptavidin-coated plates, mouse monoclonal anti-Sestrin 2 antibody (SIGMA) was then added to a final concentration of 1 μg / ml for 1 hour with shaking at 350 rpm. Wells were again washed in TBS-T, and a secondary anti-mouse SULFO-tag antibody (MSD) was added to a final concentration of 1 μg / ml for 1 hour with shaking at 350 rpm. Finally, the wells were washed three times with TBS-T, 2X read buffer (MSD) was added, and the plate was immediately read on the MSD instrument.If the samples were captured by a bare plate coated with mouse anti-Sestrin 2 antibody, after washing, a streptavidin secondary SULFO-tag antibody (MSD) was added at a final concentration of 1 μg / ml for 1 h with shaking, followed by washing and incubation with read buffer before analysis.

[0615] In another version of this assay, crude lysates overexpressing Flag-Sestrin2 were analyzed and captured or detected with a mouse monoclonal anti-Flag antibody (SIGMA) using the same MSD-based protocol as above.

[0616] For all assays, compounds that significantly reduced the signal corresponding to Sestrin 2 immunoreactivity were considered as leucine mimetics, whereas compounds that significantly increased the signal were considered as potential leucine antagonists.

[0617] Table 3 shows the activities of selected compounds of the present invention. Compound numbers correspond to the compound numbers in Tables 1 and 2. Compounds with an activity designated "A" achieved a % activity of ≥ 40% relative to leucine, compounds with an activity designated "B" achieved a % activity of ≤ -40% relative to leucine, and compounds with an activity designated "C" achieved a % activity between -40 and 40% relative to leucine. At the concentrations indicated, compounds with an activity designated "D" achieved a 0.5-2 fold shift relative to the DMSO control, compounds with an activity designated "E" achieved a 2.1-5 fold shift relative to DMSO, and compounds with an activity designated "F ... at the concentrations indicated. achieved a 5.1- to 10-fold shift relative to DMSO, and compounds with activity designated "G" achieved a 10.1- to 14-fold shift relative to DMSO.

[0618] Activity in terms of % activity relative to leucine assay was determined using Assay Method 1. Activity in terms of cell-based mTORC1 activation assay was determined using Assay Method 2. [Table 3-1] [Table 3-2]

[0619] Table 4 shows selected compounds of the invention that are active in the ALPHALisa cell-based assay (Method 5). Compound numbers correspond to the compound numbers in Tables 1 and 2. Compounds listed in Table 4 are mTORC1 activators and have >2-fold activity relative to the positive leucine control. [Table 4-1] [Table 4-2]

[0620] Table 5 shows selected compounds of the present invention that were active in the thermal shift assay (Method 6). Compound numbers correspond to the compound numbers in Tables 1 and 2. Compounds listed in Table 5 showed a positive shift of 2 degrees or greater. [Table 5-1] [Table 5-2]

[0621] The present invention provides, for example: (Item 1) Compounds of Formula I: [ka] or a pharmaceutically acceptable salt thereof, R1 is H or C 1~6 is alkyl, R 2 is R, -(CH2) n -phenyl, -C(O)R, -SOR or -C(O)N(R), n is 0, 1 or 2; Each R is independently hydrogen, -CN, or a saturated or unsaturated C 1~6 an optionally substituted group selected from aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms, or a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R 3 is ring A, -C(O)R, -C(O)OR, -C(O)N(R), -SOH, -SON(R), -S(O)R, -S(O)R, -S(O)R, -OR, or -B(OR), where two OR groups on the same boron together with their intervening atoms form a 5-8 membered saturated or partially unsaturated monocyclic ring having, in addition to the boron and two oxygens, 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 3 and R 4 together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen or sulfur; L is a covalent bond or a straight or branched C optionally substituted with 1 to 9 fluoro groups. 1~6 is an alkylene chain, Ring A is an optionally substituted ring selected from phenyl or an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R 4 is R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4together form an optionally substituted 5-6 membered ring having 0-1 heteroatoms selected from nitrogen, oxygen or sulfur; R 5 is H or C 1~4 alkyl) A compound of Formula I, wherein said compound of Formula I is other than those depicted in Table 2. (Item 2) R 1 is H. (Item 3) R 1 C 1~6 The compound according to item 1, wherein the aryl group is alkyl. (Item 4) R 2 But -(CH2) n -phenyl. (Item 5) R 2 is —C(O)R, —S0R or —C(O)N(R) (Item 6) R 2 ...

Claims

1. Compounds of Formula I: 【Chemistry 130】 or a pharmaceutically acceptable salt thereof, R １ is H or C １～６ is alkyl, R ２ is ═O and —(CH ２ ) ０～４ O (CH ２ ) ０～１ Saturated C substituted by Ph １ Hydrocarbons, -(CH ２ ) ｎ -phenyl, -C(O)R, -SO ２ R or -C(O)N(R) ２ and n is 0, 1 or 2; Each R is independently hydrogen, —CN, or a saturated or unsaturated C １～６ a group selected from aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms, or a 4-8 membered saturated or partially saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R ３ is —C(O)OR, L is a covalent bond or a linear or branched C substituted with 1 to 9 fluoro groups. １～６ is an alkylene chain, R ４ is R, -CF ３ , -OR, -N(R) ２ , -Si(R) ３ or -SR, or R ３ and R ４ together form a 5- to 6-membered ring having 0-1 heteroatoms selected from nitrogen, oxygen, or sulfur; R ５ is H or C １～４ alkyl).

2. R １ 2. The compound of claim 1, wherein is H, or a pharmaceutically acceptable salt thereof.

3. R １ is C １～６ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R is 1 or 2;

4. R ２ But -(CH ２ ) ｎ 2. The compound of claim 1, wherein R is -phenyl, or a pharmaceutically acceptable salt thereof.

5. R ２ is -C(O)R, -SO ２ R or -C(O)N(R) ２ 2. The compound of claim 1, wherein:

6. R ２ But -(CH ２ )-phenyl or —C(O)CH ３ 2. The compound of claim 1, wherein:

7. R ３ 7. The compound of claim 1, wherein is —C(O)OH, or a pharmaceutically acceptable salt thereof.

8. 8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is a covalent bond.

9. L is a straight-chain or branched C substituted with 1 to 4 fluoro groups １～６ 8. The compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein:

10. R ４ But, -CF ３ 10. The compound of claim 1, wherein the aryl group is -OR or -SR.

11. R ４ The compound of claim 10, wherein is isopropyl, tert-butyl, cyclopropyl, cyclobutyl, sec-butyl, methoxyl, or methylthioyl.

12. R ５ 12. The compound of claim 1, wherein R is H, or a pharmaceutically acceptable salt thereof.

13. R ５ 12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein is methyl.

14. 14. A pharmaceutically acceptable composition comprising a compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

15. The composition of claim 14 in combination with an additional therapeutic agent.

16. 16. The composition of claim 15, wherein the additional therapeutic agent is an antiproliferative compound.

17. A composition for modulating Sestrin-GATOR2 interaction, thereby indirectly and selectively modulating mTORC1 activity in a biological sample, characterized in that the composition comprises a compound described in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and the compound described in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof is contacted with the biological sample.

18. 15. The pharmaceutically acceptable composition of claim 14 for modulating Sestrin-GATOR2 interaction, thereby indirectly and selectively modulating mTORC1 activity in a patient.

19. 15. The pharmaceutically acceptable composition of claim 14 for treating an mTORC1-mediated disorder in a patient in need thereof.

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