Modulators of the sestrin-gator2 interaction and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of the application filed on April 25, 2018, with application number 201880039788.3 and invention title "Regulator of SESTRIN-GATOR2 Interaction and Its Use". Technical Field

[0002] This invention relates to compounds and methods suitable for modulating Sestrin-GATOR2 interactions, thereby selectively and indirectly regulating mTORC1 activity. The invention also provides pharmaceutically acceptable compositions comprising the compounds of this invention and methods for treating various conditions using said compositions. Background Technology

[0003] The mechanism of action of rapamycin complex 1 (mTORC1) protein kinase is to sense a wide variety of environmental cues, such as growth factors, cellular stress, and key growth regulators of nutrients and energy levels. Upon activation, mTORC1 phosphorylates substrates that enhance anabolic processes, such as mRNA translation and lipid synthesis, and restrict catabolism, such as autophagy. mTORC1 dysregulation occurs in a wide range of diseases, including diabetes, epilepsy, neurodegeneration, immune responses, musculoskeletal growth inhibition, and cancer (Howell et al., (2013) Biochemical Society transactions 41, 906-912; Kim et al., (2013) Molecules and cells 35, 463-473; Laplante and Sabatini, (2012) Cell 149, 274-293).

[0004] Multiple upstream inputs (including growth factors and energy levels) signal to mTORC1 via the TSC complex that regulates Rheb (a smaller 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, 566-571; Stocker et al., (2003) Nature Cell Biology 5,559-565; Tee et al., (2002) Proc Natl Acad Sci USA 99, 13571-13576. Amino acids do not appear to signal to mTORC1 via the TSC-Rheb axis but rather via heterodimeric Rag GTPases, which are composed of RagA or RagB bound to RagC or RagD, respectively (Hirose et al., (1998) Journal of Cell Science 111(Pt 1), 11-21; Kim et al., (2008) Nature Cell Biology 10, 935-945; Nobukuni et al., (2005) Proc Natl Acad Sci USA 102, 14238-14243; Roccio et al., (2005) Oncogene 25, 657-664; Sancak et al., (2008) Science (New York, NY) 320, 1496-1501; Schürmann et al., (1995) The Journal of Biological Chemistry). 270, 28982-28988; Sekiguchi et al., (2001) The Journal of biological chemistry 276, 7246-7257; Smith et al., (2005) The Journal of biological chemistry 280, 18717-18727).Rag GTPases control the subcellular localization of mTORC1, and amino acids promote its recruitment 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) 320, 1496-1501). Several positive components upstream of the Rag GTPase pathway have been identified. The regulator complex localizes Rag GTPase to the lysosomal surface and, together with vacuole ATPase, promotes the exchange of GTP with GDP on RagA / B (Bar-Peled et al., (2012) Cell 150, 1196-1208; Sancak et al., (2010) Cell 141, 290-303; Zoncu et al., (2011) Science Signaling 334, 678-683). Different FLCN-FNIP complexes act on RagC / D and stimulate it to hydrolyze GTP to GDP (Tsun et al., 2013). When RagA / B is loaded with GTP and RagC / D is loaded with GDP, the heterodimer binds and recruits mTORC1 to the lysosomal surface, where it can contact its activator, Rheb GTPase.

[0005] Recent work has identified the GATOR1 multiprotein complex as a major negative regulator of the amino acid sensing pathway, and its absence renders mTORC1 signaling completely insensitive to amino acid deficiencies (Bar-Peled et al., (2013) Science 340, 1100-1106; Panchaud et al., (2013) Science Signaling 6, ra42). GATOR1 is composed of DEPDC5, Nprl2, and Nprl3, and is the GTPase activator (GAP) of RagA / B. The GATOR2 multiprotein complex, with five known subunits (WDR24, WDR59, Mios, Sec13, and Seh1L), is a positive component of the GATOR1 pathway and is upstream of or parallel to GATOR1, but its molecular function remained unknown until recently (Bar-Peled et al., (2013) Science 340, 1100-1106).

[0006] Recently, additional information regarding the mTORC1 pathway has been elucidated by identifying the binding of GATOR2 to one or more of Sestrin and demonstrating that the resulting Sestrin-GATOR2 complex regulates the subcellular localization and activity of mTORC1. Specifically, the presence of the Sestrin-GATOR2 complex inhibits the mTORC1 pathway and reduces mTORC1 activity by preventing mTORC1 translocation to the lysosomal membrane. The interaction between GATOR2 and Sestrin, particularly Sestrin1 and Sestrin2, is counteracted by amino acids, especially leucine and (to a lesser extent) isoleucine, methionine, and valine. In the presence of leucine, GATOR2 does not interact with Sestrin1 or Sestrin2, and mTORC1 is able to migrate to the active lysosomal membrane. Sestrin1 and Sestrin2 directly bind leucine and (to a lesser extent) isoleucine and methionine (Chantranupong et al., (2014) Cell Rep.; 9(1):1-8). Sestrin1 or Sestrin2 needs to bind leucine to disrupt its interaction with GATOR2 and subsequent activation of mTORC1. Sestrin2 mutants that cannot bind leucine cannot signal the presence of leucine to mTORC1, and cells and their homologs that are depleted of Sestrin2 make mTORC1 insensitive to the absence of leucine (Wolfson et al., (2015) Science pii:ab2674 [electronic version before print]).

[0007] Sestrin consists of three proteins (Sestrin1, Sestrin2, and Sestrin3) associated with poorly characterized molecular functions (Buckbinder et al., (1994) Proc Natl Acad Sci USA 91, 10640-10644; Budanov et al., (2002) Cell 134, 451-460; Peeters et al., (2003) Human Genetics 112, 573-580). Sestrin2 inhibits mTORC1 signaling and has been proposed to activate upstream AMPK in TSC and interact with TSC (Budanov and Karin, (2008) Cell 134, 451-460). However, subsequent studies have found that Sestrin2 inhibits mTORC1 in the absence of AMPK (Peng et al., (2014) Cell 159(1):122-33), further highlighting the important role of the GATOR2 complex in regulating mTORC1 in response to Sestrin2.

[0008] Modulating the Sestrin-GATOR2 complex represents a potential therapeutic target for selectively and indirectly modulating mTORC1 activity. Summary of the Invention

[0009] The compounds of this invention and their pharmaceutically acceptable compositions have been found to be effective as Sestrin-GATOR2 modulators. These compounds have the general formula I:

[0010]

[0011] Or its pharmaceutically acceptable salt, wherein the variables are as defined and described herein.

[0012] The compounds of this invention and pharmaceutically acceptable compositions thereof are suitable for treating a variety of diseases, conditions, or illnesses associated with mTORC1. These diseases, conditions, or illnesses include diabetes, epilepsy, neurodegeneration, immune responses, musculoskeletal growth inhibition, and cell proliferation disorders (e.g., cancer), such as those described herein. Attached Figure Description

[0013] Figure 1 The study timeline for Example A is shown below. Male Sporgdory rats were acclimatized for 5 days and administered the drug on day 0, as described in Table 6. A female urine olfactory test (FUST) was performed 24 hours after administration (day 1). Motor activity (LMA) was assessed on day 2 (48 hours after administration). Rats were fasted overnight for 20 hours, followed by a novel restraint feeding test (NSFT) (72 hours after administration).

[0014] Figure 2 This demonstrates the role of Ket and NV-5138 (I-90) in FUST. All data are presented as mean ± SEM (n = 8 / treatment group). *p < 0.05 and **p < 0.01 indicate significant differences in unpaired two-tailed Students' t-test.

[0015] Figure 3 The effects of Ket and NV-5138 (I-90) on LMA assessment are demonstrated. Ketamine catalyst (Sal); NV-5138 catalyst (Veh); Ketamine (Ket); NV-5138 (NV). The number of infrared beam breaks over a 30-minute time period is shown. All data are expressed as mean ± SEM (n = 8 / treatment group). No significant differences were observed between groups by unpaired two-tailed Steudent's t test.

[0016] Figure 4Demonstrates the role of Ket and NV-5138 (I-90) in the novel restraint feeding test (NSFT). Ketamine mediator (Sal); NV-5138 mediator (Veh); Ketamine (Ket); NV-5138 (NV). Feeding wait times are shown in seconds. All data are presented as mean ± SEM (n = 8 / treatment group). **p < 0.01 indicates a significant difference in the unpaired two-tailed Steudon's t test.

[0017] Figure 5 The study timeline for Example B is shown below. Male Sporgodory rats were acclimatized for 5 days and administered the drug on day 0, as described in Table 7. One hour after administration (day 0 + 1 hour), rats in groups 1, 2, 3, and 4 were sacrificed, and prefrontal cortex (PFC) samples were collected for synaptosome preparation and Western blotting analysis. Twenty-four hours after administration (day 1), rats in groups 5, 6, 7, and 8 were sacrificed, and PFC samples were collected for synaptosome preparation and Western blotting analysis.

[0018] Figure 6 shows a schematic diagram of the PFC anatomy in the rat brain. A) Dorsal view of the rat brain, with two black lines indicating the anatomical locations to separate the PFC region of the brain. B) Sagittal view of the rat brain, with black diagonal lines representing incisions made to remove the olfactory bulb and two black vertical lines representing incisions separating the PFC region.

[0019] Figure 7 illustrates the effects of NV-5138 (I-90) and Ket on the levels of pmTOR, pp70S6K, and p4E-BP1 in crude synaptosomes prepared from PFC one hour after drug administration. Fold changes in pmTOR (A), pp70S6K (B), and p4E-BP1 (C) are shown (normalized to control for Veh or Sal). Three representative Western blots from a total of n=6 used for statistical analysis are shown as the above curves for pmTOR (A), pp70S6K (B), and p4E-BP1 (C). All data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences in unpaired two-tailed Steudon's t-tests.

[0020] Figure 8 shows the effects of Ket and NV-5138 (I-90) on the levels of GluR1, synaptoprotein 1, and PSD95 in crude synaptosomes prepared by PFC 24 hours after drug administration. The fold changes in GluR1 (A), synaptoprotein 1 (B), and PSD95 (C) in PFC-purified crude synaptosomes (normalized to the corresponding control groups Sal or Veh) are shown. Three representative Western blots from a total of n=6 used for statistical analysis illustrate the above curves for GluR1 (A), synaptoprotein 1 (B), and PSD95 (C). All data are mean ± SEM. *p<0.05 and **p<0.01 indicate significant differences in the unpaired two-tailed Students' t-test.

[0021] Figure 9 The timeline study of Example C is shown. Male Sporgodory rats were acclimatized for 5 days and administered the drug on day 0, as described in Table 8. One hour after administration (day 0 + 1 hour), rats in groups 1 and 2 were sacrificed, and plasma and dissected brain regions were collected for compound content determination and Western blot analysis.

[0022] Figure 10 A schematic diagram showing the dissected brain regions of a rat. A sagittal view of the rat brain, with white arrows indicating the regions dissected for Western blotting analysis.

[0023] Figure 11 This study demonstrates the effect of NV-5138 (I-90) on pS6 levels in multiple brain regions 1 hour after administration. Bar charts show the effect compared to Veh (normalized to 1 in various regions of the rat brain). S240 / 244 fold change of pS6. A representative Western blot (WB) for quantitative analysis is shown on the graph. All data are mean ± SEM. *p < 0.05 indicates a significant difference in unpaired two-tailed Steudent's t-tests.

[0024] Figure 12 The timetable study of Example D is shown. Male Sporgodory rats were acclimatized for 5 days and administered the drug on day 0, as described in Table 9. One hour after administration (day 0 + 1 hour), rats in groups 1, 2, and 3 were sacrificed, and plasma, brain, and peripheral tissues were collected and processed for Western blotting analysis.

[0025] Figure 13This study demonstrates the effects of Leu and NV-5138 (I-90) on pS6 levels in the brain and selected peripheral organs 1 hour after oral administration. Bar charts show the fold change in pS6 levels of Veh normalized to rat brain and peripheral tissues: gastrocnemius muscle, anterior tibialis abdominis muscle, and epididymal fat. Representative Western blots are shown on the graphs. All data are mean ± SEM (n = 10 / group). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant differences in unpaired two-tailed Steudon's t-tests.

[0026] Figure 14 The timeline study of Example E is shown. Dosing was administered on day 0, as described in Table 10. The chronic unpredictable stress (CUS) program began 20 days prior to administration and ended on day 5 (groups 3 and 4, n = 14 / group). Two groups of rats (groups 1 and 2, n = 14 / group) were typically housed for 25 days and used as the non-stress (NS) group. The first dose was administered on day 0. Twenty-four hours after administration (day 1), a sucrose preference test (SPT) was performed after 6 hours of dehydration. Rats were then fasted overnight for 20 hours, followed by an NSFT (48 hours after administration). A second dose of Veh or NV-5138 was administered on day 5, followed by sacrifice for 24 hours, after which brain harvesting, synaptic preparation, and Western blotting were performed.

[0027] Figure 15 shows a schematic diagram of the PFC anatomy in a rat brain. A) Dorsal view of the rat brain, with two black lines indicating the anatomical locations that separate the PFC region of the brain. B) Sagittal view of the rat brain, with black diagonal lines representing incisions made to remove the olfactory bulb and two black vertical lines representing incisions that separate the PFC region.

[0028] Figure 16 The body weights of NS and CUS rats are shown at 21 days prior and on day 0. Both NS and CUS rats were treated and weighed weekly. All data are presented as mean ± SEM (n = 14 / treatment group). ****p < 0.0001 indicates a significant difference in a two-way ANOVA followed by multiple Durkheimer comparisons.

[0029] Figure 17 This study demonstrates the effect of NV-5138 (I-90) on SPT in NS and CUS rats. Rats were treated with Veh or NV-5138 (160 mg / kg) on ​​day 0 at the end of the NS and CUS programs, and SPT was performed 24 hours after administration. At the end of 1 hour exposure, the volume ratio of 1% sucrose to water for all four groups (preferred) is presented. All data are mean ± SEM (n = 14 / treatment group). *p < 0.05 indicates significant differences in a two-way ANOVA followed by multiple Durkheimer comparisons.

[0030] Figure 18 This study demonstrates the effect of NV-5138 (I-90) on NSFT in NS and CUS rats. Rats were treated with Veh or NV-5138 at the end of their NS and CUS programs, and NSFT was performed 48 hours after administration. Bar charts show the feeding wait time during NSFT. All data are presented as mean ± SEM (n = 14 / treatment group). **p < 0.01 and ****p < 0.0001 indicate significant differences in a two-way ANOVA followed by multiple Durkheimer comparisons.

[0031] Figure 19 This study demonstrates the effect of NV-5138(I-90) on the expression of GluR1 and PSD95 in synaptosomes prepared from PFCs of NS and CUS rats.

[0032] Figure 20 The timetable study of Example F is presented. After 5 days of environmental acclimatization, bilateral IT cannulas were implanted into the PFC of all rats 14 days prior, followed by a 2-week recovery period. On day 0, test items were administered as shown in Study Design Table 11.

[0033] Figure 21 This study demonstrates the effect of mTORC1 inhibition in PFC on the pharmacological efficacy of oral NV-5138 in FST. Rats were administered Veh-NV-5138 or NV-5138 (160 mg / kg) orally for 30 minutes, followed by administration of Veh-R or R via IT. FST was performed 24 hours after oral administration. All data are presented as mean ± SEM (n = 6–7 / treatment group). ***p < 0.001 and ****p < 0.0001 indicate significant differences in a one-way ANOVA followed by multiple Durkheimer comparisons.

[0034] Figure 22 The effects of treatments assessed using LMA are demonstrated. Rats were administered Veh-R or R via intraperitoneal administration (IT) 30 minutes after oral administration of Veh-NV-5138 or NV-5138 (I-90; 160 mg / kg). LMA was measured 48 hours after oral administration. All data are presented as mean ± SEM (n = 6–7 / treatment group). No significant differences were observed between groups by one-way ANOVA followed by multiple Durkheimer comparison tests.

[0035] Figure 23 illustrates the effect of mTORC1 inhibition in PFC on the pharmacological efficacy of oral NV-5138 (I-90). Rats were administered Veh-R or R via IT 30 minutes after oral administration of Veh-NV-5138 or NV-5138 (160 mg / kg). NSFT was performed 72 hours after oral administration, following a 20-hour fasting period. Bar charts show NFST (A) and food consumption (B). All data are presented as mean ± SEM (n = 6–7 / treatment group). *p < 0.05 indicates significant differences in a one-way ANOVA followed by multiple comparisons by Duker.

[0036] Figure 24 This study demonstrates the time-dependent behavioral testing of Example G. Male Sporgdolly rats were all acclimatized to their environment 5 days after transport. On day 0, test items were administered as shown in Study Design Table 12. FST was performed on days 3 and 7, and NSFT was performed on day 10 after a 20-hour overnight fast.

[0037] Figure 25 The effects of Ket and NV-5138 (I-90) on FST are shown on days 3 and 7 post-dose. Bar charts show FST performed on days 3 and 7 post-dose. Ketamine mediator (Sal); NV-5138 mediator (Veh); ketamine (Ket); NV-5138 (NV). All data are presented as mean ± SEM. *p<0.05 and **p<0.01 indicate significant differences in unpaired two-tailed Steudent's t-tests.

[0038] Figure 26 shows the effects of Ket and NV-5138 (I-90) on NSFT on day 10 post-administration. Ketamine mediator (Sal); NV-5138 mediator (Veh); ketamine (Ket); NV-5138 (NV). Bar graphs show NSFT (A) and cage food consumption (B) on day 10 post-administration (groups 3, 4, 5, and 6). All data are presented as mean ± SEM. No significant differences were observed between groups by unpaired two-tailed Steudent's t test.

[0039] Figure 27 The study timeline for Example H is shown below. Male Sporgodory rats were acclimatized for 5 days and administered the drug on day 0, as described in Table 13. Twenty-four hours after drug administration (day 1), the rats were sacrificed, brain sections were prepared, and EPSC was recorded. The fixed sections were then analyzed by microscopy for spine density and morphology.

[0040] Figure 28The frequencies of 5-HT and hypothalamic-secreting hormone (Hcrt)-induced EPSCs are shown. Bar charts show 5-HT-EPSCs and Hcrt-EPSCs in neurons 24 hours post-drug administration, recorded in PFCs via whole-cell patch-clamp. Cell numbers shown are from n = 8 rats. All data are presented as mean ± SEM. **p < 0.01 and ***p < 0.001 indicate significant differences in unpaired two-tailed Steudon's t-tests.

[0041] Figure 29 illustrates the effect of NV-5138 (I-90) on dendrite morphology. A) High-magnification two-photon images of representative Z-stacked protrusions of distal, intermediate, and proximal chain segments of apical clusters of neurobiotin-labeled V pyramidal cells 24 hours after administration. B) Quantitative bar graphs of short, thick, long, and mushroom-shaped spikes per μm from 4 and 5 animals, respectively, for the mediator and NV-5138 groups. All data are presented as mean ± SEM. *p<0.05 indicates significant differences in unpaired two-tailed Steudon's t-tests.

[0042] Figure 30 Whole-cell voltage clamp traces of 5-HT and Hcrt-induced EPSCs in sections from rats treated with the medium or NV-5138 (I-90) 24 hours after administration are shown.

[0043] Figure 31 The cumulative probability distribution of EPSC induced by 5-HT and Hcrt is shown, as well as the effect of NV-5138(I-90) on frequency rather than amplitude.

[0044] Figure 32 The study timeline for Example J is shown. Male Sporgodory rats were acclimatized to their environment for 5 days. One day prior, the rats underwent pre-swimming. Starting on day 0, the rats received Ket or Sal every other day for 7 days (day 0 to day 6), or NV-5138 once daily for 7 days (day 0 to day 6), as described in Table 14.

[0045] Figure 33 Demonstrate the role of Ket and NV-5138 (I-90) in FST. NV-5138 mediator (Veh); physiological saline (Sal); ketamine (Ket, 10 mg / kg), NV-5138 (“NV”; 40 or 80 mg / kg).

[0046] Figure 34 Demonstrate the role of Ket and NV-5138 (I-90) in LMA. NV-5138 mediator (Veh); normal saline (Sal); ketamine (Ket, 10 mg / kg), NV-5138 (“NV”; 40 or 80 mg / kg).

[0047] Figure 35 Demonstrates the effects of Ket and NV-5138 (I-90) in NSFT and cage-fed control groups. NV-5138 mediator (Veh); physiological saline (Sal); ketamine (Ket, 10 mg / kg), NV-5138 (“NV”; 40 or 80 mg / kg).

[0048] Figure 36 This study demonstrates the effects of Ket (0.3 mg / kg) and NV-5138 (I-90; 160 mg / kg) compared to dichlorodioxide (1 mg / kg) in the human threat assay in marmosets. Data are presented as mean ± SEM (n = 10 / treatment group). ***P < 0.001 relative to the corresponding mediator group, by one-way ANOVA followed by the dunnette test. *Indicates that vomiting was observed in 6 of the 12 animals administered in this group.

[0049] Figure 37 This study demonstrates the effects of Ket (0.3 mg / kg) and NV-5138 (I-90; 160 mg / kg) compared to dichlorodioxide (1 mg / kg) in motor control in marmosets under the human threat test. Data are presented as mean ± SEM (n = 10 / treatment group). ***P < 0.001 relative to the corresponding mediator group, by one-way ANOVA followed by the dunnette test. *Indicates that vomiting was observed in 6 of the 12 animals administered in this group. Detailed Implementation

[0050] 1. General description of certain embodiments of the present invention:

[0051] The compounds and compositions thereof of the present invention are used as Sestrin-GATOR2 modifiers. In some embodiments, the present invention provides compounds of formula I:

[0052]

[0053] Or its pharmaceutically acceptable salt, wherein:

[0054] R 1 For H or C 1-6 alkyl;

[0055] R 2 For R, -(CH2) n -Phenyl, -C(O)R, -SO2R or -C(O)N(R)2;

[0056] n is 0, 1, or 2;

[0057] Each R is independently hydrogen, -CN, or an optionally substituted group selected from the following: saturated or unsaturated C 1-6 Aliphatic group, phenyl, 4- to 7-membered saturated or partially unsaturated monocyclic carbon ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0058] R 3 The rings are A, -C(O)R, -C(O)OR, -C(O)N(R)2, -SO3H, -SO2N(R)2, -S(O)R, -S(O)ring A, -OR, or -B(OR)2, wherein the two OR groups on the same boron atom are bonded together with their intermediate atoms to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur, excluding boron and two oxygen atoms, or R 3 and R 4 They combine to form a substituted 5- or 6-membered ring having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur;

[0059] L is a covalent bond or optionally a straight or branched C with 1 to 9 fluorine groups substituted. 1-6 Alkylene chain;

[0060] Ring A is a optionally substituted ring selected from phenyl or a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur;

[0061] R 4 For R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4 They combine to form optionally substituted 5- to 6-membered rings having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur; and

[0062] R 5 For H or C 1-4 alkyl.

[0063] 2. Compounds and their definitions:

[0064] The compounds of this invention include those generally described herein and further illustrated by the classes, subclasses and species disclosed herein. Unless otherwise specified, the following definitions shall apply as used herein. For the purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999 and “March's Advanced Organic Chemistry”, 5th edition, eds. Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0065] As used herein, the terms "aliphatic" or "aliphatic group" mean a fully saturated or branched (i.e., unbranched) or branched substituted or unsubstituted hydrocarbon chain containing one or more unsaturated units, or a fully saturated or branched monocyclic or bicyclic hydrocarbon (also referred to herein as "carbocyclic," "cycloaliphatic," or "cycloalkyl") that is not aromatic and has a single connection point to the remainder of the molecule. Unless otherwise stated, an aliphatic group contains 1 to 6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 4 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 3 aliphatic carbon atoms, and in other embodiments, an aliphatic group contains 1 to 2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocyclic" or "cycloalkyl") refers to a fully saturated or branched monocyclic C3-C6 hydrocarbon containing one or more unsaturated units, but not aromatic and having a single connection point to the remainder of the molecule. Suitable aliphatic groups include (but are not limited to) straight-chain or branched substituted or unsubstituted alkyl, alkenyl, alkynyl, and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0066] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any quaternized form of basic nitrogen; or a substituted nitrogen of a heterocycle, such as N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or NR). + (e.g., in N-substituted pyrroleyl groups).

[0067] As used in this article, the term "unsaturated" refers to a portion having one or more unsaturated units.

[0068] As used in this article, the term "divalent C" 1-8 (or C) 1-6 "Saturated or unsaturated, straight or branched hydrocarbon chains" refers to divalent alkylene, alkenyl, and ynylene chains as defined herein, whether straight or branched.

[0069] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2). n - where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. The substituted alkylene chain is a polymethylene in which one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents include those described below with respect to substituted aliphatic groups.

[0070] The term "alkenyl" refers to a divalent alkenyl group. A substituted alkenyl chain is a polymethylene group containing at least one double bond, wherein one or more hydrogen atoms are substituted by substituents. Suitable substituents include those described below with respect to substituted aliphatic groups.

[0071] The term "halogen" refers to F, Cl, Br, or I.

[0072] The term "aryl," used alone or as part of a larger portion of "aralkyl," "arylalkoxy," 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" is used interchangeably with the term "aromatic ring." In some embodiments of the invention, "aryl" refers to an aromatic ring system comprising (but not limited to) phenyl, biphenyl, naphthyl, anthracene, and similar groups, which may have one or more substituents. As used herein, the scope of the term "aryl" also includes groups in which an aromatic ring is fused to one or more non-aromatic rings, such as dihydroindenyl, phthalimide, naphthylimide, phenidyl, or tetrahydronaphthyl and similar groups.

[0073] The terms "heteroaryl" and "heteroaryl-", such as "heteroarylalkyl" or "heteroarylalkoxy", used alone or as part of a larger portion, refer to a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and having one to five heteroatoms in addition to a carbon atom. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur; and any quaternized form of basic nitrogen. Heteroaryl groups include (but are not limited to) thiophene, furanyl, pyrrole, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indoleazinyl, purine, etc. Pyridyl and pteridinyl. As used herein, the terms "heteroaryl" and "heteroary-" also include groups in which a heteroaryl ring is fused to one or more aryl, cycloaliphatic, or heterocyclic rings, wherein a free radical or linker is located on the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothiopheneyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, The heteroaryl group can be phyrinyl, phthalazinyl, quinazolinyl, quinolinyl, 4H-quinazinyl, carbazoyl, acridineyl, benazinoyl, benazinoyl, benazinoyloxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. The heteroaryl group can be monocyclic or bicyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl," or "heteroaromatic," any of which contains an optionally substituted ring. The term "heteroarylalkyl" refers to a heteroaryl-substituted alkyl group, wherein the alkyl and heteroaryl portions are optionally substituted independently.

[0074] As used herein, the terms “heterocycle,” “heterocyclic group,” “heterocyclic radical,” and “heterocycle” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic portion that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above, in addition to a carbon atom. When referring to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen may be N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or... + NR (e.g., in N-substituted pyrroleyl groups).

[0075] Heterocycles can be attached to their side groups at any heteroatom or carbon atom to produce a stable structure, and any ring atom can optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include (but are not limited to) tetrahydrofuranyl, tetrahydrothiophenylpyrrolyl, piperidinyl, pyrrololinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolyl, piperazine, dioxacyclohexyl, dioxopentyl, diazapyryl, oxazopentyl, thiazapyryl, morpholinyl, and pyridyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical” are used interchangeably herein and also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indololinyl, 3H-indolyl, chromanyl, phenidyl, or tetrahydroquinolinyl. The heterocyclic group can be monocyclic or bicyclic. The term “heterocyclic alkyl” refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl group and the heterocyclic moiety are optionally substituted independently.

[0076] As used herein, the term “partially unsaturated” refers to a ring moiety containing at least one double or triple bond. As defined herein, the term “partially unsaturated” is intended to encompass a ring having multiple unsaturated sites, but not necessarily to include aryl or heteroaryl moieties.

[0077] As described herein, the compounds of the present invention may contain an "optionally substituted" portion. Generally, the term "substituted," whether or not preceded by the term "optionally," means that one or more hydrogens of the specified portion are replaced by suitable substituents. Unless otherwise specified, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. The combinations of substituents contemplated in this invention are preferably combinations that form stable or chemically viable compounds. As used herein, the term "stable" means that the compound does not undergo substantial changes when subjected to conditions permissible for its production, detection, and (in some embodiments) its recovery, purification, and use for one or more of the purposes disclosed herein.

[0078] The suitable monovalent substituent on the substituted carbon atom of the "optionally substituted" group is independently a halogen; -(CH2) 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 Ro -O-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 CH(OR o )2;-(CH2) 0- 4SR o ;-(CH2) 0-4 Ph, which can be transmitted via R o Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be transmitted via R o Substitution; -CH=CHPh, which can be obtained via R o Substitution; -(CH2) 0-4 O(CH2) 0-1 -pyridyl group, which can be transmitted via R o Substitution; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2) 0-4 N(R o )C(O)NR o 2; -N(R) o )C(S)NR o 2;-(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2; -N(R) o )N(R o )C(O)OR o ;-(CH2) 0-4 C(O)R o ;-C(S)R o ;-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 C(O)SR o ;-(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o;-OC(O)(CH2) 0-4 SR-;SC(S)SR o ;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NR o 2; -C(S)NR o 2;-C(S)SR o ;-SC(S)SR o ;-(CH2) 0-4 OC(O)NR o 2; -C(O)N(OR) o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR) o )R o ;-(CH2) 0-4 SSR o ;-(CH2) 0-4 S(O)2R o ;-(CH2) 0-4 S(O)2OR o ;-(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2;-(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2; -N(R) o )S(O)2R o ;-N(OR) o )R o ;-C(NH)NR o 2; -P(O)2R o ;-P(O)R o 2; -OP(O)R o 2; -OP(O)(OR o )2;-SiR o 3; -(C 1-4 (linear or branched alkylene)ON(R) o )2; or -(C 1-4 (straight-chain or branched alkylene)C(O)ON(R) o )2, where each R o It can be substituted and independently formed as hydrogen or C as defined below. 1-6 Aliphatic groups, -CH2Ph, -O(CH2) 0-1Ph, -CH2- (5- to 6-membered heteroaryl ring) or 5- to 6-membered saturated, partially unsaturated or aromatic ring having 0 to 4 independent heteroatoms selected from nitrogen, oxygen or sulfur, or regardless of the above definition, two separate R groups. o It combines with its intermediate atom to form a 3- to 12-member saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0079] R o Suitable monovalent substituents (or two separate Rs) o The ring formed with its intermediate atoms is independently a halogen, -(CH2). 0-2 R · -(halogenated R) · -(CH2) 0-2 OH, -(CH2) 0-2 OR · -(CH2) 0-2 CH(OR · )2;-O(halogenated R · -CN, -N3, -(CH2) 0-2 C(O)R · -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR · -(CH2) 0-2 SR · -(CH2) 0- 2SH、-(CH2) 0-2 NH2、-(CH2) 0-2 NHR · -(CH2) 0-2 NR · 2, -NO2, -SiR · 3. -OSiR · 3. -C(O)SR · -(C 1-4 (straight-chain or branched alkylene)C(O)OR · or -SSR · , where each R · It is either unsubstituted or substituted with only one or more halogens when placed before the "halogen group", and is independently selected from C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R o Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0080] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following: =O, =S, =NNR * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * -O(C(R) * 2)) 2-3 O- or -S(C(R) * 2)) 2-3 S-, where each individual R appears * Selected from hydrogen, and substituted C as defined below. 1-6 An aliphatic group or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents for adjacent substituted carbons bonded to the "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each individual R appears * Selected from hydrogen, and substituted C as defined below. 1-6 Aliphatic group or unsubstituted 5- to 6-membered saturated, partially unsaturated or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0081] R * Suitable substituents on the aliphatic group include halogens, -R · -(halogenated R) · -OH, -OR · -O(halogenated R) · -CN, -C(O)OH, -C(O)OR · -NH2, -NHR · -NR · 2 or -NO2, where each R · It is unsubstituted or, if placed before the "halogen group", substituted with only one or more halogens, and is independently C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0082] Suitable substituents on the substituted nitrogen gas of the "optionally substituted" group include

[0083] Among them each Independently, hydrogen, or substituted C as defined below 1-6 Aliphatic group, unsubstituted -OPh, or unsubstituted 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, or, regardless of the above definition, two separate groups. It combines with its intermediate atom to form an unsubstituted 3- to 12-member saturated, partially unsaturated, or aryl monocyclic or bicyclic ring with 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

[0084] Suitable substituents on the aliphatic group are independently halogens, -R · -(halogenated R) · -OH, -OR · -O(halogenated R) · -CN, -C(O)OH, -C(O)OR · -NH2, -NHR · -NR · 2 or -NO2, where each R · It is unsubstituted or, if placed before the "halogen group", substituted with only one or more halogens, and is independently C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated ring or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0085] As used herein, the term "pharmaceutically acceptable salt" refers to salts that, to the extent reasonably medically permissible, are suitable for contact with human and lower animal tissues without adverse toxicity, irritation, anaphylactic reactions, or the like, and for which the benefit / risk ratio is proportionate. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention comprise those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, p-pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and similar salts.

[0086] Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and nitrogen salts. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and similar salts. Other pharmaceutically acceptable salts include (where appropriate) non-toxic ammonium, quaternary ammonium, and amine cations formed using relative ions (e.g., halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate).

[0087] Unless otherwise stated, the structures described herein are also intended to include all isomers (e.g., enantiomers, diastereomers, and geometric isomers (or configurational isomers)); for example, R and S configurations of each asymmetry center, Z and E double bond isomers, and Z and E configurational isomers. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and geometric isomers (or configurational isomers), are within the scope of the present invention. Unless otherwise stated, all tautomers of the compounds of the present invention are within the scope of the present invention. Furthermore, unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds containing hydrogen replaced by deuterium or tritium, or carbon replaced by... 13 C or 14 C-enriched carbon substitutions and compounds having the structure of this invention are within the scope of this invention. Such compounds are suitable for use as, for example, analytical tools, as probes in bioanalysis, or as therapeutic agents according to this invention.

[0088] As used herein, the term "leucine mimic" is defined as a compound that, at 25 μM, reduces the amount of Sestrin2 bound to GATOR2 by at least about 40% relative to leucine. In some embodiments, the "leucine mimic" reduces the amount of Sestrin2 bound to GATOR2 by at least about 100%, at least about 150%, or at least about 200%.

[0089] As used herein, the term "leucine antagonist" is defined as a compound that, at 25 μM, increases the amount of Sestrin2 bound to GATOR2 by at least about 40% (expressed as -40% of leucine activity) relative to leucine. In some embodiments, the "leucine antagonist" increases the amount of Sestrin2 bound to GATOR2 by at least about 100%, at least about 150%, or at least about 200%.

[0090] As used herein, the terms “measurable affinity” and “measurable inhibition” refer to a measurable change in the binding of GATOR2 in a sample comprising the compound or composition thereof of the present invention and Sestrin2, GATOR2 and leucine, and an equivalent sample comprising Sestrin2, GATOR2 and leucine in the absence of said compound or composition thereof.

[0091] 3. Description of exemplary embodiments:

[0092] In some embodiments, the present invention provides compounds of formula I:

[0093]

[0094] Or its pharmaceutically acceptable salt, wherein:

[0095] R 1 For H or C 1-6 alkyl;

[0096] R 2 For R, -(CH2) n -Phenyl, -C(O)R, -SO2R or -C(O)N(R)2;

[0097] n is 0, 1, or 2;

[0098] Each R is independently hydrogen, -CN, or an optionally substituted group selected from the following: saturated or unsaturated C 1-6 Aliphatic group, phenyl, 4- to 7-membered saturated or partially unsaturated monocyclic carbon ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0099] R 3 The rings are A, -C(O)R, -C(O)OR, -C(O)N(R)2, -SO3H, -SO2N(R)2, -S(O)R, -S(O)ring A, -OR, or -B(OR)2, wherein the two OR groups on the same boron atom are bonded together with their intermediate atoms to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur, excluding boron and two oxygen atoms, or R 3 and R 4 They combine to form a substituted 5- or 6-membered ring having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur;

[0100] L is a covalent bond or optionally a straight or branched C with 1 to 9 fluorine groups substituted. 1-6 Alkylene chain;

[0101] Ring A is a optionally substituted ring selected from phenyl or a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur;

[0102] R 4 For R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4 They combine to form optionally substituted 5- to 6-membered rings having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur; and

[0103] R 5 For H or C 1-4 alkyl.

[0104] In some embodiments, the provided Formula I compounds are not those described in Table 2 below.

[0105] As generally defined above, R 1 For H or C 1-6 Alkyl group. In some embodiments, R 1 For H. In other embodiments, R 1 C 1-6 Alkyl group. In some embodiments, R 1 methyl. In some embodiments, R 1 It is isobutyl. In some embodiments, R... 1 Selected from those described in Table 1 below. In some embodiments, R 1 Selected from those described in Table 2 below.

[0106] As generally defined above, R 2 For R, -(CH2) n -Phenyl, -C(O)R, -SO2R, or -C(O)N(R)2. In some embodiments, R 2 R. In some embodiments, R 2 -(CH2) n -Phenyl. In some embodiments, R 2 For -C(O)R. In some embodiments, R 2 For -SO2R. In some embodiments, R 2 It is -C(O)N(R)2. In some embodiments, R 2 methyl. In some embodiments, R 2 It is -(CH2)-phenyl. In some embodiments, R 2 It is -C(O)CH3. In some embodiments, R 2 Selected from those described in Table 1 below. In some embodiments, R 2 Selected from those described in Table 2 below.

[0107] 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.

[0108] As generally defined above, R 3 The rings are A, -C(O)R, -C(O)OR, -C(O)N(R)2, -SO3H, -SO2N(R)2, -S(O)R, -S(O)ring A, -OR, or -B(OR)2, wherein the two -OR groups on the same boron atom are bonded together with their intermediate atoms to form a 5- to 8-membered monocyclic saturated or partially unsaturated ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur, excluding boron and two oxygen atoms, or R. 3 and R 4They combine to form optional substituted 5- to 6-membered rings with 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur.

[0109] In some embodiments, R 3 It is -C(O)OH. In some embodiments, R 3 It is -C(O)N(R)2. In some embodiments, R 3 It is -SO3H. In some embodiments, R 3 It is -SO2N(R)2. In some embodiments, R 3 The form is -B(OR)2, wherein two -OR groups on the same boron atom are bonded together with their intermediate atoms to form a 5- to 8-membered monocyclic saturated, partially unsaturated, or heterocyclic ring having 0 to 3 independent heteroatoms selected from nitrogen, oxygen, or sulfur, in addition to boron and two oxygen atoms. In some embodiments, R 3 and R 4 They combine to form optional substituted 5- to 6-membered rings with 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur.

[0110] In some embodiments, R 3 Ring A is a ring selected from phenyl, optionally substituted, or a 5- to 6-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, ring A is an optionally substituted phenyl ring. In some embodiments, ring A is a 5-membered heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, ring A is a 5-membered heteroaryl ring selected from imidazolyl, isoxazolyl, 1H-pyrroleyl (e.g., maleic anhydride), pyrazolyl, oxazolyl, tetrazolyl, thiazolyl, and triazolyl. In some embodiments, ring A is a 6-membered heteroaryl ring having 1 to 2 nitrogen atoms. In some embodiments, ring A is a 6-membered ring selected from pyridyl and pyrimidinyl. In some embodiments, ring A is selected from those depicted in Table 1 below.

[0111] In some embodiments, R 3 For (pinacolyl)boron. In some embodiments, R 3 Selected from those described in Table 1 below. In some embodiments, R 3 Selected from those described in Table 2 below.

[0112] As generally defined above, L is a covalently bonded or optionally substituted with 1 to 4 fluorine groups, either straight or branched. 1-6 Alkylene chain. In some embodiments, L is a covalent bond. In some embodiments, L is a straight-chain or branched C optionally substituted with 1 to 4 fluorine groups. 1-6Alkylene chain. In some embodiments, L is methylene. In some embodiments, L is n-butylene. In some embodiments, L is ethylene. In some embodiments, L is n-propylene. 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.

[0113] In some embodiments, L is a branched C optionally substituted with 1 to 4 fluorine groups. 1-6 Alkylene chain. In some embodiments, L is -C(CH3)2-. In other embodiments, L is -C(CH3)(CF3)-.

[0114] As generally defined above, R 4 For R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR, or R 3 and R 4 These are combined to form optionally substituted 5- to 6-membered rings having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R 4 R. In some embodiments, R 4 For -CF3. In some embodiments, R 4 For -OR. In some embodiments, R 4 It is -N(R)2. In some embodiments, R 4 It is -Si(R)3. In some embodiments, R 4 For -SR. In some embodiments, R 4 It is isopropyl. In some embodiments, R... 4 It is tert-butyl. In some embodiments, R 4 It is cyclopropyl. In some embodiments, R 4 It is cyclobutyl. In some embodiments, R 4 It is sec-butyl. In some embodiments, R 4 It is methoxylated. In some embodiments, R 4 It is methylthioyl. In some embodiments, R 3 and R 4 These are combined to form optionally substituted 5- to 6-membered rings having 0 to 1 heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R 4 Selected from those described in Table 1 below. In some embodiments, R 4 Selected from those described in Table 2 below.

[0115] As generally defined above, R 5 For H or C 1-4 Alkyl group. In some embodiments, R 5 For H. In some embodiments, R 5C 1-4 Alkyl group. In some embodiments, R 5 methyl. In some embodiments, R 5 Selected from those described in Table 1 below. In some embodiments, R 5 Selected from those described in Table 2 below.

[0116] In some embodiments, the present invention provides compounds of formula II:

[0117]

[0118] Or a pharmaceutically acceptable salt thereof, wherein the variables are as defined above and described in the examples provided herein (in individual and in combination).

[0119] In some embodiments, the present invention provides compounds of formula III:

[0120]

[0121] Or its pharmaceutically acceptable salt, wherein:

[0122] Q is either -C(R')2- or -NH-;

[0123] R x and R y Each of them is hydrogen, or R x and R y Together they form = O;

[0124] It can be a double bond or a single bond;

[0125] Each R is independently hydrogen, -CN, or an optionally substituted group selected from the following: C 1-6 Aliphatic group, phenyl, 4- to 7-membered saturated or partially unsaturated monocyclic carbon ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0126] Each R' is independently hydrogen, halogen, -CN, or an optionally substituted group selected from the following: C 1-6 Aliphatic group, phenyl, 4- to 7-membered saturated or partially unsaturated monocyclic carbon ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0127] L is a covalent bond or optionally a straight or branched C with 1 to 9 fluorine groups substituted. 1-6 Alkylene chain;

[0128] R4 'is R, -CF3, -OR, -N(R)2, -Si(R)3 or -SR; and

[0129] R 5 'For H, -OR or C 1-4 alkyl.

[0130] In some embodiments, Q is -NH-. In some embodiments, Q is -CH2-. In some embodiments, Q is -CHF-.

[0131] In some embodiments, L is -CH2-.

[0132] In some embodiments, each R x and R y It is hydrogen. In some embodiments, R is... x and R y They combine to form =O.

[0133] In some embodiments, R 5 'For H. In some embodiments, R 5 'is -OH.

[0134] In some embodiments, It is a single key. In some embodiments, It is a double bond.

[0135] In some embodiments, the present invention provides compounds of formula IV-a, IV-b, or IV-c:

[0136]

[0137] Or its pharmaceutically acceptable salt, wherein:

[0138] R 1 It is H or C1-6 alkyl;

[0139] R 2 For R, -(CH2) n -Phenyl, -C(O)R, -SO2R or -C(O)N(R)2;

[0140] Each R 4 "Independently R, halogen, or -CF3;

[0141] Each R is independently hydrogen, -CN, or an optionally substituted group selected from the following: saturated or unsaturated C 1-6 Aliphatic group, phenyl, 4- to 7-membered saturated or partially unsaturated monocyclic carbon ring, 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, or 4- to 8-membered saturated or partially saturated heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and

[0142] L 1 C is a covalently bonded, straight-chain or branched C that is optionally substituted with 1 to 9 fluorine groups. 1-6 Alkylene chain.

[0143] In some embodiments, R 1 For H. In some embodiments, R 1 C 1-6 alkyl.

[0144] In some embodiments, R 1 Selected from those described in Table 1 below.

[0145] In some embodiments, R 2 R. In some embodiments, R 2 -(CH2) n -Phenyl. In some embodiments, R 2 It is -C(O)R.

[0146] In some embodiments, R 2 Selected from those described in Table 1 below.

[0147] In some embodiments, each R 4 "Independently R, halogen, or -CF3. In some embodiments, R..." 4 " is R. In some embodiments, R 4 "is a halogen. In some embodiments, R..." 4 "For -CF3. In some embodiments, R 4 "Selected from those described in Table 1 below."

[0148] In some embodiments, L 1 Straight or branched C atoms that are covalently bonded or optionally substituted with 1 to 9 fluorine groups. 1-6 Alkylene chain. In some embodiments, L 1 It is a covalent bond. In some embodiments, L 1 A straight-chain or branched C-type carbon optionally substituted with 1 to 9 fluorine groups. 1-6 Alkylene chain. In some embodiments, L 1 Selected from those described in Table 1 below.

[0149] Exemplary compounds of the present invention are described in Table 1 below.

[0150] Table 1. Exemplary Compounds

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Exemplary compounds of the present invention are described in Table 2 below.

[0178] Table 2. Exemplary Compounds

[0179]

[0180]

[0181] In some embodiments, the present invention provides the compounds set forth in Table 1 above, or pharmaceutically acceptable salts thereof. In some embodiments, the present invention provides the compounds set forth in Table 2 above, or pharmaceutically acceptable salts thereof.

[0182] 4. Use, allocation and distribution

[0183] Pharmaceutically acceptable compositions

[0184] According to another embodiment, the present invention provides a composition comprising a compound of the present invention or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier, adjuvant, or mediator. The amount of the compound in the composition of the present invention is such that it can effectively and measurably inhibit or activate Sestrin-GATOR2 interaction in a biological sample or patient. In some embodiments, the amount of the compound in the composition of the present invention is such that it can effectively and measurably inhibit or activate Sestrin-GATOR2 interaction in a biological sample or patient. In some embodiments, the composition of the present invention is formulated for administration to a patient requiring such a composition. In some embodiments, the composition of the present invention is formulated for oral administration to a patient.

[0185] As used herein, the term "patient" refers to an animal, preferably a mammal, and most preferably a human.

[0186] The term "pharmaceutically acceptable carrier, adjuvant, or catalyst" refers to a non-toxic carrier, adjuvant, or catalyst that does not impair the pharmacological activity of the compound it is formulated with. Pharmaceutically acceptable carriers, adjuvants, or catalysts that can be used in the compositions of this invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffering substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0187] The compositions of this invention can be administered orally, non-enterically, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or by means of an implantable reservoir. As used herein, the term "non-enteric" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheathic, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions of this invention can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injectable formulation can also be a sterile injectable solution or suspension in a non-toxic, non-enteric acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among acceptable mediators and solvents, water, Ringer's solution, and isotonic sodium chloride solution can be used. Additionally, sterile, non-volatile oils are conventionally used as solvents or suspension media.

[0188] For this purpose, any mild, non-volatile oil, including synthetic monoglycerides or diglycerides, may be used. For example, fatty acids of oleic acid and their glycerol derivatives are suitable for preparing injectable formulations, such as pharmaceutically acceptable natural oils, such as olive oil or castor oil, especially in their polyoxyethylene form. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms (including emulsions and suspensions). 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 formulation purposes.

[0189] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form, including (but not limited to) capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may also be added.

[0190] Alternatively, the pharmaceutically acceptable compositions of the present invention can be used for administration in the form of rectal suppositories. These suppositories can be prepared by mixing the pharmaceutical agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0191] The pharmaceutically acceptable compositions of the present invention can also be administered topically, especially when the therapeutic target comprises areas or organs easily accessible by topical application (including diseases of the eyes, skin, or lower intestine). Suitable topical formulations can be readily prepared for each of these areas or organs.

[0192] For local application to the lower intestine, it can be achieved in the form of rectal suppositories (see above) or in the form of suitable enema formulations. Topical percutaneous patches may also be used.

[0193] For topical application, the pharmaceutically acceptable compositions provided may be formulated into suitable ointment forms containing active ingredients 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 paraffin, white paraffin, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the pharmaceutically acceptable compositions provided may be formulated into suitable lotion or cream forms containing active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include (but are not limited to) mineral oil, sorbitan monostearate, polysorbate 60, hexadecyl wax, hexadecyl stearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water.

[0194] For ophthalmic use, the pharmaceutically acceptable composition provided may be formulated as a micron-sized suspension, with or without a preservative (e.g., benzyl chloride), in pH-adjusted isotonic sterile saline, or preferably as a solution in pH-adjusted isotonic sterile saline. Alternatively, for ophthalmic use, the pharmaceutically acceptable composition may be formulated as an ointment (e.g., paraffin).

[0195] The pharmaceutically acceptable compositions of the present invention can also be administered via nasal aerosol or inhaler. These compositions are prepared according to techniques well known in pharmaceutical formulation and can be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, bioavailability enhancers, fluorocarbons, and / or other conventional solvents or dispersants.

[0196] Most preferably, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations may or may not be administered with food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0197] The amount of the compounds of the present invention that can be combined with carrier materials to produce compositions in a single dosage form will vary depending on the host being treated and the specific administration method. Preferably, the provided compositions should be formulated such that an inhibitor can be administered to patients receiving these compositions at a dose between 0.01 mg / kg body weight / day and 100 mg / kg body weight / day.

[0198] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health, sex, diet, administration time, excretion rate, drug combination, the judgment of the treating physician, and the severity of the specific disease being treated. The amount of the compound of the invention in the composition also depends on the specific compound in the composition.

[0199] Use of compounds and pharmaceutically acceptable compositions

[0200] The compounds and compositions described herein are generally suitable for inhibiting or activating Sestrin-GATOR2 interactions. In some embodiments, the provided compounds or compositions thereof are activators of Sestrin-GATOR2 interactions.

[0201] The activity of compounds used as inhibitors or activators of Sestrin-GATOR2 interaction in this invention can be analyzed in vitro, in vivo, or in cell lines. In vitro analysis includes assays to determine the inhibition or activation of Sestrin-GATOR2 interaction. Alternative in vitro analyses quantify the ability of inhibitors or activators to reduce or increase Sestrin binding to GATOR2. Detailed conditions for analyzing compounds used as inhibitors or activators of Sestrin-GATOR2 interaction in this invention are described in the following examples.

[0202] As used herein, the terms "treatment," "treat," and "treating" refer to reversing or alleviating a disease or condition as described herein, or one or more of its symptoms, delaying its onset, or inhibiting its progression. In some embodiments, treatment may be administered after one or more symptoms have already appeared. In other embodiments, treatment may be administered when symptoms are absent. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0203] The provided compounds are inhibitors or activators of Sestrin-GATOR2 interaction and are therefore suitable for treating one or more conditions associated with mTORC1 activity. Accordingly, in some embodiments, the present invention provides a method for treating mTORC1-mediated conditions, comprising the step of administering to a patient in need a compound of the present invention or a pharmaceutically acceptable composition thereof.

[0204] As used herein, the term "mTORC1-mediated" condition, disease, and / or illness means any disease or other harmful condition in which mTORC1 is known to play a role. Therefore, another embodiment of the invention relates to treating one or more diseases in which mTORC1 is known to play a role, or reducing their severity.

[0205] In some embodiments, the method of activating mTORC is used to treat or prevent depression. (See Ignácio et al., (2015) Br J Clin Pharmacol. November 27). Therefore, in some embodiments, the present invention provides a method for treating or preventing depression in a patient in need, comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the depression is treatment-resistant depression (“TRD”). In some embodiments, treatment-resistant depression is resistant to first-line treatment. In some embodiments, treatment-resistant depression is resistant to second-line treatment.

[0206] In some embodiments, the present invention provides a method for treating depression in a patient in need, wherein the patient's depression scale score is reduced by 50%. In some embodiments, the patient's depression scale score is reduced by 50% within six weeks of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within four weeks of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within two weeks of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within one week of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within seven days of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within six days of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within five days of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within four days of administration of a compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score decreased by 50% within three days of administration of the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score decreased by 50% within two days of administration of the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score decreased by 50% within one day of administration of the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score decreased by 50% within twenty-four hours of administration of the compound or pharmaceutically acceptable composition. In some embodiments, the depression scale score is selected from the Montgomery-Asberg Depression Rating Scale (MADRS), the Hamilton Depression Rating Scale (HAMD-6), the List of Self-Rating Scales for Depressive Symptoms (IDS-SR), and the Clinical Global Impression Severity Scale (CGI-S).

[0207] In some embodiments, the present invention provides a method for treating depression in a patient in need, comprising the step of orally administering to the patient a provided compound or a pharmaceutically acceptable composition thereof, wherein the patient's depression scale score reduction is equivalent to that of ketamine administered via intraperitoneal injection. In some embodiments, the reduction in depression scale score is produced by a single oral administration. In some embodiments, the reduction in depression scale score is produced by multiple oral administrations.

[0208] In some embodiments, the method of activating mTORC1 is used to induce rapid-acting antidepressant activity. Therefore, in some embodiments, the present invention provides a method for inducing rapid-acting antidepressant activity in a patient with TRD of need, comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the rapid-acting antidepressant activity occurs within two weeks of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within one week of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within seven days of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within six days of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within five days of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within four days of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within three days of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within two days of administration of the compound or composition. In some embodiments, the rapid-acting antidepressant activity occurs within one day of administration of the compound or composition. In some embodiments, rapid-acting antidepressant activity occurs within 24 hours of administration of the compound or composition.

[0209] In some embodiments, the present invention provides a method for inducing long-acting, durable antidepressant activity in a patient in need of depression, comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the patient in need suffers from TRD. In some embodiments, the long-acting, durable antidepressant activity persists for at least twenty-four hours following a single administration of the provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the long-acting, durable antidepressant activity persists for more than one day. In some embodiments, the long-acting, durable antidepressant activity persists for at least two days. In some embodiments, the long-acting, durable antidepressant activity persists for at least three days. In some embodiments, the long-acting, durable antidepressant activity persists for at least four days. In some embodiments, the long-acting, durable antidepressant activity persists for at least five days. In some embodiments, the long-acting, durable antidepressant activity persists for at least six days. In some embodiments, the long-acting, durable antidepressant activity persists for at least seven days.

[0210] In some embodiments, the present invention provides a method for inducing rapid onset and long-lasting antidepressant activity.

[0211] In some embodiments, the present invention provides a method for evoking a positive behavioral response in an individual, comprising the step of administering to the individual a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the positive behavioral response is associated with improved mood. In some embodiments, the positive behavioral response is associated with reduced anxiety. In some embodiments, the positive behavioral response corresponds to improved mood. In some embodiments, the positive behavioral response is associated with improved stress resilience.

[0212] In some embodiments, the present invention provides a method for inducing a rapidly acting positive behavioral response in an individual, comprising the step of administering to the individual a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the positive behavioral response occurs within twenty-four hours of administration. In some embodiments, the positive behavioral response occurs within one day of administration. In some embodiments, the positive behavioral response occurs within two days of administration. In some embodiments, the positive behavioral response occurs within three days of administration. In some embodiments, the positive behavioral response occurs within four days of administration. In some embodiments, the positive behavioral response occurs within five days of administration. In some embodiments, the positive behavioral response occurs within six days of administration. In some embodiments, the positive behavioral response occurs within seven days of administration. In some embodiments, the positive behavioral response occurs within one week of administration.

[0213] In some embodiments, the present invention provides a method for inducing a long-lasting positive behavioral response in an individual, comprising the step of administering to the patient a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the long-lasting positive behavioral response lasts for more than one day. In some embodiments, the long-lasting positive behavioral response lasts for at least two days. In some embodiments, the long-lasting positive behavioral response lasts for at least three days. In some embodiments, the long-lasting positive behavioral response lasts for at least four days. In some embodiments, the long-lasting positive behavioral response lasts for at least five days. In some embodiments, the long-lasting positive behavioral response lasts for at least six days. In some embodiments, the long-lasting positive behavioral response lasts for at least seven days.

[0214] In some embodiments, the present invention provides a method for inducing a rapid-onset and long-lasting positive behavioral response.

[0215] In some embodiments, the present invention provides a method for improving and / or reversing behavioral and synaptic deficits caused by chronic unpredictable stress (CUS) in patients in need, comprising the step of administering a provided compound or a pharmaceutically acceptable composition thereof to the patient. In some embodiments, the method improves and / or reverses behavioral deficits caused by CUS. In some embodiments, the method improves and / or reverses synaptic deficits caused by CUS. In some embodiments, the synaptic deficits caused by CUS are reduced postsynaptic protein expression. In some embodiments, the reduced postsynaptic protein expression is reduced expression of GLUR1 or PSD95.

[0216] In some embodiments, the method of activating mTORC1 is used to treat or prevent autism severity. (See Novarino et al., (2012) Science, October 19, 338:6105, pp. 394-397). Therefore, in some embodiments, the present invention provides a method for treating or preventing a degree of autism in an individual in need, comprising the step of administering to said individual a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the autism is genetic autism.

[0217] In some embodiments, the present invention provides a method for treating a patient with genetic autism in need, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient. SHANK3 haploinadequacy is a pathogenic factor for the neurological characteristics of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (Bidinosti et al. (2016) Science Reports 351, 1199-1203). Downregulation of mTORC1 in SHANK3-deficient neurons is attributed to enhanced phosphorylation via its kinase, Cdc2-like kinase 2, and activation of the serine / threonine protein phosphatase 2A (PP2A) regulatory subunit B56b (Bidinosti et al. (2016) Science Reports 351, 1199-1203). SHANK3 mutant mice exhibit autistic traits (Yang et al. (2012) The Journal of Neuroscience 32, 6525-6541). Patients with autistic traits and motor delay carry a detrimental allozonant mutation in the SLC7A5 gene. Solute carrier transporter 7a5 (SLC7A5) (a large neutral amino acid transporter located at the blood-brain barrier (BBB)) plays an essential role in maintaining the standard levels of BCAAs in the brain. Intraventricular administration of leucine improves aberrant behavior in adult mutant mice (Tarlungeanu et al. (2016) Cell 167, 1481-1494).

[0218] In some embodiments, the present invention provides a method for treating lysosomal storage diseases or conditions (“LSD”) in patients in need, comprising the step of administering a provided compound or a pharmaceutically acceptable composition thereof to the patient. LSD is a group of inherited metabolic disorders resulting from lysosomal dysfunction. Lysosomal storage diseases are caused by lysosomal dysfunction, typically due to a deficiency of a single enzyme required for lipid, glycoprotein (glycoprotein-containing), or so-called mucopolysaccharide metabolism. In some embodiments, the present invention provides a method for treating lipid storage diseases in patients in need, comprising the step of administering a provided compound or a pharmaceutically acceptable composition thereof to the patient. In some embodiments, lipid storage diseases are selected from sphingolipid metabolism disorders (e.g., ganglioside storage diseases, Gaucher disease, Niemann-Pick disease, or metachromatic leukodystrophy). In some embodiments, the present invention provides a method for treating ganglioside storage diseases (e.g., Tay-Sachs disease or white matter disorders). In some embodiments, the present invention provides a method for treating mucopolysaccharidosis in patients in need, comprising the step of administering a provided compound or a pharmaceutically acceptable composition thereof to the patient. In some embodiments, mucopolysaccharidosis is Hunter syndrome or Hurler disease.

[0219] In some embodiments, the present invention provides a method for treating JNCL (Batten Disease) in a patient in need, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient. JNCL is caused by the deletion of exons 7 and 8 of the CLN3 gene, which produces a nonfunctional protein. Battenin (a full-length protein encoded by CLN3) is a transmembrane protein located in late endosomes and lysosomes, which has been shown to help regulate pH, amino acid homeostasis, and vesicle transport (Pearce et al. (1999) Nature Genetics 22,1; Fossale et al. (2004) BMC Neuroscience 10,5). mTOR activation requires intracellular nutrients provided by autophagy, which is reduced in in vitro and in vivo JNCL models due to the lack of functional battenin (Cao et al. (2006) Journal of Biological Chemistry 281,29).

[0220] In some embodiments, the present invention provides a method for treating cystinosis in a patient in need, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient. Cystinosis is an autosomal recessive disease affecting individuals with two allelic mutations in the CSTN gene; a deficiency of the lysosomal cystine transporter cystinosin in the lysosomal cystine effluent leads to cystine crystal formation in the renal epithelial tubules and loss of renal function. Studies have shown a lack or reduction of mTORC1 signaling in cells lacking CSTN and mislocalized mTOR (Ivanova et al. (2016) J Inherit Metab Dis. 39(3), 457-64; Andrzejewska et al. (2016) J Am Soc Nephrol. 27(6), 1678-1688e). These defects may not be repaired by cysteine ​​(Ivanova et al. (2016) J Inherit Metab Dis. 39(3), 457-64; Andrzejewska et al. (2016) J Am Soc Nephrol. 27(6), 1678-1688e). Cystinosin has also been found to bind to mTORC1 pathway components v-ATPase, Rags, and regulators (Andrzejewska et al. (2016) J Am Soc Nephrol. 27(6), 1678-1688e). CTNS-deficient cells exhibit increased autophagosome numbers and associated protein-mediated reduced autophagy (Napolitano et al. (2015) EMBOMol Med. 7(2), 158-74).

[0221] In some embodiments, the present invention provides a method for treating Fabry disease in patients of need, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient. In Fabry disease, α-galactose deficiency leads to the lysosomal accumulation of acylsphingolipid trihexosyllipoprotein. In Fabry disease cell models, reduced mTOR activity and increased autophagy have been observed in vitro and in vivo, wherein α-galactose is blocked by shRNA (Liebau et al. (2013) PLoS 8, e63506). Hyperactive autophagy has also been observed in mouse brains, wherein α-galactose is gene knocked out (Nelson et al. (2014) Acta Neuropathologica Communications 2, 20).

[0222] In some embodiments, the present invention provides a method for treating type IV mucolipid storage disease (MLIV) in patients of need, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient. In MLIV, mutations in the TRPML1 lysosomal Ca(2+) channel result in disordered lysosomal membrane transport. Knockout of the MLIV gene in Drosophila leads to upregulation of autophagy and decreased mTOR activity, both of which can be reversed by genetically activating mTORC1 or by feeding the animals a high-protein diet (Wong et al. (2012) Curr Biol. 22(17), 1616-1621). Increased autophagy has also been observed in fibroblasts from MLIV patients (Vergarajauregui et al. (2008) Human Molecular Genetics 17, 2723-2737).

[0223] In some embodiments, the present invention provides a method for treating intellectual disability in a patient in need, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the patient. In Homo sapiens, the cereblon mutation is linked to mild autosomal recessive non-syndrome intellectual disability. In a mouse model of intellectual disability with cereblon gene knockout, cereblon deletion activates AMPK, inhibits mTOR, and reduces protein translation in the cerebellum (Lee et al. (2014) J Biol Chem. 289, 23343-52; Xu et al. (2013) J Biol Chem. 288, 29573-85).

[0224] In some embodiments, the present invention provides a method for increasing neuronal protein expression in an individual, comprising the step of administering the provided compound or a pharmaceutically acceptable composition thereof to the individual. In some embodiments, the increase in neuronal protein expression occurs in postsynaptic neurons. In some embodiments, the increase in neuronal protein expression includes increasing the expression of brain-derived neurotrophic factor (BDNF). In some embodiments, the increase in neuronal protein expression includes increasing the expression of glutamate receptor 1 (GluR1). In some embodiments, the increase in neuronal protein expression includes increasing the expression of synaptic proteins. In some embodiments, the increase in neuronal protein expression includes increasing the expression of PSD95.

[0225] In some embodiments, the present invention provides a method for enhancing synaptic development in an individual, comprising the step of administering to the individual a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, enhanced synaptic development involves synaptic remodeling. In some embodiments, enhanced synaptic development involves inducing dendritic spines. In some embodiments, inducing dendritic spines results in an increase in dendritic spine density. In some embodiments, the dendritic spines are elongated spines. In some embodiments, the dendritic spines are mushroom-shaped spines.

[0226] In some embodiments, the present invention provides a method for enhancing synaptic function in an individual, comprising the step of administering to the individual a provided compound or a pharmaceutically acceptable composition thereof. In some embodiments, the enhanced synaptic function in the individual involves an increase in excitatory postsynaptic currents (EPSCs).

[0227] The pharmaceutically acceptable compositions of the present invention may be administered to humans and other animals, depending on the severity of the infection being treated, via oral, rectal, non-intestinal, intracisional, vaginal, intraperitoneal, topical (e.g., by powder, ointment, or drops), buccal, as an oral or nasal spray, or similar methods. In some embodiments, the compounds of the present invention may be administered orally or non-intestinally once or more times daily at dose levels of about 0.01 mg / kg to about 50 mg / kg, and preferably about 1 mg / kg to about 25 mg / kg of individual body weight, to achieve the desired therapeutic effect.

[0228] Liquid dosage forms intended for oral administration include (but are not limited to) pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the field, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol; and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0229] Injectable formulations can be formulated using suitable dispersants, wetting agents, and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, non-enteric-acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Among acceptable mediators and solvents, water, Ringer's solution, USP, and isotonic sodium chloride solution can be used. Additionally, sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild, non-volatile oil containing synthetic monoglycerides or diglycerides can be used. Furthermore, fatty acids, such as oleic acid, are used in the preparation of injectable formulations.

[0230] Injectable formulations can be sterilized, for example, by filtration via a bacterial retention filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0231] To prolong the effects of the compounds of this invention, it is generally necessary to slow the absorption of compounds administered subcutaneously or intramuscularly. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of the compound depends on its solubility, which in turn may depend on crystal size and crystal form. Alternatively, delayed absorption of compounds administered non-enterovenously can be achieved by dissolving or suspending the compound in an oil-based medium. Injectable storage forms are manufactured by forming microcapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolic acid. The release rate of the compound can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir-type injectable formulations are also prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues.

[0232] Compositions for rectal or vaginal administration are preferably suppositories prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers (e.g., cocoa butter, polyethylene glycol); or suppository waxes that are solid at ambient temperature but liquid at body temperature and thus melt in the rectal or vaginal cavity to release the active compound.

[0233] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound may be mixed with: at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate; and / or a) fillers or expanders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solvent inhibitors, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) humectants, such as cetyl alcohol and glyceryl monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

[0234] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or glutenose and high molecular weight polyethylene glycol and its analogues. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in pharmaceutical formulation techniques. They may optionally contain emulsifiers and may also have compositions that release or optionally delay the release of the active ingredient only or preferentially in a portion of the intestine. Examples of usable encapsulation compositions include polymeric substances and waxes. Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose and high molecular weight polyethylene glycol and its analogues.

[0235] The active compound may also be present in microencapsulation with one or more excipients as noted above. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules may be prepared with coatings and shells, such as enteric coatings, release-controlled coatings, and other coatings well known in pharmaceutical compounding techniques. In such solid dosage forms, the active compound may be mixed with at least one inert diluent (e.g., sucrose, lactose, or starch). As is common practice, such dosage forms may also include substances other than inert diluents, such as tablet-making lubricants and other tablet-making aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage form may also include a buffer. It may optionally contain an emulsifier and may also have a composition that releases or optionally releases the active ingredient only or preferentially in a portion of the intestine. Examples of encapsulation compositions that may be used include polymers and waxes.

[0236] Dosage forms for topical or transdermal administration of the compounds of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient is mixed with a pharmaceutically acceptable carrier and any desired preservatives or buffers under sterile conditions, as needed. Ocular formulations, ear drops, and eye drops are also covered within the scope of this invention. Additionally, this invention covers the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared 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 by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0237] According to one embodiment, the present invention relates to a method for modulating Sestrin-GATOR2 interaction, thereby selectively and indirectly modulating mTORC1 activity in a biological sample, comprising the step of contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0238] As used herein, the term “biological sample” includes (but is not limited to) cell cultures or extracts thereof; biopsy material or extracts thereof obtained from mammals; and blood, saliva, urine, feces, semen, tears or other bodily fluids or extracts thereof.

[0239] Another embodiment of the invention relates to a method for modulating Sestrin-GATOR2 interaction, thereby selectively and indirectly modulating mTORC1 activity in a patient, comprising the step of administering the compound of the invention or a composition comprising the compound to the patient.

[0240] According to another embodiment, the present invention relates to a method for modulating Sestrin-GATOR2 interaction, thereby indirectly and selectively modulating the mTORC1 activity of a patient, comprising the step of administering to the patient a compound of the present invention or a composition comprising the compound. In other embodiments, the present invention provides a method for treating mTORC1-mediated conditions in patients in need, comprising the step of administering to the patient a compound of the present invention or a pharmaceutically acceptable composition thereof. Such conditions are described in detail herein.

[0241] Depending on the specific condition or disease to be treated, additional therapeutic agents typically administered for treating said condition may also be present in the compositions of the present invention. As used herein, additional therapeutic agents typically administered for treating a specific disease or condition are referred to as “the disease or condition to be treated”.

[0242] In some embodiments, the provided compound is administered in combination with an antidepressant. Antidepressants are well known to those skilled in the art and include selective serotonin reuptake inhibitors (“SSRIs”, such as sertraline, escitalopram, citalopram, fluvoxamine, fluoxetine, paroxetine) and antidepressants (such as bupropion, venlafaxine, mirtazapine, duloxetine, amitriptyline, imipramine, selegiline, nortriptyline, trazodone, desvenlafaxine, and aripiprazole).

[0243] In some embodiments, the provided compound is administered in combination with additional therapeutic agents or methods suitable for treating one or more LSDs. In some embodiments, the provided compound is administered in combination with enzyme replacement therapy, chemopreservative protein therapy, bone marrow transplantation, substrate reduction therapy, α-L-iduroside, recombinant human N-acetylgalactosamine glycoside-4-sulfatase (arylsulfatase B), sphingolipid biosynthesis inhibitors, N-butyldeoxynojirimycin (megrub), and inhibitors of hydrophobic imine sugars or α-galactose A (e.g., 1-deoxy-galactonojirimycin).

[0244] These additional agents may be administered separately from the composition containing the compound of the present invention as part of a multiple-dose regimen. Alternatively, these agents may be part of a single dosage form, mixed with the compound of the present invention in a single composition. If administered as part of a multiple-dose regimen, the two active agents may be provided simultaneously, sequentially, or at intervals (typically within 5 hours of each other).

[0245] As used herein, the terms "combination" and related terms refer to the simultaneous or sequential administration of a therapeutic agent according to the invention. For example, the compound of the invention may be administered simultaneously or sequentially with another therapeutic agent in individual unit dosage forms or together in a single unit dosage form. Therefore, the present invention provides a single unit dosage form comprising the compound of the invention, other therapeutic agents, and pharmaceutically acceptable carriers, adjuvants, or mediators.

[0246] The amounts of the compounds of the present invention that can be combined with a carrier to produce a single dosage form, and additional therapeutic agents (in those compositions including additional therapeutic agents as described above), will vary depending on the host being treated and the specific administration method. Preferably, the compositions of the present invention should be formulated such that the compounds of the present invention can be administered at doses between 0.01 mg / kg body weight / day and 100 mg / kg body weight / day.

[0247] In compositions that include an additional therapeutic agent, the additional therapeutic agent and the compound of the present invention can act synergistically. Therefore, the amount of the additional therapeutic agent in such compositions will be less than that required in a single therapy using only the therapeutic agent. In such compositions, the additional therapeutic agent can be administered at doses ranging from 0.01 micrograms / kg body weight / day to 1,000 micrograms / kg body weight / day.

[0248] The amount of additional therapeutic agent present in the compositions of the present invention will not exceed the amount typically administered in the form of a composition comprising the therapeutic agent as the sole active agent. Preferably, the amount of additional therapeutic agent in the disclosed compositions will be in the range of about 50% to 100% of the amount typically present in a composition comprising the pharmaceutical agent as the sole active agent.

[0249] 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 cannulas. Vascular stents have been used, for example, to overcome restenosis (re-narrowing of the vessel wall after injury). However, patients using vascular stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition including a kinase inhibitor. Implantable devices coated with the compounds of the present invention are another embodiment of the present invention.

[0250] example

[0251] As depicted in the following examples, in some exemplary embodiments, the compounds are prepared according to the following general procedure. It should be understood that while the general method describes the synthesis of certain compounds of the present invention, the following general method and other methods known to those skilled in the art can be applied to all compounds as described herein and to subclasses and types of each of these compounds.

[0252] List of abbreviations used in the experimental section.

[0253] 4A MS: Molecular sieve

[0254] AcOH: Acetic acid

[0255] ACN: Acetonitrile

[0256] Anhyd: Waterless

[0257] Aq: water based

[0258] Bn: benzyl

[0259] Boc: tert-butoxycarbonyl

[0260] CbzCl: Benzoic acid chloroformate

[0261] Cbz-OSU: N-(Benzyloxycarbonyloxy)succinimide

[0262] Cu(OAc)₂: Copper(II) acetate

[0263] d: sky

[0264] DAST: Diethylaminosulfuric acid trifluoride

[0265] DBU: 1,8-diazobicyclo[5.4.0]undec-7-ene

[0266] DCE: 1,2-Dichloroethane

[0267] DCM: Dichloromethane

[0268] DEA: Diethylamine

[0269] DIBAL-H: Diisobutylaluminum hydride

[0270] DIPEA: N,N-Diisopropylethylamine

[0271] DMA: N,N-dimethylacetamide

[0272] DMAP: 4-Dimethylaminopyridine

[0273] DMF: N,N-Dimethylformamide

[0274] DMSO: Dimethyl sulfoxide

[0275] DPPA: Diphenylphosphoazide

[0276] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0277] ee: enantiomeric excess

[0278] ESI: Electrospray Ionization

[0279] Et3N: Triethylamine

[0280] Et2O: Diethyl ether

[0281] EtOAc: Ethyl acetate

[0282] EtOH: Ethanol

[0283] Fmoc: fluorenylmethoxycarbonyl

[0284] Fmoc-OSu: N-(9-fluorenylmethoxycarbonyl)succinimide

[0285] h: hours

[0286] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0287] HCOONH4: Ammonium formate

[0288] HPLC: High Performance Liquid Chromatography

[0289] IBX: 2-Iodooxybenzoic acid

[0290] IPA: Isopropyl alcohol

[0291] KOAc: Potassium acetate

[0292] M: Moore

[0293] Me: Methyl

[0294] MeOH: Methanol

[0295] mins: minutes

[0296] mL: milliliters

[0297] mM: millimoles

[0298] mmol: millimole

[0299] MTBE: Methyl tert-butyl ether

[0300] NaBH3CN: Sodium cyanoborohydride

[0301] Na2CO3: Sodium carbonate

[0302] NaHCO3: Sodium bicarbonate

[0303] NMP: N-methylpyrrolidine

[0304] NMR: Nuclear Magnetic Resonance

[0305] ℃: degrees Celsius

[0306] PBS: Phosphate-buffered saline

[0307] Pd / C: Palladium / Carbon

[0308] Pd(OH)₂ / C: Pearlman's catalyst; PE: Petroleum ether

[0309] PhNH2: Aniline

[0310] PPh3: Triphenylphosphine

[0311] Rel: relative

[0312] rt: room temperature

[0313] sat: saturation

[0314] SFC: Supercritical Fluid Chromatography

[0315] SOCl2: thionyl chloride

[0316] TBAB: Tetra-n-Butylammonium Bromide

[0317] tBuOK: Potassium tert-butoxide

[0318] TEA: Triethylamine

[0319] Tf: Trifluoromethanesulfonate

[0320] TfAA: Trifluoromethanesulfonic anhydride

[0321] TFA: Trifluoroacetic acid

[0322] TIPS: Triisopropylsilane

[0323] THF: Tetrahydrofuran

[0324] TMSCN: Trimethylsilane Cyanide

[0325] pTSA: p-Toluenesulfonic acid

[0326] TsOH: p-Toluenesulfonic acid

[0327] The preparation of representative, non-limiting examples of the provided compounds is described below.

[0328] Example 1: (S)-2-(dimethylamino)-4-methylpentanoic acid [I-1].

[0329]

[0330] Synthesis process:

[0331]

[0332] Program and features:

[0333] Step 1: (S)-2-(dimethylamino)-4-methylpentanoic acid:

[0334] Formaldehyde (38%, 24.0 g) and Pd / C (10%, 500 mg) were added to a solution of (S)-2-amino-4-methylpentanoic acid (2.0 g, 15.24 mmol), and the resulting solution (60 mL) was filtered. The mixture was hydrogenated at room temperature for two days and filtered to remove the catalyst. The filtrate was concentrated to dryness, and EtOH (30 mL) was added to the residue. The mixture was stirred for 1 hour and filtered. The filtrate was concentrated to give (S)-2-(dimethylamino)-4-methylpentanoic acid (1.3 g, 8.16 mmol, 53%) as a white powder. ESI-MS (EI) + ,m / z):160.2[M+H] + . 1 H-NMR (400MHz, MeOD-d4): δ3.47 (dd, J = 4.4Hz, 10.0Hz, 1H), 2.85 (S, 6H), 1.89-1.74 (m, 2H), 1.62-1.55 (m, 1H), 1.00 (dd, J = 2.8Hz, 6.8Hz, 6H).

[0335] Examples 2 and 3: (S)-2-amino-7,7,7-trifluoroheptanoate [I-2] and (R)-2-amino-7,7,7-trifluoroheptanoate [I-3].

[0336]

[0337] Synthesis process:

[0338]

[0339] Program and features:

[0340] Step 1: 1,1,1-Trifluoro-5-iodopentane:

[0341] Under ice bath conditions, I₂ (4.45 g, 17.5 mmol) was added to a solution of 5,5,5-trifluoropentane-1-ol (2.0 g, 14.0 mmol), imidazole (1.48 g, 21.7 mmol), and PPh₃ (5.5 g, 21.0 mmol) in DCM (40 mL). The mixture was warmed to room temperature and stirred overnight. Et₂O (50 mL) was added to the mixture, followed by stirring for 10 minutes. The mixture was filtered, and the filtrate was evaporated at 65 °C to remove the solvent at atmospheric pressure. The residue was diluted with Et₂O (30 mL), the mixture was filtered, and the filtrate was used for the next step.

[0342] Step 2: (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester and (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester:

[0343] At -10°C, KOH (50%, 20 mL) was added to a solution of 2-(diphenylmethyleneamino)acetic acid tert-butyl ester (2.0 g, 6.78 mmol) and TBAB (109 mg, 0.339 mmol) in toluene (35 mL) and DCM (15 mL). After 5 minutes, 1,1,1-trifluoro-5-iodopentane was added dropwise to the above solution in Et₂O (30 mL) over 5 minutes. The mixture was stirred at -10°C to 0°C for 1 hour. The solution was diluted with water (200 mL) and extracted with EA (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 10) and subsequently by chiral preparative HPLC [column, R,R-spiro-ol 4.6 × 250 mm 5 μm; solvent, MeOH (0.2% methanol ammonia)] to give (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%) and (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%).

[0344] (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%). ESI-MS (EI+, m / z): 243.1 [M+H]+. 1 H-NMR (500MHz, CDCl3): δ8.64 (d, J = 8.0Hz, 2H), 7.43-7.46 (m, 3H), 7.38-7.39 (m, 1H), 7.31-7.34 (m, 2H), 7 .15-7.17(m,2H),3.91(dd,J=5.5Hz,7.5Hz,1H),2.00-2.05(m,2H),1.88-1.92(m,2H),1.31-1.52(m,13H).

[0345] (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol, 7.1%). ESI-MS (EI+, m / z): 243.1 [M+H]+. 1H-NMR (500MHz, CDCl3): δ8.64 (d, J = 7.0Hz, 2H), 7.43-7.46 (m, 3H), 7.38-7.39 (m, 1H), 7.31-7.34 (m, 2H), 7 .15-7.17(m,2H),3.92(dd,J=5.5Hz,7.5Hz,1H),2.00-2.05(m,2H),1.88-1.92(m,2H),1.31-1.52(m,13H).

[0346] Step 3: (S)-2-amino-7,7,7-trifluoroheptanoate salt [I-2]:

[0347] A solution of (S)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol) in 6M HCl (10 mL) and dioxane (5 mL) was heated to 100 °C for 17 hours. The solution was extracted with Et₂O (10 mL × 2), and the aqueous phase was concentrated to dryness to give (S)-2-amino-7,7,7-trifluoroheptanoate salt (I-2) (82.7 mg, 0.35 mmol, 74%) as a white solid. ESI-MS (EI+, m / z): 200.1 [M+H]+. ¹H NMR (500 MHz, D₂O) δ 3.93 (t, J = 6.0 Hz, 1H), 2.10–2.15 (m, 2H), 1.83–1.90 (m, 2H), 1.40–1.56 (m, 4H).

[0348] Step 4: (R)-2-amino-7,7,7-trifluoroheptanoate salt [I-3]:

[0349] A solution of (R)-2-(diphenylmethyleneamino)-7,7,7-trifluoroheptanoate tert-butyl ester (200 mg, 0.48 mmol) in 6 M HCl (10 mL) and dioxane (5 mL) was heated to 100 °C for 17 hours. The solution was extracted with Et₂O (10 mL × 2), and the aqueous phase was concentrated to dryness to give (R)-2-amino-7,7,7-trifluoroheptanoate salt (I-3) (91.6 mg, 0.39 mmol, 82%) as a white solid. ESI-MS (EI+, m / z): 200.1 [M+H]+. ¹H NMR (500 MHz, D₂O) δ 3.92 (t, J = 6.0 Hz, 1H), 2.09–2.14 (m, 2H), 1.82–1.89 (m, 2H), 1.39–1.55 (m, 4H).

[0350] Examples 4 and 5: (S)-2-amino-4,4,4-trifluorobutyric acid [I-4] and (R)-2-amino-4,4,4-trifluorobutyric acid [I-5].

[0351]

[0352] Synthesis process:

[0353]

[0354] Program and features:

[0355] Step 1: (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid

[0356] N-(benzyloxycarbonyloxy)succinimide (1.75 g, 7.01 mmol) was slowly added to a solution of 2-amino-4,4,4-trifluorobutyric acid (1.0 g, 6.36 mmol) and NaHCO3 (589 mg, 7.01 mmol) in acetone (60 mL), and the resulting solution (60 mL) was filtered at 0 °C. The mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with CH2Cl2 (2 × 100 mL), and the aqueous layer was acidified to approximately pH 4 with HCl (3 M), followed by extraction with EtOAc (3 × 150 mL). The organic phase was dried over Na2SO4, and the solvent was evaporated under vacuum. The crude product was purified by chiral preparative HPLC (column, AY-H, 4.6 × 250 mm 5 μm; solvent, EtOH) to give (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol, 37.8%) and (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol, 37.8%) as white solids. ESI-MS (EI+, m / z): 314.0 [M+Na]+.

[0357] (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid. 1 H-NMR (500MHz, DMSO-d6): δ13.20 (s, 1H), 7.84 (d, J = 9.0Hz, 1H), 7.40-7.30 (m, 5H), 5.06 (s, 2H), 4.31-4.27 (m, 1H), 2.85-2.58 (m, 2H).

[0358] (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid. 1H-NMR (500MHz, DMSO-d6): δ13.21 (s, 1H), 7.85 (d, J = 8.5Hz, 1H), 7.38-7.30 (m, 5H), 5.06 (s, 2H), 4.31-4.27 (m, 1H), 2.83-2.59 (m, 2H).

[0359] Step 2: (S)-2-amino-4,4,4-trifluorobutyric acid [I-4].

[0360] A mixture of (S)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol) and Pd / C (10%) (200 mg) in MeOH (50 mL) was stirred for 2 hours at room temperature under a hydrogen atmosphere. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (S)-2-amino-4,4,4-trifluorobutyric acid (I-4) as a white solid (250 mg, 1.59 mmol, 66.3%). ESI-MS (EI+, m / z): 158.1 [M+H]+. 1H-NMR (500 MHz, DMSO-d6+1drop TFA+1drop D2O): δ 4.32 (t, J = 6.0 Hz, 1H), 3.03–2.82 (m, 2H).

[0361] Step 3: (R)-2-amino-4,4,4-trifluorobutyric acid [I-5].

[0362] A mixture of (R)-2-(benzyloxycarbonylamino)-4,4,4-trifluorobutyric acid (700 mg, 2.40 mmol) and Pd / C (10%) (200 mg) in MeOH (50 mL) was stirred for 2 hours at room temperature under a hydrogen atmosphere. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (R)-2-amino-4,4,4-trifluorobutyric acid (I-5) as a white solid (250 mg, 1.59 mmol, 66.3%). ESI-MS (EI) + ,m / z):158.1[M+H] + . 1 ¹H-NMR (500MHz, DMSO-d⁶ + 1 drop TFA + 1 drop D₂O): δ 4.31 (t, J = 6.0 Hz, 1H), 3.03-2.83 (m, 2H).

[0363] Examples 6 and 7: (S)-2-amino-5,5,5-trifluorovaleric acid [I-6] and (R)-2-amino-5,5,5-trifluorovaleric acid [I-7].

[0364]

[0365] Synthesis process:

[0366]

[0367] Program and features:

[0368] Step 1: 4,4,4-Trifluorobutyraldehyde:

[0369] Under ice bath conditions, IBX (13.0 g, 46.9 mmol) was added to a solution of 4,4,4-trifluorobut-1-ol (4.0 g, 31.3 mmol) in DMSO (80 mL). The mixture was heated to room temperature and stirred overnight. The reaction mixture was poured into water (200 mL) and extracted with Et₂O (100 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was used for the next step.

[0370] Step 2: 2-(phenylmethylamino)-5,5,5-trifluoropentadienonitrile:

[0371] Under ice bath conditions, benzylamine (4 mL), AcOH (3.0 mL), and subsequently TMSCN (3.5 mL) were added to the above solution of 4,4,4-trifluorobutyraldehyde in Et₂O (200 mL). The mixture was heated to room temperature and stirred overnight. The solution was diluted with water (200 mL) and extracted with EtOAc (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(phenylmethylamino)-5,5,5-trifluoropentadienonitrile (6.7 g, crude material) as a brown solid, which was used in the next step. ESI-MS (EI+, m / z): 243.1 [M+H]+.

[0372] Step 3: 2-(phenylmethylamino)-5,5,5-trifluorovaleric acid:

[0373] A solution of 2-(phenylmethylamino)-5,5,5-trifluoropentanoic acid (6.7 g, crude) in concentrated HCl (80 mL) and AcOH (30 mL) was heated to 95 °C for 17 hours. The solution was concentrated to dryness, diluted, filtered (100 mL), and mixed with ACN (50 mL). The pH was adjusted to 3-4 with saturated NaHCO3 solution. The mixture was filtered and dried to give 2-(phenylmethylamino)-5,5,5-trifluoropentanoic acid (3.5 g, 13.4 mmol, 43%, 3 steps) as a white solid. ESI-MS (EI) + ,m / z):262.1[M+H] + .

[0374] Step 4: 2-Amino-5,5,5-trifluorovaleric acid:

[0375] A mixture of 2-(phenylmethylamino)-5,5,5-trifluorovaleric acid (3.3 g, 12.6 mmol) and Pd(OH)₂ / C (20%, 400 mg) in AcOH (60 mL) was stirred at 30 °C for 17 hours. The mixture was filtered, and the filtrate was concentrated to dryness to give 2-amino-5,5,5-trifluorovaleric acid (3.0 g, crude substance) as a brown solid. ESI-MS (EI+, m / z): 172.2 [M+H]⁺.

[0376] Step 5: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid:

[0377] Under ice bath conditions, Cbz-OSu (3.45 g, 13.9 mmol) was added to a solution of 2-amino-5,5,5-trifluorovaleric acid (3.0 g, crude) in saturated NaHCO3 solution (100 mL) and acetone (100 mL). After 2 hours, the mixture was adjusted to pH 3 with 6 M HCl, extracted with EtOAc (50 mL × 2), and the organic phase was washed with water (50 mL) and brine (100 mL), dried (Na2SO4), and concentrated under vacuum. The solution was then subjected to chromatography (silica, ethyl acetate / petroleum ether = 1 / 2) followed by chiral-preparative HPLC [column, AY-H 4.6 × 250 mm 5 μm; solvent, MeOH (0.5%)]. The crude product was purified by NH4OH to give (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps) as white solids.

[0378] (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps). ESI-MS (EI+, m / z): 328.0 [M+Na]+. 1H-NMR (500 MHz, DMSO-d6): δ 12.86 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.31–7.39 (m, 5H), 5.05 (s, 2H), 4.05–4.10 (m, 1H), 2.34–2.41 (m, 1H), 2.21–2.29 (m, 1H), 1.84–1.97 (m, 2H).

[0379] (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (1.50 g, 4.92 mmol, 28%, 2 steps) ESI-MS (EI+, m / z): 328.0 [M+Na]+. 1H-NMR (500 MHz, DMSO-d6): δ 12.85 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.30–7.39 (m, 5H), 5.05 (s, 2H), 4.05–4.10 (m, 1H), 2.34–2.41 (m, 1H), 2.21–2.29 (m, 1H), 1.84–1.97 (m, 2H).

[0380] Step 6: (S)-2-amino-5,5,5-trifluorovaleric acid [I-6]:

[0381] A mixture of (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (500 mg, 1.64 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred at room temperature under hydrogen atmosphere for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (S)-2-amino-5,5,5-trifluorovaleric acid (I-6) (200 mg, 1.17 mmol, 71%) as a white solid. ESI-MS (EI) + ,m / z):172.1[M+H] + . 1 H-NMR (400MHz, DMSO-d6): δ8.38 (s, 3H), 4.05 (d, J = 4.4Hz, 1H), 2.34-2.55 (m, 2H), 1.95-20.9 (m, 2H).

[0382] Step 7: (R)-2-amino-5,5,5-trifluorovaleric acid [I-7]:

[0383] A mixture of (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluorovaleric acid (500 mg, 1.64 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred at room temperature under hydrogen atmosphere for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (R)-2-amino-5,5,5-trifluorovaleric acid (I-7) as a white solid (160 mg, 0.94 mmol, 57%). 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).

[0384] 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].

[0385]

[0386] Synthesis process:

[0387]

[0388] Program and features:

[0389] Step 1: (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid and (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid

[0390] At 0 °C, benzoyl chloroformate (554 mg, 3.25 mmol) was slowly added to a solution of 2-amino-6,6,6-trifluorohexanoic acid (556 mg, 2.5 mmol) and 1 M NaOH (25 mL, 25 mmol) in THF (25 mL), and the mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with DCM (2 × 100 mL), and the aqueous layer was acidified to approximately pH 4 with HCl (3 M), followed by extraction with EtOAc (3 × 50 mL). The organic phase was dried over Na₂SO₄ and the solvent was evaporated under vacuum. The crude product was purified by chiral preparative HPLC (column: AY-H (250 × 4.6 mm 5 μm); mobile phase: n-hexane (0.1% DEA): EtOH (0.1% DEA) = 90:10) to give (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]+.

[0391] (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid, 1H-NMR (500MHz, DMSO-d6): δ 12.68 (s, 1H), 7.66 (d, J = 7.5Hz, 1H), 7.38–7.32 (m, 5H), 5.04 (s, 2H), 4.00–3.96 (m, 1H), 2.28–2.19 (m, 2H), 1.80–1.51 (m, 4H).

[0392] (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid, 1H-NMR (500MHz, DMSO-d6): δ 12.68 (s, 1H), 7.67 (d, J = 8.5Hz, 1H), 7.38–7.30 (m, 5H), 5.04 (s, 2H), 4.00–3.96 (m, 1H), 2.33–2.15 (m, 2H), 1.82–1.51 (m, 4H).

[0393] Step 2: (S)-2-amino-6,6,6-trifluorohexanoic acid [I-8].

[0394] A mixture of (S)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (200 mg, 0.63 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred for 2 hours at room temperature under a hydrogen atmosphere. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (S)-2-amino-6,6,6-trifluorohexanoic acid (I-8) as a white solid (56.2 mg, 0.30 mmol, 48.2%). ESI-MS (EI... + ,m / z):186.1[M+H] + . 1 ¹H-NMR (500MHz, DMSO-d⁶ + 1 drop TFA + 1 drop D₂O): δ 3.99 (t, J = 5.5 Hz, 1H), 2.32–2.30 (m, 2H), 1.91–1.83 (m, 2H), 1.70–1.57 (m, 2H).

[0395] Step 3: (R)-2-amino-6,6,6-trifluorohexanoic acid [I-9].

[0396] A mixture of (R)-2-(benzyloxycarbonylamino)-6,6,6-trifluorohexanoic acid (250 mg, 0.78 mmol) and Pd / C (10%) (50 mg) in MeOH (20 mL) was stirred for 2 hours at room temperature under a hydrogen atmosphere. The mixture was filtered, and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated to give (R)-2-amino-6,6,6-trifluorohexanoic acid (I-9) as a white solid (48.8 mg, 0.26 mmol, 33.8%). ESI-MS (EI) + ,m / z):186.1[M+H] + . 1H-NMR (500MHz, DMSO-d6+1drop TFA+1drop D2O): δ3.98(t,J=6.5Hz,1H), 3.33-2.28(m,2H), 1.93-1.81(m,2H), 1.71-1.54(m,2H).

[0397] Example 11: (S)-2-(phenylmethylamino)-4-methylpentanoic acid [I-11].

[0398]

[0399] Synthesis process:

[0400]

[0401] Program and features:

[0402] Step 1: (S)-2-(phenylmethylamino)-4-methylpentanoic acid benzoate:

[0403] Benzaldehyde (0.26 g, 2.4 mmol) and potassium acetate (0.4 g, 4.1 mmol) were added to a stirred solution of L-leucine benzyl ester p-toluenesulfonate (800 mg, 2.0 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 min. Then, sodium cyanoborohydride (0.2 g, 3.0 mmol) was added, and the mixture was stirred again at room temperature for 5 h. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL × 2), washed with the resulting solution, filtered (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(phenylmethylamino)-4-methylpentanoic acid benzyl ester (200 mg, 0.64 mmol, 32%) as a colorless oil. MS(EI+,m / z):312.3[M+H]+. 1H-NMR (500MHz,,MeOD): δ7.41~7.49(m,10H),5.34(dd,J=12.0Hz,45.0Hz,2H),4.23(q ,J=12.0Hz,2H),4.07~4.09(m,3H),1.68~1.85(m,3H),0.94(dd,J=8.5Hz,20.5Hz,6H).

[0404] Step 2: (S)-2-(phenylmethylamino)-4-methylpentanoic acid [I-11]:

[0405] Add 1M NaOH (0.5 mL) to a stirred solution of (S)-2-(phenylmethylamino)-4-methylpentanoic acid methyl ester (50 mg, 0.16 mmol) in MeOH (5 mL). Stir the reaction mixture at room temperature for 4 hours. Concentrate the resulting solution and purify the residue by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(phenylmethylamino)-4-methylpentanoic acid (I-11) as a white solid (21 mg, 0.095 mmol, 58%). MS (EI+, m / z): 222.2 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ9.32 (s, 1H), 7.43~7.50 (m, 5H), 4.17 (dd, J=13.0 Hz, 44.0Hz, 2H), 3.82 (t, J = 6.5Hz, 1H), 1.68 ~ 1.76 (m, 3H), 0.85 ~ 0.90 (m, 6H).

[0406] Example 12: (S)-4-methyl-2-(2-phenylacetamido)valerate [I-12]:

[0407]

[0408] Synthesis process:

[0409]

[0410] Program and features:

[0411] Step 1: (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid methyl ester:

[0412] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), 2-phenylacetic acid (260 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid benzyl ester (300 mg, 0.88 mmol, 70%) as a white solid. MS (EI+, m / z): 340.2 [M+H]+.

[0413] Step 2: (S)-4-methyl-2-(2-phenylacetamido)valerate [I-12]:

[0414] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid methyl ester (250 mg, 0.74 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-4-methyl-2-(2-phenylacetamido)pentanoic acid (I-12) as a white solid (100 mg, 0.40 mmol, 54%). MS (EI+, m / z): 250.2 [M+H]+. 1H-NMR (500MHz, MeOD): δ7.24-7.32 (m, 5H), 4.44 (t, J = 7.5 Hz, 1H), 3.58 (s, 2H), 1.64-1.68 (m, 3H), 0.96 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H).

[0415] Example 13: (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]:

[0416]

[0417] Synthesis process:

[0418]

[0419] Program and features:

[0420] Step 1: (S)-2-(isopropylamino)-4-methylpentanoic acid benzoate:

[0421] Acetone (177 mg, 3.05 mmol) and potassium acetate (0.5 g, 5.08 mmol) were added to a stirred solution of L-leucine benzyl ester p-toluenesulfonate (1.0 g, 2.53 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 min. Sodium cyanoborohydride (0.24 g, 3.81 mmol) was then added, and the mixture was stirred again at room temperature for 3 h. The mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with EtOAc (50 mL × 2), washed with the resulting solution, filtered (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(isopropylamino)-4-methylpentanoic acid benzyl ester (200 mg, 0.76 mmol, 30%) as a colorless oil. MS(EI+,m / z):264.3[M+H]+. 1H-NMR (500MHz,,MeOD): δ7.22~7.29(m,5H),5.07(dd,J=11.5Hz,17.0Hz,2H),3.33 (dd, J=6.5Hz, 8.5Hz, 1H), 2.54~2.59(m,1H), 1.30~1.48(m,3H), 0.72~0.94(m,12H).

[0422] Step 2: (S)-2-(isopropylamino)-4-methylpentanoic acid [I-13]:

[0423] A catalytic amount of Pd / C (10%, 50 mg) was added to a stirred solution of (S)-2-(isopropylamino)-4-methylpentanoic acid methyl ester (200 mg, 0.76 mmol) in MeOH (10 mL). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 24 hours. The resulting solution was filtered and the filtrate was concentrated to give (S)-2-(isopropylamino)-4-methylpentanoic acid (I-13) as a white solid (100 mg, 0.57 mmol, 76%). MS (EI+, m / z): 174.3 [M+H]+. 1H-NMR (500MHz, MeOD): δ3.56 (dd, J=6.0Hz, 8.5Hz, 1H), 3.33~3.40 (m, 1H) ,1.75~1.86(m,2H),1.53~1.58(m,1H),1.31~1.36(m,6H),0.96~1.02(m,6 H).3.85(dd,J=5.5Hz,8.5Hz,1H),2.87(q,J=6.0Hz,1H),2.68(dd,J=7.5H z,12.0Hz,1H),1.92~1.99(m,1H),1.65~1.78(m,3H),0.88~0.96(m,12H).

[0424] Example 14: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0425]

[0426] Synthesis process:

[0427]

[0428] Program and features:

[0429] Step 1: (S)-2-(isobutylamino)-4-methylpentanoic acid benzoate:

[0430] Isobutyraldehyde (0.22 g, 3.05 mmol) and potassium acetate (0.5 g, 5.08 mmol) were added to a stirred solution of L-leucine benzyl ester p-toluenesulfonate (1.0 g, 2.53 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 minutes, followed by the addition of sodium cyanoborohydride (0.24 g, 3.81 mmol). The mixture was stirred for another 5 hours at room temperature. The mixture was quenched with a saturated solution of NaHCO3 (50 mL), extracted with EtOAc (50 mL × 2), washed with the resulting solution, filtered (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by preparative HPLC (Boston C18 21×250mm 10μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-(isobutylamino)-4-methylpentanoic acid benzoate (300 mg, 1.08 mmol, 50%) as a colorless oil. MS (EI+, m / z): 278.2 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ9.16 (s, 1H), 9.14 (d, J = 17.5Hz, 2H), 7.42-7.43 (m, 5H), 5.28 (q, J = 12.0Hz, 2H), 4.0 8-4.09(m,1H),2.87-2.89(m,1H),2.65-2.66(m,1H),1.91-1.95(m,1H),1.62~1.71(m,3H),0.88~0.94(m,12H).

[0431] Step 2: (S)-2-(isobutylamino)-4-methylpentanoic acid [I-14]:

[0432] A catalytic amount of Pd / C (10%, 50 mg) was added to a stirred solution of (S)-2-(isobutylamino)-4-methylpentanoic acid methyl ester (300 mg, 1.08 mmol) in MeOH (10 mL). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 24 hours. The resulting solution was filtered and the filtrate was concentrated to give (S)-2-(isobutylamino)-4-methylpentanoic acid (I-14) as a white solid (150 mg, 0.8 mmol, 74%). MS (EI+, m / z): 188.3 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ8.82(s,2H),3.85(dd,J=5.5Hz,8.5Hz,1H),2.87(q,J=6.0Hz,1H ), 2.68 (dd, J=7.5Hz, 12.0Hz, 1H), 1.92~1.99 (m, 1H), 1.65~1.78 (m, 3H), 0.88~0.96 (m, 12H).

[0433] Example 15: (S)-2-benzoylamino-4-methylpentanoic acid [I-15]:

[0434]

[0435] Synthesis process:

[0436]

[0437] Program and features:

[0438] Step 1: (S)-2-benzoylamino-4-methylpentanoic acid methyl ester:

[0439] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), benzoic acid (223 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-benzoylamino-4-methylpentanoic acid benzyl ester (300 mg, 0.92 mmol, 73%) as a white solid. MS (EI+, m / z): 326.2 [M+H]+.

[0440] Step 2: (S)-2-benzoylamino-4-methylpentanoic acid [I-15]:

[0441] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-benzoylamino-4-methylpentanoic acid methyl ester (100 mg, 0.46 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-benzoylamino-4-methylpentanoic acid (I-15) as a white solid (100 mg, 0.42 mmol, 65%). MS (EI+, m / z): 236.2 [M+H]+. 1H-NMR (400MHz, MeOD): δ7.87 (t, J = 6.5 Hz, 2H), 7.47-7.57 (m, 3H), 4.69 (dd, J = 4.0 Hz, 11.0 Hz, 1H), 1.75-1.84 (m, 3H), 1.01 (dd, J = 6.5 Hz, 10.5 Hz, 6H).

[0442] Example 16: (S)-2-isobutyrylamino-4-methylpentanoic acid [I-16]:

[0443]

[0444] Synthesis process:

[0445]

[0446] Program and features:

[0447] Step 1: (S)-2-isobutyrylamino-4-methylpentanoic acid benzoate:

[0448] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), isobutyric acid (168 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-isobutyrylamino-4-methylpentanoic acid benzyl ester (300 mg, 1.03 mmol, 81%) as a white solid. MS (EI+, m / z): 292.2 [M+H]+.

[0449] Step 2: (S)-2-isobutyrylamino-4-methylpentanoic acid [I-16]:

[0450] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-(cyclohexylformamido)-4-methylpentanoic acid methyl ester (200 mg, 0.69 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-isobutyrylamino-4-methylpentanoic acid (100 mg, 0.50 mmol, 73%) as a white solid. MS (EI+, m / z): 202.2 [M+H]+. 1H-NMR (400MHz, MeOD): δ4.43 (t, J = 6.4 Hz, 1H), 2.49-2.56 (m, 1H), 1.60-1.74 (m, 3H), 1.12 (dd, J = 2.4Hz, 6.8Hz, 6H), 0.96 (dd, J = 6.4Hz, 16.0Hz, 6H).

[0451] Example 17: (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid [I-17]:

[0452]

[0453] Synthesis process:

[0454]

[0455] Program and features:

[0456] Step 1: (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid methyl ester:

[0457] Cyclohexanesulfonyl chloride (278.53 mg, 1.52 mmol) was added to a solution of (S)-2-amino-4-methylpentanoic acid benzoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and Et3N (642.89 mg, 6.35 mmol) in DMF (3 mL) cooled in an ice bath. The mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), washed with the resulting solution, filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-(cyclohexanesulfonylamino)-4-methylpentanoic acid benzoate (200 mg, 0.544 mmol, 98%) as a white solid. ESI-MS(EI+,m / z):368.3[M+H]+. 1H-NMR (500MHz, DMSO-d6) δ7.70 (d, J=9.0Hz, 1H), 7.38 (t, J=6.5Hz, 4H), 7.37-7. 32(m,1H),5.14(q,J=12.5Hz,2H),3.91(td,J=5.0Hz,9.5Hz,1H),2.69-2.74(m,1 H),2.05(d,J=12.5Hz,1H),1.97(d,J=12.5Hz,1H),1.74-1.67(m,2H),1.57-1.51 (m,2H),1.50-1.44(m,1H),1.36-0.99(m,5H),0.87(dt,J=10.5Hz,J=20.5Hz,6H).

[0458] Step 2: (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid [I-17]:

[0459] Pd / C (20 mg, 10%) was added to a solution of (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid benzoate (192 mg, 0.552 mmol) in EtOH (3 mL). The reaction mixture was stirred under hydrogen at 50 °C for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-(cyclohexanesulfonamide)-4-methylpentanoic acid (I-17) as a white solid (23.3 mg, 0.084 mmol, 100%). ESI-MS (EI)+ ,m / z):300.2[M+Na] + . 1 H NMR(500MHz,DMSO-d6)δ12.75(s,1H),7.47(d,J=9.0Hz,1H),3.76(td,J=5.0Hz,9.5Hz,1H),2.82-2.69(m,1H),2.1 8-1.97(m,2H),1.82-1.69(m,3H),1.61(d,J=12.5Hz,1H),1.54-1.40(m,2H),1.39-1.07(m,5H),0.95-0.80(m,6H).

[0460] Example 18: (S)-4-methyl-2-(phenylmethylsulfonylamino)valerate [I-18]:

[0461]

[0462] Synthesis process:

[0463]

[0464] Program and features:

[0465] Step 1: (S)-4-methyl-2-(phenylmethylsulfonylamino)pentanoic acid methyl ester:

[0466] To a solution of (S)-2-amino-4-methylpentanoic acid benzoate 4-methylbenzenesulfonate (500 mg, 1.27 mmol) and Et3N (642.89 mg, 6.35 mmol) in DMF (3 mL) 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), washed with the resulting solution, filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-4-methyl-2-(phenylmethanesulfonylamino)pentanoic acid benzoate (149 mg, 0.396 mmol, 90%) as a white solid. ESI-MS(EI+,m / z):398.0[M+Na]+. 1H-NMR(500MHz,DMSO-d6)δ7.81(d,J=8.5Hz,1H),7.52-7.18(m,9H),5.15(s,2H),4.28(dd,J=13.5 Hz, 44.5Hz, 2H), 3.87 (dd, J = 8.0Hz, 15.0Hz, 1H), 1.57-1.15 (m, 4H), 0.82 (dd, J = 4.5Hz, 6.0Hz, 6H).

[0467] Step 2: (S)-4-methyl-2-(phenylmethylsulfonylamino)valerate [I-18]:

[0468] Pd / C (20 mg, 10%) was added to a solution of (S)-4-methyl-2-(phenylmethylsulfonylamino)pentanoic acid methyl ester (121 mg, 0.322 mmol) in EtOH (3 mL). The reaction mixture was stirred under hydrogen at 50 °C for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(phenylmethylsulfonylamino)pentanoic acid (I-18) as a white solid (41.2 mg, 0.144 mmol, 100%). ESI-MS (EI+, m / z): 308.0 [M+Na]+. 1HNMR(500MHz,DMSO-d6)δ12.77(s,1H),7.59(d,J=8.5Hz,1H),7.47-7.25(m,5H),4.30(dd,J=13.5Hz,37.0Hz,2H),3 .75(dd,J=7.5Hz,15.5Hz,1H),1.65(dt,J=6.5Hz,13.5Hz,1H),1.45(t,J=7.2Hz,2H),0.85(dd,J=1.5Hz,6.5Hz,6H).

[0469] Example 19: (S)-4-methyl-2-(methanesulfonylamino)valerate [I-19]:

[0470]

[0471] Synthesis process:

[0472]

[0473] Program and features:

[0474] Step 1: (S)-4-methyl-2-(methylsulfonamide)pentanoic acid methyl ester:

[0475] Methylbenzenesulfonate (500 mg, 1.27 mmol) and Et3N (642.89 mg, 6.35 mmol) were added to a solution of (S)-2-amino-4-methylpentanoate benzoate (4-methylbenzenesulfonate) in ice-bath-cooled DMF (3 mL), and the mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), washed with the resulting solution, filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-4-methyl-2-(methanesulfonylamino)pentanoate benzoate (192 mg, 0.641 mmol, 98%) as a white solid. ESI-MS (EI) + ,m / z):323.0[M+Na] + . 1 H-NMR(500MHz,DMSO-d6)δ7.79(d,J=8.8Hz,1H),7.42-7.36(m,4H),7.37-7.32(m,1H),5.16(s,2H),3.97(t d, J=6.0Hz, 9.0Hz, 1H), 2.85 (s, 3H), 1.68 (dq, J=6.5Hz, 13.0Hz, 1H), 1.54-1.46 (m, 2H), 0.91-0.82 (m, 6H).

[0476] Step 2: (S)-4-methyl-2-(methanesulfonamide)valerate [I-19]:

[0477] Pd / C (20 mg, 10%) was added to a solution of (S)-4-methyl-2-(methanesulfonamide)pentanoic acid benzoate (149 mg, 0.497 mmol) in EtOH (3 mL). The reaction mixture was stirred at 50 °C under a hydrogen atmosphere for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(methanesulfonamide)pentanoic acid (I-19) as a white solid (31.4 mg, 0.150 mmol, 100%). ESI-MS (EI... + ,m / z):232.1[M+Na] + . 1H NMR(500MHz,DMSO-d6)δ12.82(s,1H),7.56(d,J=9.0Hz,1H),3.82(dd,J=8.0Hz,15.5Hz,1H) ,2.88(s,3H),1.72(dt,J=6.5Hz,13.0Hz,1H),1.48(t,J=7.0Hz,2H),0.89(t,J=7.0Hz,6H).

[0478] Example 20: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0479]

[0480] Synthesis process:

[0481]

[0482] Program and features:

[0483] Step 1: (S)-4-methyl-1-oxo-1-(phenylamino)pent-2-ylcarbamate:

[0484] At room temperature, aniline (702 mg, 7.55 mmol), HATU (1.72 g, 4.52 mmol), and Et3N (1.14 g, 11.31 mmol) were added to a solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 3.77 mmol) in DMF (20 mL). After 2 hours, the solution was diluted with EtOAc (80 mL), washed with the resulting solution, filtered (80 mL × 3), washed with brine (80 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 3) to give (S)-4-methyl-1-oxo-1-(phenylamino)pent-2-ylcarbamate (350 mg, 1.03 mmol, 27%) as a white solid. ESI-MS (EI+, m / z): 341.1 [M+H]+.

[0485] Step 2: (S)-2-amino-4-methyl-N-phenylpentanamide [I-20]:

[0486] A mixture of (S)-4-methyl-1-oxo-1-(phenylamino)pentan-2-ylcarbamate (350 mg, 1.03 mmol) and Pd / C (10%, 50 mg) in MeOH (10 mL) was stirred at room temperature under hydrogen for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-amino-4-methyl-N-phenylpentanamide (I-20) as a white solid (100 mg, 0.49 mmol, 47%). ESI-MS (EI+, m / z): 207.2 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ9.86 (s, 1H), 7.63 (dd, J = 1.0Hz, 8.5Hz, 2H), 7.31-7.27 (m, 2H), 7.03 (t, J = 7.5Hz, 1H), 3. 31(dd,J=5.0Hz,8.5Hz,1H),1.80-1.71(m,1H),1.50-1.44(m,1H),1.35-1.29(m,1H),0.90(dd,J=6.5Hz,14.0Hz,6H).

[0487] Example 21: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0488]

[0489] Synthesis process:

[0490]

[0491] Program and features:

[0492] Step 1: (S)-4-methyl-1-(methylamino)-1-oxopent-2-ylcarbamate:

[0493] At 25 °C, a solution of (S)-2-(benzyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 3.77 mmol) in DMF (20 mL) was added with MeNH₂HCl (509 mg, 7.54 mmol), HATU (1.72 g, 4.52 mmol), and Et₃N (1.14 g, 11.31 mmol). After 2 hours, the solution was diluted with EtOAc (80 mL), washed with the resulting solution, filtered (80 mL × 3), washed with brine (80 mL), dried (Na₂SO₄), filtered again, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 3) to give (S)-4-methyl-1-(methylamino)-1-oxopentan-2-ylcarbamate (550 mg, 1.98 mmol, 52%) as a colorless oil. ESI-MS(EI+,m / z):279.2[M+H]+.

[0494] Step 2: (S)-2-amino-N,4-dimethylpentanamide [I-21]:

[0495] A mixture of (S)-4-methyl-1-(methylamino)-1-oxopentan-2-ylcarbamate (300 mg, 1.08 mmol) and Pd / C (10%) (50 mg) in MeOH (10 mL) was stirred at room temperature under hydrogen for 2 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-2-amino-N,4-dimethylpentanamide (I-21) (152 mg, 1.05 mmol, 98%) as a colorless oil. ESI-MS (EI+, m / z): 145.3 [M+H]+. 1H-NMR (500MHz, DMSO-d6): δ7.80 (s, 1H), 3.10 (dd, J=5.0Hz, 9.0Hz, 1H), 2.57 (dd, J=3.0Hz, 5.0H z,3H),1.81(s,2H),1.66-1.69(m,1H),1.34-1.39(m,1H),1.16-1.22(m,1H),0.81-0.87(m,6H).

[0496] Example 22: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-22]:

[0497]

[0498] Synthesis process:

[0499]

[0500] Program and features:

[0501] Step 1: (S)-2-(cyclohexylformamido)-4-methylpentanoic acid benzoate:

[0502] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), cyclohexanecarboxylic acid (244 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-(cyclohexylformamide)-4-methylpentanoic acid benzyl ester (300 mg, 0.91 mmol, 71%) as a white solid. MS (EI+, m / z): 332.3 [M+H]+.

[0503] Step 2: (S)-2-(cyclohexylformamido)-4-methylpentanoic acid [I-22]:

[0504] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-(cyclohexylformamido)-4-methylpentanoic acid methyl ester (200 mg, 0.60 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-(cyclohexylformamido)-4-methylpentanoic acid (I-22) as a white solid (100 mg, 0.41 mmol, 69%). MS (EI+, m / z): 242.3 [M+H] + . 1H-NMR (500MHz, CD3OD): δ4.43 (t, J=7.5Hz, 1H), 2.29 (td, J=8.0Hz, 11.0Hz, 1H), 1.74-1.85 (m ,4H),1.63-1.72(m,4H),1.43-1.49(m,2H),1.26-1.36(m,3H),0.96(dd,J=6.0Hz,20.5Hz,6H).

[0505] Example 25: (S)-4-methyl-2-(phenylsulfonamide)valerate [I-25]:

[0506]

[0507] Synthesis process:

[0508]

[0509] Program and features:

[0510] Step 1: (S)-4-methyl-2-(phenylsulfonamide)pentanoic acid methyl ester:

[0511] To a solution of (S)-2-amino-4-methylpentanoic acid methylbenzenesulfonate (300 mg, 0.762 mmol) and Et3N (385.73 mg, 3.81 mmol) in DMF (3 mL) cooled in an ice bath, benzenesulfonyl chloride (148.12 mg, 0.838 mmol) was added. The mixture was stirred at 25 °C for 2 hours. The solution was diluted with ethyl acetate (10 mL), washed with the resulting solution, filtered (10 mL × 3), washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product (280 mg, purity: 85%, yield: 74%) was used directly in the next step. ESI-MS (EI+, m / z): 384.1 [M+Na]+.

[0512] Step 2: (S)-4-methyl-2-(phenylsulfonamide)valerate [I-25]:

[0513] Pd / C (20 mg, 10%) was added to a solution of (S)-4-methyl-2-(phenylsulfonamide)pentanoic acid methyl ester (200 mg, 0.553 mmol) in EtOH (3 mL). The reaction mixture was stirred at 50 °C under a hydrogen atmosphere for 4 hours. The mixture was filtered, and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated to give (S)-4-methyl-2-(phenylsulfonamide)pentanoic acid (I-25) as a white solid (63.7 mg, 0.234 mmol, 100%). ESI-MS (EI) + ,m / z):294.0[M+Na] + . 1 H-NMR (500MHz, DMSO-d6) δ12.61(s,1H),8.16(d,J=8.6Hz,1H),7.79-7.73(m,2H),7.62(t,J=7.3Hz,1H),7.56(t,J=7.4Hz,2H), 3.63(dd,J=8.5Hz,14.5Hz,1H),1.53(td,J=6.5Hz,13.5Hz,1H),1.41-1.31(m,2H),0.79(d,J=6.6Hz,3H),0.66(d,J=6.5Hz,3H).

[0514] Example 26: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]:

[0515]

[0516] Synthesis process:

[0517]

[0518] Program and features:

[0519] Step 1: (S)-4-methyl-2-(phenylamino)pentanoic acid methyl ester:

[0520] A mixture of L-leucine benzyl ester p-toluenesulfonate (200 mg, 0.51 mmol), phenylboronic acid (186 mg, 1.52 mmol), and Cu(OAc)₂ (462 mg, 2.54 mmol) in DCM (10 mL) was added with 4 AMS (1.0 g) and Et₃N (155 mg, 1.52 mmol) and stirred at room temperature for 18 hours. The mixture was quenched with the resulting solution, filtered (50 mL), extracted with EtOAc (50 mL × 2), washed with the resulting solution, filtered (50 mL), and washed with brine (50 mL). The organic phase was concentrated and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 20) to give (S)-4-methyl-2-(phenylamino)pentanoic acid benzyl ester (100 mg, 0.34 mmol, 66%) as a colorless oil. MS (EI+, m / z): 298.2 [M+H]+.

[0521] Step 2: (S)-4-methyl-2-(phenylamino)pentanoic acid [I-26]:

[0522] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-4-methyl-2-(phenylamino)pentanoic acid methyl ester (100 mg, 0.34 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 2 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-4-methyl-2-(phenylamino)pentanoic acid (I-26) as a white solid (30 mg, 0.15 mmol, 43%). MS (EI+, m / z): 208.1 [M+H]+. 1H-NMR (400MHz, CDCl3): δ7.23(t,J=8.0Hz,2H),6.83(t,J=7.6Hz,1H),6.66(d,J=8.0Hz,2H),3.9 9(d,J=8.4Hz,1H), 2.87(q,J=6.0Hz,1H), 1.72~1.86(m,2H), 1.62~1.68(m,1H), 0.85~1.03(m,6H).

[0523] Example 36: (S)-2-acetamido-4-methylpentanoic acid [I-36]:

[0524]

[0525] Synthesis process:

[0526]

[0527] Program and features:

[0528] Step 1: (S)-2-acetamido-4-methylpentanoic acid benzoate:

[0529] DIPEA (410 mg, 3.18 mmol) was added to a solution of L-leucine benzyl ester p-toluenesulfonate (500 mg, 1.27 mmol), acetic acid (114 mg, 1.91 mmol), and HATU (726 mg, 1.91 mmol) in DMF (10 mL), and the solution was stirred at room temperature for 2 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-2-acetamido-4-methylpentanoic acid benzyl ester (300 mg, 1.14 mmol, 89%) as a white solid. MS (EI+, m / z): 264.2 [M+H]+.

[0530] Step 2: (S)-2-acetamido-4-methylpentanoic acid [I-36]:

[0531] A catalytic amount of Pd / C (10%, 20 mg) was added to a stirred solution of (S)-2-acetamido-4-methylpentanoic acid methyl ester (250 mg, 0.74 mmol) in EtOH (10 mL). The reaction mixture was stirred at 50 °C for 3 hours under a hydrogen atmosphere. The resulting solution was filtered and concentrated to give (S)-2-acetamido-4-methylpentanoic acid (I-36) as a white solid (100 mg, 0.57 mmol, 81%). MS (EI+, m / z): 174.2 [M+H] + . 1 H-NMR (500MHz, MeOD): δ4.43 (dd, J = 6.0 Hz, 9.5 Hz, 1H), 2.00 (s, 3H), 1.61-1.73 (m, 3H), 0.97 (dd, J = 6.0 Hz, 17.5 Hz, 6H).

[0532] Example 45: (S,E)-2-(4-methoxy-4-oxobut-2-enoylamino)-4-methylpentanoic acid [I-45]:

[0533]

[0534] Synthesis process:

[0535]

[0536] Program and features:

[0537] Step 1: (S,E)-2-(4-methoxy-4-oxobut-2-enoylamino)-4-methylpentanoic acid [I-45]:

[0538] SOCl2 (1.83 g, 15.38 mmol) was added to a solution of (E)-4-methoxy-4-oxobut-2-enoic acid (1.0 g, 7.69 mmol) in DCM (30 mL), followed by DMF (0.1 mL). The solution was heated to 40 °C for 4 hours. The solution was concentrated to dryness to obtain an oil. The oil was diluted with DCM (10 mL). (S)-2-amino-4-methylpentanoic acid (1.0 g, 7.62 mmol) was added dropwise to a solution of acetone (20 mL) and saturated Na2CO3 solution (20 mL) cooled in an ice bath. After 1 hour, the solution was adjusted to pH 2 with 6 M HCl solution, extracted with EtOAc (40 × 2), washed with the resulting solution, filtered (80 mL × 3), washed with brine (80 mL), dried (Na2SO4), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, MeOH / DCM = 1 / 20) to give (S,E)-2-(4-methoxy-4-oxobut-2-enoylamino)-4-methylpentanoic acid (I-45), a yellow oil (1.0 g, 4.11 mmol, 53%). ESI-MS (EI...) + ,m / z):244.2[M+H] + . 1 H-NMR (400MHz, CDCl3): δ7.32(d,J=15.2Hz,1H),7.05(d,J=15.2Hz,1H),6.85-6.8 9(m,2H),7.30-7.46(m,1H),3.82(s,1H),1.63-1.78(m,3H),0.97(d,J=4.8Hz,6H).

[0539] 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]:

[0540]

[0541] Synthesis process:

[0542]

[0543] Program and features:

[0544] Step 1: Ethyl 2,2-difluoro-3-methylbutyrate:

[0545] A mixture of ethyl 3-methyl-2-oxobutyrate (10 g, 0.069 mol) and DAST (16.8 g, 0.10 mol) was stirred at room temperature for 12 hours. After TLC analysis, the reaction mixture was slowly added dropwise to a cold, saturated aqueous solution of sodium bicarbonate. The mixture was extracted with Et2O (300 mL × 2), and the organic layer was washed with brine, dried, and concentrated to give crude ethyl 2,2-difluoro-3-methylbutyrate (8.3 g) for direct use in the next step.

[0546] Step 2: 2,2-Difluoro-3-methylbutanal:

[0547] Under argon atmosphere at -78°C, a solution of DIBAL-H in hexane (1.0 M, 69 mL, 69.0 mmol) was added dropwise to a solution of crude ethyl 2,2-difluoro-3-methylbutyrate (8.3 g) in CH₂Cl₂ (200 mL), and the mixture was stirred at -78°C for 30 min. After TLC analysis, the reaction mixture was quenched with saturated citric acid and extracted with Et₂O. The extract was washed with saturated citric acid and brine, dried over Na₂SO₄, and concentrated under reduced pressure to obtain 4.2 g of the oily aldehyde 2,2-difluoro-3-methylbutyraldehyde, which was used immediately in the next step without purification.

[0548] Step 3: 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide:

[0549] The solution of crude 2,2-difluoro-3-methylbutanal (4.2 g) in 50 mL MeOH was cooled to 0 °C. Acetic acid (ice, 2.1 mL) was added dropwise over a 15-minute period while maintaining the temperature at approximately 0 °C, followed by the addition of trimethylsilane cyanide (4.2 mL). The reaction mixture was heated to 25 °C and stirred overnight. The filtered, cooled solution (200 mL) was added to the reaction mixture and extracted with dichloromethane (2 × 200 mL). The dichloromethane layer was washed with the resulting solution, filtered (2 × 100 mL), and then washed with brine (2 × 50 mL). The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (2.8 g) which was used immediately in the next step without purification. ESI-MS (EI+, m / z): 238.2 [M+H]+.

[0550] Step 4: 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid:

[0551] A solution of crude 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (2.8 g) in 50 mL concentrated hydrochloric acid and 10 mL HOAc was stirred at 90 °C for 24 hours and then concentrated. The residue was purified by preparative HPLC to give 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (513 mg) as a white solid. The pure product was purified by chiral HPLC to give (R)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (80 mg) and (S)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanilide (63 mg), both of which were white solids. ESI-MS (EI+, m / z): 258.2 [M+H]+.

[0552] Step 5-A: (R)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-46]:

[0553] At room temperature, HCOONH4 (98 mg, 1.56 mmol) and Pd / C (100 mg) were added to a solution of (R)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid (80 mg, 0.31 mmol) in 20 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give (R)-2-amino-3,3-difluoro-4-methylpentanoic acid (I-46) (23 mg, 44%) as a white solid; 1H-NMR (500 MHz, D2O): δ 4.27 (dd, J = 24.0, 3.5 Hz, 1H), 2.55–2.42 (m, 1H), 1.04 (d, J = 7.0 Hz, 3H), 0.993 (d, J = 6.5 Hz, 3H).

[0554] Step 5-B: (S)-2-amino-3,3-difluoro-4-methylpentanoic acid [I-47]:

[0555] At room temperature, HCOONH4 (77 mg, 1.22 mmol) and Pd / C (100 mg) were added to a solution of (S)-2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid (63 mg, 0.24 mmol) in 15 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give (S)-2-amino-3,3-difluoro-4-methylpentanoic acid (I-47) (14 mg, 34%) as a white solid; 1H-NMR (500 MHz, D2O): δ 4.27 (dd, J = 24.0, 3.5 Hz, 1H), 2.55–2.42 (m, 1H), 1.04 (d, J = 7.0 Hz, 3H), 0.993 (d, J = 6.5 Hz, 3H).

[0556] Example 147: (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147].

[0557]

[0558] Synthesis process:

[0559]

[0560] Program and features:

[0561] Step 1: (S)-4-methyl-1-(methanesulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0562] TEA (1.3 g, 12.9 mmol) was added to a solution of (S)-2-(tert-butyloxycarbonylamino)-4-methylpentanoic acid (1.0 g, 4.32 mmol), methanesulfonamide (452 ​​mg, 4.75 mmol), and HATU (1.8 g, 4.75 mmol) in DMF (30 mL), and the solution was stirred at room temperature for 17 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain (S)-4-methyl-1-(methanesulfonylamino)-1-oxopentan-2-ylcarbamate tert-butyl ester (130 mg, 0.42 mmol, 8.9%) as a white solid. MS (EI-, m / z): 307.0 [MH] - .

[0563] Step 2: (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147]:

[0564] A solution of (S)-4-methyl-1-(methanesulfonylamino)-1-oxopentan-2-ylcarbamate tert-butyl ester (130 mg, 0.42 mmol) in Et2O (15 mL) was added and stirred in 4 M HCl / dioxane (5 mL) for 3 hours at room temperature. The solid was filtered to give (S)-2-amino-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-147] (32 mg, 0.13 mmol, 31%) as a white solid. ESI-MS (EI+, m / z): 209.1 [M+H] + . 1H NMR (500MHz, CD3OD) δ3.96 (t, J = 3.0Hz, 1H), 3.32 (s, 3H), 1.74-1.79 (m, 3H), 1.02-1.05 (m, 6H).

[0565] Example 193: (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193].

[0566]

[0567] Synthesis process:

[0568]

[0569] Program and features:

[0570] Step 1: (S)-4,4-dimethyl-1-(N-methylmethanesulfonylamino)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0571] HATU (900 mg, 2.36 mmol) was added to a solution of (S)-2-(tert-butoxycarbonylamino)-4,4-dimethylvaleric acid (500 mg, 1.97 mmol) in DCM (60 mL), and the mixture was stirred at room temperature for 2 hours. Subsequently, Cs₂CO₃ (1.92 g, 5.91 mmol) and N-methylmethanesulfonamide (322 mg, 2.95 mmol) were added to the mixture, and the mixture was stirred 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 (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 5) to give (S)-4,4-dimethyl-1-(N-methylmethanesulfonylamino)-1-oxopent-2-ylcarbamate tert-butyl ester (420 mg, 1.25 mmol, 63%) as a yellow oil. ESI-MS (EI+, m / z): 359.1 [M+Na] + .

[0572] Step 2: (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193]:

[0573] A solution of (S)-4,4-dimethyl-1-(N-methylmethanesulfonylamino)-1-oxopentan-2-ylcarbamate tert-butyl ester (420 mg, 1.25 mmol) in Et2O (20 mL) was added and stirred in 4 M HCl / dioxane (10 mL) for 17 hours at room temperature. The solid was filtered to give (S)-2-amino-N,4,4-trimethyl-N-(methanesulfonyl)pentanamide hydrochloride [I-193] (250 mg, 0.13 mmol, 71%) as a white solid. ESI-MS (EI+, m / z): 237.1 [M+H] +. 1H NMR (500MHz, DMSO) δ8.55(s,3H),4.59(s,1H),3.50(s,3H),3.26(s,3H),1.81-1.85(m,1H),1.63-1.67(m,1H),0.95(s,9H).

[0574] Example 192: 2-Amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192].

[0575]

[0576] Synthesis process:

[0577]

[0578] Program and features:

[0579] Step 1: 4-Fluoro-4-methyl-1-(methylsulfonamide)-1-oxopent-2-ylcarbamate tert-butyl ester:

[0580] HATU (451 mg, 1.19 mmol) was added to a solution of tert-butyl 4-fluoro-4-methyl-1-(methanesulfonamide)-1-oxopentan-2-ylcarbamate (270 mg, 1.08 mmol) in DCM (50 mL), and the mixture was stirred at room temperature for 2 hours. Subsequently, Cs₂CO₃ (1.06 g, 3.24 mmol) and methanesulfonamide (206 mg, 2.17 mmol) were added to the mixture, and the mixture was stirred overnight at room temperature. The solution was diluted with water (200 mL) and extracted with DCM (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 5) to give (S)-4,4-dimethyl-1-(N-methylmethanesulfonylamino)-1-oxopentan-2-ylcarbamate tert-butyl ester (200 mg, 0.6 mmol, 55%) as a yellow oil. ESI-MS (EI+, m / z): 344.1 [M+NH₄] + .

[0581] Step 2: 2-Amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192].

[0582] A solution of (S)-4,4-dimethyl-1-(N-methylmethanesulfonylamino)-1-oxopentan-2-ylcarbamate tert-butyl ester (200 mg, 0.6 mmol) in Et2O (20 mL) was added and stirred in 4 M HCl / dioxane (10 mL) for 17 hours at room temperature. The solid was filtered to give 2-amino-4-fluoro-4-methyl-N-(methanesulfonyl)pentanamide hydrochloride [I-192] (89.8 mg, 0.34 mmol, 57%) as a white solid. ESI-MS (EI+, m / z): 227.1 [M+H] + . 1H NMR (500MHz, DMSO) δ8.44(s,3H),4.02(s,1H),3.25(s,3H),2.16-2.25(m,1H),2.03-2.10(m,1H),1.43(s,3H),1.38(s,3H).

[0583] Example 190: (S)-2-((S)-2-amino-4,4-dimethylpentanoylamino)-4-methylpentanoic acid methyl ester hydrochloride [I-190].

[0584]

[0585] Synthesis process:

[0586]

[0587] Program and features:

[0588] Step 1: Methyl (S)-2-((S)-2-(tert-Butoxycarbonylamino)-4,4-dimethylpentanoylamino)-4-methylpentanoate:

[0589] HATU (900 mg, 2.3 mmol) was added to a solution of (S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanoic acid (500 mg, 2.0 mmol) in DCM (80 mL), and the mixture was stirred at room temperature for 2 hours. Subsequently, Cs₂CO₃ (1.95 g, 6.0 mmol) and (S)-2-amino-4-methylpentanoic acid methyl ester hydrochloride (555 mg, 3.0 mmol) were added to the mixture, and the mixture was 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 (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 5) to give methyl (S)-2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanoylamino)-4-methylpentanoate (500 mg, 1.34 mmol, 67%) as a white solid. ESI-MS (EI+, m / z): 317.2 [M-56] + .

[0590] Step 2: (S)-2-((S)-2-amino-4,4-dimethylpentanoylamino)-4-methylpentanoic acid methyl ester hydrochloride [I-190].

[0591] A solution of (S)-2-((S)-2-(tert-butoxycarbonylamino)-4,4-dimethylpentanoylamino)-4-methylpentanoate methyl ester (500 mg, 1.34 mmol) in Et₂O (20 mL) was added and stirred in 4 M HCl / dioxane (10 mL) for 17 hours at room temperature. The solid was filtered to give a white solid of (S)-2-(S)-2-amino-4,4-dimethylpentanoylamino)-4-methylpentanoate methyl hydrochloride [I-190] (300 mg, 0.97 mmol, 73%). ESI-MS (EI+, m / z): 273.2 [M+H] + . 1H NMR(500MHz,DMSO)δ9.07-9.09(d,J=7.5Hz,1H),8.42(s,3H),4.29-4.34(m,1H),3 .82(m,1H),3.60(s,3H),1.72-1.83(m,2H),1.50-1.62(m,3H),0.86-0.91(m,15H).

[0592] Example 122: (S)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-122].

[0593]

[0594] Synthesis process:

[0595]

[0596] Program and features:

[0597] Step 1: (S)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-122]:

[0598] A solution of (S)-2-amino-4,4-dimethylvalerate (100 mg, 0.69 mmol) in MeOH (10 mL) was added and stirred at 80 °C with 4 M HCl / dioxane (10 mL) for 24 hours. The mixture was concentrated and the residue was stirred with Et2O to give a white solid of (S)-2-amino-4,4-dimethylvalerate methyl hydrochloride [I-122] (23.6 mg, 0.12 mmol, 20%). ESI-MS (EI+, m / z): 160.1 [M+H]+. 1H-NMR (500 MHz, CD3OD): δ 4.02–4.04 (m, 1H), 3.86 (s, 3H), 1.97–2.02 (m, 1H), 1.64–1.68 (m, 1H), 1.03–1.05 (d, 9H).

[0599] Example 123: (R)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-123].

[0600]

[0601] Synthesis process:

[0602]

[0603] Program and features:

[0604] Step 1: (R)-2-amino-4,4-dimethylvalerate methyl ester hydrochloride [I-123]:

[0605] SOCl2 (0.5 mL) was added to a mixture of (R)-2-amino-4,4-dimethylvalerate (50 mg, 0.34 mmol) and dry MeOH (10 mL), and stirred at room temperature for 17 hours. The mixture was concentrated and the residue was stirred with Et2O to give (R)-2-amino-4,4-dimethylvalerate methyl hydrochloride [I-123] (34.2 mg, 0.17 mmol, 50%) as a white solid. 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).

[0606] Example 205: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205].

[0607]

[0608] Synthesis process:

[0609]

[0610] Program and features:

[0611] Step 1: 2-(tert-Butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid:

[0612] A mixture of 2-amino-5,5,5-trifluoro-4-methylpentanoic acid (250 mg, 1.35 mmol), Boc₂O (353 mg, 1.62 mmol), and NaOH (80 mg, 2.0 mmol) was dissolved in dioxane (10 mL) and H₂O (2 mL). The mixture was stirred at room temperature for 3 hours. The solution was diluted with water (200 mL) and extracted with DCM (50 mL). The organic phase was washed with water (20 mL × 2) and brine (10 mL), dried (Na₂SO₄), filtered, and concentrated to give crude 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (385 mg) as a colorless oil. ESI-MS (EI) + ,m / z):307.9[M+Na] + .

[0613] Step 2: 2-(tert-Butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid 2,5-dioxopyrrolidine-1-ester:

[0614] A mixture of 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (385 mg, 1.35 mmol), 1-hydroxypyrrolidine-2,5-dione (197 mg, 1.71 mmol), and DCC (353 mg, 1.71 mmol) was dissolved in DCM (15 mL). The mixture was stirred at room temperature for 17 hours. The mixture was filtered, and the filtrate was washed with brine (20 mL), dried (Na₂SO₄), filtered again, and concentrated to give crude 2-(tert-butoxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid 2,5-dioxopyrrolidine-1-ester (400 mg) as a white solid. ESI-MS (EI) + ,m / z):282.9[M-100] + .

[0615] Step 3: 1-Cyanamido-5,5,5-trifluoro-4-methyl-1-oxopent-2-ylcarbamate tert-butyl ester:

[0616] A mixture of 2-(tert-butyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid 2,5-dioxopyrrolidine-1-ester (300 mg, 0.78 mmol), cyanamide (66 mg, 1.57 mmol), and NaOH (156 mg, 3.9 mmol) was dissolved in THF (16 mL). The mixture was stirred at 0 °C for 0.5 h and then at room temperature for 17 h. The solution was purified by preparative HPLC (Boston C1821 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give tert-butyl 1-cyanoamino-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] + .

[0617] Step 4: 2-Amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205]:

[0618] A solution of tert-butyl 1-cyanoamino-5,5,5-trifluoro-4-methyl-1-oxopentan-2-ylcarbamate (45 mg, 0.14 mmol) in Et2O (20 mL) was added and stirred in 4 M HCl / dioxane (10 mL) for 24 hours at room temperature. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give 2-amino-N-cyano-5,5,5-trifluoro-4-methylpentanamide hydrochloride [I-205] (12.3 mg, 0.05 mmol, 27%) as a white solid. MS (EI+, m / z): 210.1 [M+H] + . 1H NMR (500MHz, CD3OD) δ4.06-4.09(m,1H), 2.43-2.65(m,1H), 1.67-1.85(m,2H), 1.18-1.22(m,3H).

[0619] Example 206: 2-Amino-3-(1-methylcyclobutyl)propionic acid [I-206].

[0620]

[0621] Synthesis process:

[0622]

[0623] Program and features:

[0624] Step 1: N-methoxy-N,1-dimethylcyclobutaneformamide:

[0625] TEA (30.3 g, 0.3 mol) was added to a solution of 1-methylcyclobutanecarboxylic acid (11.6 g, 0.1 mol), N,O-dimethylhydroxylamine hydrochloride (19.5 g, 0.2 mol), and HATU (42 g, 0.11 mol) in DMF (300 mL), and the solution was stirred at room temperature for 17 hours. The solution was diluted with water (600 mL) and extracted with EtOAc (400 mL × 2). The organic phase was washed with 1 N HCl, saturated NaHCO3, and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give N-methoxy-N,1-dimethylcyclobutanecarboxamide (12.2 g, 0.07 mol, 75%) as a colorless oil. ESI-MS (EI) + ,m / z):158.2[M+H] + .

[0626] Step 2: 1-Methylcyclobutane formaldehyde:

[0627] At 0°C and under N2, 1M LiAlH4 (19mL, 19mmol) was added dropwise to a solution of N-methoxy-N,1-dimethylcyclobutaneformamide (2.0g, 12.7mmol) in anhydrous THF (20mL). The mixture was heated to room temperature and stirred for 2 hours. The solution was slowly quenched with saturated seignette salt and extracted with Et2O (100mL). The organic phase was washed with water (100mL × 2) and brine (100mL), dried (Na2SO4), filtered, and used for the next step.

[0628] Step 3: (Z)-2-(tert-Butoxycarbonylamino)-3-(1-Methylcyclobutyl)acrylate:

[0629] At 0 °C, t-BuONa (844 mg, 8.79 mmol) was added to a solution of witting reagent (2.15 g, 5.86 mmol) in anhydrous THF (80 mL) and stirred for 1 hour. Subsequently, a solution of 1-methylcyclobutaneformaldehyde was added and stirred at room temperature for 17 hours. The solution was extracted with EtOAc (100 mL × 2). The organic phase was washed with brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 30) to give (Z)-2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)acrylate tert-butyl ester (700 mg, 2.2 mmol) as a colorless oil. ESI-MS (EI)+ ,m / z):200.2[M-56×2] + .

[0630] Step 4: 2-(tert-Butoxycarbonylamino)-3-(1-Methylcyclobutyl)propionate tert-butyl ester:

[0631] A mixture of (Z)-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 hours. The mixture was filtered, and the filtrate was concentrated to dryness to give tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propionate (600 mg, crude product) as a colorless oil. ESI-MS (EI) + ,m / z):158.2[M-156] + .

[0632] Step 5: 2-Amino-3-(1-methylcyclobutyl)propionic acid [I-206]:

[0633] A solution of tert-butyl 2-(tert-butoxycarbonylamino)-3-(1-methylcyclobutyl)propionate (600 mg, crude product) in Et2O (20 mL) was added and stirred at room temperature with 4 M HCl / dioxane (10 mL) for 17 hours. The solution was concentrated to give 2-amino-3-(1-methylcyclobutyl)propionic acid. MS (EI) + ,m / z):158.0[M+H] + .

[0634] 1 H NMR (500MHz, D2O) δ3.91 (t, J = 7.5Hz, 1H), 2.06-2.02 (m, 1H), 1.88-1.64 (m, 7H), 1.15 (s, 3H).

[0635] Example 93: S-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93].

[0636]

[0637] Synthesis process:

[0638]

[0639] Program and features:

[0640] The procedure for 2-amino-3-(1-methylcyclobutyl)propionic acid is the same as in Example 8.

[0641] Step 6: 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid:

[0642] 2-Amino-3-(1-methylcyclobutyl)propionic acid (300 mg, crude product) and CbzOSu (714 mg, 2.8 mmol) were stirred at room temperature in a mixture of acetone (10 mL) and saturated NaHCO3 (3 mL) for 5 hours. The solution was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to obtain 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (160 mg, 0.54 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + .

[0643] Step 7: (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid:

[0644] 2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (160 mg, 0.54 mmol) was purified by chiral HPLC to give (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (50 mg, 0.17 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + .

[0645] Step 8: (S)-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93]:

[0646] A mixture of (S)-2-(benzyloxycarbonylamino)-3-(1-methylcyclobutyl)propionic acid (50 mg, 0.17 mmol) and Pd / C (10%, 10 mg) in MeOH (10 mL) was stirred at room temperature for 1 hour. The solution was purified by preparative HPLC (Boston C1821 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-amino-3-(1-methylcyclobutyl)propionic acid [I-93] (2 mg, 0.01 mmol) as a white solid. MS (EI+, m / z): 292.0 [M+H] + . 1HNMR(500MHz,D2O)δ3.76-3.79(t,1H),1.96-2.00(m,1H),1.61-1.86(m,7H),1.11(s,3H).

[0647] Example 204: 2-Amino-3-(trimethylsilyl)propionate [I-204]

[0648]

[0649] Synthesis process:

[0650]

[0651] Program and features:

[0652] Step 1: 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester:

[0653] A solution of 2-(diphenylmethyleneamino)-tert-butyl acetate (2.5 g, 8.47 mmol) in THF (20 mL) was cooled to -78 °C, followed by dropwise addition of LiHMDS (8.47 mL, 8.47 mmol) under N2. The solution was stirred at -78 °C for 1 hour. (iodomethyl)trimethylsilane (1.8 g, 8.47 mmol) was added dropwise. The solution was stirred overnight at -78 °C to room temperature. The solution was washed with brine (25 mL × 2), dried (Na2SO4), concentrated, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 30) to give 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate-tert-butyl ester (2.3 g, 6.04 mmol, 71%) as a yellow solid. ESI-MS (EI+, m / z): 382.3 [M+H] + .

[0654] Step 2: 2-Amino-3-(trimethylsilyl)propionate [I-204]:

[0655] A solution of tert-butyl 2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate (500 mg, 1.31 mmol) in 4 M HCl / dioxane (6 mL) was stirred at room temperature for 17 hours. DCM (80 mL) was added. The solid was filtered to give a white solid of 2-amino-3-(trimethylsilyl)propionate [I-204] (113 mg, 0.57 mmol, 44%). ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500MHz, CD3OD) δ13.78(br,1H),8.33(br,1H),3.75(m,1H),1.00-1.14(m,2H),0.06(s,9H).

[0656] Example 201: (S)-2-amino-3-(trimethylsilyl)propionate [I-201].

[0657]

[0658] Synthesis process:

[0659]

[0660] Program and features:

[0661] Step 1: (S)-2-amino-3-(trimethylsilyl)propionate [I-201]:

[0662] A solution of (S)-2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester (300 mg, 0.79 mmol) in 4 M HCl / dioxane (3 mL) was stirred at room temperature for 17 hours. DCM (40 mL) was added. The solid was filtered to give (S)-2-amino-3-(trimethylsilyl)propionate salt [I-201] (92 mg, 0.47 mmol, 62%) as a white solid. ESI-MS (EI+, m / z): 162.2 [M+H] + . 1H NMR (500MHz, CD3OD) δ13.76(br,1H),8.38(br,1H),3.76(m,1H),1.02-1.16(m,2H),0.06(s,9H).

[0663] Example 200: (R)-2-amino-3-(trimethylsilyl)propionate [I-200].

[0664]

[0665] Synthesis process:

[0666]

[0667] Program and features:

[0668] Step 1: (R)-2-amino-3-(trimethylsilyl)propionate [I-200]:

[0669] A solution of (R)-2-(diphenylmethyleneamino)-3-(trimethylsilyl)propionate tert-butyl ester (300 mg, 0.79 mmol) in 4 M HCl / dioxane (3 mL) was stirred at room temperature for 17 hours. DCM (40 mL) was added. The solid was filtered to give (R)-2-amino-3-(trimethylsilyl)propionate salt [I-200] (80 mg, 0.41 mmol, 52%) as a white solid. ESI-MS (EI+, m / z): 162.2 [M+H] +. 1H NMR (500MHz, CD3OD) δ13.77(br,1H),8.33(br,1H),3.76(m,1H),1.02-1.14(m,2H),0.06(s,9H).

[0670] Example 194: (S)-2-amino-4-fluoro-4-methylvaleric acid [I-194].

[0671]

[0672] Synthesis process:

[0673]

[0674] Program and features:

[0675] Step 1: (S)-2-amino-4-fluoro-4-methylpentanoic acid [I-194]:

[0676] A mixture of (S)-2-amino-4-fluoro-4-methylpentanoic acid ethyl ester hydrochloride (65 mg, 0.31 mmol), LiOH·H₂O (29 mg, 0.69 mmol), and H₂O (2 mL) was stirred at room temperature for 2.5 h. Subsequently, 1 N HCl was added to adjust the pH to 3. The mixture was purified directly by reversed-phase HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-2-amino-4-fluoro-4-methylpentanoic acid [I-194] (40 mg, 0.27 mmol, 87%) as a white solid. MS (EI+, m / z): 150.3 [M+H] + . 1H NMR (500MHz, CD3OD) δ 8.10 (br, 2H), 3.79 (m, 1H), 2.19-2.26 (m, 1H), 1.97-2.05 (m, 1H), 1.42 (d, Jz = 3.5Hz, 3H), 1.37 (d, Jz = 4.0Hz, 3H).

[0677] Example 94: (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)but-1-amine [I-94].

[0678]

[0679] Synthesis process:

[0680]

[0681] Program and features:

[0682] Step 1: (S)-1-cyano-3,3-dimethylbutylcarbamate tert-butyl ester:

[0683] A solution of (S)-1-amino-4,4-dimethyl-1-oxopentan-2-ylcarbamate tert-butyl ester (500 mg, 2.1 mmol) in DMF (10 mL) was mixed with cyanuryl chloride (450 mg, 2.5 mmol) and stirred at room temperature for 2 hours. The mixture was then diluted with brine (100 mL), extracted with ethyl acetate (50 mL), dried (Na₂SO₄), and concentrated to give crude (S)-1-cyano-3,3-dimethylbutylcarbamate tert-butyl ester (500 mg) as a yellow dopant. ESI-MS (EI+, m / z): 249.2 [M+Na] + .

[0684] Step 2: (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate tert-butyl ester:

[0685] A mixture of (S)-1-cyano-3,3-dimethylbutylcarbamate (500 mg crude), ZnBr2 (900 mg, 4.0 mmol), and NaN3 (260 mg, 4.0 mmol) in DMF (20 mL) was stirred at 100 °C for 17 hours. The mixture was then diluted with brine (200 mL), extracted with ethyl acetate (60 mL), dried (Na2SO4), and concentrated to give crude (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate (400 mg) as a yellow dopant. ESI-MS (EI+, m / z): 214.3 [M+H-56] + .

[0686] Step 3: ((S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)but-1-amine [I-94]:

[0687] A solution of (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butylcarbamate tert-butyl (crude substance 300 mg) was stirred in 4M HCl / dioxane (3.5 mL) for 17 hours at room temperature. The solution was then concentrated and purified directly by reversed-phase HPLC (Boston C18 21×250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give (S)-3,3-dimethyl-1-(2H-tetrazol-5-yl)butyl-1-amine 2,2,2-trifluoroacetate [I-94] (30 mg, 0.11 mmol, 9%, 3 steps) as a white solid. MS (EI+, m / z): 170.2 [M+H] +. 1H NMR (500MHz, CD3OD) δ 8.18 (br, 3H), 4.48 (m, 1H), 2.14 (m, 1H), 1.73 (dd, Jz = 3.5, 16.5Hz 1H), 0.72 (s, 9H).

[0688] Example 175: Synthesis of 2-amino-5,5,5-trifluoro-4-methoxyvalerate [I-175]:

[0689]

[0690] Synthesis process:

[0691]

[0692] Program and features:

[0693] Step 1: (S)-4-methyl-2-(phenylmethylsulfonylamino)pentanoic acid methyl ester:

[0694] IBX (20.2 g, 72.29 mmol) was added to a solution of 3-(benzyloxy)prop-1-ol (10.0 g, 60.24 mmol) in DMSO (100 mL) under ice bath conditions. The mixture was heated to room temperature and stirred at that temperature for 17 hours. The reaction mixture was poured into water (300 mL) and extracted with EA (200 mL × 2). The organic phase was washed with water (200 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was concentrated. The crude product was purified by SGC to obtain a pale yellow liquid (8.0 g, 81%).

[0695] 1H NMR (500MHz, CDCl3) δ9.77(s,1H),7.36-7.26(m,5H),4.53(s,2H),3.8-3.83(m,2H),2.71-2.68(m,2H).

[0696] Step 2: (4-(benzoxy)-1,1,1-trifluorobut-2-yloxy)trimethylsilane:

[0697] At room temperature, trimethyl(trifluoromethyl)silane (10.4 g, 73.2 mmol) was added to a solution of 3-(benzoxy)propionaldehyde (4.0 g, 24.4 mmol) in THF (50 mL), followed by the addition of CsF (0.37 g, 2.44 mmol). The resulting solution was stirred at room temperature for 2 hours. The solution was then quenched with water (100 mL) and extracted with EA (100 mL × 2). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated. The crude product was purified by ISCO biotage to obtain a colorless liquid (4-(benzoxy)-1,1,1-trifluorobut-2-yloxy)trimethylsilane (4.5 g, 60%).

[0698] 1H NMR (500MHz, CDCl3) δ7.38-7.29(m,5H),4.51(t,J=12Hz,2H),4.23-4.19(m ,1H),3.59-3.57(m,2H),2.04-2.01(m,1H),1.78-1.73(m,1H),0.13(s,9H).

[0699] Step 3: 4-(benzooxy)-1,1,1-trifluorobut-2-ol:

[0700] A solution of 4-(benzoxy)-1,1,1-trifluorobut-2-ol (4.5 g, 14.7 mmol) in HCl solution (3 M in MeOH, 50 mL) was stirred for 2 hours at room temperature. The solution was then concentrated and purified by ISCO baitezid to obtain 4-(benzoxy)-1,1,1-trifluorobut-2-ol (2.75 g, 80%) as a colorless liquid.

[0701] Step 4: ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene:

[0702] At 0 °C, t-BuOK (1.58 g, 14.1 mmol) was added to a solution of 4-(benzyloxy)-1,1,1-trifluorobut-2-ol (2.75 g, 11.75 mmol) in THF (100 mL), and the mixture was stirred at the same temperature for 30 min. MeI (2.17 g, 15.28 mmol) was then added, and the mixture was stirred for another 1 h at room temperature. The reaction mixture was quenched with water (100 mL) and extracted with EA (100 mL × 2). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated. The crude mixture was purified by ISCO baitezid to obtain ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene (2.04 g, 70%) as a colorless liquid.

[0703] 1H NMR (500MHz, CDCl3) δ7.38-7.29(m,5H),4.53(t,J=12Hz,2H),3.78-3.74(m ,1H),3.66-3.57(m,2H),3.5(s,3H),2.03-1.96(m,1H),1.78-1.57(m,1H).

[0704] Step 5: 4,4,4-Trifluoro-3-methoxybut-1-ol:

[0705] A solution of ((4,4,4-trifluoro-3-methoxybutoxy)methyl)benzene (2.04 g, 8.23 ​​mmol) and Pd / C (0.5 g) in MeOH (30 mL) was stirred at room temperature for 2 hours, followed by filtration and concentration to obtain 4,4,4-trifluoro-3-methoxybut-1-ol as a colorless liquid. This crude substance was used directly in the next step.

[0706] Step 6: 4,4,4-Trifluoro-3-methoxybutyraldehyde:

[0707] Under ice bath conditions, IBX (2.76 g, 9.88 mmol) was added to a solution of 4,4,4-trifluoro-3-methoxybut-1-ol (1.3 g crude, from the last step) in DMSO (20 mL). The mixture was heated to room temperature and stirred at that temperature for 17 hours. The reaction mixture was poured into water (80 mL) and extracted with Et2O (80 mL × 2). The organic phase was washed with water (80 mL × 3) and brine (80 mL), and the solution was used directly for the next step.

[0708] Step 7: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentadienonitrile:

[0709] Under ice bath conditions, benzylamine (2 mL), AcOH (2.0 mL), and subsequently TMSCN (3 mL) were added to the above solution of 4,4,4-trifluoro-3-methoxybutyraldehyde in Et₂O (160 mL). The mixture was heated to room temperature and stirred at said temperature for 17 hours. The solution was diluted with water (200 mL) and extracted with EA (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentadienonitrile (2.0 g, crude substance) as a brown, thick, oily substance for the next step. ESI-MS (EI) + (m / z):

[0710] Step 8: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxyvalerate:

[0711] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentanilic acid (2.0 g, crude) in concentrated HCl (30 mL) and AcOH (10 mL) was heated to 100 °C for 17 hours. The solution was concentrated to dryness, diluted with H₂O (100 mL) and ACN (50 mL), and the pH was adjusted to 3 to 4 with saturated NaHCO₃ solution. The mixture was filtered and dried to give 0.8 g, 35%, 4 steps, as a brown solid of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxypentanilic acid. ESI-MS (EI + ,m / z):[M+H] + .

[0712] Step 9: 2-Amino-5,5,5-trifluoro-4-methoxyvalerate [I-175]:

[0713] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methoxyvalerate (300 mg, 1.03 mmol) and HCOONH4 (650 mg, 10.3 mmol) in MeOH (10 mL) was stirred at 60 °C for 2 hours, followed by filtration and concentration. The crude substance was purified by reverse-phase benzo[a]tezidine to obtain 2-amino-5,5,5-trifluoro-4-methoxyvalerate [I-175] as a white solid.

[0714] 1H NMR (500MHz, methanol-d4) δ 4.23-4.19 (m, 1H), 3.96-3.88 (m, 1H), 3.64-3.6 (m, 3H), 2.29-2.22 (m, 1H), 2.04-1.97 (m, 1H).

[0715] Example 176: 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0716]

[0717] Synthesis process:

[0718]

[0719] Program and features:

[0720] Step 1: Diethyl 2-(2,3-dimethylbut-2-yl)malonate:

[0721] A solution of diethyl 2-(propane-2-yl)malonate (2 g, 10.0 mmol) in THF (60 mL) was cooled to 0 °C, followed by copper iodide (I) (2.9 g, 15.0 mmol). The mixture was stirred at 0 °C for 0.5 h. Then, isopropyl magnesium bromide (1 mol / L, 30.0 mL, 30.0 mmol) was added dropwise to the mixture at 0 °C. The mixture was stirred at 0 °C for 2 h. The mixture was quenched with HCl (1 mol / L) and extracted with EtOAc (60 mL × 2). The organic phase was separated, washed with water (100 mL × 2) and brine (130 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give diethyl 2-(2,3-dimethylbutane-2-yl)malonate (2.4 g, 10.0 mmol, 98%) as a yellow solid. ESI-MS (EI) + ,m / z):245.3[M+H] + .

[0722] Step 2: 2-(2,3-Dimethylbut-2-yl)malonic acid:

[0723] A mixture of diethyl 2-(2,3-dimethylbut-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 hours. The mixture was cooled, acidified with HCl (1 mol / L), and partitioned between EtOAc (30 mL) and water (30 mL). The organic phase was separated, washed with water (50 mL × 2) and brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(2,3-dimethylbut-2-yl)malonate (1.8 g, 10.0 mmol, 95%) as a yellow oil. ESI-MS (EI) + ,m / z):212.2[M+H] + .

[0724] Step 3: 3,3,4-Trimethylvaleric acid:

[0725] A solution of 2-(2,3-dimethylbut-2-yl)malonic acid (1.8 g, 10.0 mmol) in DMSO (30 mL) was heated to 120 °C and maintained for 12 hours. The mixture was cooled and partitioned between EtOAc (50 mL) and water (60 mL). The organic phase was separated, washed with water (60 mL × 2) and brine (60 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 3,3,4-trimethylvaleric acid (1.4 g, 10.0 mmol, 95%) as a yellow oil. ESI-MS (EI-, m / z): 143.2 [MH]+ .

[0726] Step 4: N-methoxy-N,3,3,4-tetramethylpentanamide:

[0727] At 20 °C, N,O-dimethylhydroxylamine hydrochloride (1.2 g, 12.0 mmol) was added to a solution of 3,3,4-trimethylpentanoic acid (1.4 g, 10.0 mmol) in 30 mL of DMF, followed by DIEA (3.8 g, 30.0 mmol). Then HATU (5.8 g, 15.0 mmol) was added. The mixture was heated to 25 °C with stirring and maintained for 18 hours. The reaction mixture was quenched with water, followed by methyl tert-butyl ether (50 mL × 2). The phases were separated, the organic layer was washed with brine (80 mL × 3), dried over Na₂SO₄, filtered, and concentrated under vacuum 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] + .

[0728] Step 4: 3,3,4-Trimethylpentanal:

[0729] At 0°C, LiAlH4 (1 g, 0.03 mol) was added to a solution of N-methoxy-N,3,3,4-tetramethylpentanamide (1.9 g, 0.01 mol) in 30 mL of THF. The mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched with water, followed by methyl tert-butyl ether (50 mL × 2). The phases were separated, and the organic layer was washed with brine (80 mL × 3), dried over Na2SO4, and filtered. The filtrate contained 3,3,4-trimethylpentanal (1.3 g, 95%), which was used directly as a colorless solution in the next step.

[0730] Step 5: 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile:

[0731] Under ice bath conditions, benzylamine (1.6 mL), AcOH (1.0 mL), and subsequently TMSCN (1.8 mL) were added to the above solution of 3,3,4-trimethylpentanal in methyl tert-butyl ether (120 mL). The mixture was heated to 25 °C and stirred overnight. The solution was diluted with water (60 mL) and extracted with EtOAc (30 mL). The organic phase was washed with water (50 mL × 2) and brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile (2 g, crude) as a brown oil for the next step. ESI-MS (EI+, m / z): 245.4 [M+H] + .

[0732] Step 6: 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid:

[0733] A solution of 2-(phenylmethylamino)-4,4,5-trimethylhexanonitrile (2 g, crude) in concentrated HCl (60 mL) and AcOH (10 mL) was heated to 95 °C for 18 hours. The solution was then cooled to 15 °C, and the pH was adjusted to 3-4 with a saturated NaHCO3 solution. The mixture was filtered and dried to obtain 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid (0.6 g, 2.3 mmol, 30%, 3 steps) as a white solid. ESI-MS (EI...) + ,m / z):264.4[M+H] + .

[0734] 2-Amino-4,4,5-trimethylhexanoic acid [I-176]:

[0735] At room temperature, HCOONH4 (0.13 g, 2.0 mmol) and Pd / C (30 mg) were added to a solution of 2-(phenylmethylamino)-4,4,5-trimethylhexanoic acid (78 mg, 0.3 mmol) in 8 mL of MeOH. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give 2-amino-6,6,6-trifluoro-4-methylhexanoic acid [I-176] (40 mg, 90%) as a white solid; ESI-MS (EI... + ,m / z):174.3[M+H] + ;1H NMR (500MHz, MeOD) δ3.56 (dd, J = 7.2, 4.9 Hz, 1H), 2.12 (dd, J = 14.7, 4.9 Hz, 1H), 1.66-1.51 (m, 2H), 0.97 (d, J = 14.9 Hz, 6H), 0.92 (dd, J = 6.8, 3.6 Hz, 6H).

[0736] Example 178: 2-Amino-4,4-dimethylheptanoic acid [I-178]

[0737]

[0738] Synthesis process:

[0739]

[0740] Program and features:

[0741] The program is the same as that used in Example 176.

[0742] 2-Amino-4,4-dimethylheptanoic acid [I-178]:1 H NMR (500MHz, MeOD-d4) δ3.77 (t, J = 6Hz, 1H), 2.09-2.05 (m, 1H), 1.6-1.56 (m, 1H), 1.37-1.26 (m, 4H), 1.01-0.92 (m, 9H).

[0743] 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].

[0744]

[0745] Synthesis process:

[0746]

[0747] Program and features:

[0748] The program is the same as that used in Example 176.

[0749] 2-Amino-4,4-dimethylheptanoic acid [I-195]: 1 H NMR(500MHz,D2O)δ3.87(t,J=6.0Hz,1H),1.93(dd,J=15.0Hz,J=5.5Hz,1H),1.57(dd,J=1 5.0Hz, J=6.5Hz, 1H), 1.22-1.26 (m, 2H), 0.86 (d, (dd, J=2.0Hz, 6H), 0.76 (t, J=7.5Hz, 3H).

[0750] (S)-2-amino-4,4-dimethylhexanoic acid [I-120]: 1 H NMR (500MHz, MeOD-d4) δ3.43 (dd, J=7.0Hz, J=5.0Hz, 1H), 1.95 (dd, J=15.0Hz, J=5.0Hz, 1H), 1.42 ( dd,J=15.0Hz,J=7.0Hz,1H),1.23-1.28(m,2H),0.87(d,(dd,J=4.5Hz,6H),0.80(t,J=7.5Hz,3H).

[0751] (R)-2-amino-4,4-dimethylhexanoic acid [I-191]: 1H NMR (500MHz, MeOD-d4) δ3.43 (dd, J=7.0Hz, J=5.0Hz, 1H), 1.95 (dd, J=15.0Hz, J=5.0Hz, 1H), 1.42 ( dd,J=15.0Hz,J=7.0Hz,1H),1.23-1.28(m,2H),0.87(d,(dd,J=4.5Hz,6H),0.80(t,J=7.5Hz,3H).

[0752] Example 177: 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0753]

[0754] Synthesis process:

[0755]

[0756] Program and features:

[0757] Step 1: N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide:

[0758] A mixture of 2-chloro-N-methoxy-N-methylacetamide (13.7 g, 0.1 mol) and triphenylphosphine (26.2 g, 0.1 mol) in acetonitrile (200 mL) was heated to 80 °C and maintained for 20 hours. The mixture was cooled and concentrated to remove the solvent below 40 °C. The residue was dissolved in dichloromethane (200 mL), followed by 2N KOH (100 mL). The resulting mixture was stirred at 20 °C for 1 hour. The phases were separated, and the organic layer was washed with brine (200 mL × 3), dried over Na₂SO₄, and filtered. The filtrate was concentrated under vacuum to give N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide (36 g, 0.1 mol, 98%) as a yellow solid. ESI-MS (EI) + ,m / z):364.4[M+H] + .

[0759] Step 2: (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide:

[0760] A mixture of N-methoxy-N-methyl-2-(biphenyl-15-phosphine)acetamide (36.3 g, 0.1 mol) and 4,4,4-trifluorobutyl-2-one (25.2 g, 0.2 mol) in tetrahydrofuran (500 mL) was heated to 70 °C and maintained for 7 days. The mixture was cooled and concentrated to remove the solvent under vacuum at below 40 °C. The residue was purified by silica gel column (200 g, 200 to 300 mesh, UV 254 nm) eluted with 0 to 35% ethyl acetate / petroleum ether to give (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpent-2-enamide (6 g, 0.03 mol, 28%) as a yellow oil. ESI-MS (EI) + ,m / z):212.2[M+H] + .

[0761] Step 3: 5,5,5-Trifluoro-N-methoxy-N,3-dimethylpentanamide:

[0762] A mixture of (E)-5,5,5-trifluoro-N-methoxy-N,3-dimethylpentan-2-enamide (6 g, 0.03 mol) and Pd / C (10%, 400 mg) in THF (100 mL) was stirred at 30 °C for 18 hours. The mixture was filtered, and the filtrate was concentrated to dryness under vacuum 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] + .

[0763] Step 4: 5,5,5-Trifluoro-3-methylpentanal:

[0764] At 0 °C, LiAlH4 (1 g, 0.03 mol) was added to a solution of 5,5,5-trifluoro-N-methoxy-N,3-dimethylpentanamide (6 g, 0.03 mol) in 100 mL of THF. The mixture was stirred at 0 °C for 1 hour. The reaction mixture was quenched with water, followed by methyl tert-butyl ether (60 mL × 2). The phases were separated, and the organic layer was washed with brine (80 mL × 3), dried over Na2SO4, and filtered. The filtrate yielded 5,5,5-trifluoro-3-methylpentanal (4.5 g, 95%) as a colorless solution, which could be used directly in the next step.

[0765] Step 5: 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanenitrile:

[0766] Acetylmethane (5 mL), AcOH (4.0 mL), and subsequently TMSCN (5 mL) were added to the above solution of 5,5,5-trifluoro-3-methylpentanal in methyl tert-butyl ether (200 mL) under ice bath conditions. The mixture was heated to 20 °C and stirred overnight. The solution was diluted with water (100 mL) and extracted with EtOAc (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanenitrile (6 g, crude) as a brown oil for the next step. ESI-MS (EI+, m / z): 271.3 [M+H] + .

[0767] Step 6: 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanoic acid:

[0768] A solution of 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanonitrile (3 g, crude) in concentrated HCl (100 mL) and AcOH (20 mL) was heated to 100 °C for 17 hours. The solution was then cooled to 15 °C, and the pH was adjusted to 3-4 with a saturated NaHCO3 solution. The mixture was filtered and dried to give 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanoic acid (1 g, 13.4 mmol, 33%, 3 steps) as a white solid. ESI-MS (EI...) + ,m / z):290.3[M+H] + .

[0769] 2-Amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177]:

[0770] HCOONH4 (0.13 g, 2.0 mmol) and Pd / C (30 mg) were added to a solution of 2-(phenylmethylamino)-6,6,6-trifluoro-4-methylhexanoic acid (88 mg, 0.31 mmol) in 8 mL of MeOH at room temperature. The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to give a crude product, which was purified by reversed-phase silica gel chromatography to give 2-amino-6,6,6-trifluoro-4-methylhexanoic acid [I-177] (45 mg, 84%) as a white solid; ESI-MS (EI) + ,m / z):200.2[M+H] +;1H NMR (500MHz, DMSO) δ3.15(d,J=5.7Hz,1H),2.39-2.24(m,1H),2.19-1.96(m,2H),1.82-1.66(m,1H),1.63-1.35(m,1H),0.98(dd,J=16.5,6.2Hz,3H).

[0771] Example 179: (S)-2-amino-5-fluoro-4-(fluoromethyl)valerate [I-179]

[0772]

[0773] Synthesis process:

[0774]

[0775] Program and features:

[0776] Step 1: 5-(benzoxymethyl)-2,2-dimethyl-1,3-dioxane:

[0777] NaH (60% in oil, 0.12 g, 3.0 mmol) was added to a solution of (2,2-dimethyl-1,3-dioxane-5-yl)methanol (0.29 g, 2.0 mmol) in DMF (10 mL) at 0 °C. The mixture was stirred at 0 °C for 0.2 h. Subsequently, (bromomethyl)benzene (0.45 g, 2.6 mmol) was added. The mixture was heated to 10 °C and held for 3 h, then maintained for 18 h. The reaction mixture was quenched with ice water, followed by EtOAc (60 mL). The phases were separated, and the organic layer was washed with brine (60 mL × 3), dried over Na₂SO₄, and filtered. The filtrate was concentrated and the residue was purified by passing it through a silica gel column (20 g, UV 254 nm, eluting with 10% to 50% EtOAc / PE) to give 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane (1), (0.46 g, 0.2 mol, 95%), as a colorless oil. ESI-MS (EI + ,m / z):237.3[M+H] + .

[0778] Step 2: 2-(benzyloxymethyl)propane-1,3-diol:

[0779] To a solution of 5-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxane (930 mg, 3.94 mmol) in MeOH (20 mL), 2 mL of 3N HCl aqueous solution was added. The mixture was stirred at 50 °C for 2 hours. The reaction mixture was concentrated and diluted with DCM (20 mL), washed with brine (15 mL), dried, and evaporated to give a crude colorless oil (780 mg, 100%). ESI-MS (EI) + ,m / z):197[M+H] + .

[0780] Step 3: ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene:

[0781] DAST (1.9 g, 11.8 mmol) was added dropwise to a pre-cooled solution of 2-(benzyloxymethyl)propane-1,3-diol (780 mg, 3.94 mmol) in DCM (20 mL) at -78 °C. The mixture was stirred at 20 °C for 24 h. The reaction mixture was quenched at -78 °C with 10 mL of saturated aqueous NaHCO3 solution. The DCM phase was separated and washed with brine, dried over MgSO4, filtered through a short silica gel pad, and subsequently concentrated to give a crude colorless oil (800 mg, 100%). ESI-MS (EI) + ,m / z):223[M+Na] + . 1 H NMR (500MHz, CDCl3) δ7.37-7.28(m,5H), 4.65-4.57(m,2H), 4.55-4.48(m,4H), 3.57(d,J=6.2Hz,2H), 2.50-2.34(m,1H).

[0782] Step 4: 3-Fluoro-2-(fluoromethyl)prop-1-ol:

[0783] At -78°C, BCl3 / toluene (1M, 6 mL, 6.0 mmol) was added dropwise to a pre-cooled solution of ((3-fluoro-2-(fluoromethyl)propoxy)methyl)benzene (800 mg, 3.94 mmol) in DCM (20 mL). The mixture was stirred for 2 hours from -78°C to 0°C. The reaction mixture was quenched with H2O (0.5 mL) at -78°C. The DCM phase was dried over MgSO4, filtered, and the solution (approximately 20 mL) was used directly in the next step.

[0784] Step 5: 3-Fluoro-2-(fluoromethyl)propyl trifluoromethanesulfonate:

[0785] At -40°C, py (380 mg, 4.8 mmol) was added dropwise to a pre-cooled solution of 3-fluoro-2-(fluoromethyl)prop-1-ol (8 mL from the solution in step 4, 1.6 mmol), followed by Tf₂O (1.36 g, 4.8 mmol). The mixture was stirred at -30°C for 1 hour. The reaction mixture was quenched at -40°C with brine (20 mL). The DCM phase was separated and dried over MgSO₄, filtered, and subsequently concentrated to give a crude brown oil (200 mg, 51%) for direct use in the next step.

[0786] Step 6: 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)pentanoic acid tert-butyl ester:

[0787] LDA (2.5 M in THF / toluene / hexane, 1.28 mL, 3.2 mmol) was added to a pre-cooled solution of tert-butyl 2-(diphenylmethyleneamino)acetate (944 mg, 3.2 mmol) in THF (20 mL) at -78 °C over 25 minutes. The mixture was stirred for 10 minutes at this temperature. 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 directly on a cooling bath and stirred for another 1 hour. The reaction mixture was quenched with saturated aqueous NH4Cl solution (20 mL), extracted with MTBE (30 mL × 2), washed with H2O and brine (50 mL each), dried, and concentrated to give a crude substance, which was purified twice by chromatography (silica gel, PE to 5% EA / PE) to give the desired product (22 mg, 6.9%) as a white solid. ESI-MS (EI) + ,m / z):388[M+H] + . 1 H NMR (500MHz, DMSO) δ7.56-7.45(m,6H),7.41(t,J=7.4Hz,2H),7.18(d,J=6.3Hz,2H),4.50-4.17( m, 4H), 3.91 (dd, J = 7.7, 5.5Hz, 1H), 2.11-1.97 (m, 1H), 1.87 (dd, J = 12.7, 5.5Hz, 2H), 1.38 (s, 9H).

[0788] Step 7: (S)-2-amino-5-fluoro-4-(fluoromethyl)valerate:

[0789] A solution of tert-butyl 2-(diphenylmethyleneamino)-5-fluoro-4-(fluoromethyl)valerate (55 mg, 0.14 mmol) in 3N HCl / MeOH (2 mL) was stirred for 20 hours at room temperature. The reaction mixture was concentrated and washed with Et2O to obtain a crude solid, which was then dissolved in DCM / TFA (1:1, 2 mL) and stirred at room temperature for 20 hours. The reaction mixture was evaporated and washed with Et2O to obtain a crude solid, which was then dissolved in 6N HCl (1 mL) and stirred at 80 °C for 2 hours. The reaction mixture was evaporated and lyophilized to obtain a crude product, which was purified by RP-Bitaizine using 3 mM HCl / H2O to give the desired product (8.3 mg, 29%) as a white solid. ESI-MS (EI) + ,m / z):168[M+H] + . 1 H NMR (500MHz, DMSO) δ7.85 (bs, 3H), 4.48 (dd, J = 48.3, 14.2Hz, 4H), 3.46-3.36 (m,1H),2.47-2.26(m,1H),1.78(dt,J=14.3,7.3Hz,1H),1.63-1.53(m,1H).

[0790] Example 187: (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0791]

[0792] Synthesis process:

[0793]

[0794] Program and features:

[0795] Step 1: (S)-3-amino-5,5-dimethyl-dihydrofuran-2(3H)-one [I-187]:

[0796] Concentrated HCl (1 mL) and SOCl2 (0.2 mL) were added to a round-bottom flask containing (S)-2-amino-4-methylpentan-4-enoic acid (100 mg). The mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated and washed with Et2O to give a crude solid, which was purified by RP-Bitaizine using 0.025% TFA / H2O / MeCN to give the desired product (20.2 mg, 11.4%) as a white solid. ESI-MS (EI) + ,m / z):130.1[M+H] + . 1H NMR (500MHz, DMSO) δ8.80 (bs, 3H), 4.58 (dd, J = 11.2, 9.3Hz, 1H), 2.53-2.48 (m, 1H), 2.13 (t, J = 11.7Hz, 1H), 1.45 (s, 3H), 1.40 (s, 3H).

[0797] Example 90: Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-90]:

[0798]

[0799] Synthesis process:

[0800]

[0801] Program and features:

[0802] Step 1: Diethyl 2-(1,1,1-trifluoropropionic-2-ylidene)malonate:

[0803] TiCl4 (65.8 mL, 600 mmol) was added dropwise to THF (1 L) over an ice bath for 20 minutes, followed by CCl4 (30 mL). Diethyl malonate (48.0 g, 300 mmol) and 1,1-difluoroprop-2-one (56.4 g, 600 mmol) were then added to the mixture. The mixture was allowed to warm to room temperature and stirred overnight. Pyridine (200 mL) was added dropwise over 20 minutes in an ice bath. The reaction mixture was poured into water (2 L), filtered, and the filtrate was extracted with EtOAc (500 mL × 2). The organic phase was washed with water (600 mL), 1 M HCl (600 mL × 2), water (600 mL), saturated NaHCO3 (600 mL), and brine (600 mL), dried (Na2SO4), filtered, concentrated under vacuum, and purified by chromatography (silica, 0% to 5% ethyl acetate / petroleum ether) to give diethyl 2-(1,1-difluoropropionic-2-yl)malonate (60.9 g, 258 mmol, 86%) as a colorless liquid. ESI-MS (EI) + ,m / z):237.0[M+H] + . 1 H-NMR (500MHz, CDCl3): δ6.97 (t, J = 55.5Hz, 1H), 4.25-4.33 (m, 4H), 2.03 (s, 3H), 1.29-1.34 (m, 6H).

[0804] Step 2: Diethyl 2-(1,1-difluoro-2-methylprop-2-yl)malonate:

[0805] MeMgI (42.3 mL, 130.5 mmol) was added dropwise to a mixture of 2-(1,1-difluoro-2-methylpropion-2-yl)malonate (10.0 g, 42.3 mmol) and CuI (12.1 g, 63.5 mmol) in DCM (100 mL) and THF (25 mL) for 1 hour at -20 °C. The solution was poured into ice water (200 mL) and treated with 100 mL of saturated NH4Cl solution. The mixture was stirred for 30 minutes and filtered. The filtrate was extracted with DCM (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give a brown liquid of 2-(1,1-difluoro-2-methylpropion-2-yl)malonate (10.1 g, 40.2 mmol, 95%), which was used in the next step. ESI-MS (EI + ,m / z):253.1[M+H] + . 1 H-NMR (500MHz, CDCl3): δ6.05 (t, J=57.5Hz, 1H), 4.17-4.23 (m, 4H), 3.49 (s, 1H), 1.22-1.28 (m, 6H), 1.20 (s, 6H).

[0806] Step 3: 4,4-Difluoro-3,3-dimethylbutyric acid:

[0807] A mixture of diethyl 2-(1,1-difluoro-2-methylprop-2-yl)malonate (6.1 g, 24.2 mmol) and LiOH·H₂O (5.1 g, 121 mmol) in DMSO (50 mL) and H₂O (0.5 mL) was heated to 90 °C for 17 hours. The mixture was diluted with water (200 mL), extracted with DCM (100 mL), the pH of the aqueous phase was adjusted to 3-4 with 6 M HCl solution, extracted with DCM (100 mL × 2), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 4,4-difluoro-3,3-dimethylbutyric acid (3.6 g, crude product) as a brown liquid. ESI-MS (EI) + ,m / z):151.1[MH]-.

[0808] Step 4: 4,4-Difluoro-N-methoxy-N,3,3-trimethylbutyramide:

[0809] After stirring at room temperature for 17 hours, Et3N (7.18 g, 71.1 mmol) was added to a solution of 4,4-difluoro-3,3-dimethylbutyric acid (3.6 g, crude), N,O-dimethylhydroxylamine hydrochloride (4.6 g, 47.4 mmol), and HATU (10.8 g, 28.4 mmol) in DMF (50 mL). The mixture was filtered, and the filtrate was diluted with water (200 mL), extracted with Et2O (100 mL × 2), washed with water (100 mL), 1 M HCl (100 mL), and brine (100 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give a brown liquid of 4,4-difluoro-N-methoxy-N,3,3-trimethylbutyramide (3.1 g, 15.9 mmol, 66%, 2 steps). ESI-MS (EI) + ,m / z):196.0[M+H] + . 1 H-NMR (500MHz, CDCl3): δ5.95 (t, J=57.5Hz, 1H), 3.69 (s, 3H), 3.17 (s, 3H), 2.51 (s, 2H), 1.12 (s, 6H).

[0810] Step 5: 4,4-Difluoro-3,3-dimethylbutanal:

[0811] Under ice bath conditions, LiAlH4 (24 mL, 24 mmol) was added dropwise to a solution of 4,4-difluoro-N-methoxy-N,3,3-trimethylbutyramide (3.1 g, 15.9 mmol) in THF (80 mL). After 1 hour, the mixture was quenched with citric acid solution (100 mL), the solution was extracted with Et2O (100 mL × 2), the organic phase was washed with brine (100 mL), dried (Na2SO4), and the solution was used for the next step.

[0812] Step 6: 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanilonitrile:

[0813] Under ice bath conditions, benzylamine (3 mL), AcOH (3 mL), and subsequently TMSCN (3 mL) were added to the above solution of 4,4-difluoro-3,3-dimethylbutanal in Et₂O (200 mL). The solution was stirred at 0 to room temperature for 17 hours and then diluted with EtOAc (100 mL). The solution was washed with H₂O (100 mL × 2) and then concentrated to give 3.2 g of crude 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanilide as a brown liquid. ESI-MS (EI) + ,m / z):253.0[M+H] + .

[0814] Step 7: 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanoic acid:

[0815] A solution of 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanonic acid (1.8 g, crude) in concentrated HCl (50 mL) and AcOH (10 mL) was heated to 100 °C for 64 hours. The mixture was concentrated to remove the solvent, the pH was adjusted to 12 with 1 M NaOH solution, and extracted with PE (100 mL). The aqueous phase was adjusted to pH 5 to 6 with 6 M HCl. A white solid was formed, filtered, and the filter cake was washed with water (50 mL) and dried under vacuum to give 2-(phenylmethylamino)-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

[0816] Step 8: 2-Amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0817] A mixture of 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (1.3 g, 4.80 mmol), HCOONH4 (1.51 g, 24 mmol), and Pd / C (10%, 200 mg) in MeOH (50 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated to give 2-amino-5,5-difluoro-4,4-dimethylpentanoic acid (1.0 g, crude substance) as a white solid. ESI-MS (EI) + ,m / z):182.0

[0818] Step 9: 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid

[0819] CbzOSu (2.39 g, 9.6 mmol) was added to a solution of 2-amino-5,5-difluoro-4,4-dimethylvaleric acid (1.0 g, crude substance) and NaHCO3 (1.27 g, 14.4 mmol) in acetone (30 mL) and H2O (30 mL) under ice bath conditions. After stirring for 17 hours, the mixture was adjusted to pH 3-4 with 1M HCl solution, and the solution was extracted with EtOAc (50 mL × 2), washed with brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by reversed-phase silica gel chromatography followed by chiral preparative HPLC [column, CC₄ 4.6 × 250 mm 5 μm; solvent, MeOH (0.2% methanol-ammonia)] to give (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid (400 mg, 1.27 mmol, 26%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid (380 mg, 1.21 mmol, 25%, 2 steps) as two colorless oils. ESI-MS (EI) + ,m / z):316.0

[0820] Step 10: (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid:

[0821] At room temperature, a solution of (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid (400 mg, 1.27 mmol) and Pd / C (10%, 50 mg) in MeOH (30 mL) was stirred under hydrogen atmosphere for 2 hours. The mixture was filtered, concentrated under vacuum, and purified by reversed-phase silica gel chromatography to give (S)-2-amino-5,5-difluoro-4,4-dimethylvaleric acid (115.7 mg, 0.64 mmol, 50%). ESI-MS (EI... + ,m / z):182.0 1 H-NMR (500MHz, MeOD-d4): δ5.60(t,J=56.5Hz,1H),3.97(t,J=6.0Hz,1H),2.07(dd , J=15.5Hz, J=5.5Hz, 1H), 1.77 (dd, J=15.5Hz, J=6.5Hz, 1H), 0.96 (d, J=9.5Hz, 6H).

[0822] Example 88: Synthesis of (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid [I-88]:

[0823]

[0824] Synthesis process:

[0825]

[0826] Program and features:

[0827] Step 1: (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide:

[0828] Under ice bath conditions, concentrated H2SO4 (10 mL) was added dropwise to a solution of 2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanonitrile (1.2 g, 4.76 mmol) in DCM (20 mL) over 5 minutes, and the mixture was heated to room temperature and stirred for 6 hours. The mixture was poured into ice water (100 mL), the pH of the solution was adjusted to 8-9 with 10% NaOH solution, and then extracted with EtOAc (100 mL × 2). The organic phase was washed with water (100 mL) and brine (100 mL), dried (Na₂SO₄), filtered, concentrated under vacuum, and purified by chromatography (0% to 5% MeOH / DCM) followed by chiral preparative HPLC [column, CC₄ 4.6 × 250 mm 5 μm; solvent, MeOH (0.2% methanol-ammonia)] to give (S)-2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanamide (400 mg, 1.48 mmol, 31%) and (R)-2-(phenylmethylamino)-5,5-difluoro-4,4-dimethylpentanamide (380 mg, 1.41 mmol, 30%) as two colorless liquids. ESI-MS (EI) + ,m / z):253.0[M+H] + .

[0829] Step 2: (S)-2-amino-5,5-difluoro-4,4-dimethylpentanamide:

[0830] A mixture of (S)-2-(benzylamino)-5,5-difluoro-4,4-dimethylpentanamide (200 mg, 0.74 mmol), HCOONH4 (233 mg, 3.7 mmol), and Pd / C (10%, 40 mg) in MeOH (15 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give (S)-2-amino-5,5-difluoro-4,4-dimethylpentanamide trifluoroacetic acid (128 mg, 0.44 mmol, 59%) as a white solid. ESI-MS (EI) + ,m / z):181.0[M+H] + . 1H-NMR (500MHz, MeOD-d4): δ5.66 (t, J=56.5Hz, 1H), 3.95 (dd, J=8.0Hz, J=5.0Hz, 1H), 2.1 4(dd, J=10.0Hz, J=8.0Hz, 1H), 1.83 (dd, J=14.5Hz, J=5.5Hz, 1H), 1.12 (d, J=15.0Hz, 6H).

[0831] Example 185: Synthesis of methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate [I-185]:

[0832]

[0833] Synthesis process:

[0834]

[0835] Program and features:

[0836] The procedure for 2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylvaleric acid is the same as in Example 90.

[0837] Step 1: Methyl (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate:

[0838] A solution of (S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid (150 mg, 0.476 mmol), HATU (199 mg, 0.524 mmol), (S)-2-amino-4-methylpentanoic acid methyl ester hydrochloride (104 mg, 0.571 mmol), and DIPEA (123 mg, 0.952 mmol) was stirred for 1 hour at room temperature, followed by quenching with ice water (20 mL), extraction with EA (2 × 30 mL), drying, filtration, and concentration. The crude substance was purified by reversed-phase silica gel chromatography using a tyrosine ester to obtain (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid)-4-methylpentanoic acid methyl ester (95 mg, 45%) as a white solid. ESI-MS (EI) + ,m / z):443.0

[0839] Step 2: Methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate:

[0840] A solution of methyl (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate (95 mg, 0.215 mmol) and Pd / C (30 mg) in THF (5 mL) was stirred for 2 hours at room temperature, followed by filtration and concentration. The crude substance was purified by reversed-phase silica gel chromatography using a tyrosine ester to obtain methyl (S)-2-((S)-2-amino-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate (45 mg, 69%) as a white solid. ESI-MS (EI + ,m / z):309.0

[0841] 1 H-NMR (500MHz, DMSO-d6):9.11(d,J=7Hz,1H),8.41(s,3H),5.81(t,J=56.5Hz,1H),4.34-4.31(m,1H),3.8 9-3.81(m,1H),3.62(s,3H),1.98-1.93(m,1H),1.76-1.73(m,1H),1.65-1.54(m,3H),0.93-0.81(m,12H).

[0842] Example 184: Synthesis of methyl (S)-2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate [I-184]:

[0843]

[0844] The program is the same as examples 90 and 185.

[0845] Methyl (S)-2-((R)-2-amino-5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate: ESI-MS (EI + ,m / z):309.0

[0846] 1 H-NMR (500MHz, DMSO-d6):9.18(d,J=7Hz,1H),8.38(s,3H),5.79(t,J=56.5Hz,1H),4.37-4.32(m,1H),3.8 5-3.78(m,1H),3.58(s,3H),1.97-1.92(m,1H),1.77-1.72(m,1H),1.61-1.51(m,3H),0.94-0.82(m,12H).

[0847] Example 145: Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid, (2R,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid, (2R,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid and (2S,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid: [3d; I-145]; [3c; I-146]; [3a; I-167]; [3b; I-250]

[0848]

[0849] Synthesis process:

[0850]

[0851] Program and features:

[0852] Step 1: Synthesis of (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid, (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid, (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid and (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid:

[0853] CbzOSu (970 mg, 3.9 mmol) was added to a solution of 2-amino-5,5,5-trifluoro-4-methylpentanoic acid (600 mg, 3.2 mmol) in acetone (10 mL) and saturated aqueous solution of NaHCO3 (10 mL). The mixture was stirred at room temperature for 3 hours. Subsequently, EtOAc (20 mL) and H2O (20 mL) were added, the aqueous solution was separated and further extracted with EtOAc (2 × 20 mL), the extracts were combined and washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated, and the residue was purified by preparative HPLC to give 2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylpentanoic acid (750 mg) as a white solid. The product was purified by chiral HPLC to yield four isomers: (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (150 mg, 15%), (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (40 mg, 3.9%), (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (50 mg, 4.9%), and (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (80 mg, 7.8%), all of which were white solids. ESI-MS (EI+, m / z): 342.0 [M+Na]+.

[0854] Step 2-A: Synthesis of (2S,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0855] A solution of (2S,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (150 mg, 0.47 mmol) and Pd / C (75 mg) in MeOH (15 mL) was stirred for 3 hours at room temperature. The reaction mixture was filtered and concentrated to give (2S,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (51.7 mg, 59%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + . 1 H-NMR (500MHz, MeOD): δ 3.64-3.60 (m, 1H), 2.77-2.71 (br, 1H), 2.24-2.18 (m, 1H), 1.76-1.69 (m, 1H), 1.25 (d, J = 7.0Hz, 3H).

[0856] Step 2-B: Synthesis of (2R,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0857] A solution of (2R,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (40 mg, 0.12 mmol) and Pd / C (20 mg) in MeOH (4 mL) was stirred for 3 hours at room temperature. The reaction mixture was filtered and concentrated to give (2R,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (13.3 mg, 60%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + . 1 H-NMR (500MHz, MeOD): δ3.51-3.47(m,1H), 2.64-2.58(br,1H), 2.12-2.06(m,1H), 1.63-1.57(m,1H), 1.13(d,J=7.0Hz,3H).

[0858] Step 2-C: Synthesis of (2R,4R)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0859] A solution of (2R,4R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (50 mg, 0.16 mmol) and Pd / C (25 mg) in MeOH (5 mL) was stirred for 3 hours at room temperature. The reaction mixture was filtered and concentrated to give (2R,4R)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (18.0 mg, 61%) as a white solid. ESI-MS (EI) + ,m / z):186.1[M+H] + . 1 H-NMR (500MHz, MeOD): δ3.51-3.47(m,1H), 2.46-2.44(br,1H), 1.95-1.87(m,2H), 1.11(d,J=7.0Hz,3H).

[0860] Step 2-D: Synthesis of (2S,4S)-2-amino-5,5,5-trifluoro-4-methylpentanoic acid:

[0861] A solution of (2S,4S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-methylvaleric acid (80 mg, 0.25 mmol) and Pd / C (40 mg) in MeOH (8 mL) was stirred for 3 hours at room temperature. The reaction mixture was filtered and concentrated to give (2S,4S)-2-amino-5,5,5-trifluoro-4-methylvaleric acid (38.1 mg, 82%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + .1 H-NMR (500MHz, MeOD): δ3.51-3.47(m,1H), 2.46-2.44(br,1H), 1.95-1.87(m,2H), 1.11(d,J=7.0Hz,3H).

[0862] Example 128: (S)-2-amino-5,5,5-trifluoro-4,4-dimethylvaleric acid (I-128):

[0863]

[0864] Synthesis process:

[0865]

[0866] Program and features:

[0867] The program used is the same as that used in Example 187.

[0868] (S)-2-amino-5,5,5-trifluoro-4,4-dimethylvaleric acid: ESI-MS (EI + ,m / z):200.1 1 H-NMR (500MHz, D2O): δ3.94 (t, J=5.5Hz, 1H), 2.23 (dd, J=15.5Hz, J=5.5Hz, 1H), 1.90 (dd, J=15.5Hz, J=6.0Hz, 1H), 1.13 (d, J=8.5Hz, 6H).

[0869] Example 188: (S)-2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate methyl ester [I-188]:

[0870]

[0871] Synthesis process:

[0872]

[0873] Program and features:

[0874] The procedure for 2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylvaleric acid is the same as in Example 90.

[0875] Step 1: Methyl (S)-2-((R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate:

[0876] A solution of (S)-2-(benzyloxycarbonylamino)-5,5,5-difluoro-4,4-dimethylpentanoic acid (150 mg, 0.45 mmol), HATU (188 mg, 0.495 mmol), (S)-2-amino-4-methylpentanoic acid methyl ester hydrochloride (123 mg, 0.675 mmol), and DIPEA (175 mg, 1.35 mmol) was stirred at room temperature for 1 hour, followed by quenching with ice water (20 mL), extraction with EA (2 × 30 mL), drying, filtration, and concentration. The crude substance was purified by reversed-phase silica gel chromatography using a tyrosine ester to obtain (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5-difluoro-4,4-dimethylpentanoic acid)-4-methylpentanoic acid methyl ester (120 mg, 58%) as a white solid. ESI-MS (EI) + ,m / z):461.0

[0877] Step 2: Methyl (S)-2-((R)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate:

[0878] A solution of methyl (S)-2-((S)-2-(benzyloxycarbonylamino)-5,5,5-difluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate (120 mg, 0.26 mmol) and Pd / C (30 mg) in THF (10 mL) was stirred at room temperature for 2 hours, followed by filtration and concentration. The crude substance was purified by reversed-phase silica gel chromatography using a tyrosine ester to obtain methyl (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate (49 mg, 57%) as a white solid. ESI-MS (EI) + ,m / z):326.0

[0879] 1 H-NMR(500MHz,MeOD-d4):4.47(t,J=7.5Hz,1H),3.99-3.97(m,1H),3.77(s,3H),2.33-2 .28(m,1H),1.95-1.91(m,1H),1.69-1.68(m,3H),1.24-1.17(m,6H),1.00-0.94(m,6H).

[0880] Example 189: Methyl (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate [I-189]:

[0881]

[0882] Synthesis process:

[0883] The program used is the same as that used in Example 188.

[0884] Program and features:

[0885] Example 189: Methyl (S)-2-((S)-2-amino-5,5,5-trifluoro-4,4-dimethylpentanoylamino)-4-methylpentanoate [I-189]: 1 H-NMR(500MHz,MeOD-d4):4.52(t,J=7.5Hz,1H),4.02-3.99(m,1H),3.73(s,3H),2.35-2.30 (m,1H),1.95-1.91(m,1H),1.78-1.67(m,3H),1.25(s,3H),1.17(s,3H),1.01-0.97(m,6H).

[0886] Example 108: (S)-2-amino-6-fluorohexanoic acid [I-108].

[0887]

[0888] Example 109: (R)-2-amino-6-fluorohexanoic acid [I-109].

[0889]

[0890] Synthesis process:

[0891]

[0892] Program and features:

[0893] Step 1: 2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester:

[0894] A mixture of 1-fluoro-4-iodobutane (2.0 g, 9.90 mmol), 2-(diphenylmethyleneamino)-tert-butyl acetate (2.43 g, 8.25 mmol), TBAB (266 mg, 0.83 mmol), and KOH (50% aqueous solution) (10 mL) in DCM (10 mL) and toluene (25 mL) was stirred at 50 °C for 16 hours. The solution was purified by SGC (silica, ethyl acetate / petroleum ether = 1 / 5) to give 2-(diphenylmethyleneamino)-6-fluorohexanoate-tert-butyl ester (0.91 g, 2.47 mmol, 30%) as a colorless oil. MS (EI+, m / z): 370.2 [M+H] + .

[0895] Step 2: (S)-2-amino-6-fluorohexanoic acid [I-108]:

[0896] A solution of tert-butyl (S)-2-(diphenylmethyleneamino)-6-fluorohexanoate (360 mg, 0.97 mmol) was stirred in dioxane (10 mL) and HCl (6 M aqueous solution) for 16 hours at room temperature. The mixture was extracted with ether and water. The aqueous layer was extracted with EA after pH adjustment to 3-4. The organic layer was concentrated to give (S)-2-amino-6-fluorohexanoic acid [I-108] (125 mg, 0.84 mmol, 86%) as a white solid. ESI-MS (EI+, m / z): 150.3 [M+H] + . 1H NMR(500MHz,D2O)δ4.469(t,J=6.0Hz,1H),4.351(t,J=6.0Hz,1H),3.950(t,J =6.0Hz,1H),1.904-1.820(m,2H),1.690-1.588(m,2H),1.456-1.388(m,2H).

[0897] Step 2: (R)-2-amino-6-fluorohexanoic acid [I-109]:

[0898] A solution of (R)-2-(diphenylmethyleneamino)-6-fluorohexanoate tert-butyl ester (300 mg, 0.81 mmol) in dioxane (10 mL) and HCl (6 M aqueous solution) was stirred at room temperature for 16 hours. The mixture was extracted with ether and water. The aqueous layer was extracted with EA after pH adjustment to 3-4. The organic layer was purified by HPLC to give (R)-2-amino-6-fluorohexanoic acid [I-109] (35 mg, 0.23 mmol, 29%) as a white solid. ESI-MS (EI+, m / z): 150.2 [M+H] + . 1H NMR(500MHz,D2O)δ4.505(t,J=6.0Hz,1H),4.410(t,J=6.0Hz,1H),3.823(t,J =6.0Hz,1H),1.906-1.827(m,2H),1.722-1.639(m,2H),1.485-1.399(m,2H).

[0899] Example 198: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (I-198):

[0900]

[0901] Synthesis process:

[0902]

[0903] Program and features:

[0904] Step 1: 4,4,4-Trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide:

[0905] Over a period of 1 hour, concentrated H₂SO₄ (100 mL) was slowly added dropwise to a stirred solution of hexafluoroacetone trihydrate (30 g, 136 mmol), while gaseous hexafluoroacetone was introduced into a solution of N-methoxy-N-methyl-2-(triphenylphosphine)-acetamide (10 g, 27.5 mmol) in 200 mL of THF. The mixture was stirred at room temperature for 16 hours. Petroleum ether (200 mL) was then added, and a white precipitate was filtered off. The filtrate was concentrated, and the residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 5 / 1 to 3 / 1) to give 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide (6.2 g, 24.7 mmol, 90%) as a pale oil. ESI-MS (EI) + ,m / z):252.1[M+H] + . 1 H-NMR (500MHz, CDCl3): δ7.15(s,1H),3.67(s,3H),3.26(s,3H).

[0906] Step 2: 4,4,4-Trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butyramide:

[0907] A mixture of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)but-2-enamide (4.5 g, 17.9 mmol), Pd(OH)₂ / C (620 mg), and MeOH (100 mL) was stirred at room temperature under a hydrogen atmosphere for 16 hours. The mixture was then filtered and concentrated to give 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butanamide (1.8 g, 7.1 mmol, 40%) as a pale oil. ESI-MS (EI) was used to analyze the mixture. + ,m / z):254.1[M+H] + .

[0908] Step 3: 4,4,4-Trifluoro-3-(trifluoromethyl)butanal:

[0909] Under ice bath conditions, LiAlH4 (8.5 mL, 8.5 mmol) was added dropwise to a solution of 4,4,4-trifluoro-N-methoxy-N-methyl-3-(trifluoromethyl)butyramide (1.8 g, 7.1 mmol) in THF (50 mL). After 1 hour, the mixture was quenched with citric acid solution (100 mL), the solution was extracted with Et2O (100 mL × 2), the organic phase was washed with brine (100 mL), dried (Na2SO4), and the solution was used for the next step.

[0910] Step 4: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanilonitrile:

[0911] Under ice bath conditions, benzylamine (2 mL), AcOH (2 mL), and subsequently TMSCN (2 mL) were added to the above solution of 4,4,4-trifluoro-3-(trifluoromethyl)butanal in Et₂O (200 mL). The solution was stirred at 0 to room temperature for 17 hours and then diluted with EtOAc (100 mL). The solution was washed with H₂O (100 mL × 2) and then concentrated to give 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)pentanilonitrile (2.1 g, crude substance) as a brown liquid. ESI-MS (EI) + ,m / z):311.2[M+H] + .

[0912] Step 5: 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0913] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (2.1 g, crude) in concentrated HCl (50 mL) and AcOH (10 mL) was heated to 100 °C for 40 hours. The mixture was concentrated to remove the solvent, the pH was adjusted to 12 with 1 M NaOH solution, extracted with PE (100 mL), and the pH of the aqueous phase was adjusted to 5 to 6 with 6 M HCl to form a white solid. The solid was filtered, and the filter cake was washed with water (50 mL) and dried under vacuum to give 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (1.0 g, 3.0 mmol, 42%, 3 steps) as a white solid. ESI-MS (EI) + ,m / z):272.0

[0914] Step 6: Methyl 2-(benzylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0915] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (800 mg, 2.4 mmol) in HCl / MeOH (50 mL, 2 M) was heated to 75 °C for 17 hours. The solution was concentrated and purified by preparative HPLC (Boston C1821 × 250 mm 10 μm mobile phase: A: 0.1% TFA; B: ACN) to give methyl 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (120 mg, 0.35 mmol, 15%) as a colorless oil. ESI-MS (EI) + ,m / z):344.1[M+H]+ .

[0916] Step 7: Methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate in trifluoroacetic acid:

[0917] A mixture of methyl 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (100 mg, 0.30 mmol), HCOONH4 (92 mg, 1.5 mmol), and Pd / C (10%, 20 mg) in MeOH (10 mL) was heated to 65 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give methyl 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate trifluoroacetic acid (76 mg, 0.21 mmol, 70%) as a white solid. ESI-MS (EI) + ,m / z):254.1[M+H] + . 1 H NMR (500MHz, MeOD-d4) δ4.26 (dd, J=7.5Hz, J=6.0Hz, 1H), 3.91 (m, 4H), 2.49 (dd, J=8.5Hz, J=5.0Hz, 1H), 2.33-2.37 (m, 1H).

[0918] Example 164: (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (I-164):

[0919]

[0920] Synthesis process:

[0921]

[0922] Program and features:

[0923] Step 1: 2-Amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0924] A solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (480 mg, 1.46 mmol) and Pd(OH)₂ / C (20%, 100 mg) in AcOH (15 mL) was stirred at 35 °C under hydrogen atmosphere for 17 hours. The mixture was filtered and the filtrate was concentrated under vacuum to give 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (460 mg, crude product) as a white solid. ESI-MS (EI) + ,m / z):240.2[M+H] + .

[0925] Step 2: (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0926] CbzOSu (727 mg, 2.92 mmol) was added to a solution of 2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate (460 mg, crude) and NaHCO3 (368 mg, 4.38 mmol) in acetone (30 mL) and H2O (30 mL) under ice bath conditions. After 17 hours, the pH of the reaction mixture was adjusted to 3-4 with 1M HCl solution, and the solution was extracted with EtOAc (50 mL × 2), washed with brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by reversed-phase silica gel chromatography followed by chiral preparative HPLC [column, CC4 4.6 × 250 mm 5 μm; solvent, MeOH (0.2% methanol-ammonia)] to give (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (27 mg, 0.072 mmol, 5%, 2 steps) and (R)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (22 mg, 0.059 mmol, 4%, 2 steps) as two colorless oils. ESI-MS (E I+ ,m / z):396.0[M+Na] + .

[0927] Step 3: (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate:

[0928] A mixture of (S)-2-(benzyloxycarbonylamino)-5,5,5-trifluoro-4-(trifluoromethyl)valerate (27 mg, 0.072 mmol) and Pd / C (10%, 5 mg) in MeOH (10 mL) was stirred for 1 hour at room temperature. The solution was filtered and purified by reversed-phase silica gel chromatography to give (S)-2-amino-5,5,5-trifluoro-4-(trifluoromethyl)valerate [I-164] (8.5 mg, 0.036 mmol, 49%) as a white solid. MS (EI) + ,m / z):240.2[M+H] + . 1 H NMR(500MHz,D2O)δ3.74-3.80(m,2H),2.88-2.31(m,1H),1.91-2.20(m,1H).

[0929] Example 203: 2-Amino-4-cyclopentylbutyric acid [I-203]:

[0930]

[0931] Synthesis process:

[0932]

[0933] Program and features:

[0934] Step 1: 2-Cyclopentylacetaldehyde:

[0935] Under ice bath conditions, IBX (7.35 g, 26.3 mmol) was added to a solution of 3-cyclopentylprop-1-ol (2.0 g, 17.5 mmol) in DMSO (40 mL). The mixture was heated to room temperature and stirred overnight. The reaction mixture was poured into water (200 mL) and extracted with Et₂O (100 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was used for the next step.

[0936] Step 2: (Z)-2-(tert-Butoxycarbonylamino)-4-cyclopentylbut-2-enoic acid tert-butyl ester:

[0937] Under ice bath conditions, NaOt-Bu (785 mg, 8.2 mmol) was added to a solution of Viti reagent (2.5 g, 6.8 mmol) in THF (50 mL). After 1 hour, 2-cyclopentylacetaldehyde was added to the above solution in Et2O (200 mL). The mixture was heated to room temperature and stirred overnight. The solution was diluted with water (200 mL) and extracted with EA (100 mL × 2). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, concentrated under vacuum, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 20) to give (Z)-2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoic acid tert-butyl ester (1.0 g, 3.1 mmol, 45%, 2 steps) as a colorless liquid. ESI-MS (EI) + ,m / z):326.2[M+H] + .

[0938] Step 3: 2-(tert-Butoxycarbonylamino)-4-cyclopentylbutyrate tert-butyl ester:

[0939] A mixture of (Z)-2-(tert-butoxycarbonylamino)-4-cyclopentylbut-2-enoate tert-butyl ester (240 mg, 0.74 mmol), HCOONH4 (233 mg, 3.7 mmol), and Pd / C (10%, 30 mg) in MeOH (15 mL) was heated to reflux for 4 hours. The mixture was filtered and concentrated, diluted with Et2O (50 mL), washed with water (50 mL) and brine (50 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give 2-(tert-butoxycarbonylamino)-4-cyclopentylbutyrate tert-butyl ester (224 mg, 0.69 mmol, 93%) as a colorless liquid. ESI-MS (EI) + ,m / z):328.2[M+H] + .

[0940] Step 4: 2-Amino-4-cyclopentylbutyric acid:

[0941] A solution of tert-butyl 2-(tert-butoxycarbonylamino)-4-cyclopentylbutyrate (224 mg, 0.69 mmol) in 6 M HCl (20 mL) and dioxane (10 mL) was heated to 70 °C for 2 hours. The mixture was concentrated under vacuum, diluted with water (30 mL), extracted with Et₂O (20 mL × 2), and the filtrate was concentrated to dryness to give 2-amino-4-cyclopentylbutyric acid (114.9 mg, 0.52 mmol, 81%) as a white solid. ESI-MS (EI) + ,m / z):172.3[M+H] + . 1 H-NMR (500MHz, D2O): δ3.91 (t, J=6.0Hz, 1H), 1.82-1.89 (m, 2H), 1.66-1.72 (m, 3H), 1.28-1.52 (m, 6H), 1.00-1.01 (m, 2H).

[0942] Example 202: 2-Amino-5-cyclopentylpentanoic acid [I-202]:

[0943]

[0944] Synthesis process:

[0945]

[0946] Program and features:

[0947] Step 1: 3-Cyclopentylpropionaldehyde:

[0948] Under ice bath conditions, IBX (3.28 g, 11.7 mmol) was added to a solution of 3-cyclopentylprop-1-ol (1.0 g, 7.8 mmol) in DMSO (20 mL). The mixture was heated to room temperature and stirred overnight. The reaction mixture was poured into water (100 mL) and extracted with Et₂O (60 mL × 2). The organic phase was washed with water (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and the solution was used for the next step.

[0949] Step 2: (E)-2-(tert-Butoxycarbonylamino)-5-cyclopentylpent-2-enoic acid tert-butyl ester:

[0950] Under ice bath conditions, NaOt-Bu (157 mg, 1.63 mmol) was added to a solution of Viti reagent (500 mg, 1.36 mmol) in THF (15 mL). After 1 hour, 3-cyclopentylpropanal was added to the above solution in Et2O (100 mL). The mixture was heated to room temperature and stirred overnight. The solution was diluted with water (200 mL) and extracted with EtOAc (100 mL). The organic phase was washed with water (100 mL × 2) and brine (100 mL), dried (Na2SO4), filtered, concentrated under vacuum, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 20) to give (E)-2-(tert-butoxycarbonylamino)-5-cyclopentylpent-2-enoic acid tert-butyl ester (250 mg, 0.74 mmol, 9.5%, 2 steps) as a colorless liquid. ESI-MS (EI) + ,m / z):340.2[M+H] + .

[0951] Step 3: 2-(tert-Butoxycarbonylamino)-5-cyclopentylvaleric acid tert-butyl ester:

[0952] 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 for 17 hours at room temperature under hydrogen atmosphere. 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] + .

[0953] Step 4: 2-Amino-5-cyclopentylpentanoic acid:

[0954] A solution of 2-(tert-butoxycarbonylamino)-5-cyclopentylpentanoic acid ester (250 mg, 0.73 mmol) in 6M HCl (20 mL) and dioxane (10 mL) was heated to 80 °C for 5 hours. The mixture was concentrated under vacuum, diluted with water (30 mL), extracted with Et₂O (20 mL × 2), and the filtrate was concentrated to dryness to give 2-amino-5-cyclopentylpentanoic acid (115 mg, 0.52 mmol, 71%) as a white solid. ESI-MS (EI) + ,m / z):186.2[M+H] + . 1 H-NMR (400MHz, D2O): δ3.84(t,J=6.0Hz,1H),1.79-1.84(m,2H),1.61-1.67(m,3H),1.25-1.49(m,8H),0.95-0.99(m,2H).

[0955] Example 197: Synthesis of 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide [I-197]:

[0956]

[0957] Synthesis process:

[0958]

[0959] Program and features:

[0960] Step 1: 2-(phenylmethylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0961] A mixture of 2-(phenylmethylamino)-3,3-difluoro-4-methylpentanoic acid (80 mg, 0.31 mmol), N-methylcyclopentanamide (62 mg, 0.62 mmol), HATU (141 mg, 0.37 mmol), and Et3N (94 mg, 0.93 mmol) in DMF (2 mL) was stirred at room temperature for 3 hours. The mixture was purified by preparative HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% TFA; B: ACN) to give 2-(phenylmethylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (45 mg, 0.13 mmol, 43%) as a white solid. ESI-MS (EI) + ,m / z):339.0

[0962] Step 2: 2-Amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide:

[0963] A mixture of 2-(benzylamino)-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (45 mg, 0.13 mmol), HCOONH4 (41 mg, 0.65 mmol), and Pd / C (10%, 10 mg) in MeOH (5 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give 2-amino-N-cyclopentyl-3,3-difluoro-N,4-dimethylpentanamide (16.3 mg, 0.066 mmol, 49%) as a white solid. ESI-MS (EI) + ,m / z):249.2 1HNMR (500MHz, MeOD-d4) δ5.26 (dd, J=15.5Hz, J=6.0Hz, 0.5H), 5.08 (dd, J=16.5Hz, J=5.0Hz, 1H), 4.28-4 .31(m,0.5H),2.97(d,J=48.5Hz,3H),2.38(m,1H),1.65-1.99(m,8H),11.16(dt,J=6.5Hz,J=3.0Hz,6H).

[0964] Example 196: 2-Amino-5-fluoro-4,4-dimethylvaleric acid [I-196].

[0965]

[0966] Synthesis process:

[0967]

[0968] Program and features:

[0969] Step 1: 3-Hydroxy-N-methoxy-N,2,2-trimethylpropionamide:

[0970] A mixture of 3-hydroxy-2,2-dimethylpropionic acid (10 g, 84.7 mmol), N,O-dimethylhydroxylamine hydrochloride (16.4 g, 101.7 mmol), EDCI (24.4 g, 127.1 mmol), HOBT (17.2 g, 127.1 mmol), and DIPEA (28 mL, 169.5 mmol) in DMF (200 mL) was stirred at room temperature for 16 hours. The reaction mixture was extracted with EtOAc (200 mL × 3) and water (100 mL), and the combined organic layers were washed with 1N HCl (30 mL × 2), 1N NaHCO3 (30 mL × 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-trimethylpropionamide (6.9 g, 50%) as a colorless oil. ESI-MS (EI + ,m / z):162.2[M+H] + .

[0971] Step 2: 3-Fluoro-N-methoxy-N,2,2-trimethylpropionamide:

[0972] DAST (7.4 mL, 55.9 mmol) was added dropwise to a mixture of 3-hydroxy-N-methoxy-N,2,2-trimethylpropionamide (4.5 g, 27.9 mmol) in DCM (40 mL) cooled to -78 °C. The mixture was then stirred at room temperature for 1 to 2 hours, cooled again to -78 °C, and DAST (4 mL, 27.9 mmol) was added dropwise. The reaction mixture was stirred at room temperature for another 1 hour. The reaction mixture was cooled to -78 °C, and saturated NH4Cl (15 mL) was slowly added, followed by DCM (50 mL). The organic layer was separated, washed with saturated NH4Cl (30 mL) and brine (30 mL × 2), dried, and concentrated to produce a residue, which was purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 4) to give 3-fluoro-N-methoxy-N,2,2-trimethylpropionamide (1.9 g, 28%) as a colorless oil. ESI-MS (EI) + ,m / z):164.2[M+H] + .

[0973] Step 3: 3-Fluoro-2,2-Dimethylpropionaldehyde:

[0974] LiAlH4 (6.1 mL, 61.3 mmol, 1 M in THF) was added dropwise to a mixture of 3-fluoro-N-methoxy-N,2,2-trimethylpropionamide (1.0 g, 61.3 mmol) and THF (10 mL) cooled to 0 °C. The mixture was then stirred at the stated temperature for 0.5 to 1 hour. Saturated NH4Cl (10 mL) was slowly added, and the mixture was extracted with Et2O (20 mL × 3), washed with water (15 mL × 2) and brine (15 mL), dried, and used directly for the next step. ESI-MS (EI) was performed. + ,m / z): No MS.

[0975] Step 4: (Z)-2-(tert-Butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoic acid tert-butyl ester:

[0976] A mixture of 3-fluoro-2,2-dimethylpropanal (approximately 630 mg, 6.1 mmol, from the Et₂O solution from the previous step), 2-(tert-butoxycarbonylamino)-2-diethoxyphosphoryl-tert-butyl acetate (2.25 g, 6.1 mmol), and t-BuONa (1.2 g, 12.3 mmol) in THF (15 mL) was stirred at room temperature for 16 hours. Saturated NH₄Cl (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The extract was combined with the organic layer, washed with water (15 mL) and brine (15 mL), dried, and concentrated to produce a residue, which was purified by chromatography (silica, petroleum ether to DCM) to give (Z)-2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpent-2-enoic acid tert-butyl ester (190 mg, 0.60 mmol, 8%) as a white solid. ESI-MS (EI+, m / z): 206 [M-111] + .

[0977] Step 5: 2-(tert-Butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoate tert-butyl ester:

[0978] A mixture of (Z)-2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentan-2-enoate tert-butyl ester (190 mg, 0.60 mmol) and Pd / C (10%, 30 mg) in IPA (15 mL) was stirred at room temperature under hydrogen atmosphere for 17 hours. The mixture was filtered and concentrated to give 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanate tert-butyl ester (200 mg, crude product) as a colorless liquid. ESI-MS (EI...) + ,m / z):342.2[M+Na] + .

[0979] Step 6: 2-Amino-5-fluoro-4,4-dimethylpentanoic acid trifluoroacetic acid:

[0980] A solution of 2-(tert-butoxycarbonylamino)-5-fluoro-4,4-dimethylpentanoic acid tert-butyl ester (200 mg, crude) in 6M HCl (20 mL) and dioxane (10 mL) was heated to 50 °C for 17 hours. The mixture was concentrated under vacuum, diluted with water (30 mL), extracted with Et2O (20 mL × 2), and the filtrate was concentrated under vacuum and purified by reversed-phase silica gel chromatography to give 2-amino-5-cyclopentylpentanoic acid trifluoroacetic acid (31.7 mg, 0.11 mmol, 19%) as a white solid. ESI-MS (EI) + ,m / z):164.2[M+H] + . 1 H-NMR (500MHz, D2O): δ4.16 (d, J = 47.5Hz, 1H), 3.97 (t, J = 5.5Hz, 1H), 2.03 (dd, J = 15.5 Hz, J=5.5Hz, 1H), 1.71 (dd, J=15.5Hz, J=6.0Hz, 1H), 0.91 (dd, J=15.0Hz, J=2.0Hz, 6H).

[0981] Example 186: Synthesis of 2,4-diamino-4-methylpentanoic acid [I-186]:

[0982]

[0983] Synthesis process:

[0984]

[0985] Program and features:

[0986] Step 1: 4-(methoxy(methyl)amino)-2-methyl-4-oxobutyl-2-ylcarbamate tert-butyl ester:

[0987] DIPEA (1.49 g, 11.53 mmol) was added to a solution of 3-(tert-butoxycarbonylamino)-3-methylbutyric acid (1 g, 4.61 mmol), N,O-dimethylhydroxylamine hydrochloride (536 mg, 5.53 mmol), and HATU (2.26 g, 5.99 mmol) in DMF (15 mL). The solution was stirred at room temperature for 2 hours, then the mixture was diluted with brine (100 mL) and extracted with EtOAc (50 mL × 2). The organic layers were combined, concentrated, and purified by chromatography (silica, ethyl acetate / petroleum ether = 1 / 3) to give tert-butyl 4-(methoxy(methyl)amino)-2-methyl-4-oxobut-2-ylcarbamate (1.0 g, 3.8 mmol, 82%) as a colorless oil. ESI-MS (EI) + ,m / z):261.2[M+H]+ .

[0988] Step 2: 2-Methyl-4-oxobutyl-2-ylcarbamate tert-butyl ester:

[0989] At room temperature, LiAlH4 (16 mL, 1 M in THF) was added to a solution of 3.8 g (14.6 mmol) of 4-(methoxy(methyl)amino)-2-methyl-4-oxobut-2-ylcarbamate in 50 mL of THF. The solution was stirred at room temperature for 2 hours, quenched with Na2SO4·10H2O, filtered, and washed with THF to give a yellow solution of 2-methyl-4-oxobut-2-ylcarbamate (approximately 14 mmol in 110 mL THF). MS (EI) + ,m / z):146.3[M+H-56] + .

[0990] Step 3: 4-(benzylamino)-4-cyano-2-methylbut-2-ylcarbamate tert-butyl ester:

[0991] BnNH2 (2.2 mL) and AcOH (2.2 mL) were added to a solution of tert-butyl 2-methyl-4-oxobutyl-2-ylcarbamate (crude material, about 14 mmol in 110 mL THF). The solution was stirred at room temperature for 10 min. TMSCN (2.2 mL) was added. The mixture was stirred at room temperature for 17 h. Subsequently, the reaction mixture was concentrated and passed by chromatography (silica, ethyl acetate / petroleum ether = 1 / 4) to give tert-butyl 4-(benzylamino)-4-cyano-2-methylbutyl-2-ylcarbamate (670 mg, 2.11 mmol, 15%) as a yellow dopant. MS (EI) + ,m / z):318.3[M+H] + .

[0992] Step 4: tert-butyl 5-amino-4-(benzylamino)-2-methyl-5-oxopent-2-ylcarbamate:

[0993] 30% H2O2 (0.64 mL, 5.67 mmol) was added to a mixture of 4-(phenylmethylamino)-4-cyano-2-methylbut-2-ylcarbamate (640 mg, 2.00 mmol), K2CO3 (550 mg, 3.98 mmol), and DMSO (16 mL), and the mixture was stirred at room temperature for 17 hours. The reaction mixture was then diluted with H2O (200 mL) and extracted with EtOAc (100 mL × 2). The combined organic layers were concentrated to give 2-(phenylmethylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (crude, 890 mg) as a yellow dopant. MS (EI+, m / z): 336.0 [M+H] + .

[0994] Step 5: 2-(phenylmethylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid:

[0995] A mixture of tert-butyl 5-amino-4-(phenylmethylamino)-2-methyl-5-oxopentan-2-ylcarbamate (crude, 890 mg, approx. 2.0 mmol), KOH (406 mg, 7.25 mmol), ethane-1,2-diol (9 mL), and H₂O (9 mL) was stirred at 100 °C for 5 hours. The reaction mixture was then diluted with brine (200 mL), extracted with THF / EA at a ratio of 2:1 (90 mL × 5), the organic layers were combined, concentrated, and purified by reversed-phase HPLC (Boston C18 21 × 250 mm 10 μm, mobile phase: A: 0.1% trifluoroacetic acid; B: acetonitrile) to give 2-(phenylmethylamino)-4-(tert-butyloxycarbonylamino)-4-methylpentanoic acid (120 mg, 0.36 mmol, 18%) as a white solid. MS (EI+, m / z): 337.3 [M+H] + .

[0996] Step 6: 2-Amino-4-(tert-Butoxycarbonylamino)-4-methylpentanoic acid:

[0997] A mixture of 2-(phenylmethylamino)-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (140 mg, 0.42 mmol), HCOONH4 (132 mg, 2.1 mmol), and Pd / C (10%, 20 mg) in MeOH (15 mL) was heated to 60 °C for 1 hour. The mixture was filtered, and the filtrate was concentrated and purified by reversed-phase silica gel chromatography to give 2-amino-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (60 mg, 0.24 mmol, 58%) as a white solid. ESI-MS (EI) + ,m / z):247.2

[0998] Step 7: 2,4-Diamino-4-methylpentanoic acid:

[0999] A solution of 2-amino-4-(tert-butoxycarbonylamino)-4-methylpentanoic acid (60 mg, 0.24 mmol) in 6 M HCl (10 mL) and dioxane (0 mL) was stirred at room temperature for 17 hours. The solution was concentrated under vacuum to give 2,4-diamino-4-methylpentanoic acid (51.8 mg, 0.236 mmol, 97%) as a white solid. ESI-MS (EI) + ,m / z):147.1 1H NMR (500MHz, D2O) δ4.04 (dd, J = 9.5Hz, J = 3.5Hz, 1H), 2.32 (dd, J = 15.0Hz, J = 9.5Hz, 1H), 1.94 (dd, J = 15.0Hz, J = 3.0Hz, 1H), 1.38 (dd, J = 9.5Hz, J = 5.0Hz, 6H).

[1000] Example 199: Synthesis of 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine [I-199]:

[1001]

[1002] Synthesis process:

[1003]

[1004] Program and features:

[1005] Step 1: N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide:

[1006] A mixture of 2-chloro-N-methoxy-N-methylacetamide (13.7 g, 0.1 mol) and triphenylphosphine (26.2 g, 0.1 mol) in acetonitrile (200 mL) was heated to 80 °C and maintained for 20 hours. The mixture was cooled and concentrated to remove the solvent below 40 °C. The residue was dissolved in dichloromethane (200 mL), followed by 2N KOH (100 mL). The resulting mixture was stirred at 20 °C for 1 hour. The organic layer was washed with brine (200 mL × 3), dried over Na₂SO₄, and filtered. The filtrate was concentrated under vacuum to give N-methoxy-N-methyl-2-(triphenyl-15-phosphine)acetamide (36 g, 0.1 mol, 98%) as a yellow solid. ESI-MS (EI) + ,m / z):364.4[M+H] + .

[1007] Step 2: (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide:

[1008] A mixture of N-methoxy-N-methyl-2-(biphenyl-15-phosphine)acetamide (36.3 g, 0.1 mol) and 1,1,1-trifluoroprop-2-one (22.4 g, 0.2 mol) in tetrahydrofuran (500 mL) was heated to 20 °C and maintained for 20 hours. The mixture was cooled and concentrated to remove the solvent under vacuum at below 40 °C. The residue was purified by silica gel column (200 g, 200 to 300 mesh, UV 254 nm) eluted with 0 to 25% ethyl acetate / petroleum ether to give (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbut-2-enamide (19.5 g, 0.1 mol, 98%) as a yellow oil. ESI-MS (EI + ,m / z):198.2[M+H] + .

[1009] Step 3: 4,4,4-Trifluoro-N-methoxy-N,3-dimethylbutyramide:

[1010] A mixture of (E)-4,4,4-trifluoro-N-methoxy-N,3-dimethylbutyr-2-enamide (2 g, 0.01 mol) and Pd / C (10%, 200 mg) in THF (50 mL) was stirred at 26 °C for 18 hours. The mixture was filtered, and the filtrate was concentrated to dryness under vacuum to give 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutyramide (2 g, 0.01 mol, 98%) as a yellow oil. ESI-MS (EI...) + ,m / z):200.2[M+H] + .

[1011] Step 4: 4,4,4-Trifluoro-3-methylbutanal:

[1012] At 0 °C, LiAlH4 (0.4 g, 0.01 mol) was added to a solution of 4,4,4-trifluoro-N-methoxy-N,3-dimethylbutyramide (2 g, 0.01 mol) in 40 mL of THF. The mixture was stirred at 0 °C for 1 hour. The reaction mixture was quenched with water, followed by methyl tert-butyl ether (30 mL × 2). The organic layer was washed with brine (50 mL × 3), dried over Na2SO4, and filtered. The filtrate yielded 1.4 g of crude 4,4,4-trifluoro-3-methylbutyraldehyde, which was used directly as a colorless solution in the next step.

[1013] Step 5: 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanilide:

[1014] Under ice bath conditions, benzylamine (1.5 mL), AcOH (1.0 mL), and subsequently TMSCN (1.5 mL) were added to the above solution of 4,4,4-trifluoro-3-methylbutanal in methyl tert-butyl ether (100 mL). The mixture was heated to 20 °C and stirred overnight. The solution was diluted with water (30 mL) and extracted with EtOAc (30 mL). The organic phase was washed with water (30 mL × 2) and brine (50 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum to give 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanilide (2.6 g, crude substance) as a brown oil for the next step. ESI-MS (EI) + ,m / z):257.3[M+H] + .

[1015] Step 6: N-Benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine:

[1016] NH4Cl (0.15 g, 0.003 mol) was added to a solution of 2-(phenylmethylamino)-5,5,5-trifluoro-4-methylpentanonitrile (0.3 g, crude) in DMF (10 mL), and NaN3 (0.21 g, 0.003 mol) was heated to 95 °C for 18 hours. The solution was cooled to 15 °C and extracted with EtOAc (20 mL). The organic phase was washed with water (20 mL × 2) and brine (20 mL), dried (Na2SO4), filtered, and concentrated under vacuum to give N-phenylmethyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine (0.1 g, 0.5 mmol, 33%, 3 steps) as a white solid. ESI-MS (EI) + ,m / z):300.3[M+H] + .

[1017] 4,4,4-Trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-aminetrifluoroacetic acid:

[1018] At room temperature, HCOONH4 (0.17 g, 2.7 mmol) and Pd / C (30 mg) were added to a solution of N-benzyl-4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-amine (160 mg, 0.54 mmol) in MeOH (15 mL). The mixture was stirred at 60 °C for 2 hours. The reaction mixture was filtered and concentrated to produce a crude product, which was purified by reversed-phase silica gel chromatography to give 4,4,4-trifluoro-3-methyl-1-(2H-tetrazol-5-yl)but-1-aminetrifluoroacetic acid (72.8 mg, 0.23 mmol, 42%) as a white solid; ESI-MS (EI) + ,m / z):210.2[M+H] + ; 1H NMR (500MHz, DMSO-d6) δ4.67-4.93 (m, 1H), 2.31-2.41 (m, 1H), 2.00-2.12 (m, 2H), 0.99 (dd, J = 16.8, J = 6.4Hz, 6H).

[1019] Example 210: Western blot analysis

[1020] The screening analysis measured the in vitro activity of the GATOR2 / Sestrin2 complex purified by immunoprecipitation of a stable FLAG-WDR24-expressing complex from HEK293T cells. HEK293T cells (293T) were engineered to stably express N-terminally labeled FLAG-WDR24 via lentiviral transduction. Lentiviral transfection was performed using the lentiviral transfer vector pLJM60 with a ΔVPR envelope, followed by plasmid encapsulation into HEK-293T cells using XTremeGene 9 transfection agent (Roche Diagnostics) via CMVVSV-G. Twenty-four hours post-transfection, the medium was replaced with Durbecco's Modified Eagle's media (DMEM) supplemented with 30% inactivated embryonic serum. Virus-containing supernatants were collected at 48 and 72 hours post-transfection and transferred 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 agglutinin and then spin-infected for 1 hour at 2,200 rpm by centrifugation. Twenty-four hours post-infection, the virus was removed and cells treated with appropriate antibiotics were selected. Cells were then grown in DMEM supplemented with 10% fetal bovine serum and antibiotics.

[1021] To screen for leucine mimicry compounds, 2,000,000 FLAG-WDR24 cells expressing 293T were seeded in 10 cm tissue culture plates. After 72 hours, the cells were placed in standard RPMI medium (AARPMI, US Biological Life Sciences) without amino acid formulation and supplemented with 5 mM glucose for 1 hour, followed by lysis in lysis buffer (40 mM HEPES, 1% Triton, 10 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 2.5 mM MgCl2 and protease inhibitor). To isolate the FLAG-WDR24 / endogenous-Sestrin2 complex, 1 ml of crude lysate (equivalent to 2-4 mg of total protein) was immunoprecipitated with 30 μl of anti-tag resin (SIGMA) at 4 °C for 2 hours. The lysate was washed twice with 0.5 M NaCl in cold lysis buffer and resuspended in 1 ml of cold cytosol buffer (40 mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100). Subsequently, test compounds or controls (filtered solutions or leucine) were added to each immunoprecipitated sample at various concentrations and incubated at 4 °C under rotation for 60 minutes. After incubation, the samples were centrifuged to aggregate the FLAG-WDR24 / endogenous-Sestrin2 complex bound to the anti-tag resin. The supernatant was completely removed, and the resin was resuspended in SDS-PAGE sample buffer and boiled for 5 minutes. The samples were then processed by SDS-PAGE and blotted with anti-FLAG (SIGMA) and anti-Sestrin2 (cell signaling technology) antibodies, as described in L. Chantranupong et al., Cell Reports 9:1-8 (2014).

[1022] The obtained protein blot was scanned and used The imaging platform quantified the band intensities corresponding to Sestrin2 and FLAG-WDR24. To determine the amount of Sestrin2 bound to GATOR2 under various conditions, the Sestrin2 band intensity was normalized to the FLAG-WDR24 band intensity. For each batch of compounds tested, a negative control (filtered solution) and a positive control (leucine, 25 μM, SIGMA) were also provided. Endogenous Sestrin2 bound to FLAG-WDR24 was normalized to represent 100% activity by depleting leucine. Compounds were analyzed twice, and the activity of each compound was quantified as a percentage of leucine activity and averaged. Repeated attempts of analysis yielded a 20% standard deviation of the mean leucine activity compared to water; therefore, a compound that reduced the amount of Sestrin2 bound to GATOR2 by at least 40% at 25 μM after two repetitions was considered statistically significant and characterized as a leucine mimic. Some compounds increased the amount of Sestrin2 bound to FLAG-WDR24. Compounds that increase the amount of Sestrin2 bound to GATOR2 by more than 40% (expressed as less than -40% leucine activity) are characterized as leucine antagonists.

[1023] Example 211. A method for identifying compounds that mimic or antagonize leucine activity after interaction with Sestrin2 and Sestrin2 / GATOR2.

[1024] INTRODUCTION

[1025] In the absence of sufficient leucine, Sestrin1 and Sestrin2 interact with GATOR2 via the GATOR2 components WDR24 and Seh1L. Under sufficient leucine conditions, leucine directly binds to Sestrin2, thereby inducing the dissociation of Sestrin2 from GATOR2. The purpose of the following method is to identify compounds that mimic the role of leucine in binding to Sestrin2 and cleaving the Sestrin2 / GATOR2 relationship. Furthermore, the method aims to identify compounds that antagonize leucine binding to Sestrin2 and prevent the dissociation of Sestrin2 from GATOR2 in response to leucine.

[1026] Method 1 (in vitro PPI assay)

[1027] The screening analysis measured the in vitro compound activity of the GATOR2 / Sestrin2 complex purified by immunoprecipitation of cells stably expressing Flag-WDR24 from HEK293T cells. HEK293T cells (293T) were engineered to stably express N-terminally labeled Flag-WDR24 via lentiviral transduction. Lentiviral cells were generated by co-transfection with the lentiviral transfer vector pLJM60 and a ΔVPR envelope, followed by CMV VSV-G plasmid encapsulation into HEK-293T cells using XTremeGene 9 transfection agent. Twenty-four hours post-transfection, the medium was replaced with DMEM supplemented with 30% inactivated embryonic serum. Virus-containing supernatants were collected at 48 and 72 hours post-transfection and transferred 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 phacoamic acid and spin-infected for 1 hour by centrifugation at 2,200 rpm. Twenty-four hours after infection, the virus was removed and cells were selected with appropriate antibiotics. The cells were then grown in DMEM supplemented with 10% fetal bovine serum and antibiotics.

[1028] To screen for leucine mimicry compounds, 2,000,000 Flag-WDR24 cells expressing 293T were seeded in 10 cm tissue culture plates. After 72 hours, the cells were placed in standard RPMI medium (AARPMI, US Biological Life Sciences) without amino acid formulation and supplemented with 5 mM glucose for 1 hour, followed by lysis in lysis buffer (40 mM HEPES, 1% Triton, 10 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 2.5 mM MgCl2 and protease inhibitor). The Flag-WDR24 / endogenous Sestrin2 complex was isolated as follows: 1 ml of crude lysate (equivalent to 2-4 mg total protein) was immunoprecipitated for 2 hours at 4°C with 30 μl of anti-tag resin (SIGMA). The lysate was washed twice with 0.5 M NaCl in cold lysis buffer and resuspended in 1 ml of cold cytosol buffer (40 mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100). The compound was then added to each sample at a given concentration of 25 μM and incubated at 4°C under rotation for 30 minutes. After incubation, the samples were centrifuged to aggregate the Flag-WDR24 / endogenous Sestrin2 complex bound to the anti-tag 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 processed by SDS-PAGE and Western blotted with anti-Flag (SIGMA) and anti-Sestrin2 (cell signaling technology) antibodies, as described in L. Chantranupong et al., Cell Reports 9:1-8 (2014).

[1029] The obtained protein blot was obtained by scanning and using The imaging platform quantified the band intensities corresponding to Sestrin2 and Flag-WDR24. To determine the amount of Sestrin2 bound to GATOR2 under various conditions, the Sestrin2 band intensity was normalized to the Flag-WDR24 band intensity. For each batch of compounds tested, negative controls (water) and positive controls (leucine, 25 μM, SIGMA) were also provided. Endogenous Sestrin2 bound to Flag-WDR24 was normalized to represent 100% activity by leucine depletion. Compounds were analyzed twice, and the activity of each compound was quantified as a percentage of leucine activity and averaged. A table listing the quantitative data of the tested compounds is presented in Table 3. The repeated attempts of the analysis yielded a 20% standard deviation of the mean leucine activity compared to water; therefore, test compounds that reduced the amount of Sestrin2 bound to GATOR2 by at least 40% at 25 μM were considered statistically significant and referred to as leucine mimics. Some compounds increased the amount of Sestrin2 bound to Flag-WDR24 (shown as negative percentage activity of leucine in Table 3). Compounds exhibiting less than -40% leucine activity were also sampled and referred to as leucine antagonists.

[1030] Method 2 (cell-based mTORCl activation)

[1031] To demonstrate the efficacy of compounds identified as leucine mimics in intact cells, mTORC1 signaling in response to compound treatment following leucine deficiency was measured by Western blotting. Following leucine deficiency, mTORC1 activation was achieved by adding exogenous leucine, as described in Wang, S., Tsun, Z. et al., Science 347(6218):188-194 (2015), when signaling was measured 10 to 90 minutes after leucine addition, as described. Therefore, a similar assay was designed to test whether compounds were identified as leucine mimics that activate mTORC1 in a similar manner. In short, 800,000 HEK293T cells were seeded in the wells of a 6-well plate in DMEM supplemented with 10% fetal bovine serum and antibiotics. The following day, the cells were placed in modified DMEM without leucine (Thermo Scientific) or serum for 1 hour, followed by the addition of a leucine mimic (n=3) at a given concentration for a period greater than 10 minutes. Cells were subsequently lysed, treated with SDS-PAGE, and Western blotted with antibodies against mTORC1 substrates phosphorylated S6 kinase (Thr389) and phosphorylated 4EBP1 (Thr37 / 46) (cell signaling technology) and loaded with controls (β-actin, Santa Cruz Biotechnology), as described in Kang, SA et al., Science 341(6144):364-374 (2013). The intensity of the imaging platform corresponding to the phosphorylated substrate bands was then normalized to the actin bands. Compounds that significantly enhanced mTORC1 signaling compared to untreated leucine-deficient cells (Student's t-test, p < 0.05) were considered active in the cells. As a positive control, leucine was added to leucine-deficient cells at 100 μM for 60 minutes.

[1032] Method 3 (cell-based mTORCl activation)

[1033] To demonstrate the efficacy of compounds identified as leucine antagonists or to determine whether weaker leucine mimics enhance leucine activity in intact cells, the same paradigm as described above was repeated, but with the following modifications: cells were placed in leucine-depleted DMEM medium (as described in Method 3) for 60 minutes, followed by a period of time of 60 minutes or longer for the application of the compounds (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 significantly reduced the amount of actin-normalized phosphorylated substrates of mTORC1 in response to 30 μM or 100 μM leucine (Student's t-test, p<0.05) were considered active in cells. Compounds that statistically significantly increased the levels of actin-normalized phosphorylated substrates of mTORC1 in response to 30 μM or 100 μM leucine (Studen's t-test, p < 0.05) were considered leucine enhancers in cells. As a control, leucine-deficient cells were pretreated with water before leucine supplementation. Alternatively, potential leucine antagonists were analyzed in HEK293T cells in the same manner described above, but without leucine deficiency and stimulation. Western blotting was performed to determine whether baseline mTORC1 signaling was attenuated after compound treatment under adequately supplied culture conditions.

[1034] Method 4

[1035] The ability of compounds to modulate the interaction between Sestrin2 and GATOR2 in cells was measured by repeating the analyses described in Methods 2 and 3, but in HEK293T cells engineered to stably express Flag-WDR24 seeded in 10 cm tissue culture dishes. The interaction between endogenous Sestrin2 and Flag-WDR24 was measured from cell lysates after compound treatment (n=3), as described in Method 1. Briefly, to measure the amount of endogenous Sestrin2 bound to Flag-WDR24 after cell treatment, samples were immunoprecipitated with anti-tag resin and processed for 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 (Studden's t-test, p<0.05) were used as samples.

[1036] Method 5 (ALPHA LISA cell-based assay)

[1037] To demonstrate the efficacy of compounds identified as leucine mimics in intact cells in a plate-based format, mTORC1 signaling in response to compound treatment following leucine deficiency was measured via AlphaLISA. In summary, 1,000,000 HEK293T cells were seeded in T-75 cell culture flasks in DMEM supplemented with 10% fetal bovine serum. After reaching confluence, the cells were placed in modified DMEM (Thermo Scientific) without leucine and with 10% disorbed fetal bovine serum for 1 hour. The cells were then trypsinized and reseeded at 50,000 cells / well in 96-well black clear-bottomed culture plates in DMEM (Thermo Scientific) without leucine and with 10% disorbed fetal bovine serum. Cells were allowed to adhere to the plate for 2 hours, followed by the addition of the compound at a given concentration (n=4) for a period longer than 1 hour. After the time point was reached, cells were lysed and analyzed using the p-p70 S6K(Thr389) SureFire Ultra AlphaLISA kit according to the manufacturer's instructions (http: / / www.perkinelmer.com / CMSResources / Images / 44-176283MAN_SureFire_TGR70S_p70_pT389.pdf). Compounds that significantly increased mTORC1 signaling compared to untreated leucine-deficient cells (Studen's t-test, p<0.05) were considered mTORC1 activators. Compounds that significantly decreased mTORC1 signaling compared to untreated leucine-deficient cells (Studen's t-test, p<0.05) were considered inhibitors in the cells. As a positive control, leucine was added at 100 μM to leucine-deficient cells for the same duration as the compound treatment.

[1038] Method 6, Thermal Shift Protocol (Tm Shift):

[1039] Full-length codon-optimized human Sestrin2 was fused to the N-terminus of a His-MBP tag and cloned into the pMAL6H-C5XT bacterial expression vector. This vector was transferred into *E. coli* LOBSTR(DE3) cells (Kerafast). Cells were grown to 0.6 OD at 37°C, followed by protein production induction at 18°C ​​for 12–14 h with 0.2 mM IPTG. Cells were collected by centrifugation at 6,000 g, resuspended in lysis buffer (50 mM potassium phosphate, pH 8.0, 500 mM NaCl, 30 mM imidazole, 1 mM DTT, 10 μg / ml nuclease, and 1 mM PMSF) and lysed by sonication. Lysate was removed by centrifugation at 10,000 g for 20 min. Sestrin2 protein was isolated from the soluble eluate to near 100% purity via His-tag affinity trapping, followed by ion exchange and size exclusion chromatography. For thermal migration analysis, Sestrin2 protein was diluted to 2 mg / mL in dilution buffer (10 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA). Prior to thermal migration analysis, 2 μL of Sestrin2 protein was combined with 8 μL of ROX dye (Thermo Fisher), 1 μL of mordant or compound, and 14 μL of dilution buffer per well of a 96-well plate, and incubated on ice for 1 hour to allow compound binding. Thermal migration analysis was subsequently performed on an Agilent MX3005p, with each compound analyzed three times at 10 μM, 100 μM, and 1000 μM. The melting temperature shift of Sestrin2 incubated with leucine was shifted from 2.16 to 11.61 degrees Celsius in a dose-related manner. Positive shifts of 2 degrees or greater were considered statistically significant based on the CV% variability of repeated thermal migration measurements of Sestrin2 incubated with mordant.

[1040] Method 7, Indirect Ligand Binding Assay (ILBA)

[1041] Sestrin2 binding to leucine or other ligands can be detected in intact cells, in vitro, or via immunoassay of purified proteins using rabbit monoclonal anti-Sestrin2 antibodies from Cell Signaling Technology (CST, catalog number 8487). CST antibody binding to native (non-denatured) Sestrin2 is modulated by binding leucine in a manner that reduces antibody affinity after leucine binding. Similarly, the affinity of CST antibodies for native Sestrin2 decreases after compounds bind to native Sestrin in a manner similar to leucine binding. Conversely, compounds that destabilize Sestrin2, as measured by thermal shift analysis, increase the affinity of CST antibodies for non-denatured Sestrin2. Therefore, various formats of this Indirect Ligand Binding Assay (ILBA) are developed, which measure the affinity of CST anti-Sestrin2 antibodies after leucine or compound binding. In one version, analysis was performed using crude lysates from human cell lines after a period of less than 1 hour of amino acid lysis (cell lysis in 1% Triton, 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 40 mM HEPES [pH 7.4], 150 mM NaCl, and 2.5 mM MgCl2). The lysates were then incubated on ice or at room temperature for 1 hour with leucine or other compounds. Following compound incubation, samples were subjected to CST anti-Sestrin2 antibody immunoprecipitation for 1.5 hours, followed by incubation with protein-A agarose for 30 minutes, as described in L. Chantranupong et al., Cell Reports 9:1-8 (2014). The agarose conjugated antibody-protein complex was precipitated by centrifugation, and the flow-through was subjected to a second round of immunoprecipitation with rabbit polyclonal anti-Sestrin2 antibody (ProteinTech, #10795-1-AP) to determine the total Sestrin2 protein content between equal samples. SDS-PAGE was performed on the immunoprecipitated samples, followed by Western blotting of the mouse monoclonal anti-Sestrin2 antibody from SIGMA (catalog number WH0083667M3). Leucine binding induced a significant decrease of 50% or more in the band intensity corresponding to Sestrin2 on the immunoblot of samples immunoprecipitated with the anti-Sestrin2 antibody from CST, but did not cause any change in the Sestrin2 band intensity on the immunoblot of samples immunoprecipitated with the Protein Tech antibody. This version of the analysis also measured the increased instability of Sestrin2 induced by incubation with the compound. The analysis was performed in the same manner, but compounds that destabilized Sestrin2 (as measured by thermal migration analysis) resulted in increased intensity of the immunoblot band corresponding to Sestrin2 immunoprecipitated using the CST antibody.

[1042] This analysis was also performed in cultured human cells overexpressing Sestrin2, which is fused to the Flag tag at its N-terminus. In this version of the analysis, the procedure remained the same, but Western blotting was performed using a mouse anti-Flag antibody (#F3165, SIGMA). No decrease in CST antibody affinity was observed after leucine or γ-methylleucine binding when ILBA was performed with the point-mutated form of Sestrin2 that cannot bind leucine.

[1043] In another version of the analysis, cultured human cells were subjected to a combination of amino acid stimulation for less than one hour, followed by stimulation with leucine or a compound. One hour after stimulation, the cells were lysed and processed as described above, except for the one-hour ligand binding step.

[1044] Indirect ligand binding assays were also performed in a multi-well format using the ALPHAlisa technique (Perkin Elmer). This version of the assay required a biotinylated anti-Sestrin2 antibody, an anti-streptolysin donor bead (Perkin Elmer) coupled with an anti-Flag receptor bead (Perkin Elmer) for detecting overexpressed Flag-Sestrin2, or a mouse anti-Sestrin2 antibody (SIGMA) coupled with an anti-mouse receptor bead (Perkin Elmer) for detecting endogenous Sestrin2.

[1045] The analysis was performed as described above, but with the following modifications: For the leucine or compound-binding fraction being analyzed, crude lysate produced from cells temporarily or stably overexpressing human Flag-Sestrin2, after 1 hour of amino acid deficiency, was diluted in lysis buffer to 0.8 mg / ml total protein and arranged in multi-well plates, such as 96-well plates. For the detection of endogenous Sestrin2, the crude lysate was diluted in lysis buffer to 4 mg / ml total protein. Leucine or the compound was added to each well and the plate was incubated on ice or at room temperature with gentle agitation for 1 hour. For the assay to detect endogenous Sestrin2, during the ligand binding step, biotinylated anti-Sestrin2 antibody (CST) was diluted to 5 nM in ALPHAlisa immunoassay buffer (Perkin Elmer), and 5 nM mouse anti-Sestrin2 antibody (SIGMA) was combined with 4× preform (40 μg / ml) of anti-mouse receptor beads. For Flag-Sestrin2 detection, 4× preforms (40 μg / ml) of anti-tag receptor beads were prepared in immunoassay buffer. Following the ligand binding step, 5 μL of the lysate was combined with 10 μL of biotinylated anti-Sestrin2 antibody, 12.5 μL of a mouse Sestrin2 antibody / anti-mouse receptor bead mixture or anti-Flag receptor beads, and 10 μL of ALPHAISA immunoassay buffer and incubated at room temperature for 1 hour. Finally, before reading the plate on an Envision plate reader in the dark, 12.5 μL of streptavidin donor beads (160 μg / ml in immunoassay buffer) was added for an additional hour.

[1046] As described, but using purified Sestrin2 protein diluted in immunoassay buffer at a final reactant concentration of 3 ng / ml for ALPHAISA analysis.

[1047] Finally, prior to lysis, ALPHAISA was performed under amino acid-deficient conditions using lysates from cells treated with leucine or a compound. Cell-based processing was performed in multi-well plates, with each ALPHAISA reaction using 15 μL of lysate (1 mg / ml total protein) combined with 10 μL of biotinylated antibody, 12.5 μL of antibody / receptor bead mixture, and 12.5 μL of streptavidin donor bead mixture.

[1048] Indirect ligand binding analysis was also performed using capture-based methods, such as sandwich ELISA as used in the field. In one version of the analysis, the MULTI- generated by Meso-Scale Discovery (MSD) was used. ILBA was performed using the MSD system, which is based on electrochemiluminescence detection of antibodies bound to the analytes. Prior to lysis, ILBA was performed with a crude lysate expressing endogenous Sestrin2, either in vitro or in cells, to overexpress Flag-Sestrin2 and leucine. For in vitro ILBA of endogenous Sestrin2, a crude lysate (0.8 mg / ml total protein) was prepared and leucine was bound in the same manner described for ALPHAISA ILBA. After ligand binding was complete, biotinylated anti-Sestrin2 antibody from CST was added to each well until a final concentration of 0.25 μg / ml was achieved, and the wells were incubated at 4°C with gentle agitation for 1 hour. Sample collection from the wells of the 96-well plate was achieved using either an MSD plate coated with streptavidin or a naked MSD plate coated with mouse anti-Sestrin2 antibody from SIGMA. 25 μL of sample was collected per well, followed by incubation at 350 rpm with shaking for 1 hour. After sample capture, the wells were washed three times with Tris-buffered saline containing 0.1% Tween (TBS-T). If the sample was captured on an anti-Sestrin2 plate, mouse monoclonal anti-Sestrin2 antibody (SIGMA) was subsequently added at 350 rpm with shaking for 1 hour to a final concentration of 1 μg / ml. The wells were washed again with TBS-T, and anti-mouse second SULFO-TAG antibody (MSD) was added at 350 rpm with shaking for 1 hour. Finally, the wells were washed three times with TBS-T and 2× Read buffer (MSD) was added, followed by reading the plate on the MSD instrument. If the sample was captured after washing with a naked culture plate coated with mouse anti-Sestrin2 antibody, anti-Sestrin2 antibody (MSD) was added at a final concentration of 1 μg / ml with shaking for 1 hour, followed by washing and incubation with Read buffer prior to analysis.

[1049] In another version of the analysis, crude lysates of Flag-Sestrin2 overexpression were analyzed and captured or detected using the same MSD-based protocol as described above with mouse monoclonal anti-Flag antibody (SIGMA).

[1050] For all analyses, compounds that significantly reduced the signal corresponding to Sestrin2 immunoreactivity were considered leucine mimics, while compounds that significantly increased the signal were considered potential leucine antagonists.

[1051] Table 3 shows the activities of the selected compounds of this invention. The compound numbers correspond to the compound numbers in Tables 1 and 2. Compounds with activity indicated as "A" have an activity % ≥ 40% relative to leucine; compounds with activity indicated as "B" have an activity % ≤ -40% relative to leucine; and compounds with activity indicated as "C" have an activity % relative to leucine between -40% and 40%. At specified concentrations, compounds with activity indicated as "D" show a shift of 0.5 to 2 times relative to the DMSO control; compounds with activity indicated as "E" show a shift of 2.1 to 5 times relative to DMSO; compounds with activity indicated as "F" show a shift of 5.1 to 10 times relative to DMSO; and compounds with activity indicated as "G" show a shift of 10.1 to 14 times relative to DMSO.

[1052] The percentage activity relative to leucine was determined using analytical method 1. The activity based on cell-mediated mTORC1 activation was determined using analytical method 2.

[1053] Table 3. Analytical data of exemplary compounds

[1054]

[1055]

[1056] Table 4 shows the selected compounds of the present invention that are active in ALPHALISA cell-based analysis (Method 5). The compound numbers correspond to the compound numbers in Tables 1 and 2. The compounds listed in Table 4 are mTORC1 activators and have >2 times the activity relative to the positive leucine control.

[1057] Table 4. Exemplary compounds with activity in ALPHALISA cell-based analyses

[1058]

[1059]

[1060] Table 5 shows the selected compounds of the present invention that are active in thermal migration analysis (Method 6). The compound numbers correspond to the compound numbers in Tables 1 and 2. The compounds listed in Table 5 show a positive migration of 2 degrees or more.

[1061] Table 5. Exemplary compounds exhibiting activity in thermal migration analysis

[1062]

[1063]

[1064] General materials and methods for in vivo testing

[1065] Animal Use: Upon arrival at Yale University (New Haven CT), male Spurgeon Dalmatian rats (Charles River Laboratories, Wilmington, MA) weighing 175-200g were housed in groups and acclimatized for 5 days prior to the start of the experimental studies. Rats were provided with free access to food and water, except during the fasting periods specified in the protocol. Clinical signs of the animals were monitored daily. All rodent procedures were overseen by a qualified veterinarian. All personnel were trained by the Yale Animal Care and Use Committee (IACUC). All animal procedures at Yale University were strictly conducted in accordance with the IACUC of the National Institutes of Health and approved by the Yale Animal Care and Use Committee.

[1066] Behavioral analysis using the female urine sniffing test (FUST): The FUST was performed 24 hours after administration, following the published procedure (Malkesman, O. et al., Biol Psychiatry 67(9):864-71(2010)). In short, rats were acclimatized in their cages to a swab soaked in tap water for 60 minutes. Subsequently, the rats were exposed to a second swab soaked in tap water, and 45 minutes later, to a third swab containing fresh rat urine from estrous female rats aged 11 to 14 weeks. For each animal, the total time spent sniffing the cotton tip applicator was quantified over 5 minutes.

[1067] Behavioral analysis using the Liver Activity Assessment (LMA): LMA was assessed in an open space using an automated activity meter equipped with parallel infrared beam arrays, according to the published procedure (Warner-Schmidt, JL & Duman, RSPNAS 104(11):4647-52(2007)). For each animal, the number of beam breaks was recorded at 30-minute intervals.

[1068] Behavioral analysis was performed using the novel restraint feeding test (NSFT): The NSFT was performed as previously described (Warner-Schmidt, JL & Duman, RSPNAS 104(11):4647-52 (2007)). Rats were fasted in their cages for 20 hours and then placed in an open plastic-glass enclosure (76.5cm × 76.5cm × 40cm) with a small amount of food in the center. The animals were allowed to explore the open space for 8 minutes and the feeding wait time was recorded.

[1069] Behavioral analysis using the sucrose preference test (SPT): Rats were acclimatized to a palatable 1% sucrose solution for 48 hours to avoid novelty phobia. Rats were treated with NV-5138 or Veh at the end of Day 0 and underwent the SPT 24 hours after administration on Day 1. For the SPT, rats were dehydrated for 6 hours and exposed to two bottles containing equal volumes of 1% sucrose or water for 60 minutes. The ratio of the volume of sucrose-water consumed to the total water consumed during the 1-hour test period was defined as sucrose preference (e.g., a ratio of 1 would indicate that the rat consumed only 1% sucrose, while a ratio of 0.5 would indicate that the rat drank equal amounts of 1% sucrose and water).

[1070] Long-term unpredictable stress (CUS) conditions: rats were exposed to 12 unpredictable stressors in a variable sequence, with preventative adaptation as described (Li, N. et al., Biol Psychiatry 69(8):754-61(2011)). The following twelve stressors were applied (twice daily for 25 days): rotating cage, light on, light off, cold stress, separation, swimming stress, food and water deprivation, wet bedding, stroboscope, cage tilting, odor exposure, and group housing. Animals in the non-stress (NS) group were typically housed without external stressors. Both NS and CUS rats were treated and weighed weekly.

[1071] Human Threat Test (HTT) in marmosets: Marmosets are periodically stimulated in the presence of a human observer over a prolonged period of time. Such long-term stimulation is known to increase plasma cortisol levels, and the subsequent enhancement of hypothalamic-pituitary-adrenal (HPA) function contributes to the pathophysiology of depression.

[1072] Example A: Novel inhibition of behavioral changes in feeding and female urine sniffing tests following a single administration of the compound or ketamine.

[1073] Study Design: After a 5-day acclimatization period, thirty-two (32) male Spurgeon Dolly rats weighing between 175 and 200 g were randomly assigned to four study groups (n = 8 / treatment group). On day 0, rats in groups 1 and 2 received a single dose of saline (Sal) or ketamine (Ket) via intraperitoneal injection (ip). Rats in groups 3 and 4 received a single dose of NV-5138 mediator (Veh, 0.5% methylcellulose / 0.1% Tween-80) or NV-5138 (160 mg / kg) via oral tube feeding. All rats underwent FUST 24 hours after administration. LMA was measured in an open space for all rats 48 hours after administration. Rats were then fasted for 20 hours and underwent NSFT 72 hours after administration. The study design is presented in Table 6. The timing of test item administration and the three behavioral tests are summarized in Table 6. Figure 1 middle.

[1074] Preparation of test articles: Ket (Sigma, catalog number K1884) was dissolved in Sal at a concentration of 10 mg / mL. For Groups 1 and 2, 1 mL / kg of Sal or Ket was administered via intraperitoneal injection, respectively. NV-5138 (Navitor, batch number 06) was prepared by dissolving Veh (0.5% methylcellulose / 0.1% Tween-80) at a concentration of 50 mg / mL. Veh or NV-5138 was administered orally to the study animals in Groups 3 and 4 via tube feeding at a dose based on animal weight (3.2 mL / kg). Test articles were prepared on the day of administration.

[1075] Results: Summary of FUST results on Day 1 Figure 2 In the study, Ket treatment significantly increased the time spent by male rats sniffing female urine by 2.1 times (17.4±3.9 s vs. 36.5±7.9 s in Ket group 2 vs. Sal group 1, p<0.05). Similarly, NV-5138 treatment significantly increased the time spent by male rats sniffing female urine by 2.9 times (33.8±2.9 s vs. 11.6±6.2 s in NV-5138 group 4 vs. Veh group 3, p<0.01). The results of LMA on day 2 are summarized in […]. Figure 3 The mean number of beam breaks was quantified. No significant difference in LMA was observed between groups using the unpaired 2-tailed Steudon's t-test. Results of the NSFT on day 3 are summarized in... Figure 4 Compared to group 1 (Sal), a significant 31% reduction in feeding wait time was observed in group 2 (Ket) (p<0.01). Similarly, compared to group 3 (Veh), a significant 36% reduction in feeding wait time was observed in group 4 (NV) (p<0.01).

[1076] Table 6: Example of a Study Design A

[1077]

[1078] Example B: Comparative effects of single-dose NV-5138 and ketamine on the mTORC1 signaling pathway and synaptic protein expression in synaptosome formulations derived from the rat prefrontal cortex.

[1079] Study Design: After a 5-day acclimatization period, forty-eight (48) male Spögdolly rats weighing between 175 and 200 g were randomly assigned to eight study groups (n = 6 / group). On day 0, rats in groups 3 and 7 received a single dose of Sal, while rats in groups 4 and 8 received a single dose of Ket (10 mg / kg) via intraperitoneal injection. Rats in groups 1 and 5 received a single dose of Veh, while rats in groups 2 and 6 received a single dose of NV-5138 (160 mg / kg) via oral tube feeding. One hour after administration, rats in groups 1 through 4 were euthanized by awake decapitation, and PFC was subsequently collected. Crude synaptosomes were prepared from PFC and three mTORC1 substrates (pmTOR, pp70S6K, and p4E-BP1), and the corresponding total protein loading controls (mTOR, p70S6K, and GAPDH) were quantified by Western blotting. Twenty-four hours after administration, rats in groups 5 through 8 were sacrificed by awake decapitation, and PFCs were collected. Crude synaptosomes were prepared from PFC and synaptic proteins (GluR1 and PSD95), and the total protein loading control (GAPDH) was quantified by Western blotting. The study design is presented in Table 7. The timing of test item administration and the sacrifice of rats for Western blotting are provided in [Table 7]. Figure 5 middle.

[1080] Preparation of Ket and NV-5138 for administration: Dissolve Ket (Sigma, catalog number K1884) in Sal at a concentration of 10 mg / mL. Administer 1 mL / kg volume of NV-5138 (Navitor, batch number 06) prepared by dissolving in Veh at a concentration of 50 mg / mL via intraperitoneal injection. Administer orally via tube feeding at a dose based on animal weight (3.2 mL / kg). Prepare test items on the day of administration.

[1081] Prefrontal cortical synaptosome preparation: The brains of rats in all groups (n=6 / group) were dissected and washed in PBS. PFCs were collected as shown in Figure 6 and homogenized at 4°C in homogenization buffer (a mixture of 0.32M sucrose, 20mM HEPES, 1mM EDTA, 5mM NaF, 1mM NaVO3 and protease inhibitors at pH 7.4 (Roche; #19543200)). The homogenate was centrifuged at 2,800 rpm for 10 min at 4°C, then the supernatant was removed and centrifuged again at 12,000 rpm for 10 min at 4°C. The resulting aggregates containing coarse synapses were resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM NaVO3, 5 mM NaF, and a mixture of protease inhibitors) and sonicated on ice at 50% amplitude for 20 seconds. Protein concentrations were determined using a Bradford assay. All samples were mixed with loading buffer (60 mM Tris-HCl pH 6.8, 20 mM DTT, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) and stored at -20°C until Western blotting analysis.

[1082] Western blot analysis: Western blot analysis of GluR1, PSD95, and GAPDH was performed as previously described. In short, the synaptosome preparation (15 μg total protein) was loaded into a 10–15% SDS-PAGE gel for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane in 10× premixed electrophoresis buffer (containing 25 mM Tris, 192 mM glycine, pH 8.3; Bio-Rad). The membrane was then blotted at room temperature with blocking buffer (2% BSA in PBS-T (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, and 0.1% phosphate)... Tween-20 was used to block PVDF membranes for 1 hour, followed by overnight incubation at 4°C in blocking buffer with the following primary antibodies: 1:1000 rabbit anti-pmTOR (cell signaling; #5536), 1:1000 rabbit anti-mTOR (cell signaling; #2972), 1:1000 rabbit anti-pp70S6K (cell signaling; #9205), and 1:1000 rabbit anti-p70S6K (cell signaling). Rabbit anti-p4E-BP1 (cell signaling; #2708), rabbit anti-GluR1 (cell signaling; #13185), rabbit anti-synaptic 1 (cell signaling; #5297), rabbit anti-PSD95 (cell signaling; #9644), and rabbit anti-GAPDH (cell signaling; #5174) were diluted 1:1000. The following day, the membrane was washed three times in PBS-T buffer and incubated for 1 hour with horseradish peroxidase conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Laboratories Inc.) diluted 1:5000 to 1:10000. After a final three washes with PBS-T buffer, enhanced chemiluminescence was used to detect the bands. The blots were then incubated in regeneration buffer (2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris-HCl, pH 6.8) at 50–55 °C for 30 min, followed by washing three times with PBS-T buffer. The regenerated blots were held in blocking solution for 1 h and incubated with primary antibodies targeting the corresponding protein or total GAPDH content of the loaded control. Densitometry analysis of phosphorylation and total immunoreactivity of each protein was performed using NIH ImageJ software. The resulting densitometry readings were used to generate the ratio of phosphorylated protein to its corresponding total protein content or, as indicated, GAPDH. The resulting ratios were further normalized to the Sal or Veh treatment control group for each protein.

[1083] Results: Western blotting (WB) analysis results of pmTOR, pp70S6K, and P4E-BP, normalized to controls Veh or Sal, are summarized in Figure 7. NV-5138 and Ket administration significantly increased the levels of pmTOR and p4E-BP1 in crude synaptosomes prepared from PFC 1 hour after administration. Furthermore, NV-5138 (but not Ket) significantly increased the level of pp70S6K 1 hour after administration. WB analysis results of synaptic proteins GluR1 and synaptic protein 1 are summarized in Figure 8. NV-5138 and Ket administration significantly increased the levels of GluR1 and synaptic protein 1 in crude synaptosomes prepared from PFC 24 hours after administration. Furthermore, Ket significantly increased the level of PSD95 24 hours after administration, while there was a trend towards increased expression after NV-5138 administration.

[1084] Table 7: Example of Research Design B

[1085]

[1086] Example C: Effects of a single Roal dose of NV-5138 on the mTORC1 signaling pathway in multiple regions of the rat brain

[1087] Study Design: After a 5-day acclimatization period, ten (10) male rats weighing between 175 and 200 g were randomly assigned to two study groups (n = 5 / group). Group 1 received a single dose of Veh via oral tube feeding, and Group 2 received a single dose of NV-5138 (160 mg / kg prepared in Veh) via oral tube feeding. One hour after administration, rats were euthanized by awake decapitation, and plasma was collected for NV-5138 exposure analysis, except for microdissection of the PFC, hippocampus, striatum, neocortex, and cerebellum. Total protein extracts were prepared from the collected tissues and submitted for Western blotting analysis, followed by quantitative analysis of the selected mTORC1 substrate. The study design is presented in Table 8. The timetable for test item administration and the euthanasia of rats via Western blotting are provided in Table 8. Figure 9 middle.

[1088] NV-5138 (160 mg / mL): NV-5138 (Navitor, batch number 09) was prepared at a concentration of 160 mg / mL by dissolving in Veh. The administration volume (10 mL / kg) based on animal weight was administered orally to the study animals in Group 2 via tube feeding. Test items were prepared on the day of administration.

[1089] Western blot analysis: Synaptosome preparations (15 μg total protein) were loaded and separated on NuPAGE 4-12% Bis-Tris gels and transferred to PVDF membranes (Immobilon-FL PVDF membrane, Millipore) using CAPS buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 12.5% ​​ethanol, pH 10). After transfer, the membrane was incubated in Odyssey blocking buffer (Licor) for 1 hour at room temperature. After blocking, the membrane was incubated overnight at 4°C with the primary antibody. The primary antibody used was rabbit anti-antibody at a 1:1000 dilution in Odyssey blocking buffer. S400 / 440 pS6 (cell signaling; #5364) and 1:10000 mouse anti-α-tubulin (Sigma; #T5168). The following day, the membrane was washed three times in 1×TBS-Tween (25 mM Tris, pH 7.4, 3.0 mM KCl, 140 mM NaCl, and 0.05% Tween-20) and incubated for 30 minutes in Odyssey blocking buffer with dye-conjugated secondary antibodies (goat anti-mouse IRdye680 and goat anti-rabbit IRdye800 from LI-COR), followed by three washes in 1×TBS-Tween. Signal quantification was performed using an Odyssey infrared imaging system (LI-CORBioscience). The resulting densitometer readings were used to generate the ratio of phosphorylated proteins to α-tubulin. The resulting ratio was further normalized to the mordant-treated control group.

[1090] Prefrontal cortical synaptosome preparation: One hour after administration, rats were euthanized by awake decapitation, and plasma and brains were collected. The brains of each group (n=5, Veh; n=5, NV) were dissected and washed in PBS. Figure 10The PFC, striatum, hippocampus, neocortex, and cerebellum were collected and homogenized at 4°C in a homogenization buffer (0.32M sucrose, a mixture of 20mM HEPES, 1mM EDTA, 5mM NaF, 1mM NaVO3, and a protease inhibitor in pH 7.4 (Roche; #19543200)). The homogenate was centrifuged at 2,800 rpm at 4°C for 10 minutes, the supernatant was removed, and the homogenate was centrifuged again at 12,000 rpm at 4°C for 10 minutes. The resulting aggregates were resuspended in a lysis buffer (50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.1% SDS, 2mM EDTA, 1mM NaVO3, 5mM NaF, and a protease inhibitor in a mixture) and sonicated on ice at 50% amplitude for 20 seconds. Total protein concentration was determined by Bradford assay, and all samples were mixed with loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) and stored at -20°C until Western blot analysis.

[1091] Compound Analysis: To determine the concentration of compounds in plasma, proteins were precipitated from 50 μL of tissue homogenate containing 150 μL of internal standard (tolbutamide) in acetonitrile, followed by centrifugation at 3000 rpm for 10 min. One hundred μL of the resulting supernatant was added to 100 μL of water, thoroughly mixed, and injected onto an LC-MS / MS system using the following procedure for assessing compound concentration:

[1092] Phenomenex LUX cellulose column (4.6 × 150 mm, 5 μm)

[1093] • Mobile phase A - 0.1% formic acid aqueous solution

[1094] • Mobile phase B - Acetonitrile containing 0.1% formic acid

[1095] ·gradient:

[1096] Initial -40% A

[1097] ο2 minutes - 40% A

[1098] ο2.1 minutes - 2% A

[1099] ο3 minutes - 2% A

[1100] ο3.1 minutes - 40% A

[1101] ο4 minutes - 40% A

[1102] • Flow rate 0.8 mL / min

[1103] • Column temperature 40℃

[1104] Sciex 5500 Triple Quad mass spectrometry

[1105] Results: Results of exposure to NV-5138 in brain regions summarized in Figure 11 In summary, oral administration of 160 mg / kg NV-5138 to rats with random access to food produced significant activation of mTORC1 in most, but not all, major brain regions.

[1106] Table 8: Study Design for Example C

[1107]

[1108] Example D: Effects of a single oral dose of NV-5138 or leucine on the mTORC1 signaling pathway in rat brain and selected peripheral organs.

[1109] Study Design: After a 5-day acclimatization period, thirty (30) male rats weighing between 175 and 200 g were randomly assigned to three study groups (n = 10). Rats were administered orally via tube feeding at the dosages shown in Table 9. Figure 12 The timetable shown in the figure shows the administration of test items. One hour after administration, rats were euthanized by conscious decapitation, and plasma, brain, and selected peripheral tissues were collected for compound content and Western blotting analysis. Tissue quantification of the mTORC1 substrate pS6 prepared for Western blotting was used as a measure of mTORC1 activity.

[1110] Preparation of test articles: NV-5138 (Navitor, batch number 12) and leucine (Leu, Sigma; #L8912) were prepared by dissolving in Veh (0.5% methylcellulose / 0.1% Tween-80) at concentrations of 16 mg / mL and 100 mg / mL, respectively. The administration was performed orally via tube feeding at a volume based on animal weight (10 mL / kg). Test articles were prepared on the day of administration.

[1111] Tissue preparation: One hour after drug administration, rats were euthanized by conscious decapitation, and plasma, brain, and peripheral tissues were collected and immediately frozen in liquid nitrogen. Thawed tissues were homogenized twice at 4°C in lysis buffer (cell lysis buffer: 1% Triton X-100, 50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM EDTA, 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, and one protease inhibitor strip per 50 mL of fresh solution) for 1 minute each. The lysates were then sonicated on ice at 50% amplitude for 20 seconds. Protein concentrations were determined by Bradford assay, and all samples were mixed with loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) and stored at -20°C until Western blotting analysis.

[1112] Western blot (WB) analysis: Equal volumes of each sample (15 μg total protein) were loaded and separated on NuPAGE 4–12% Bis-Tris gels. The samples were then transferred to PVDF membranes (Immobilon-FL PVDF membrane, Millipore) using CAPS buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 12.5% ​​ethanol, pH 10). After transfer, the membranes were incubated in Odyssey blocking buffer (Licor) for 1 hour at room temperature. Following blocking, the membranes were incubated overnight at 4°C with the primary antibody. The primary antibodies used were 1:1000 rabbit anti-S400 / 440pS6 (cell signaling; #5364), 1:1000 mouse anti-GAPDH (Sigma; #G8795), and 1:10000 mouse anti-α-tubulin (Sigma; #T5168) in Odyssey blocking buffer. The following day, the membrane was washed three times in 1×TBS-Tween (25 mM Tris, pH 7.4, 3.0 mM KCl, 140 mM NaCl, and 0.05% Tween-20) and incubated for 30 minutes in Odyssey blocking buffer with dye-coupled secondary antibodies (goat anti-mouse IRdye680 and goat anti-rabbit IRdye800 from LI-COR), followed by three washes in 1×TBS-Tween. Signal quantification was performed using the Odyssey infrared imaging system (LI-COR Bioscience). The resulting densitometer readings were used to generate the ratio of phosphorylated proteins to α-tubulin or GAPDH. The resulting ratios were further normalized to the mordant-treated control group.

[1113] Compound Analysis: To determine the compound content in the tissue preparation, 70% isopropanol at a ratio of 3:1 v:w (μL:mg) was added to the tissue sample, followed by homogenization using a Biospec bead stirrer. Proteins were precipitated from 50 μL of the homogenized tissue in 150 μL of acetonitrile containing an internal standard (tolbutamide), and then centrifuged at 3000 rpm for 10 min. One hundred μL of the resulting supernatant was added to 100 μL of water, thoroughly mixed, and injected onto an LC-MS / MS system using the following procedure for assessing compound content:

[1114] Phenomenex LUX cellulose column (4.6 × 150 mm, 5 μm)

[1115] • Mobile phase A - 0.1% formic acid aqueous solution

[1116] • Mobile phase B - Acetonitrile containing 0.1% formic acid

[1117] ·gradient:

[1118] Initial -40% A

[1119] ο2 minutes - 40% A

[1120] ο2.1 minutes - 2% A

[1121] ο3 minutes - 2% A

[1122] ο3.1 minutes - 40% A

[1123] ο4 minutes - 40% A

[1124] • Flow rate 0.8 mL / min

[1125] • Column temperature 40℃

[1126] Sciex 5500 Triple Quad mass spectrometry

[1127] Results: The results of a single NV-5138 injection and its effect on mTORC1 activation are summarized in... Figure 13 In contrast, NV-5138 ...

Claims

1. Use of a compound or a pharmaceutically acceptable composition thereof in combination with one or more additional antidepressant therapeutic agents in the manufacture of a medicament for treating a disease, disorder, or condition in a patient in need thereof, wherein the disease, disorder, or condition is treatment-resistant depression, and the compound is: ###0001### or a pharmaceutically acceptable salt thereof, wherein the one or more additional antidepressant therapeutic agents are suitable for the disease being treated, and wherein the one or more additional antidepressant therapeutic agents are administered simultaneously, separately, or sequentially with the compound or a pharmaceutically acceptable composition thereof.

2. The use of claim 1, wherein the one or more additional antidepressant therapeutic agents are one or more of bupropion, venlafaxine, mirtazapine, duloxetine, amitriptyline, and imipramine.

3. The use of claim 1, wherein the one or more additional antidepressant therapeutic agents are one or more of selegiline, nortriptyline, trazodone, desvenlafaxine, and aripiprazole.

4. The use of claim 1, wherein the one or more additional antidepressant therapeutic agents are a selective serotonin reuptake inhibitor (SSRI).

5. The use of claim 4, wherein the SSRI is one or more of sertraline, escitalopram, citalopram, fluvoxamine, fluoxetine, and paroxetine.

6. The use of claim 4, wherein the SSRI is sertraline.

7. The use of claim 4, wherein the SSRI is escitalopram.

8. The use of claim 4, wherein the SSRI is citalopram.

9. The use of claim 4, wherein the SSRI is fluvoxamine.

10. The use of claim 4, wherein the SSRI is fluoxetine.

11. The use of claim 4, wherein the SSRI is paroxetine.

12. The use of claim 1, wherein the one or more additional antidepressant therapeutic agents are administered simultaneously or separately with the compound or a pharmaceutically acceptable composition thereof.

13. The use of claim 1, wherein the one or more additional antidepressant therapeutic agents are administered in individual unit dosage forms or as a single unit dosage form with the compound or a pharmaceutically acceptable composition thereof.

14. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier, adjuvant, or vehicle.

15. The use of claim 1, wherein the combination further comprises a pharmaceutically acceptable carrier, adjuvant, or vehicle.

16. The use of claim 1, wherein the treatment-resistant depression is resistant to first-line therapy.

17. The use of claim 1, wherein the treatment-resistant depression is resistant to second-line therapy. ​

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