Spiro heterocyclic CDK2 inhibitors

Abstract

The present disclosure describes compounds of Formula (I), (I), or a pharmaceutically acceptable salt thereof, pharmaceutical compositions comprising the compounds, methods of preparation, and methods of use. The compounds of the disclosure can inhibit a cyclin-dependent kinase, such as CDK2, and are believed to be useful for treating a disease or condition associated with CDK2 activity, such as a cancer.

Description

Attorney Docket No.: 064105-502001WOSPIRO HETEROCYCLIC CDK2 INHIBITORS BACKGROUND

[0001] Cell cycle dysregulation, including uncontrolled cell growth, impaired cell differentiation and abnormal apoptosis have been shown to be caused by over activity of Cyclin-dependent kinases (CDKs). CDKs are important serine / threonine protein kinases that become active when combined with a specific cyclin partner. There are various subtypes of CDKs, each having a different role during the cell cycle, with varying levels of activity during each of the phases. CDK1, CDK2, CDK4 and CDK6 have been found to be specifically important subtypes, where over activity of one or more of these subtypes may lead to dysregulation of the cell cycle and the development of a variety of cancers. The S phase of the cell cycle is responsible for DNA replication and is the phase where aberrant DNA replication may occur. The CDK2 / cyclin E complex is required for the cell cycle transition from the G1 phase to the S phase. Therefore, selective inhibition of the CDK2 / cyclin E complex can prevent aberrant DNA replication and can be used to treat certain cancers.

[0002] PCT application publications WO 2022 / 165513, WO 2024 / 026479, WO 2024 / 026481, WO 2024 / 026483, WO 2024 / 026484, and WO 2024 / 026486 reported synthesis and evaluation of selective CDK2 inhibitors.

[0003] Accordingly, there is a need for the development of compounds capable of inhibiting the activity of CDK2 / cyclin complexes, and pharmaceutical compositions thereof, for the prevention, and treatment of CDK2 related diseases or disorders.BRIEF SUMMARY

[0004] The present disclosure provides a compound of Formula I:or pharmaceutically acceptable salt thereof,whereineach X1, X2, X3is -C(Xla)2-, -NXlb-, -O-, or -S(0)o-2-;X4is -C(Xla)2-, -NXlb-, -O-, -S(0)o-2-, or is absent;provided that when one of X1and X2is -NXlb-, -O-, or -S(0)o-2-, then the other is -C(Xla)2-, and when X4is present, one of X3and X4is -NXlb-, -O-, or -S(0)o-2-, then the other is -C(Xla)2-;each Xlais independently H, C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-C6haloalkoxy, halogen, -ORla, -NRlbRlc, -CN, -C(O)Rla, -C(O)NRlbRlc, -OC(O)NRlbRlc, -N(Rla)C(O)NRlbRlc, or -S(O)2NRlaRlb, or alternatively, two Xlaattached to the same carbon combine to form C3-C6 cycloalkyl, 4- to 10-membered heterocyclyl, or oxo;Xlbis H, C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, or Ci-Ce haloalkyl;'ll represents a single or a double bond;L is -C(=O)-NH-, -NH-C(=O)-, -CH(RL)-NH-, -CH(RL)-N(CH3)-, -NH-CH(RL)-, -CH(RL)- O-, -O-CH(RL)-, or a 5-membered heteroaryl;RLis H or C1-C6alkyl;R1is C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, halogen, -ORla, -NRlbRlc, -CN, -C(O)Rla,-C(O)NRlbRlc, -OC(O)NRlbRlc, -N(Rla)C(O)NRlbRlc, or -S(O)2NRlaRlb, or alternatively, two R1attached on the same carbon combine to form C3-C6 cycloalkyl; each Rla, Rlb, and Rlcis independently H, C1-C6alkyl, or Ci-Ce haloalkyl;variable m is 1 or 2;variable n is 0, 1, 2, or 3, provided that if variable n is 0 or 1, then at least one of X2and X3is -C(Xla)2-;variable p is 0, 1, 2, 3, or 4;variable q is 0 or 1;Ring A is a C5-C8cycloalkyl or 5- to 8-membered heterocyclyl, wherein the cycloalkyl or heterocyclyl is substituted with 0, 1, 2, 3, or 4 R2;each R2is independently C1-C6alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, or halogen, or two R2attached to the same carbon combine to form oxo;R3is a 8- to 12-membered spiro heterocyclyl, which is substituted by two L3a;each L3ais independently R3a, R3a-C(=O)-, (R3a)NH-C(=O)-, (R3a)O-C(=O)-, -CH(R3a)-, or a 5-membered heteroaryl substituted by R3a;each R3ais independently Ci-Ce haloalkyl, C3-C8 cycloalkyl, 4- to 10-membered heterocyclyl, Ce-Cio aryl, or 5- to 10-membered heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, or 3 C1-C6alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, or halogen; andR4is C1-C6alkyl or Ci-Ce haloalkyl.

[0005] Also provided herein is a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof of the present disclosure.

[0006] Further provided herein is a method of inhibiting activity of a cyclin-dependent kinase (CDK) in a cell, comprising administering to the cell an effective amount of a compound or pharmaceutically acceptable salt thereof of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0007] Further provided herein is a method of treating a disease or disorder associated with CDK2 activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof of the present disclosure, or a pharmaceutical composition of the present disclosure.DETAILED DESCRIPTIONI. GENERAL

[0008] The disclosure relates generally to spiro heterocyclic compounds, pharmaceutically acceptable salts thereof, and methods and uses thereof, for inhibiting cyclin-dependent kinase 2 (CDK2) and / or CDK2 / cyclin complexes. The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description isnot intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.II. DEFINITIONS

[0009] As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0010] A dashthat is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -CONH2 is attached through the carbon atom. A dash at the front or end of a chemical group is a matter of convenience; chemical groups can be depicted with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group.

[0011] A squiggly line on a chemical group as shown below, for example,vOHindicates a point of attachment, i.e., it shows the broken bond by which the group is connected to another described group.

[0012] The prefix “Cu-Cv” indicates that the following group has from u to v carbon atoms. For example, “Ci-Cs alkyl” indicates that the alkyl group has from 1 to 8 carbon atoms.

[0013] “Alkyl” refers to a monovalent unbranched or branched saturated hydrocarbon chain. For example, an alkyl group can have 1 to 20 carbon atoms (i.e., C1-C20 alkyl), 1 to 8 carbon atoms (i.e., Ci-Cs alkyl), 1 to 6 carbon atoms (i.e., C1-C6alkyl), or 1 to 3 carbon atoms (i.e., C1-C3 alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl ( / / -Pr, / / -propyl, -CH2CH2CH3), 2-propyl (z-Pr, z-propyl, -CH(CH3)2), 1 -butyl (zz-Bu, / / -butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (z-Bu, z-butyl, -CH2CH(CH3)2), 2-butyl (.s-Bu,.s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl ( / -Bu, / -butyl, -C(CH3)3), 1 -pentyl ( / / -pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3 -pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3 -methyl- 1 -butyl (-CH2CH2CH(CH3)2), 2-methyl-l-butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3 -hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3 -methyl-3 -pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3 -pentyl (-CH(CH2CH3)CH(CH3)2), 2,3 -dimethyl-2 -butyl (-C(CH3)2CH(CH3)2), and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, pentadcyl, hexadecyl, heptadecyl and octadecyl.

[0014] “Alkenyl” refers to an unbranched or branched hydrocarbon chain containing at least two carbon atoms and at least one carbon-carbon double bond. As used herein, alkenyl can have from 2 to 20 carbon atoms (i.e., C2-20 alkenyl), 2 to 8 carbon atoms (i.e., C2-8 alkenyl), 2 to 6 carbon atoms (i.e., C2-6 alkenyl), or 2 to 4 carbon atoms (i.e., C2-4 alkenyl). Alkenyl can include any number of carbons, such as C2, C3, C4, C5, Ce, C7, Cs, C9, C10, C11, C12, Ci3, C14, C15, Ci6, C17, Cis, C19, C20, or any range therein. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more.Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1 -pentenyl, 2-pentenyl, isopentenyl, 1,3 -pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,1,3 -hexadienyl, 1,4-hexadienyl, 1,5 -hexadienyl, 2,4-hexadienyl, or 1,3, 5 -hexatrienyl.

[0015] “Alkynyl” refers to an unbranched or branched hydrocarbon chain containing at least one carbon-carbon triple bond. For example, an alkynyl group can have from 2 to 20 carbon atoms (i.e., C2-20 alkynyl), 2 to 8 carbon atoms (i.e., C2-8 alkynyl), 2 to 6 carbon atoms (i.e., C2-6 alkynyl), or 2 to 4 carbon atoms (i.e., C2-4 alkynyl). The term “alkynyl” also includes those groups having one triple bond and one double bond. Examples of C2-6alkynyl include, but are not limited to, ethynyl, prop-l-ynyl, but-l-ynyl, pent-l-ynyl, pent-4-ynyl and penta- 1,4-diynyl.

[0016] “Alkoxy” means a group having the formula -O-alkyl, in which an alkyl group, as defined above, is attached to the parent molecule via an oxygen atom. The alkyl portion of an alkoxy group can have 1 to 20 carbon atoms (i.e., C1-C20 alkoxy), 1 to 12 carbon atoms (i.e., C1-C12 alkoxy), 1 to 8 carbon atoms (i.e., Ci-Cs alkoxy), 1 to 6 carbon atoms (i.e., Ci-Ce alkoxy) or 1 to 3 carbon atoms (i.e., Ci-C3alkoxy). Examples of suitable alkoxy groups include, but are not limited to, methoxy (-O-CH3or -OMe), ethoxy (-OCH2CH3or -OEt), isopropoxy (-O-CH(CH3)2), t-butoxy (-O-C(CH3)3or -OtBu) and the like. Other examples of suitable alkoxy groups include, but are not limited to, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like.

[0017] “Halo” or “halogen” as used herein refers to fluoro (-F), chloro (-C1), bromo (-Br) and iodo (-1).

[0018] “Haloalkyl” is an alkyl group, as defined above, in which one or more hydrogen atoms of the alkyl group is replaced with a halogen atom. The alkyl portion of a haloalkyl group can have 1 to 20 carbon atoms (i.e., C1-C20 haloalkyl), 1 to 12 carbon atoms (i.e., Ci-C12 haloalkyl), 1 to 8 carbon atoms (i.e., Ci-Cs haloalkyl), 1 to 6 carbon atoms (i.e., Ci-Ce haloalkyl) or 1 to 3 carbon atoms (i.e., C1-C3 haloalkyl). The alkyl groups can be substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more halogens. Examples of suitable haloalkyl groups include, but are not limited to, -CF3, -CHF2, -CFH2, -CH2CF3, fluorochloromethyl, difluorochloromethyl, 1,1,1 -trifluoroethyl and pentafluoroethyl.

[0019] “Haloalkoxy” refers to an alkoxy group where some or all of the hydrogen atoms are substituted with halogen atoms. As for an alkyl group, haloalkoxy groups can have any suitable number of carbon atoms, such as C1-6. The alkoxy groups can be substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more halogens. When all the hydrogens are replaced with a halogen, for example by fluorine, the compounds are per-substituted, for example, perfluorinated. Haloalkoxy includes, but is not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, perfluoroethoxy, etc.

[0020] “Cycloalkyl” refers to a saturated or partially saturated cyclic alkyl group having a single ring or multiple rings, such as 2, 3, 4 or more, wherein the multiple rings can be fused, bridged, spiro, or any combination thereof. As used herein, cycloalkyl has from 3 to 20 ring carbon atoms (i.e., C3-20 cycloalkyl), 3 to 12 ring carbon atoms (i.e., C3-12 cycloalkyl), 3 to 10 ring carbon atoms (i.e., C3-10 cycloalkyl), 3 to 8 ring carbon atoms (i.e., C3-8 cycloalkyl), or 3 to 6 ring carbon atoms (i.e., C3-6 cycloalkyl). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, and cyclooctyl. Cycloalkyl groups also include partially unsaturated ring systems containing one or more double bonds, including fused ring systems with one aromatic ring and one non-aromatic ring, but not fully aromatic ring systems.

[0021] “Heterocycle” or “heterocyclyl” or “heterocycloalkyl” refer to a saturated or partially unsaturated cyclic alkyl group, with one or more ring heteroatoms independently selected from nitrogen, oxygen, sulfur and silicon. A heterocyclyl can be a single ring or multiple rings, such as 2, 3, 4 or more, wherein the multiple rings can be fused, bridged, spiro, or any combination thereof. As used herein, heterocyclyl has 3 to 20 ring atoms (i.e., 3 to 20 membered heterocyclyl), 3 to 12 ring atoms (i.e., 3 to 12 membered heterocyclyl), 3 to 10 ring atoms (i.e., 3 to 10 membered heterocyclyl), 3 to 8 ring atoms (i.e., 3 to 8 memberedheterocyclyl), 4 to 12 ring carbon atoms (i.e., 4 to 12 membered heterocyclyl), 4 to 8 ring atoms (i.e., 4 to 8 membered heterocyclyl), or 4 to 6 ring atoms (i.e., 4 to 6 membered heterocyclyl). Examples of heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, oxetanyl, dioxolanyl, azetidinyl, and morpholinyl.

[0022] “Aryl” means an aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Exemplary aryl groups include, but are not limited to, radicals derived from benzene (e.g., phenyl), naphthalene, anthracene, biphenyl, and the like.

[0023] “Heteroaryl” refers to an aromatic group, including groups having an aromatic tautomer or resonance structure, having a single ring, multiple rings, or multiple fused rings, with at least one heteroatom in the ring, i.e., one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the nitrogen or sulfur can be oxidized. Thus, the term includes rings having one or more annular O, N, S, S(O), S(O)2, and N-oxide groups. The term includes rings having one or more annular C(O) groups. As used herein, heteroaryl include 5 to 20 ring atoms (i.e., 5- to 20-membered heteroaryl), 5 to 12 ring atoms (i.e., 5- to 12-membered heteroaryl), or 5 to 10 ring atoms (i.e., 5- to 10-membered heteroaryl), and 1 to 5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and oxidized forms of the heteroatoms. Examples of heteroaryl groups include, but are not limited to, pyridin-2(lH)-one, pyridazin-3(2H)-one, pyrimidin-4(3H)-one, quinolin-2(lH)-one, pyrimidinyl, purinyl, pyridyl, pyridazinyl, benzothiazolyl, and pyrazolyl. Heteroaryl does not encompass or overlap with aryl as defined above.

[0024] “ Substituted” as used herein refers to wherein one or more hydrogen atoms of the group are independently replaced by one or more substituents (e.g., 1, 2, 3, or 4 or more) as indicated.

[0025] A “compound of the present disclosure” includes compounds disclosed herein, for example a compound of the present disclosure includes compounds of Formula 1, 1-1, la, la-1, lb, Ib-1, II, II- 1, II-2, and II-3, including the compounds of the Examples.

[0026] “ Treatment” or “treating” as used herein is an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results may include one or more of the following: (a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of thedisease or condition); (b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and / or preventing or delaying the spread (e.g., metastasis) of the disease or condition); and / or (c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0027] The term “therapeutically effective amount,” as used herein, is the amount of compound disclosed herein present in a formulation described herein that is needed to provide a desired level of drug in the bloodstream of a subject to be treated to give an anticipated physiological response or desired biological effect when such a formulation is administered by the chosen route of administration. The precise amount will depend upon numerous factors, for example the particular compound disclosed herein, the specific activity of the formulation, the delivery device employed, the physical characteristics of the formulation, its intended use, as well as subject considerations such as severity of the disease state, subject cooperation, etc., and can readily be determined by one skilled in the art based upon the information provided herein.

[0028] “Administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject. The administration can be carried out according to a schedule specifying frequency of administration, dose for administration, and other factors.

[0029] A “subject” or “patient” is meant to describe a human or vertebrate animal including a dog, cat, pocket pet, marmoset, horse, cow, pig, sheep, goat, elephant, giraffe, chicken, lion, monkey, owl, rat, squirrel, slender loris, and mouse. A “pocket pet” refers to a group of vertebrate animals capable of fitting into a commodious coat pocket such as, for example, hamsters, chinchillas, ferrets, rats, guinea pigs, gerbils, rabbits and sugar gliders.III. COMPOUNDS

[0030] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound is a compound of Formula I:whereineach X1, X2, X3is -C(Xla)2-, -NXlb-, -O-, or -S(0)o-2-;X4is -C(Xla)2-, -NXlb-, -O-, -S(0)o-2-, or is absent;provided that when one of X1and X2is -NXlb-, -O-, or -S(0)o-2-, then the other is -C(Xla)2-, and when X4is present, one of X3and X4is -NXlb-, -O-, or -S(0)o-2-, then the other is -C(Xla)2-;each Xlais independently H, C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-C6haloalkoxy, halogen, -ORla, -NRlbRlc, -CN, -C(O)Rla, -C(O)NRlbRlc, -OC(O)NRlbRlc, -N(Rla)C(O)NRlbRlc, or -S(O)2NRlaRlb, or alternatively, two Xlaattached to the same carbon combine to form C3-C6 cycloalkyl, 4- to 10-membered heterocyclyl, or oxo;Xlbis H, C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, or Ci-Ce haloalkyl;'ll represents a single or a double bond;L is -C(=O)-NH-, -NH-C(=O)-, -CH(RL)-NH-, -CH(RL)-N(CH3)-, -NH-CH(RL)-, -CH(RL)-O-, -O-CH(RL)-, or a 5-membered heteroaryl;RLis H or C1-C6alkyl;R1is Ci-C6alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, halogen, -ORla, -NRlbRlc, -CN, -C(O)Rla,-C(O)NRlbRlc, -OC(O)NRlbRlc, -N(Rla)C(O)NRlbRlc, or -S(O)2NRlaRlb, or alternatively, two R1attached on the same carbon combine to form C3-C6 cycloalkyl; each Rla, Rlb, and Rlcis independently H, C1-C6alkyl, or Ci-Ce haloalkyl;variable m is 1 or 2;variable n is 0, 1, 2, or 3, provided that if variable n is 0 or 1, then at least one of X2and X3is -C(Xla)2-;variable p is 0, 1, 2, 3, or 4;variable q is 0 or 1;Ring A is a C5-C8cycloalkyl or 5- to 8-membered heterocyclyl, wherein the cycloalkyl or heterocyclyl is substituted with 0, 1, 2, 3, or 4 R2;each R2is independently C1-C6alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, or halogen, or two R2attached to the same carbon combine to form oxo;R3is a 8- to 12-membered spiro heterocyclyl, which is substituted by two L3a;each L3ais independently R3a, R3a-C(=O)-, (R3a)NH-C(=O)-, (R3a)O-C(=O)-, -CH(R3a)-, or a 5-membered heteroaryl substituted by R3a;each R3ais independently Ci-Ce haloalkyl, C3-C8 cycloalkyl, 4- to 10-membered heterocyclyl, Ce-Cio aryl, or 5- to 10-membered heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, or 3 C1-C6alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, or halogen; andR4is C1-C6alkyl or Ci-Ce haloalkyl.

[0031] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound is a compound of Formula 1-1:

[0032] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound of Formula I has the structure of Formula la:

[0033] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound of Formula I has the structure of Formula la-1:

[0034] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound of Formula I has the structure of Formula Ib:

[0035] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound of Formula I has the structure of Formula Ib-1:

[0036] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, X1is -C(=O); and X2is -NH-. In some embodiments, X1is -CH2-; and X2is -O-. In some embodiments, X1is -CH(OH)-; and X2is -CH2-.

[0037] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, each Xlais independently H, C1-C6alkyl, C1-C6haloalkyl, halogen, or -OR1a.

[0038] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, each Xlaisindependently H, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, halogen, or -OR1a. In some embodiments, each Xlais independently H, CH3, OCH3, CF3, F, or OH.

[0039] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, Xlbis H or C1-C6alkyl. In some embodiments, X1bis H or methyl. In some embodiments, X1bis H.

[0040] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, X3is -CH2-.

[0041] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, X4is -O-.

[0042] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, X4is absent. In instances where X4is absent, X3is bound to the adjacent carbons by single bonds.

[0043] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, and / or lb, or pharmaceutically acceptable salt thereof, L is -C(=O)-NH-, -CH(RL)-NH-, -CH(RL)-O-, or a 5-membered heteroaryl. In some embodiments, L is -C(=O)-NH-, -CH2O-, -CH(Me)-O-, or triazolyl. In some embodiments, L is -C(=O)-NH-. In some embodiments, L is -CH2O- or -CH(Me)-O-. In some embodiments, L is triazolyl. In some embodiments, L is

[0044] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, and / or lb, or pharmaceutically acceptable salt thereof, RLis H.

[0045] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, and / or lb, or pharmaceutically acceptable salt thereof, R1is C1-C6alkyl, C1-C6haloalkyl, C1-C6haloalkoxy, halogen, -OR1a, -NR1bR1c, or -CN. As used herein, R1specifies substitution on the macrocycle unless already indicated by other variables, e.g., X1, X2, X3, and X4.

[0046] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, and / or lb, or pharmaceutically acceptable salt thereof, each R1is independently CH3, OCH3, CF3, F, or OH.

[0047] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, and / or lb, or pharmaceutically acceptable salt thereof, variable m is 1. In some embodiments, variable m is 2.

[0048] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, or pharmaceutically acceptable salt thereof, variable n is 0. In some embodiments, variable n is 1. In some embodiments, variable n is 2. In some embodiments, variable n is 3.

[0049] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, and / or lb, or pharmaceutically acceptable salt thereof, variable p is 0. In some embodiments, variable p is 1. In some embodiments, variable p is 2. In some embodiments, variable p is 3. In some embodiments, variable p is 4.

[0050] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, or pharmaceutically acceptable salt thereof, variable q is 0. In some embodiments, variable q is 1.

[0051] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, Ring A is cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, or tetrahydropyranyl, which are each substituted with 0, 1, 2, or 3 R2. In some embodiments, Ring A is cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, or tetrahydropyranyl, which are each substituted with 0, 1, or 2 R2. In some embodiments, Ring A is cycloheptyl, which is substituted with 0, 1, or 2 R2.

[0052] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, each R2is independently halogen. In some embodiments, R2is F.

[0053] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, R3iswherein variable t is 1 or 2; and variable w is 1 or 2.

[0054] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, R3iswherein variable t is 1 or 2; and variable w is 1 or 2.

[0055] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, variable t is 1. In some embodiments, variable t is 2.

[0056] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, variable w is 1. In some embodiments, variable w is 2.

[0057] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, R4is C1-C3 alkyl. In some embodiments, R4is methyl.

[0058] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, or pharmaceutically acceptable salt thereof, the compound has the structure of Formula II:

[0059] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, or pharmaceutically acceptable salt thereof, the compound has the structure of Formula II- 1:

[0060] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, each R3ais independently C3-C8 cycloalkyl or 5- to 10-membered heteroaryl, wherein the cycloalkyl and heteroaryl is independently substituted with 0, 1, 2, or 3 C1-C6alkyl or halogen.

[0061] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, R3ais 5- to 10-membered heteroaryl, substituted with 0, 1, 2, or 3 halogen. In some embodiments, R3ais thiazolyl, oxazolyl, pyridyl, pyrimidyl, pyrazinyl, benzoxazolyl, benzothiazolyl, thiazolo[5,4-c]pyridyl, thiazolo[4,5-d]pyrimidyl, or naphthyridyl, substituted with 0, 1, 2, or 3 F.

[0062] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, R3ais C3-C8 cycloalkyl, substituted with 0, 1, 2, or 3 C1-C6alkyl or halogen. In some embodiments, R3aiscyclopropyl substituted with 1, 2, or 3 methyl. In some embodiments, R3ais cyclopropyl substituted with two methyl.

[0063] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, or pharmaceutically acceptable salt thereof, the compound has the structure of Formula II-2:whereinR3bis a 5 -membered heteroaryl; andR3cis a C1-C6haloalkyl or C3-C4cycloalkyl substituted with 1 or 2 C1-C4alkyl.

[0064] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, or pharmaceutically acceptable salt thereof, the compound has the structure of Formula II-3:whereinRing A is cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, or tetrahydropyranyl, which are each substituted with 0, 1, or 2 R2;R3bis a 5 -membered heteroaryl; andR3cis a C1-C6haloalkyl or C3-C4cycloalkyl substituted with 1 or 2 C1-C4alkyl.

[0065] In some embodiments, R3bis thiazolyl or isothiazolyl. In some embodiments, R3bis

[0066] In some embodiments, R3cis C1-C4haloalkyl or C3-C4cycloalkyl substituted with 1 or 2 methyl. In some embodiments, R3cisIn some embodiments, R3cis

[0067] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula I, la, lb, and / or II, or pharmaceutically acceptable salt thereof, R3is

[0068] In some embodiments of the compound of the present disclosure, e.g., a compound of Formula 1, 1-1, la, la-1, lb, Ib-1, II, II-l, II-2, and / or II-3„ or pharmaceutically acceptable salt thereof, the compound is one selected from Table 1.Table 1. Compounds

[0069] Provided are also compounds described herein or pharmaceutically acceptable salts, isomers, or a mixture thereof, in which from 1 to n hydrogen atoms attached to a carbon atom can be replaced by a deuterium atom or D, in which n is the number of hydrogen atoms in the molecule. As known in the art, the deuterium atom is a non-radioactive isotope of the hydrogen atom. Such compounds can increase resistance to metabolism, and thus can be useful for increasing the half-life of the compounds described herein or pharmaceutically acceptable salts, isomer, or a mixture thereof when administered to a mammal. See, e.g., Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism,” TRENDS PHARMACOL. SCI., 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogen atoms have been replaced by deuterium.

[0070] Examples of isotopes that can be incorporated into the disclosed compounds also include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,36Cl,123I, and125I, respectively. Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of Formula I can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0071] Furthermore, the compounds of Formula I or pharmaceutically acceptable salt thereof may exist in the form of different isomers, in particular stereoisomers (including, e.g., geometric isomers (or cis / trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto / enol tautomers or thione / thiol tautomers). All such isomers of the compounds of Formula I are contemplated as being part of the present disclosure, either in admixture or in pure or substantially pure form. As for stereoisomers, the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures / racemates). The racemates can be resolved (i.e., separated) by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization. The present disclosure further encompasses any tautomers of the compounds of formula I and / or II. It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms. The formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.

[0072] The compounds of the embodiments disclosed herein, or their pharmaceutically acceptable salts can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for thepreparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. Where compounds are represented in their chiral form, it is understood that the embodiment encompasses, but is not limited to, the specific diastereomerically or enantiomerically enriched form. Where chirality is not specified but is present, it is understood that the embodiment is directed to either the specific diastereomerically or enantiomerically enriched form; or a racemic or scalemic mixture of such compound(s). As used herein, “scalemic mixture” is a mixture of stereoisomers at a ratio other than 1:1.

[0073] “Racemates” refers to a mixture of enantiomers. The mixture can comprise equal or unequal amounts of each enantiomer.

[0074] “ Stereoisomer” and “stereoisomers” refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds can exist in stereoisomeric form if they possess one or more asymmetric centers or a double bond with asymmetric substitution and, therefore, can be produced as individual stereoisomers or as mixtures. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see, e.g., Chapter 4 of ADVANCED ORGANIC CHEMISTRY, 4th ed., J. March, John Wiley & Sons, New York, 1992).

[0075] Provided are also pharmaceutically acceptable salts, hydrates, solvates, tautomeric forms, polymorphs, and prodrugs of the compounds described herein. “Pharmaceutically acceptable” or “physiologically acceptable” refer to compounds, salts, formulations, dosage forms and other materials which are useful in preparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

[0076] The compounds described herein can be prepared and / or formulated as pharmaceutically acceptable salts or when appropriate as a free base. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possess the desired pharmacological activity of the free base. These salts can be derived from inorganic ororganic acids or bases. For example, a compound that contains a basic nitrogen can be prepared as a pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthal ene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of other suitable pharmaceutically acceptable salts are found in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21stEdition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

[0077] Examples of “pharmaceutically acceptable salts” of the compounds disclosed herein also include salts derived from an appropriate base, such as an alkali metal (for example, sodium, potassium), an alkaline earth metal (for example, magnesium), ammonium and NX4+(wherein X is C1-C4 alkyl). Also included are base addition salts, such as sodium or potassium salts.IV. COMPOSITIONS

[0078] Also provided herein is a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0079] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes, but is not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and combinations thereof. The use of pharmaceutically acceptable carriers and pharmaceutically acceptable excipients for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic formulations is contemplated. Supplementary active ingredients can also beincorporated into the formulations. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

[0080] The compounds disclosed herein can be formulated with conventional carriers and excipients. Tablets can contain, for instance, excipients, glidants, fillers, binders, or a combination thereof. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. Exemplary excipients include, but are not limited to, those set forth in the “HANDBOOK OF PH RMACEUTICAL EXCIPIENTS” (1986). Excipients can include, for example, ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and combinations thereof. In some embodiments, the formulation is basic. In some embodiments, the formulation is acidic. In some embodiments, the formulation has a neutral pH. In some embodiments, the pH of the formulations is from 2 to 11 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 6-7, 6-8, 6-9, 6-10, 6-11, 7-8, 7-9, 7-10, 7-11, 8-9, 8-10, 8-11, 9-10, or 9-11).

[0081] In some embodiments, the compounds disclosed herein have pharmacokinetic properties (e.g., oral bioavailability) suitable for oral administration of the compounds.Formulations suitable for oral administration can, for instance, be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be administered, for instance, as a bolus, electuary, or paste.

[0082] A tablet can be made by compression or molding, optionally with at least accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as, for instance, a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active, dispersing agent, or a combination thereof. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets can optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.

[0083] Formulations for oral use can be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0084] For diseases of the eye or other external tissues (e.g., mouth and skin), the formulations can be applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w / w (including active ingredient(s) in a range from 0.1% to 20% in increments of 0.1% w / w such as 0.6% w / w, 0.7% w / w, etc.), from 0.2% to 15% w / w, or from 0.5% to 10% w / w. When formulated in an ointment, the active ingredients can be employed in some embodiments with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients can be formulated in a cream with an oil-in-water cream base.

[0085] In some embodiments, the aqueous phase of the cream base can include, for example, from 30% to 90% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%) w / w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. In some embodiments, the cream base can include, for instance, a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include, but are not limited to, dimethyl sulfoxide and related analogs. In some embodiments, the cream or emulsion does not include water.

[0086] The oily phase of the emulsions can be constituted from known ingredients in a known manner. In some embodiments, the phase comprises merely an emulsifier (otherwise known as an emulgent). In some embodiments, the phase comprises a mixture of at least one emulsifier with a fat, an oil, or a combination thereof. In some embodiments, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. Together, the emulsifier(s) with or without stabilizer(s) can make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base that can form the oily dispersed phase of the cream formulations.

[0087] Emulgents and emulsion stabilizers suitable for use in the formulation can include, but are not limited to, TWEEN® 60, TWEEN® 80, SPAN® 80, cetostearyl alcohol, benzylalcohol, myristyl alcohol, glyceryl mono-stearate, sodium lauryl sulfate, captisol (and other beta cyclodextrin vehicles), and combinations thereof.

[0088] The choice of suitable oils or fats for the formulation can be based on achieving the desired cosmetic properties. In some embodiments, the cream can be a non-greasy, nonstaining, and washable product with suitable consistency to avoid leakage from tubes or other containers. In some embodiments, esters can be included, such as, for example, straight or branched chain, mono- or dibasic alkyl esters such as di -isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, a blend of branched chain esters known as CRODAMOL® CAP, or a combination thereof. In some embodiments, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be included.

[0089] Effective dose of active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, the method of delivery, and the pharmaceutical composition, and will be determined by the clinician using conventional dose escalation studies. In some embodiments, the effective dose is from 0.0001 to 100 mg / kg body weight per day; for instance, from 10 to 30 mg / kg body weight per day; from 15 to 25 mg / kg body weight per day; from 10 to 15 mg / kg body weight per day; or from 20 to 30 mg / kg body weight per day. For example, the daily candidate dose for an adult human of approximately 70 kg body weight can range from 1 mg to 2000 mg (e.g., from 5 mg to 500 mg, from 500 mg to 1000 mg, from 1000 mg to 1500 mg, from 1500 mg to 2000 mg), and can take the form of single or multiple doses. For example, the daily candidate dose for an adult human of approximately 70 kg body weight can range from 1 mg to 1000 mg (e.g., from 5 mg to 500 mg), and can take the form of single or multiple doses.V. KITS

[0090] Also provided herein are kits that includes a compound disclosed herein or a pharmaceutically acceptable salt thereof. In some embodiments the kits described herein can comprise a label and / or instructions for use of the compound in the treatment of a disease or condition in a subject (e.g., human) in need thereof.

[0091] In some embodiments, the kit can also comprise one or more additional therapeutic agents and / or instructions for use of additional therapeutic agents in combination with thecompound disclosed herein in the treatment of the disease or condition in a subject (e.g., human) in need thereof.

[0092] In some embodiments, the kits provided herein comprise individual dose units of a compound as described herein, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate thereof. Examples of individual dosage units can include pills, tablets, capsules, prefilled syringes or syringe cartridges, IV bags, inhalers, nebulizers etc., each comprising a therapeutically effective amount of the compound in question, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate thereof. In some embodiments, the kit can contain a single dosage unit and in others multiple dosage units are present, such as the number of dosage units required for a specified regimen or period.

[0093] Also provided are articles of manufacture that include a compound disclosed herein, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers or tautomer thereof; and a container. In some embodiments, the container of the article of manufacture is a vial, jar, ampoule, preloaded syringe, blister package, tin, can, bottle, box, an intravenous bag, an inhaler, or a nebulizer.VI. ADMINISTRATION

[0094] One or more of the compounds of Formula I or pharmaceutically acceptable salt thereof (herein referred to as the active ingredients) are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the route may vary with for example the condition of the recipient. An advantage of the compounds herein is that they are orally bioavailable and can be dosed orally.

[0095] The compounds of the present disclosure can be administered by any route appropriate to the condition to be treated.

[0096] Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the route mayvary with for example the condition of the recipient. An advantage of certain compounds disclosed herein is that they are orally bioavailable and can be dosed orally.

[0097] A compound of the present disclosure may be administered to an individual in accordance with an effective dosing regimen for a desired period of time or duration, such as at least about one month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months or longer. In some embodiments, the compound is administered on a daily or intermittent schedule for the duration of the individual’s life.

[0098] The dosage or dosing frequency of a compound of the present disclosure may be adjusted over the course of the treatment, based on the judgment of the administering physician.

[0099] The compound may be administered to an individual (e.g., a human) in an effective amount. In some embodiments, the compound is administered once daily.

[0100] The compound can be administered by any useful route and means, such as by oral or parenteral (e.g., intravenous) administration. Therapeutically effective amounts of the compound may include from about 0.00001 mg / kg body weight per day to about 10 mg / kg body weight per day, such as from about 0.0001 mg / kg body weight per day to about 10 mg / kg body weight per day, or such as from about 0.001 mg / kg body weight per day to about 1 mg / kg body weight per day, or such as from about 0.01 mg / kg body weight per day to about 1 mg / kg body weight per day, or such as from about 0.05 mg / kg body weight per day to about 0.5 mg / kg body weight per day, or such as from about 0.3 mg to about 30 mg per day, or such as from about 30 mg to about 300 mg per day.

[0101] A compound of the present disclosure may be combined with one or more additional therapeutic agents in any dosage amount of the compound of the present disclosure (e.g., from about 1 mg to about 1000 mg of compound).VII. METHODS AND / OR USES

[0102] Without being bound by the theory, the compound of Formula 1, 1-1, la, la-1, lb, Ib-1, II, II-l, II-2, and / or II-3, or pharmaceutically acceptable salt described herein inhibits the activity of a cyclin-dependent kinase (CDK). In some embodiments, the method comprises contacting a compound of the present disclosure with a CDK. In some embodiments, the compound and the CDK are contacted in a cell. In some embodiments, the compound and theCDK are contacted in vivo. In some embodiments, the compound and the CDK are contacted in vitro. In some embodiments, the CDK is selected from CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12 and CDK13. In some embodiments, the CDK is CDK2. In some embodiments, the CDK is CDK3. In some embodiments, the CDK is CDK4. In some embodiments, the CDK is CDK6. In some embodiments, the method inhibits the activity of both CDK2 and CDK3. In some embodiments, the method inhibits the activity of CDK2 and one or both of CDK4 and CDK6.

[0103] In some embodiments, the compounds of the present disclosure inhibit the activity of one or more CDKs selected from CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12 and CDK13. In some embodiments, the compounds of the present disclosure inhibit CDK2. In some embodiments, the compounds of the present disclosure inhibit CDK3. In some embodiments, the compounds of the present disclosure inhibit CDK4. In some embodiments, the compounds of the present disclosure inhibit CDK5. In some embodiments, the compounds of the present disclosure inhibit CDK6. In some embodiments, the compounds of the present disclosure are CDK2 / 3 inhibitors. In some embodiments, the compounds of the present disclosure are CDK2 / 4 / 6 inhibitors.

[0104] In some embodiments, the present disclosure provides compounds that selectively inhibit CDK2 over other cyclin-dependent kinases (CDKs). In some embodiments, the compounds of the present disclosure selectively inhibit CDK2 over one or more other CDKs, selected from CDK1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12 and CDK13. In some embodiments, the compounds of the present disclosure selectively inhibit CDK2 over CDK4. In some embodiments, the compounds of the present disclosure selectively inhibit CDK2 over CDK6. In some embodiments, the compounds of the present disclosure selectively inhibit CDK2 over CDK4 and CDK6.

[0105] In some embodiments, the present disclosure provides compounds that selectively inhibit CDK2 / cyclin E complexes over other CDK complexes.

[0106] Any cell can be used in a method of the present disclosure. In some embodiments, the cell is a cancer cell or an immune cell. In some embodiments, the cancer cell is selected from breast cancer cell, ovarian cancer cell, bladder cancer cell, uterine cancer cell, prostate cancer cell, lung cancer cell, esophageal cancer cell, head and neck cancer cell, colorectal cancer cell, kidney cancer cell, liver cancer cell, pancreatic cancer cell, stomach cancer cell,melanoma cell, and thyroid cancer cell. In some embodiments, the immune cell is a B-cell, neutrophil, dendritic cell, or monocyte.

[0107] It has been reported in the literature that CDK2 plays a role in certain diseases or conditions, e.g., cancers, myeloproliferative disorders, autoimmune disorders, inflammatory disorders, viral infections, fibrotic disorders, and neurodegenerative disorders.

[0108] In some embodiments, the disease or disorder associated with CDK2 activity is a CDK2 -mediated disease or disorder. In some embodiments, the disease or disorder associated with CDK2 activity is a disease or disorder caused by CDK2 over-activity.

[0109] Overexpression of CDK2 is associated with abnormal regulation of the cell-cycle. The cyclin E / CDK2 complex plays an important role in regulation of the Gl / S transition, histone biosynthesis and centrosome duplication. Progressive phosphorylation of retinoblastoma (Rb) by cyclin D / Cdk4 / 6 and cyclin E / Cdk2 releases the G1 transcription factor, E2F, and promotes S-phase entry. Activation of cyclin A / CDK2 during early S-phase promotes phosphorylation of endogenous substrates that permit DNA replication and inactivation of E2F, for S-phase completion.

[0110] Cyclin E, the regulatory cyclin for CDK2, is frequently overexpressed in cancer. Cyclin E amplification or overexpression has long been associated with poor outcomes in breast cancer. (Keyomarsi et al., Cyclin E and survival in patients with breast cancer. N Engl J Med. (2002) 347:1566-75). Cyclin E2 (CCNE2) overexpression is associated with endocrine resistance in breast cancer cells and CDK2 inhibition has been reported to restore sensitivity to tamoxifen or CDK4 inhibitors in tamoxifen-resistant and CCNE2 overexpressing cellsCyclin E amplification also reportedly contributes to trastuzumab resistance in HER2+ breast cancer. Cyclin E overexpression has also been reported to play a role in basal-like and triple negative breast cancer (TNBC), as well as inflammatory breast cancer.

[0111] Amplification or overexpression of cyclin El (CCNE1) is also associated with poor outcomes in ovarian, gastric, endometrial and other cancers. See, e.g., Noske et al., Oncotarget (2017) 8: 14794-14805.

[0112] Accordingly, in some embodiments, a method of the present disclosure is a method treating, preventing or lessening the severity of a disease or disorder associated with CDK2 activity in a subject in need thereof, comprising administering to the subject a therapeuticallyeffective amount of a compound or pharmaceutically acceptable salt thereof of the present disclosure, or a pharmaceutical composition of the present disclosure. In some embodiments, the disease or disorder associated with CDK2 activity comprises a cancer, myeloproliferative disorder, autoimmune disorder, inflammatory disorder, viral infection, fibrotic disorder, or a neurodegenerative disorder.

[0113] Accordingly, the compounds of the present disclosure are believed to be useful in the treatment or prevention of cancers. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, esophagogastric cancer, head and neck cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, melanoma, thyroid cancer, and glioblastoma.

[0114] In some embodiments, the cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2. In some embodiments, the cancer has resistance or non-responsiveness to treatment with a CDK4 / 6 inhibitor.

[0115] In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is a breast cancer selected from ER-positive / HR-positive breast cancer, HER2-negative breast cancer, ER- positive / HR-positive breast cancer, HER2-positive breast cancer, triple negative breast cancer (TNBC), inflammatory breast cancer, endocrine resistant breast cancer, trastuzumab resistant breast cancer, breast cancer with primary or acquired resistance to CDK4 / CDK6 inhibition, advanced breast cancer and metastatic breast cancer. In some embodiments the breast cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2.

[0116] In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is high-grade serous ovarian cancer (HGSOC). In some embodiments the ovarian cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2.

[0117] In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is a lung cancer selected from non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, adenocarcinoma, and mesothelioma. In some embodiments, the lung cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2. In some embodiments, the lung cancer is CCNE1 amplified squamous cell carcinoma or CCNE1 amplified adenocarcinoma.

[0118] In some embodiments, the cancer is kidney cancer. In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the kidney cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2.

[0119] In some embodiments, the cancer is melanoma. In some embodiments, the melanoma is characterized by amplification or overexpression of CCNE1 and / or CCNE2. CDK2 expression is regulated by essential melanocytic transcription factor MITF. It has been found that CDK2 depletion suppresses the growth of melanoma. See, Du et al., Cancer Cell.2004 Dec; 6(6): 565-576)

[0120] In some embodiments, the disease or disorder associated with CDK2 activity is a myeloproliferative disorder.

[0121] In some embodiments, the disease or disorder associated with CDK2 activity is a neurodegenerative disease or disorder. In some embodiments, the neurodegenerative disease or disorder is Alzheimer’s disease (AD). It has been reported that neuronal cell death in subjects suffering from AD is preceded by cell cycle events. Inhibition of one or more CDKs has been reported to inhibit cell cycle events and therefore stave off neuronal cell death.

[0122] In some embodiments, the disease or disorder associated with CDK2 activity is a liver disease. In some embodiments, the disease or disorder associated with CDK2 activity is liver fibrosis. It has been reported that CCNE1 knockout mice do not develop liver fibrosis upon exposure to pro-fibrotic toxin CCl4, suggesting that liver fibrosis can be treated via administration of a CDK2 inhibitor.

[0123] In some embodiments, the disease or disorder associated with CDK2 activity is Cushing disease. Pituitary cyclin E / E2F1 signaling is a molecular mechanism underlying neuroendocrine regulation of the hypothalamic-pituitary-adrenal axis, and therefore provides a subcellular therapeutic target for CDK2 inhibitors of pituitary ACTH-dependent hypercortisolism, also known as Cushing disease.

[0124] In some embodiments, the disease or disorder associated with CDK2 activity is a kidney disease. In some embodiments, the disease or disorder associated with CDK2 activity is polycystic kidney disease. It has been reported that CDK2 / CDK5 inhibitor roscovitine yields effective arrest of cystic kidney disease in mouse models of polycystic kidney disease.

[0125] In some embodiments, the disease or disorder associated with CDK2 activity is anautoimmune disorder. CDK2 ablation has been reported to promote immune tolerance by supporting the function of regulatory T cells.

[0126] In some embodiments, the disease or disorder associated with CDK2 activity is an inflammatory disorder. Cyclin E ablation has been shown to attenuate hepatitis in mice, while p27 knockout mice display exacerbation of renal inflammation. In some embodiments, the inflammatory disorder is hepatitis.

[0127] In some embodiments, the compounds and compositions of the present disclosure are useful as male contraceptives. Based on the finding that male CDK2 knockout mice are sterile, CDK2 inhibitors have been studied as possible male contraceptives. In some embodiments, the present disclosure provides a method of reducing male fertility comprising administering to a patient in need thereof, a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising an effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0128] Thus, in one embodiment, the present disclosure relates to the compound of Formula I, la, lb, and / or II, or its pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure, for use in treatment or prevention of a disease or disorder associated with CDK2 activity.

[0129] In some embodiments, the present disclosure relates to use of the compound of Formula I, la, lb, and / or II, or its pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure, in the manufacture of a medicament for the treatment or prevention of a disease or disorder associated with CDK2 activity.

[0130] In some embodiments, the present disclosure relates to the method of treating or preventing a disease or disorder associated with CDK2 activity, the method comprising administering the compound of Formula I, la, lb, and / or II, or its pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure, to a subject in need thereof. It is to be understood that said compound or its salt or said pharmaceutical composition is to be administered in a therapeutically effective amount as described herein.VIII. EXAMPLES

[0131] The following examples are provided to further aid in understanding the embodiments disclosed in the application and presuppose an understanding of conventional methods well known to those persons having ordinary skill in the art to which the examples pertain. The particular materials and conditions described hereunder are intended to exemplify particular aspects of embodiments disclosed herein and should not be construed to limit the reasonable scope thereof.

[0132] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7thedition, Wiley-Interscience, 2013.)

[0133] Compounds as described herein can be purified by any of the means known in the art, including chromatographic means, such as high performance liquid chromatography (HPLC), preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. For example, disclosed compounds can be purified via silica gel chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 3rded., ed. L. R. Snyder, J. J. Kirkland, J. W. Dolan. John Wiley and Sons, 2011; and Thin Layer Chromatography, E. Stahl (ed.), Springer- Verlag, New York, 1969.

[0134] Compounds were characterized using standard instrumentation methods.Identification of the compound was carried out by hydrogen nuclear magnetic resonance spectrum (' H-NMR) and mass spectrum (MS). 'H-NMR was measured at 400 MHz, unless otherwise specified. In some cases, exchangeable hydrogen could not be clearly observed depending on the compound and measurement conditions. The designation br. or broad, used herein, refers to a broad signal. LCMS were performed according to the following. HPLC preparative chromatography was carried out according to the following conditions, unless otherwise specified.

[0135] General LCMS conditions:

[0136] LCMS (Shimadzu), column: YMC-Triart Cl 8, 50x4.6 mm, 5 pm, mobile phase: Solvent A: H2O / MeCN / NH4OH = 90 / 10 / 0.05; Solvent B: MeCN, Flow rate: 3 mL / min,temperature: 40°C; gradient: 0.01min @ 20% B, 1.79 min gradient (20-95% B), then 0.7 min @ 95% B.

[0137] LCMS (Shimadzu), column: YMC-Triart Cl 8, 50x4.6 mm, 5 pm, RP-18e, 50x4.6 mm, mobile phase: Solvent A: H2O (10 mmol / L NH4HCO3) / MeCN= 90 / 10 Solvent B: H2O / MeCN= 10 / 90, Flow rate: 2.5 mL / min, temperature: 40°C; gradient: 0.01 min @ 20% B, 1.79 min gradient (20-95% B), then 0.7 min @ 95% B.

[0138] LCMS (Shimadzu), column: YMC-Triart Cl 8, 50x4.6 mm, 5 pm, RP-18e, 50x4.6 mm, mobile phase: Solvent A: H2O / MeCN / FA = 90 / 10 / 0.05 Solvent B: MeCN, Flow rate: 2.5 mL / min, temperature: 40°C; gradient: 0.01 min @ 20% B, 1.79 min gradient (20-95% B), then 0.7 min @ 95% B.

[0139] General preparatory HPLC conditions: HPLC purifications were performed on a SHIMADZU LC-8A, Shimadzu LH-40 or Shimadzu LC-8A; Column: YMC-Triart Cl 8, 250x20 mm, 5 pm, YMC-Triart C8, 250x20 mm, 5 pm, YMC-Pack ODS-AQ, 250x20 mm, 5 pm or Xbridge Prep Cl 8 5 pm OBD etc., 250x20 mm, 5 pm with UV detection which were controlled by LC solution Chemstation software. H2O (FA / TFA) or H2O NH4OH / NH4HCO3) and MeOH (MeCN) as mobile phase at the indicated flow rate (15-20 mL / min) at room temperature.

[0140] The Examples provided herein describe the synthesis of compounds disclosed herein as well as intermediates used to prepare the compounds. It is to be understood that individual steps described herein may be combined. It is also to be understood that separate batches of a compound may be combined and then carried forth in the next synthetic step.

[0141] Representative syntheses of compounds of the present disclosure are described in schemes below, and the particular examples that follow.

[0142] Abbreviations. Certain abbreviations and acronyms are used in describing the experimental details. Although most of these would be understood by one skilled in the art, the following table contains a list of many of these abbreviations and acronyms.Table 2. List of abbreviations and acronyms.Intermediate 1: (S)-2-((S)-2,2-Dimethylcyclopropane-1-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acidScheme 1

[0143] Step 1: To a solution of (R)-4-phenyloxazolidin-2-one (10 g, 61.3 mmol) indry THF (200 mL) was added dropwise n-BuLi (2.5 in n-hexane, 27 mL, 67.4 mmol) under N2 at -78 °C. The reaction mixture was stirred at -78 °C for 0.5 h, before 2-bromoacetyl bromide (5.6 mL, 64.3 mmol) was added. The resulting mixture was allowed to warm to room temperature, stirred at room temperature for another 2 h, diluted with EtOAc (200 mL), quenched with saturated aqueous NH4CI solution (200 mL), and extracted with EtOAc (200 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 25% EtOAc in PE) to obtain the product ((R)-3-(2-bromoacetyl)-4-phenyloxazolidin-2-one) (15 g, 52.8 mmol, 86% yield) as a yellow solid. LCpurity: 90 % (UV at 254 nm); Mass calculated for C11H10BrNO3, [M+1]+, 284.0, found 284.0; Retention time: 1.435 min;1H NMR (400 MHz, DMSO-6) δ 7.41 - 7.37 (m, 2H), 7.34 (dd, J= 7.2, 2.8 Hz, 3H), 5.49 (dd, J= 8.7, 3.8 Hz, 1H), 4.79 (dd, J= 10.8, 5.0 Hz, 2H), 4.53 (d, J= 13.0 Hz, 1H), 4.21 (dd, J= 8.8, 4.0 Hz, 1H).

[0144] Step 2: A mixture of (R)-3-(2-bromoacetyl)-4-phenyloxazolidin-2-one (15 g, 52.8 mmol) in tri ethyl phosphite (46 mL, 264 mmol) was stirred under N2 at 50 °C for 18 h. After the solution was cooled to rt, the reaction mixture was concentrated and the resulting residue purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to obtain the product (diethyl (R)-(2-oxo-2-(2-oxo-4-phenyloxazolidin-3-yl)ethyl)phosphonate) (16.4 g, 48.0 mmol, 91% yield) as a yellow oil. LC purity: 90 % (UV at 254 nm); Mass calculated for C15H20NO6P [M+1]+, 342.1, found 342.4; Retention time: 1.428 min;1H NMR (400 MHz, CDCl3) δ 7.40 - 7.30 (m, 5H), 5.46 (dd, J= 8.8, 3.8 Hz, 1H), 4.70 (t, J= 8.8 Hz, 1H), 4.27 (dd, J= 8.8, 3.8 Hz, 1H), 4.14 - 4.07 (m, 4H), 3.78 (dq, J= 22.0, 13.8 Hz, 2H), 1.28 (dt, J = 10.2, 7.2 Hz, 6H).

[0145] Step 3: To a solution of diethyl (R)-(2-oxo-2-(2-oxo-4-phenyloxazolidin-3-yl)ethyl)phosphonate (11 g, 32.2 mmol) in THF (50 mL) was added LiHMDS (1 in THF, 32.2 mL, 32.2 mmol) dropwise under N2 at 0 °C. The resulting mixture was stirred at this temperature for 0.5 h, before a solution of tert-butyl 3 -oxoazetidine- 1 -carboxylate (8.3 g, 48.3 mmol) in THF (100 mL) was added dropwise at 0 °C. The resulting mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4CI solution (200 mL) at 0 °C and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 50% EtOAc in PE) to obtain the product (tert-butyl (R)-3-(2-oxo-2-(2-oxo-4-phenyloxazolidin-3-yl)ethylidene)azetidine-l -carboxylate) (9.0 g, 25.0 mmol, 78% yield) as a white solid. LC purity: 90% (UV at 254 nm); Mass calculated for C19H22N2O5[M+1]+, 359.1, found 359.4; Retention time: 1.653 min;1H NMR (400 MHz, DMSO-6) δ 7.41 - 7.28 (m, 5H), 7.18 (t, J= 2.2 Hz, 1H), 5.52 - 5.46 (m, 1H), 4.76 (t, J= 8.8 Hz, 1H), 4.62 (s, 4H), 4.22 - 4.16 (m, 1H), 1.38 (s, 9H).

[0146] Step 4: To a solution of tert-butyl (R)-3-(2-oxo-2-(2-oxo-4-phenyloxazolidin-3-yl)ethylidene)azeti dine- 1 -carboxylate (20 g, 55.8 mmol) and N-benzyl-1 -methoxy -N-((trimethylsilyl)methyl)methanamine (66.3 g, 279.0 mmol) in MeCN (200 mL) was added LiF (1.5 g, 55.8 mmol) at 25 °C. The reaction mixture was stirred under N2 at 80 °C for30 h, cooled to room temperature, quenched with water (400 mL) and extracted with EtOAc (300 mL x 3). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product tertbutyl (S)-6-benzyl-8-((R)-2-oxo-4-phenyloxazolidine-3-carbonyl)-2,6-diazaspiro[3.4]octane- 2-carboxylate (first eluent, 8 g, 16.3 mmol, 29% yield) as a yellow solid and tert-butyl (R)-6-benzyl-8-((R)-2-oxo-4-phenyloxazolidine-3-carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (second eluent, 6 g, 12.1 mmol, 22% yield) as a yellow solid. tert-Butyl (S)-6-benzyl-8-((R)-2-oxo-4-phenyloxazolidine-3-carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (first eluent): LC purity: 93% (UV at 254 nm); Mass calculated for C28H33N3O5 [M+1]+, 492.2, found 492.2; Retention time: 1.300 min;1H NMR (400 MHz, DMSO-6) 6 7.43 - 7.24 (m, 10H), 5.46 (dd, J= 8.4, 3.6 Hz, 1H), 4.72 (t, J= 8.8 Hz, 1H), 4.21 - 4.11 (m, 2H), 3.89 (t, J= 9.6 Hz, 1H), 3.79 (s, 1H), 3.66 - 3.59 (m, 2H), 3.52 (dd, J= 12.4, 2.8 Hz, 2H), 3.19 (dd, J= 15.2, 6.4 Hz, 1H), 2.90 (d, J= 8.8 Hz, 1H), 2.55 (d, J= 9.2 Hz, 1H), 2.31 (dd, J= 9.6, 5.4 Hz, 1H), 1.36 (s, 9H). tert-Butyl (R)-6-benzyl-8-((R)-2-oxo-4-phenyloxazolidine-3-carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (second eluent): LC purity: 70% (UV at 254 nm); Mass calculated for C28H33N3O5 [M+1]+, 492.2, found 492.2; Retention time: 1.163 min;1H NMR (400 MHz, DMSO-6) 67.41 - 7.23 (m, 10H), 5.44 (dd, J= 8.8, 4.2 Hz, 1H), 4.73 (t, = 8.8 Hz, 1H), 4.34 - 4.30 (m, 1H), 4.19 - 4.16 (m, 1H), 4.00 -3.96 (m, 1H), 3.68 - 3.62 (m, 1H), 3.58 - 3.57 (m, 2H), 3.37 - 3.34 (m, 2H), 3.03 (t, J= 8.8 Hz, 1H), 2.91 (d, J= 9.0 Hz, 1H), 2.55 - 2.53 (m, 2H), 1.36 (s, 9H).

[0147] Step 5: To a solution of tert-butyl (S)-6-benzyl-8-((R)-2-oxo-4-phenyloxazolidine- 3-carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (6 g, 12.2 mmol) in DCM (40 mL) was added TFA (40 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 0.5 h and concentrated under reduced pressure to give the product ((R)-3-((S)-6-benzyl-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one) (4 g, 10.2 mmol, 84% yield) as a yellow oil. The crude product was used directly in the next step without further purification. LC purity: 90% (UV at 254 nm); Mass calculated for C23H25N3O3 [M+1]+, 392.1, found 392.0; Retention time: 0.642 min.

[0148] Step 6: To a solution of (S)-2,2-dimethylcyclopropane-l -carboxylic acid (1.2 g, 10.2 mmol) in DMF (20 mL) was added DIPEA (6.6 g, 51.1 mmol) and HATU (3.9 g, 10.2 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 0.5 h, before (R)-3-((S)-6-benzyl-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (4 g, 10.2 mmol)was added. The reaction mixture was then stirred at 25 °C for 0.5 h, quenched with water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (100 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product ((R)-3-((S)-6-benzyl-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one) (4.5 g, 9.2 mmol, 90% yield) as a yellow solid. LC purity: 90 % (UV at 254 nm); Mass calculated for C29H33N3O4 [M+1]+, 488.2, found 488.1; Retention time: 1.052 min;1H NMR (400 MHz, DMSO-6) 6 7.42 - 7.23 (m, 10H), 5.52 - 5.44 (m, 1H), 4.73 (t, = 8.7 Hz, 1H), 4.28 - 4.15 (m, 2H), 4.13 - 4.03 (m, 1H), 3.92 - 3.85 (m, 1H), 3.77 (s, 1H), 3.61 - 3.51 (m, 2H), 3.32 (d, J= 5.2 Hz, 1H), 3.25 - 3.21 (m, 1H), 2.91 (d, J= 8.9 Hz, 1H), 2.62 - 2.54 (m, 1H), 2.41 - 2.33 (m, 1H), 1.40 - 1.34 (m, 1H), 1.12 - 1.05 (m, 5H), 0.98 (s, 1H), 0.86 - 0.82 (m, 1H), 0.69 - 0.63 (m, 1H).

[0149] Step 7: To a solution of (R)-3-((S)-6-benzyl-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (4 g, 8.2 mmol) in TFE (50 mL) was added 10% Pd / C (1 g) at 25 °C. The reaction mixture was stirred under H2 (1 atm) at 25 °C for 6 h and then filtered. The filtrate was concentrated under reduced pressure to give the crude product ((R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one) (2.5 g, 6.3 mmol, 77% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 93% (UV at 254 nm); Mass calculated for C22H27N3O4 [M+1]+, 398.2, found 398.1; Retention time: 0.771 min.

[0150] Step 8: To a solution of thiazole-5-carboxylic acid (9.1 g, 70.4 mmol) in DMF (100 mL) was added HATU (32.1 g, 84.5 mmol) and DIPEA (37 mL, 211 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 0.5 h before (R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (28 g, 70.4 mmol) was added. The reaction mixture was stirred at 25 °C for 0.5 h, quenched with water (200 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to obtain the product ((R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one) (32 g, 62.9 mmol, 89% yield) as a yellow solid. LCpurity: 90% (UV at 254 nm); Mass calculated for C26H28N4O5S [M+1]+, 509.2, found 509.2; Retention time: 1.220 min.

[0151] Step 9: To a solution of (R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3,4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (32 g, 62.9 mmol) in THF (300 mL) were added a solution of H2O2(30% in water, 15 mL) and LiOH solution (4.5 g, 189 mmol) in water (20 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h and the pH of the solution was adjusted to 4-5 with diluted aqueous HCl solution (1 N). The mixture was diluted with water (200 mL) and extracted with EtOAc (200 mL x 5). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to obtain the product ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid) (17 g, 46.7 mmol, 74% yield) as a white solid. LC purity: 90 % (UV at 254 nm); Mass calculated for C17H21N3O4S [M+1]+, 364.1, found 364.2; Retention time: 1.418 min;1H NMR (400 MHz, DMSO-6) 6 12.89 (s, 1H), 9.26 (s, 1H), 8.42 - 8.36 (m, 1H), 4.38 - 4.05 (m, 4H), 4.02 - 3.79 (m, 4H), 3.74 - 3.71 (m, 1H), 1.42 - 1.31 (m, 2H), 1.11 (t, J= 4.2 Hz, 3H), 1.05 (s, 2H), 0.87 (d, J= 4.0 Hz, 1H), 0.70 - 0.65 (m, 1H).Intermediate 2: 1-Benzyl 2-methyl (2S,3S)-3-methylaziridine-l.,2-dicarboxylateScheme 2

[0152] Step 1: To a solution of methyl L-threoninate (200 g, 1.2 mol) in DCM (4 L) were added triethylamine (844 mL, 6.0 mol) and trityl chloride (402 g, 1.4 mol) at 0 °C. The reaction was stirred at room temperature overnight, quenched with water (2 L) and extracted with DCM (2 L x 2). The combined extracts were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl trityl-L-threoninate (396 g, 949 mmol, 79% yield) as a white solid.1H NMR (400 MHz, DMSO-6) 67.43 (d, J= 7.6 Hz, 6H), 7.28 (t, J =7.6 Hz, 6H), 7.19 (t, J= 7.2 Hz, 3H), 5.02 (s, 1H), 3.98 - 3.89 (m, 1H), 3.23 - 3.17 (m, 1H), 3.04 (s, 3H), 2.70 (d, J= 10.1 Hz, 1H), 1.09 (d, J= 6.3 Hz, 3H).

[0153] Step 2: To a solution of methyl trityl-L-threoninate (390 g, 1.0 mol) and triethylamine (210.2 g, 2.0 mol) in THF (3.9 L) was added methanesulfonyl chloride (97 mL, 1.2 mol) at 0 °C. The resulting mixture was stirred at 80 °C for 30 h, cooled to room temperature, quenched with water (2 L) and extracted with EtOAc (2 L x 2). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was concentrated to obtain the desired product methyl (2S,3S)-3-methyl-l-tritylaziridine-2-carboxylate (250 g, 629 mmol, 60.6% yield) as a white solid. The crude product was used directly in the next step without further purification.1H NMR (400 MHz, DMSO-6) 67.43 (d, J= 7.6 Hz, 6H), 7.32 (t, J= 7.6 Hz, 6H), 7.25 (t, J= 7.2 Hz, 3H), 3.67 (s, 3H), 1.73 (d, J = 6.6 Hz, 1H), 1.59 - 1.53 (m, 1H), 1.29 (d, J= 5.4 Hz, 3H).

[0154] Step 3: To a solution of methyl (2S,3S)-3-methyl-l-tritylaziridine-2-carboxylate (350 g, 979 mmol) in DCM (3.5 L) and MeOH (175 mL) was added TFA (1.75 L, 22 mol) at 0 °C. The reaction mixture was stirred at room temperature for 3 h, concentrated, diluted with water (1 L) and extracted with EtOAc (1 L x 2). The aqueous layer was adjusted to pH 9 with solid NaHCO₃ at 0 °C and diluted with EtOAc (1 L). The resulting solution of methyl (2S,3S)-3-methylaziridine-2-carboxylate in EtOAc was used directly in the next step without further purification. LC purity: 70% (UV at 200 nm); Mass calculated for C5H9NO2, [M+1]+, 116.1, found 116.3; Retention time: 0.511 min.

[0155] Step 4: To a solution of methyl (2S,3S)-3-methylaziridine-2-carboxylate in EtOAc (1 L) and water (1 L) was added dropwise CbzCl (149 mL, 1.04 mol) at 0 °C. The reaction mixture was slowly warmed to room temperature, stirred overnight, quenched with saturated aqueous NH4CI solution (1 L) and extracted with EtOAc (1 L x 2). The combined organic layers were washed with water (1 L) and brine (1 L x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to give the product 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-1,2-dicarboxylate (168 g, 640 mmol, 65.3% yield) as a yellow oil.1H NMR (400 MHz, CDCI3) 87.37 - 7.33 (m, 5H), 5.15 - 5.13 (m, 2H), 3.78 (s, 3H), 3.18 (d, J= 6.6 Hz, 1H), 2.86 -2.77 (m, 1H), 1.35 (d, J= 5.6 Hz, 3H).Intermediate 3: (S)-2-((S)-2,2-DimethylcvdoDroDane-l-carbonyl)-6-(isothiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid

[0156] Step 1: To a solution of isothiazole-5-carboxylic acid (970 mg, 7.5 mmol) and DIEA (4.0 mL, 22.6 mmol) in DMF (40 mL) was added HATU (4.3 g, 11.3 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 0.5 h before (R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (3.0 g, 7.5 mmol) was added. The reaction mixture was stirred at 25 °C for another 0.5 h, quenched with water (50 mL), and extracted with EtOAc (50 mL x 3).The combined organic layers were washed with brine (40 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to give the product ((R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(isothiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one) (2.3 g, 4.5 mmol, 60% yield) as a yellow solid. LC purity: 90% (UV at 254 nm); Mass calculated for C26H28N4O5S [M+1]+, 509.2, found 509.1; Retention time: 1.235 min.

[0157] Step 2: To a solution of (R)-3-((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(isothiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (2.3 g, 4.5 mmol) in THF (50 mL) was added a solution of H2O2(30% in water, 1.4 mL) and LiOH (217 mg, 9.0 mmol) in water (4 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, the pH was adjusted to 4-5 with diluted aqueous HC1 solution (1 N), diluted with water (40 mL), and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to give the product ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(isothiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid) (1.0 g, 2.7 mmol, 60% yield) as a white solid. LC purity: 98% (UV at 254 nm); Mass calculated for C17H21N3O4S [M+1]+, 364.1, found 364.0; Retention time: 0.730 min;1H NMR (400 MHz, DMSO-6) δ 12.99 (s, 1H), 8.70 - 8.66 (m, 1H), 7.85 - 7.79 (m, 1H), 4.37 - 4.21 (m, 1H), 4.17 - 4.12 (m, 1H), 4.07 - 3.80 (m, 5H), 3.75 - 3.71 (m, 1H), 3.41 - 3.33 (m, 1H), 1.40 - 1.33 (m, 1H), 1.13 -1.09 (m, 3H), 1.07 - 1.03 (m, 3H), 0.88 - 0.83 (m, 1H), 0.72 - 0.65 (m, 1H).Intermediate 4: (S)-6-(Thiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)- 2,6-diazaspiro [3.4] octane-8-carboxylic acid

[0158] Step 1: To a solution of 3,3,3-trifluoro-2,2-dimethylpropanoic acid (3.5 g, 22.5 mmol) and DIE A (14 mL, 81.7 mmol) in DMF (100 mL) was added HATU (10.1 g, 26.6 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 30 min before (R)-3-((S)-6-benzyl-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (8 g, 20.4 mmol) was added. The reaction mixture was stirred at 25 °C for 30 min, quenched with water (500 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to give the desired product ((R)-3-((S)-6-benzyl-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one) (8.2 g, 15.5 mmol, 76% yield) as a yellow solid. LC purity: 90% (UV at 254 nm); Mass calculated for C28H30F3N3O4 [M+1]+, 530.2, found 530.3; Retention time: 1.031 min.

[0159] Step 2: To a solution of (R)-3-((S)-6-benzyl-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (8.5 g, 16.1 mmol) in TFE (200 mL, 16.1 mmol) was added 10% Pd / C (2 g). The reaction mixture was stirred under H2 (1 atm) at 25 °C for 6 h, filtered, and the cake was washed with MeOH (100 mL). The combined filtrates were concentrated under reduced pressure to give the product (R)-4-phenyl-3-((S)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)oxazolidin-2-one (6.3 g, 14.3 mmol, 89% yield) as acolorless oil. LC purity: 90% (UV at 254 nm); Mass calculated for C21H24F3N3O4 [M+1]+, 440.2, found 440.1; Retention time: 0.942 min.

[0160] Step 3: To a solution of thiazole-5-carboxylic acid (1.06 g, 8.2 mmol) and DIEA (3.6 mL, 20.5 mmol) in DMF (40 mL) was added HATU (3.6 g, 9.6 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 30 min before (R)-4-phenyl-3-((S)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3,4]octane-8-carbonyl)oxazolidin-2-one (3.0 g, 6.8 mmol) was added. The reaction mixture was stirred at 25 °C for 30 min, quenched with water (50 mL) and extracted with EtOAc (40 mL x 2). The combined organic layers were washed with brine (60 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to give the desired product ((R)-4-phenyl-3-((S)-6-(thiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)oxazolidin-2-one) (3.5 g, 6.4 mmol, 93% yield) as a yellow solid. LC purity: 90% (UV at 254 nm); Mass calculated for C25H25F3N4O5S [M+1]+, 551.2, found 551.3; Retention time: 1.269 min.

[0161] Step 4: To a solution of (R)-4-phenyl-3-((S)-6-(thiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)oxazolidin-2-one (3.5 g, 6.4 mmol) in THF (40 mL) was added a solution of H2O2(30% in water, 3 mL) and LiOH·H2O (538 mg, 12.8 mmol) in water (6 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and the pH was adjusted to 4-5 with diluted aqueous HC1 solution (1 N). The mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL x 5). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to give the product ((S)-6-(thiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid) (1.2 g, 3.0 mmol, 47% yield) as a white solid. LC purity: 95% (UV at 254 nm); Mass calculated for C16H18F3N3O4S [M+1]+, 406.1, found 406.0; Retention time: 0.716 min;1H NMR (400 MHz, DMSO-6) 6 12.99 (s, 1H), 9.26 (s, 1H), 8.37 (d, J= 15.4 Hz, 1H), 4.58 -4.38 (m, 2H), 4.10 (s, 1H), 3.91 (d, J= 9.4 Hz, 2H), 3.84 - 3.67 (m, 2H), 3.37 (d, J= 6.8 Hz, 1H), 3.32 - 3.29 (m, 1H), 1.34 (d, J= 7.0 Hz, 6H).Intermediate 5: (S)-6-(Isothiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid

[0162] Step 1: To a solution of (R)-4-phenyl-3-((S)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)oxazolidin-2-one (30 g, 68 mmol) and DIEA (47 mL, 272 mmol) in DMF (400 mL) were added HATU (36 g, 95 mmol) and isothiazole-5-carboxylic acid (10.5 g, 81.5 mmol) at 0 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (500 mL) and extracted with EtOAc (300 mL x 3). The combined organic layers were washed with brine (300 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to give the desired product (R)-3-((S)-6-(isothiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (30 g, 55 mmol, 81% yield) as a white solid. LC purity: 90% (UV at 220 nm); Mass calculated for C25H25F3N4O5S [M+1]+, 551.2, found 551.3; Retention time: 1.502 min.

[0163] Step 2: To a solution of (R)-3-((S)-6-(isothiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carbonyl)-4-phenyloxazolidin-2-one (30 g, 54.5 mmol) in THF (300 mL) was added a solution of H2O2(30% in water, 13.5 mL) and LiOH·H2O (4.6 g, 109 mmol) in water (20 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, the pH was adjusted to 4-5 with HC1 solution (1.0 N), diluted with water (50 mL) and extracted with EtOAc (50 mL x 5). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.The remaining residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to give the crude product, which was further separated with SFC (A for CO2 and B for MeOH (0.1% 7 mol / L NH3 in MeOH)) to obtain the desired product (S)-6-(isothiazole-5-carbonyl)-2-(3,3,3-trifluoro-2,2-dimethylpropanoyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (9 g, 22 mmol, 40% yield). LC purity: 98% (UV at 220 nm); Mass calculated for C16H18F3N3O4S [M+1]+, 406.1, found 406.6; Retention time: 0.791 min;1H NMR (400 MHz, DMSO-6) 6 13.01 (s, 1H), 8.67 (dd, J= 6.0, 1.8 Hz, 1H), 7.80 (dd, J= 13.6, 1.6 Hz, 1H), 4.23 - 3.71 (m, 6H), 3.42 - 3.24 (m, 3H), 1.34 (d, J= 8.0 Hz, 6H).Example 1. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2.,6-diazaspiro [3.4] octane-8-carboxamide

[0164] Scheme 3

[0165] Step 1: To a stirred suspension of methyl cyclohexanecarboxylate (5.1 mL, 35.2 mmol) in THF (50 mL) was added LDA (2 in THF, 26 mL, 52.7 mmol) at -65 °C under N2. The resulting mixture was stirred at this temperature for 1 h under N2 before 3-bromoprop-l-ene (4.6 mL, 52.7 mmol) was added. The reaction mixture was stirred at 25 °C overnight, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic phases were washed with brine (60 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 1 -allylcyclohexane- 1 -carboxylate (5.5 g, 27.2 mmol, 77% yield) as a yellow oil.1H NMR (400 MHz, CDCI3) 85.67 - 5.57 (m, 1H), 5.02 - 4.87 (m, 2H), 3.59 (s, 3H), 2.16 (d, J = 7.4 Hz, 2H), 1.99 - 1.93 (m, 2H), 1.55 - 1.41 (m, 3H), 1.31 - 1.13 (m, 5H).

[0166] Step 2: To a solution of methyl 1 -allylcyclohexane- 1 -carboxylate (4.5 g, 24.7 mmol) in THF (45 mL) was added LAH (2.5 in THF, 4.9 mL, 12.3 mmol) dropwise at -60 °C. The mixture was stirred at -60 °C for 2 h. The reaction mixture was diluted with THF (100 mL), quenched with Na2SO410 H2O, and stirred at room temperature for 30 min. The suspension was filtered, and the cake was washed with THF (50 mL x 2). The combinedfiltrates were concentrated in vacuo to obtain the product (l-allylcyclohexyl)methanol (3.7 g, 21.6 mmol, 87% yield) as a yellow oil.1H NMR (400 MHz, CDCl3) 65.85 - 5.74 (m, 1H), 5.04 - 4.95 (m, 2H), 3.35 (s, 2H), 2.05 (d, J= 7.6 Hz, 2H), 1.41 - 1.34 (m, 5H), 1.32 - 1.23 (m, 5H).

[0167] Step 3: To a solution of (l-allylcyclohexyl)methanol (3.4 g, 22.0 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (5.5 g, 22.0 mmol) in chloroform (40 mL) was added BF3·Et2O (2.8 mL, 22.0 mmol) at 0 °C. The reaction mixture was stirred under nitrogen at 0 °C for 2 h. After evaporation, the remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3.6 g, 8.7 mmol, 40% yield) as a yellow oil. LC purity: 90% (UV at 220 nm); Mass calculated for C23H33NO5 [M+1]+, 404.2, found 404.2; Retention time: 2.147 min.

[0168] Step 4: To a solution of methyl O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4 g, 9.9 mmol) in THF (20 mL) and water (20 mL) was added LiOH (475 mg, 19.8 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h and the pH was adjusted to 4-5 with diluted aqueous HC1 solution (1.0 N). The mixture was diluted with H2O (20 mL) and extracted with DCM (40 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to obtain the product O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (3.9 g, 9.1 mmol, 92% yield) as a white solid. The crude product was used directly in the next step without further purification. LC purity: 90%; Mass calculated for C22H31NO5 [M+1]+, 390.2, found 390.4; Retention time: 2.039 min.

[0169] Step 5: To a solution of O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (3.9 g, 10.1 mmol) and DIE A (7.1 mL, 40.5 mmol) in DMF (40 mL) were added HATU (4.6 g, 12.1 mmol) and prop-2-en-l -amine (866 mg, 15.2 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2S,3R)- 1 -(allylamino)-3 -(( 1 -allylcy cl ohexyl)m ethoxy)- 1 -oxobutan-2-yl)carbamate (1.9 g, 3.6 mmol, 35% yield) as a white solid. LC purity: 80% (UV at 220 nm); Mass calculated for C25H36N2O4 [M+1]+, 429.2, found 429.1; Retention time: 2.151 min.

[0170] Step 6: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((l-allylcyclohexyl)methoxy)-l-oxobutan-2-yl)carbamate (500 mg, 1.2 mmol) in DCM (500 mL) were added Grubbs 2ndgeneration catalyst(99 mg, 0.12 mmol). The reaction mixture was stirred under N2 at 60 °C for 24 h. After evaporation, the remaining residue was purified by flash chromatography (silica gel, 0 - 25% EtOAc in PE) to obtain the product benzyl ((9R,10S, Z)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (140 mg, 0.3 mmol, 27% yield) as a brown solid. LC purity: 90% (UV at 220 nm); Mass calculated for C23H32N2O4 [M+1]+, 401.2, found 401.2; Retention time: 1.924 min.

[0171] Step 7: To a solution of benzyl ((9R,10S, Z)-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (140 mg, 0.3 mmol) in THF (5 mL) was added 10% Pd / C (30 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C overnight, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated to give the crude product ((9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-ll-one) (90 mg, 0.3 mmol, 86% yield) as a colorless oil. The crude product was used directly in the next step without further purification. LC purity: 90% (UV at 220 nm); Mass calculated for C15H28N2O2 [M+1]+, 269.2, found 269.5; Retention time: 0.667 min.

[0172] Step 8: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (48 mg, 0.13 mmol) and DIEA (0.09 mL, 0.53 mmol) in DMF (3 mL) were added HATU (60 mg, 0.16 mmol) and (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l l-one (53 mg, 0.12 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1%FA) to obtain the product ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R, 10S)-9-methyl- 11 -oxo-8-oxa- 12-azaspiro[5.10]hexadecan- 10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide) (12 mg, 0.02 mmol, 14% yield) as a white solid. LC purity: 98% (UV at 254 nm); Mass calculated for C32H47N5O5S [M+1]+, 614.3, found 614.6; Retention time: 1.628 min;1H NMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.37 (d, J= 9.0 Hz, 1H), 7.78 (s, 1H), 4.44 - 4.18 (m, 4H), 4.16 - 3.80 (m, 7H), 3.55 -3.42 (m, 3H), 3.05 - 3.01 (m, 1H), 1.84 - 1.74 (m, 1H), 1.50 - 1.33 (m, 11H), 1.25 - 1.19 (m, 6H), 1.16 - 1.09 (m, 6H), 1.07 - 0.99 (m, 2H), 0.80 - 0.74 (m, 1H).Example 2. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((9R,10S, Z)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0173] Scheme 4

[0174] Step 1: To a solution of benzyl ((9R,10S, Z)-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (140 mg, 0.38 mmol) in DCM (3 mL) were added BCI3 (0.35 mL, 3.8 mmol) at -30 °C. The reaction mixture was stirred under N2 at25 °C for 12 h, quenched with saturated aqueous NaHCCl4solution (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were washed with water (10 mL) and brine (10 mL x 2), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give the crude product ((9R,10S, Z)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadec-14-en-ll-one) (100 mg, 0.3 mmol, 80% yield) as a colorless oil. LC purity: 80% (UV at 220 nm); Mass calculated for C15H26N2O2 [M+1]+, 267.2, found 267.2;Retention time: 0.567 min.

[0175] Step 2: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (40 mg, 0.11 mmol) and DIEA (0.08 mL, 0.44 mmol) in DMF (3 mL) were added HATU (50 mg, 0.13 mmol) and (9R, 10S, Z)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadec-14-en-l 1-one (32 mg, 0.12 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R, 10S, Z)-9-methyl- 11 -oxo-8-oxa- 12-azaspiro[5.10]hexadec- 14-en- 10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide) (13 mg, 0.02 mmol, 19% yield) as a white solid. LC purity: 98% (UV at 254 nm); Mass calculated for C32H45N5O5S [M+1]+, 612.3, found 612.0; Retention time: 1.628 min;1H NMR (400 MHz, MeOD-4) 69.15 (s, 1H), 8.37 (d, J= 8.8 Hz, 1H), 8.20 - 7.95 (m, 2H), 5.84 - 5.76 (m, 1H), 5.59 (t, J = 11.6 Hz, 1H), 4.48 - 4.35 (m, 2H), 4.30 - 4.21 (m, 2H), 4.18 - 4.04 (m, 3H), 4.00 - 3.84 (m, 4H), 3.55 - 3.44 (m, 2H), 2.79 (t, J= 5.2 Hz, 1H), 2.61 - 2.48 (m, 1H), 1.79 - 1.75 (m, 1H), 1.65 - 1.29 (m, 12H), 1.25 - 1.15 (m, 6H), 1.13 - 0.97 (m, 4H), 0.81 - 0.73 (m, 1H).Example 3. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.11]heptadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0176] Scheme 5

[0177] Step 1: To a solution of methyl O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (5 g, 12.4 mmol) in THF (30 mL) was added dropwise a solution of lithium hydroxide (594 mg, 24.8 mmol) in water (15 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. The mixture was then adjusted to pH 4 with diluted aqueous HC1 solution (2 A) and extracted with EtOAc (10 mL x 3). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was evaporated to give the crude product O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (4.5 g, 10.4 mmol, 84% yield) as a yellow oil. The crude product was used directly in the next step without further purification. LC purity: 90% (UV at 220 nm); Mass calculated for C22H31NO5 [M+1]+, 390.2, found 390.1; Retention time: 1.972 min.

[0178] Step 2: To a solution of O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (2.5 g, 6.4 mmol), but-3-en-l -amine (0.6 g, 8.3 mmol), and DIEA (3.4 mL, 19.3 mmol) in DMF (25 mL) was added HATU (3.7 g, 9.6 mmol) at 0 °C. The reaction mixturewas gradually warmed to room temperature and stirred for 2 h, quenched with saturated aqueous NH4CI solution (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with water (50 mL) and brine (50 mL x 3), dried over by anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product benzyl ((2S,3R)-3-((l-allylcyclohexyl)methoxy)-l-(but-3-en-l-ylamino)-l-oxobutan-2-yl)carbamate (1.6 g, 3.6 mmol, 56% yield) as a yellow oil. LC purity: 80% (UV at 220 nm); Mass calculated for C26H38N2O4 [M+1]+, 443.3, found 443.2; Retention time: 2.203 min.

[0179] Step 3: To a solution of benzyl ((2S,3R)-3-((l-allylcyclohexyl)methoxy)-l-(but-3-en-l-ylamino)-l-oxobutan-2-yl)carbamate (1.5 g, 3.4 mmol) in DCM (3 mL) was added Grubbs 2ndgeneration catalyst (863 mg, 1 mmol). The reaction mixture was stirred at 60 °C under N2 for 16 h and concentrated. The remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to give the product (benzyl ((9R,10S, Z)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.11]heptadec-15-en-10-yl)carbamate) (400 mg, 1.0 mmol, 28% yield) as a white solid. LC purity: 80% (UV at 220 nm); Mass calculated for C24H34N2O4 [M+1]+, 415.3, found 415.2; Retention time: 1.933 min.

[0180] Step 4: To a solution of benzyl ((9R,10S, Z)-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.11]heptadec-15-en-10-yl)carbamate (400 mg, 1.0 mmol) in THF (10 mL) was added 10% Pd / C (102 mg) and the resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C for 16 h. After filtration, the filtrate was concentrated in vacuo to give the crude product ((9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.11]heptadecan-ll-one) (270 mg, 0.96 mmol, 99% yield) as a brown oil, which was used directly in the next step without further purification. LC purity: 76% (UV at 200 nm); Mass calculated for C16H30N2O2 [M+1]+, 283.2, found 283.1; Retention time: 1.117 min.

[0181] Step 5: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (40 mg, 0.1 mmol), (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.11]heptadecan-ll-one (31 mg, 0.1 mmol), and DIE A (0.06 mL, 0.3 mmol) in DMF (3 mL) was added HATU (63 mg, 0.17 mmol) at 0 °C. The resulting mixture was stirred for 2 h, quenched with saturated aqueous NaHCCl4solution (10 mL) and extracted with EtOAc (10 mL x 2). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing0.1% FA) to obtain the product ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R, 10S)-9-methyl- 11 -oxo-8-oxa- 12-azaspiro[5.11 ]heptadecan- 10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide) (19 mg, 0.03 mmol, 27% yield) as a white solid. LC purity: 99.3% (UV at 254 nm); Mass calculated for C33H49N5O5S [M+1]+, 628.4, found 628.3; Retention time: 1.570 min;1H NMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.37 (d, J= 8.8 Hz, 1H), 7.87 - 7.76 (m, 1H), 4.40 - 4.38 (m, 1H), 4.34 - 4.32 (m, 1H), 4.30 - 4.22 (m, 2H), 4.18 - 4.04 (m, 3H), 4.01 - 3.80 (m, 4H), 3.58 - 3.37 (m, 3H), 2.90 - 2.85 (m, 1H), 1.90 - 1.78 (m, 1H), 1.71 - 1.60 (m, 2H), 1.52 - 1.32 (m, 12H), 1.24 - 1.08 (m, 12H), 1.04 - 0.98 (m, 2H), 0.81 - 0.74 (m, 1H).Example 4. (S)-2-((S)-2.,2-Dimethylcvclopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.9]pentadecan-10-yl)-6-(thiazole-5-carbonyl)-2.,6-diazaspiro [3.4] octane-8-carboxamide

[0182] Scheme 6.

[0183] Step 1: To a solution of methyl O-((l-allylcyclohexyl)methyl)-N- ((benzyloxy)carbonyl)-L-threoninate (1 g, 2.5 mmol) in THF (20 mL) was added BH3·Me2S (10 M, 1.2 mL, 12.4 mmol) at 0 °C. The reaction mixture was stirred under a nitrogenatmosphere at 0 °C for 2 h before H2O (5.0 mL) and NaBO3·4H2O (1.9 g, 12.4 mmol) were added. The resulting mixture was stirred at room temperature overnight, quenched with water (30 mL), and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 10 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-hydroxypropyl)cyclohexyl)methyl)-L-threoninate (550 mg, 1.3 mmol, 53% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) 87.42 - 7.29 (m, 5H), 5.48 (d, J= 9.8 Hz, 1H), 5.14 (s, 2H), 4.35 - 4.32 (m, 1H), 4.01 - 3.94 (m, 1H), 3.74 (s, 3H), 3.65 - 3.53 (m, 2H), 3.28 (d, J= 8.8 Hz, 1H), 2.99 (d, J= 8.8 Hz, 1H), 1.45 - 1.35 (m, 8H), 1.33 - 1.21 (m, 6H), 1.19 (d, J= 6.4 Hz, 3H).

[0184] Step 2: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-hydroxypropyl)cyclohexyl)methyl)-L-threoninate (550 mg, 1.3 mmol) in DCM (20 mL) were added TEA (0.4 mL, 2.6 mmol) and methansulfonyl chloride (0.1 mL, 1.8 mmol) at 0 °C. The reaction mixture was stirred under nitrogen atmosphere at room temperature for 2 h, quenched with H2O (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the crude product methyl N-((benzyloxy)carbonyl)-O-((l-(3-((methylsulfonyl)oxy)propyl)cyclohexyl)methyl)-L-threoninate (680 mg, 1.2 mmol, 89% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 85% (UV at 254 nm); Mass calculated for C24H37NO8S [M+1]+, 500.2, found 500.2; Retention time: 1.854 min.

[0185] Step 3: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-((methylsulfonyl)oxy)propyl)cyclohexyl)methyl)-L-threoninate (680 mg, 1.3 mmol) inDMF (20 mL) was added NaN3(2.5 eq, 221.0 mg, 3.4 mmol) at 0 °C. The reaction mixture was stirred at 60 °C under nitrogen atmosphere for 12 h, cooled to room temperature, diluted with H2O (20 mL) and EtOAc (10 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl O-((l-(3-azidopropyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (530 mg, 1.2 mmol, 87% yield) as a colorless oil. LC purity: 91% (UV at 220 nm); Mass calculated for C23H34N4O5 [M+l-28]+, 419.2, found 419.2; Retention time: 2.095 min.

[0186] Step 4: To a solution of methyl O-((l-(3-azidopropyl)cyclohexyl)methyl)-N- ((benzyloxy)carbonyl)-L-threoninate (530 mg, 1.2 mmol) in THF (12 mL) and water (4 mL)was added Ph3P (778 mg, 2.9 mmol). The reaction mixture was stirred at 60 °C for 1.5 h. After the solution was cooled to room temperature, Boc2O (392 mg, 1.8 mmol) was added, and the resulting mixture was stirred for 1 h, diluted with H2O (20 mL), and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cyclohexyl)methyl)-L-threoninate (590 mg, 1.1 mmol, 95% yield) as a colorless oil. LC purity: 90% (UV at 220 nm); Mass calculated for C28H44N2O7 [M+1]+, 521.3, found 521.3; Retention time: 2.135 min.

[0187] Step 5: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cyclohexyl)methyl)-L-threoninate (590 mg, 1.1 mmol) in THF (5 mL) and water (5 mL) was added LiOH (95 mg, 2.3 mmol). The reaction mixture was stirred at room temperature for 12 h, adjusted to pH 4-5 with aqueous HC1 solution (1 N), diluted with water (20 mL), and extracted with EtOAc (20 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give the crude product N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cyclohexyl)methyl)-L-threonine (570 mg, 1.1 mmol, 99% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 92% (UV at 220 nm); Mass calculated for C27H42N2O7 [M+1]+, 507.3, found 507.3; Retention time: 2.105 min.

[0188] Step 6: To a solution of N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cyclohexyl)methyl)-L-threonine (540 mg, 1.1 mmol) in DCM (6 mL) was added HCl-di oxane (4 N in 1,4-di oxane, 3 mL, 12 mmol). The mixture was stirred at room temperature for 2 h and concentrated in vacuo to give the crude product O-((l-(3-aminopropyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (430 mg, 1.1 mmol, 99% yield) as a light yellow oil, which was used directly in the next step without further purification. LC purity: 93% (UV at 220 nm); Mass calculated for C22H34N2O5 [M+1]+407.2, found 407.2; Retention time: 0.901 min.

[0189] Step 7: To a solution of O-((l-(3-aminopropyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (430 mg, 1.1 mmol) in DMF (80 mL) were added DIEA (0.9 mL, 5.3 mmol) and HATU (60 mg, 1.6 mmol). The resulting mixture was stirred at roomtemperature for 0.5 h, quenched with saturated aqueous NaHCCl4solution (100 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with brine (50 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((9R,10S)-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.9]pentadecan-10-yl)carbamate (300 mg, 0.8 mmol, 73% yield) as a colorless oil. LC purity: 50% (UV at 220 nm); Mass calculated for C22H32N2O4 [M+1]+, 389.2, found 389.1; Retention time: 1.691 min.

[0190] Step 8: To a solution of benzyl ((9R,10S)-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.9]pentadecan-10-yl)carbamate (300 mg, 0.8 mmol) in THF (50 mL) were added 10% Pd / C (60 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at room temperature for 12 h and filtered. The filtrate was concentrated under reduced pressure to give the crude product (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.9]pentadecan-ll-one (150 mg, 0.6 mmol, 76% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 50% (UV at 220 nm); Mass calculated for C14H26N2O2 [M+1]+, 255.2, found 255.0; Retention time: 0.521 min.

[0191] Step 9: To a solution of (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.9]pentadecan-ll-one (52 mg, 0.2 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (50 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (0.1 mL, 0.4 mmol) and HATU (78 mg, 0.2 mmol). The resulting mixture was stirred at room temperature for 1 h, quenched with saturated aqueous NaHCCl4solution (10 mL) and extracted with EtOAc (10 mL x 2). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The remaining residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-((9R, 10S)-9-m ethyl- 11 -oxo-8-oxa- 12-azaspiro[5.9]pentadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (59 mg, 0.1 mmol, 72% yield) as a white solid. LC purity: 100% (UV at 220 nm); Mass calculated for C31H45N5O5S [M+1]+, 600.3, found 600.5; Retention time: 1.195 min;1H NMR (400 MHz, MeOD-4) 69.15 (s, 1H), 8.36 (d, J= 10.9 Hz, 1H), 7.85 - 7.73 (m, 1H), 4.43 - 3.83 (m, 11H), 3.58 (d, J= 9.1 Hz, 1H), 3.52 - 3.40 (m, 1H), 3.17 - 2.99 (m, 2H), 1.63 - 1.48 (m, 3H), 1.46 - 1.33 (m, 10H), 1.25 - 1.20 (m, 3H), 1.19 - 1.13 (m, 4H), 1.10 (s, 3H), 1.07 - 1.00 (m, 2H), 0.81 - 0.73 (m, 1H).Example 5. (S)-N-((9R,10S)-3.,3-Difluoro-9-methyl-ll-oxo-8-oxa-12-azasDiro[5.10]hexadecan-10-yl)-2-((S)-2.,2-dimethylcycloDroDane-l-carbonyl)-6- (thiazole-5-carbonyl)-2.,6-diazasDiro[3.4]octane-8-carboxamide

[0192] Scheme 7.

[0193] Step 1: To a stirred suspension of methyl 4,4-difluorocyclohexane-l -carboxylate (5 g, 28.1 mmol) in THF (100 mL) was added dropwise LDA (2 in THF, 27.4 mL, 54.8 mmol) at -65 °C. The resulting mixture was stirred at this temperature for 0.5 h under N2 before 3 -bromoprop- 1-ene (1.5 eq, 3.6 mL, 42.1 mmol) was added. The resulting mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the desired product methyl l-allyl-4,4-difluorocyclohexane-l -carboxylate (5 g, 20.6 mmol, 74% yield) as a yellow oil. LC purity: 90% (UV at 220 nm); Mass calculated for C11H16F2O2, [M+1]+, 219.1, found 219.1 Retention time: 1.602 min;1H NMR (400 MHz, CDCI3) 85.72 - 5.62 (m, 1H), 5.11 - 5.00 (m, 2H), 3.70 (s, 3H), 2.28 (d, J= 7.4 Hz, 2H), 2.19 (d, J= 13.7 Hz, 2H), 2.00 - 1.98 (m, 2H), 1.87 - 1.73 (m, 2H), 1.57 - 1.49 (m, 2H).

[0194] Step 2: To a stirred suspension of methyl l-allyl-4,4-difluorocyclohexane-l-carboxylate (2 g, 9.2 mmol) in THF (40 mL) was added dropwise LAH (1 in THF, 9.2 mL, 9.2 mmol) at -65 °C under N2. The resulting mixture was stirred under N2 for 2 h, quenched with diluted HC1 solution (1 TV, 40 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the desired product (1 -allyl-4,4-difluorocyclohexyl)methanol (1.3 g, 6.2 mmol, 68% yield) as a yellow oil.1H NMR (400 MHz, CDCI3) 85.91 - 5.76 (m, 1H), 5.12 - 5.08 (m, 2H), 3.45 (s, 2H), 2.16 - 2.13 (m, H), 1.96 - 1.83 (m, 4H), 1.60 - 1.51 (m, 4H).

[0195] Step 3: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (2 g, 4.6 mmol) and (l-allyl-4,4-difluorocyclohexyl)methanol 1.3 g, 6.9 mmol) in chloroform (10 mL) was added dropwise BF3·Et2O (0.6 mL, 4.6 mmol) at 0 °C under N2. The resulting mixture was stirred at 25 °C for 2 h and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l -allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.8 g, 3.2 mmol, 70% yield) as a yellow oil. LC purity: 78% (UV at 220 nm); Mass calculated for C23H31F2NO5, [M+1]+, 440.2, found 440.2; Retention time: 1.895 min.

[0196] Step 4: To a solution of methyl O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.8 g, 4.1 mmol) in THF (10 mL) and water (10 mL) was added LiOH·H2O (196 mg, 8.2 mmol). The resulting mixture was stirred for 2 h at 25 °C, neutralized to pH 5 with dilute HC1 solution (1 N) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to obtain the product O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.0 g, 2.1 mmol, 52% yield) as a yellow oil. LC purity: 91% (UV at 220 nm); Mass calculated for C22H29F2NO5, [M+1]+, 426.2, found 426.1;Retention time: 1.680 min.

[0197] Step 5: To a solution of O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.0 g, 2.4 mmol) and prop-2-en-l -amine (160 mg, 2.8 mmol) in DCM (30 mL) were added DIEA (1.2 mL, 7.1 mmol) and HATU (1.3 g, 3.5 mmol). The resulting mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4CIsolution (30 mL) and extracted with DCM (30 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over by anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2S,3R)-3-((l-allyl-4,4-difluorocyclohexyl)methoxy)-l -(allylamino)- 1 -ox obutan-2-yl)carbamate (930 mg, 1.9 mmol, 82% yield) as a yellow oil. LC purity: 96% (UV at 220 nm); Mass calculated for C25H34F2N2O4, [M+1]+, 465.3, found 465.3; Retention time: 1.784 min.

[0198] Step 6: To a solution of benzyl ((2S,3R)-3-((l-allyl-4,4-difluorocyclohexyl)methoxy)-l -(allylamino)- 1 -ox obutan-2-yl)carbamate (940 mg, 2.0 mmol) in DCM (1000 mL) was added Grubbs 2ndgeneration catalyst (859 mg, 1.0 mmol). The resulting mixture was stirred under N2 atmosphere at 60 °C for 16 h, concentrated, and the remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((9R,10S, Z)-3,3-difluoro-9-methyl-l 1 -oxo-8 -oxa- 12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (290 mg, 0.67 mmol, 31% yield) as a black oil. LC purity: 94% (UV at 220 nm); Mass calculated for C23H30F2N2O4, [M+1]+, 437.2, found 437.2; Retention time: 1.548 min.

[0199] Step 7: To a solution of benzyl ((9R,10S, Z)-3,3-difluoro-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (290 mg, 0.67 mmol) in THF (10 mL) was added 10% Pd / C (100 mg). The reaction mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH. The filtrate was concentrated in vacuo to give the crude product (9R,10S)-10-amino-3,3-difluoro-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l l-one (170 mg, 0.4 mmol, 67% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm); Mass calculated for C15H26F2N2O2, [M+1]+, 305.2, found 305.1; Retention time: 0.715 min.

[0200] Step 8: To a solution of (9R,10S)-10-amino-3,3-difluoro-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-ll-one (30 mg, 0.1 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (36 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (0.05 mL, 0.3 mmol) and HATU (38 mg, 0.1 mmol). The reaction mixture was stirred for 2 h at 25 °C, quenched with saturated aqueous NH4CI solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% NH4HCO3) to obtain the product (S)-N-((9R, 10S)-3,3 -difluoro-9-methyl- 11 -oxo-8-oxa- 12-azaspiro[5.10]hexadecan- 10-yl)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (18 mg, 0.03 mmol, 27% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C32H45F2N5O5S, [M+1]+, 650.3, found 650.4; Retention time: 1.273 min;1H NMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.37 (d, J= 9.0 Hz, 1H), 4.43 - 4.33 (m, 2H), 4.30 - 4.19 (m, 2H), 4.15 - 4.05 (m, 3H), 4.02 - 3.84 (m, 3H), 3.70 - 3.60 (m, 1H), 3.54 -3.45 (m, 2H), 3.40 - 3.37 (m, 1H), 3.17 - 3.09 (s, 1H), 1.94 - 1.73 (s, 6H), 1.61 - 1.58 (m, 2H), 1.54 - 1.52 (m, 1H), 1.41 - 1.37 (m, 2H), 1.33 (d, J= 6.4 Hz, 3H), 1.28 - 1.25 (m, 1H), 1.22 - 1.20 (m, 3H), 1.19 (s, 1H), 1.16 (s, 3H), 1.10 (s, 2H), 1.05 - 1.02 (m, 1H), 0.81 - 0.74 (s, 1H).Example 6. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((10R,llS)-10-methyl- 12-oxo-9-oxa-13-azaspiro[6.10]heptadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0201] Scheme 8.

[0202] Step 1: To a solution of cycloheptanecarboxylic acid (5 g, 35.2 mmol) in methanol (50 mL) was added concentrated H2SO4 (5 mL, 93.8 mmol). The reaction mixture was stirred at 60 °C for 12 h. After evaporation, the residue was poured to ice water (50 mL)and extracted with DCM (10 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) toobtain the product methyl cycloheptanecarboxylate (4.4 g, 28.2 mmol, 80% yield) as a yellow oil. LC purity: 80% (UV at 254 nm); Mass calculated for C9H16O2 [M+1]+, 157.1, found 157.1; Retention time: 1.721 min.

[0203] Step 2: To a solution of methyl cycloheptanecarboxylate (2 g, 12.8 mmol) in dry THF (40 mL) was added dropwise LDA (2.0 M in THF, 48 mL, 96.1 mmol) under N2 at -78 °C and the mixture was stirred at -78 °C for 1 h. A solution of 3 -bromoprop- 1-ene (1.7 mL, 19.2 mmol) in dry THF (30 mL) was added dropwise and the mixture was stirred at room temperature for 12 h. The mixture was poured into saturated aqueous NH4CI solution (100 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 -20% EtOAc in PE) to obtain the product methyl 1 -allylcycloheptane- 1 -carboxylate (2.1 g, 10.5 mmol, 82% yield) as a yellow oil. LC purity: 66% (UV at 254 nm); Mass calculated for C12H20O2 [M+1]+, 197.1, found 197.1; Retention time: 2.123 min;1H NMR (400 MHz, DMSO-6) 85.70 - 5.51 (m, 1H), 5.08 - 4.94 (m, 2H), 3.58 (s, 3H), 2.22 (d, J= 7.4 Hz, 2H), 1.99 - 1.95 (m, 1H), 1.62 - 1.39 (m, 11H).

[0204] Step 3: To a solution of methyl 1 -allylcycloheptane- 1 -carboxylate (2.1 g, 10.5 mmol) in THF (30 mL) was added LAH (400 mg, 10.5 mmol) in portions at -78 °C. The reaction mixture was stirred at -78 °C for 3 h, diluted with THF (50 mL), quenched with Na2SO4·10H2O at -78 °C, and then stirred at room temperature for 30 min. The mixture was filtered through a Celite® pad and washed with DCM (50 mL). The combined filtrates were concentrated under reduced pressure and the resulting residue was purified by flash chromatography (silica gel, 10 - 40% EtOAc in PE) to obtain the product (1-allylcycloheptyl)methanol (1.3 g, 7.7 mmol, 73% yield) as a colorless oil. LC purity: 80% (UV at 254 nm); Mass calculated for C11H20O [M+1]+, 169.1, found 169.2; Retention time: 1.722 min.

[0205] Step 4: To a mixture of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (1.3 g, 5.1 mmol) and (1-allylcy cl oh eptyl)m ethanol (1.3 g, 7.6 mmol) in chloroform (25 mL) was added BF3·Et2O (0.64 mL, 5.1 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, concentrated, and the remaining residue was purified by flash chromatography (silica gel, 10 - 40% EtOAc in PE) to obtain the product methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.7 g, 4.1 mmol, 81%yield) as a colorless oil. LC purity: 67% (UV at 254 nm); Mass calculated for C24H35NO5 [M+1]+, 418.2, found 418.2; Retention time: 2.502 min.

[0206] Step 5: To a solution of methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.7 g, 4.1 mmol) in THF (10 mL) and water (5 mL) was added LiOH·H2O (340 mg, 8.1 mmol). The reaction mixture was stirred at room temperature for 6 h and the pH was adjusted to 4-5 with diluted HC1 solution (1 N). The resulting solution was diluted with H2O (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo to obtain the product O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.5 g, 3.7 mmol, 91% yield) as a light yellow oil, which was used directly in the next step without further purification. LC purity: 90% (UV at 254 nm); Mass calculated for C23H33NO5 [M+1]+, 404.2, found 404.1; Retention time: 2.058 min.

[0207] Step 6: To a solution of O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.5 g, 3.7 mmol) in DMF (3 mL) were added DIE A (1.3 mL, 7.4 mmol), HATU (2.1 g, 5.6 mmol), and prop-2-en-l -amine (318 mg, 5.6 mmol). The reaction mixture was stirred at 25 °C for 1 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 10 - 50% EtOAc in PE) to obtain the product benzyl ((2S,3R)-l-(allylamino)-3-((l-allylcycloheptyl)methoxy)-l-oxobutan-2-yl)carbamate (940 mg, 2.1 mmol, 57% yield) as a yellow oil. LC purity: 80% (UV at 254 nm); Mass calculated for C26H38N2O4 [M+1]+, 443.3, found 443.2; Retention time: 2.161 min.

[0208] Step 7: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((l-allylcycloheptyl)methoxy)-l-oxobutan-2-yl)carbamate (940 mg, 2.1 mmol) in DCM (1000 mL) was added Grubbs 2ndgeneration catalyst (902 mg, 1.1 mmol). The reaction mixture was stirred under N2 at 50 °C for 24 h, concentrated, and the remaining residue was purified by flash chromatography (silica gel, 10 - 30% EtOAc in PE) to obtain the product benzyl ((1 OR, 11 S, Z)- 10-methyl- 12-oxo-9-oxa- 13 -azaspiro[6.10]heptadec- 15 -en- 11 -yl)carbamate (216 mg, 0.5 mmol, 22% yield) as a brown solid. LC purity: 57% (UV at 254 nm); Mass calculated for C24H34N2O4 [M+1]+, 415.3, found 415.3; Retention time: 1.943 min.

[0209] Step 8: To a solution of benzyl ((1 OR, 11S, Z)-10-methyl- 12-oxo-9-oxa-l 3-azaspiro[6.10]heptadec-15-en-ll-yl)carbamate (216 mg, 0.5 mmol) in THF (30 mL) wasadded 10% Pd / C (166 mg). The mixture was stirred under H2atmosphere (1 atm) at room temperature for 3 h, filtered through a Celite® pad and washed with MeOH. The filtrate was concentrated under reduced pressure to give the crude product (10R,l 1S)-11 -amino- 10-methyl-9-oxa-13-azaspiro[6.10]heptadecan-12-one (103 mg, 0.4 mmol, 70% yield) as a brown oil, which was used directly in the next step without further purification. LC purity: 60% (UV at 254 nm); Mass calculated for C16H30N2O2 [M+1]+, 283.2, found 283.2; Retention time: 1.146 min.

[0210] Step 9: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (30 mg, 0.08 mmol) in DMF (3 mL) were added DIEA (22 mg, 0.17 mmol), HATU (47 mg, 0.124 mmol), and (10R,llS)-ll-amino-10-methyl-9-oxa-13-azaspiro[6.10]heptadecan-12-one (35 mg, 0.1 mmol). The reaction mixture was stirred at 25 °C for 1 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-(( 1 OR, 11 S)- 10-methyl- 12-oxo-9-oxa-13-azaspiro[6.10]heptadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (3.8 mg, 0.006 mmol, 7% yield) as a yellow solid. LC purity: 99% (UV at 254 nm); Mass calculated for C33H49N5O5S [M+1]+, 628.4, found 628.0; Retention time: 1.683 min;1H NMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.37 (d, J= 8.9 Hz, 1H), 4.42 - 4.34 (m, 2H), 4.26 - 4.24 (m, 1H), 4.16 - 3.92 (m, 7H), 3.53 - 3.42 (m, 4H), 2.90 - 2.87 (m, 1H), 2.08 (s, 1H), 1.79 - 1.74 (m, 2H), 1.61 (s, 2H), 1.42 - 1.32 (m, 11H), 1.22 - 1.19 (m, 2H), 1.17 -1.12 (m, 5H), 1.10 - 1.07 (m, 2H), 1.05 - 1.03 (m, 3H), 0.94 - 0.92 (m, 1H), 0.81 - 0.74 (m, 1H).Example 7. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((9R,10R)-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0211] Scheme 9.

[0212] Step 1: To a solution of methyl O-((l -allylcy cl ohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4 g, 9.9 mmol) in THF (40 mL) was added LAH (377 mg, 9.9 mmol) in portions at -78 °C under N2. The reaction mixture was stirred at -78 °C for 3 h, diluted with THF (40 mL), quenched with Na2SO4IOH2O at -78 °C, and then stirred at room temperature for 30 min. The reaction mixture was filtered through a Celite® pad and washed with DCM (50 mL). The combined filtrates were concentrated under reduced pressure and the resulting residue was purified by flash chromatography (silica gel, 10 - 40% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((l-allylcyclohexyl)methoxy)-l-hydroxybutan-2-yl)carbamate (1.6 g, 4.3 mmol, 42% yield) as a colorless oil. LC purity: 90% (UV at 254 nm); Mass calculated for C22H33NO4 [M+1]+, 376.2, found 376.2; Retention time: 1.975 min.

[0213] Step 2: To a solution of benzyl ((2R, 3R)-3-((l -allylcy cl ohexyl)m ethoxy)- 1-hydroxybutan-2-yl)carbamate (1.4 g, 3.4 mmol) in toluene (20 mL) were added allyl tertbutyl carbonate (1.5 g, 9.3 mmol) and Pd(PPh3)4 (431 mg, 0.4 mmol). The resulting mixture was stirred at 110 °C under N2 for 12 h, cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 10 -20% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((l-allylcyclohexyl)methoxy)-l-(allyloxy)butan-2-yl)carbamate (730 mg, 1.8 mmol, 47% yield) as a light yellow oil. LC purity: 89% (UV at 254 nm); Mass calculated for C25H37NO4 [M+1]+, 416.3, found 416.2;Retention time: 2.232 min.

[0214] Step 3: To a solution of benzyl ((2R, 3R)-3-((l -allylcy cl ohexyl)m ethoxy)- 1-(allyloxy)butan-2-yl)carbamate (730 mg, 1.8 mmol) in DCM (700 mL) was added Grubbs 2ndgeneration catalyst (746 mg, 0.9 mmol). The reaction mixture was stirred under N2 at 70 °Cfor 24 h, concentrated, and the remaining resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((9R,10R, Z)-9-methyl-8,12-dioxaspiro[5.10]hexadec-14-en-10-yl)carbamate (210 mg, 0.5 mmol, 31% yield) as a brown oil. LC purity: 44% (UV at 254 nm); Mass calculated for C23H33NO4 [M+1]+, 388.2, found 388.3; Retention time: 2.021 min.

[0215] Step 4: To a solution of benzyl ((9R,10R, Z)-9-methyl-8,12-dioxaspiro[5.10]hexadec-14-en-10-yl)carbamate (210 mg, 0.54 mmol) in THF (30 mL) was added 10% Pd / C (166 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at room temperature for 3 h, filtered through a Celite® pad and washed with MeOH. The filtrate was concentrated under reduced pressure to give the crude product (9R,10R)-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-amine (120 mg, 0.47 mmol, 92% yield) as a brown oil. LC purity: 90% (UV at 254 nm); Mass calculated for C15H29NO2 [M+1]+, 256.2, found 256.1; Retention time: 1.201 min.

[0216] Step 5: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (45 mg, 0.12mmol) and (9R,10R)-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-amine (48 mg, 0.19 mmol) in DMF (2 mL) were added DIEA (32 mg, 0.25 mmol) and HATU (71 mg, 0.19 mmol). The reaction mixture was stirred at 25 °C for 1 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R,10R)-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (11 mg, 0.02 mmol, 15% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C32H48N4O5S [M+1]+, 601.3, found 600.9; Retention time: 2.008 min;1HNMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.36 (d, J= 14.0 Hz, 1H), 4.43 - 4.20 (m, 3H), 4.18 - 4.05 (m, 3H), 4.02 - 3.81 (m, 4H), 3.71 - 3.65 (m, 1H), 3.63 - 3.37 (m, 5H), 3.11 -2.99 (m, 1H), 1.76 (s, 1H), 1.62 - 1.56 (m, 1H), 1.49 - 1.27 (m, 14H), 1.16 - 1.03 (m, 11H), 0.79 - 0.76 (m, 1H).Example 8. (9R,10S)-10-(((2-((S)-2.,2-DimethylcycloDroDane-l-carbonyl)-6-(thiazole-5-carbonyl)-2.,6-diazasDiro[3.4]octan-8-yl)methyl)amino)-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-ll-oneScheme 10

[0217] Step 1: To a stirred (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (1 g, 2.7 mmol) and K2CO3(760 mg, 5.5 mmol) in DMF (10 mL) was added Mel (0.34 mL, 5.5 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 2 h, quenched with water (20 mL) and extractedwith EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to obtain the product methyl (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylate (800 mg, 2.1 mmol, 77% yield) as a white solid. LC purity: 90% (UV at 254 nm); Mass calculated for C18H23N3O4S [M+1]+, 378.1, found 378.1; Retention time: 0.997 min.

[0218] Step 2: To a solution of methyl (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylate (1.9 g, 5 mmol) in z-PrOH (20 mL) was added LiBH4(2 M in THF, 7.6 mL, 15.1 mmol) at 0 °C under N2. The reaction mixture was stirred at 25 °C for 2 h, quenched with water (40 mL) and extracted with DCM (30 mL x 4). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to obtain the product ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-8-(hydroxymethyl)-2,6-diazaspiro[3.4]octan-6-yl)(thiazol-5-yl)methanone (1.2 g, 3.4 mmol, 68% yield) as a white solid. LC purity: 90%(UV at 254 nm); Mass calculated for C17H23N3O3S [M+1]+, 350.2, found 350.1; Retention time: 0.432 min.

[0219] Step 3: To a solution of ((S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-8-(hydroxymethyl)-2,6-diazaspiro[3.4]octan-6-yl)(thiazol-5-yl)methanone (1.2 g, 3.4 mmol) in DCM (20 mL) was added DMP (2.2 g, 5.1 mmol) at 0 °C under N2. The reaction mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NaHCCl4solution (20 mL) and saturated aqueous Na2SO3 solution (20 mL). The mixture was extracted with DCM (30 mL x 6) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to obtain the product 2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbaldehyde (1.1 g, 3.2 mmol, 92% yield) as a white solid. LC purity: 90% (UV at 254 nm); Mass calculated for C17H21N3O3S [M+1]+, 348.1, found 366.2 [M+18]+; Retention time: 0.563 min.

[0220] Step 4: To a solution of (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-ll-one (32 mg, 0.12 mmol) and 2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbaldehyde (43 mg, 0.12 mmol) in DCE (3 mL) was added AcOH (15 mg, 0. 25 mmol). The reaction mixture was stirred at room temperature for 2 h before NaBH(OAc)3 (78 mg, 0.37 mmol) was added. The resulting mixture was stirred at room temperature for 12 h, quenched with H2O (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (9R,10S)-10-(((2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octan-8-yl)methyl)amino)-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l l-one (10 mg, 0.02 mmol, 14% yield) as a white solid. LC purity: 98% (UV at 254 nm); Mass calculated for C32H49N5O4S [M+1]+, 600.4, found 600.4; Retention time: 1.289 min;1H NMR (400 MHz, MeOD-d4) δ 9.16 (s, 1H), 8.39 (s, 1H), 4.62 - 4.48 (m, 1H), 4.43 - 4.25 (m, 1H), 4.23 - 3.98 (m, 4H), 3.93 - 3.80 (m, 3H), 3.72 - 3.63 (m, 1H), 3.42 - 3.40 (m, 1H), 3.20 - 3.05 (m, 1H), 3.03 - 2.92 (m, 3H), 2.79 - 2.48 (m, 2H), 1.76 (s, 1H), 1.47 - 1.25 (m, 16H), 1.20 - 1.10 (m, 7H), 1.07 - 0.93 (m, 3H), 0.82 - 0.74 (m, 1H).Example 9. (9R,10S)-10-(4-((S)-2-((S)-2,2-DimethylcvdoDropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazasDiro[3.4]octan-8-yl)-lH-l,2,3-triazol-l-yl)-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-ll-one

[0221] Scheme 11

[0222] Step 1: To a solution of 2-((S)-2, 2-dimethylcy clopropane-1 -carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carbaldehyde (1.1 g, 3.2 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (912 mg, 4.75 mmol) in MeOH (15 mL) was added K2CO3(656 mg, 4.7 mmol) at 0 °C. The reaction mixture was stirred 30 °C for 1 h, quenched with saturated aqueous NH4CI solution (20 mL) and extracted with DCM (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to obtain the product (2-((S)-2, 2-dimethylcy clopropane-1 -carbonyl)-8-ethynyl-2,6-diazaspiro[3.4]octan-6-yl)(thiazol-5-yl)methanone (1 g, 2.6 mmol, 82% yield) as a white solid. LC purity: 80% (UV at 254 nm); Mass calculated for C18H21N3O2S [M+1]+, 344.1, found 344.1; Retention time: 0.686 min.

[0223] Step 2: To a solution of (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l 1-one (60 mg, 0.2 mmol) in acetonitrile (2 mL) and THF (2 mL) were added TEA (60 mg, 0.6 mmol) and 2-azido-l,3-dimethyl-4,5-dihydroimidazol-l-ium hexafluorophosphate (127 mg, 0.45 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 12 h, cooled to room temperature, quenched with saturated aqueous NEUCl (10 mL), and extracted with EtOAc (5 mL x 3). The combined organic layers were dried overanhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the crude product (9R,10S)-10-azido-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l 1-one (70 mg, 0.21 mmol, 96% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm); Mass calculated for C₁₅H₂₆N₄O₂ [M+1]+, 295.2, found 295.2; Retention time: 1.794 min.

[0224] Step 3: To a mixture of (2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-8-ethynyl-2,6-diazaspiro[3.4]octan-6-yl)(thiazol-5-yl)methanone (60 mg, 0.18 mmol) and (9R,10S)-10-azido-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l 1-one (57 mg, 0.19 mmol) in water (1 mL), tert-butanol (2 mL), and THF (1 mL) were added sodium ascorbate (52 mg, 0.26 mmol) and CuSO₄ (8.4 mg, 0.05 mmol). The reaction mixture was stirred under N2 at 25 °C for 2 h, filtered, quenched with water (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-TLC (DCM: MeOH = 15: 1) to give a crude product. The crude product was further purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to give the product (9R,10S)-10-(4-(2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octan-8-yl)-lH-l,2,3-triazol-l-yl)-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-l l-one (2.1 mg, 0.003 mmol, 1.8% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C₃₃H₄₇N₇O₄S [M+1]+, 638.3, found 638.2; Retention time: 1.410 min;1H NMR (400 MHz, MeOD-d₄) δ 9.18 (s, 1H), 8.42 (t, J= 9.6 Hz, 1H), 8.13 - 7.91 (m, 1H), 5.51 -5.50 (m, 1H), 4.58 (s, 1H), 4.37 - 4.22 (m, 4H), 4.09 - 4.03 (m, 2H), 3.96 - 3.74 (m, 4H), 3.56 - 3.52 (m, 1H), 3.11 (d, J= 10.0 Hz, 2H), 2.20 - 1.85 (m, 2H), 1.48 - 1.40 (m, 9H), 1.34 - 1.30 (m, 4H), 1.20 - 1.15 (m, 2H), 1.13 - 1.08 (m, 4H), 1.00 -0.93 (m, 3H), 0.88 - 0.81 (m, 3H), 0.79 - 0.72 (m, 1H).Example 10. (S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((13S,14R)-14-methyl-12-oxo-7,15-dioxa-ll-azaspiro[5.10]hexadecan-13-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0225] Scheme 12

[0226] Step 1: To a solution of NaH (60% suspension in oil, 2.3 g, 56.9 mmol) in DMF (20 mL) was added dropwise a solution of ethyl 1-hydroxycyclohexane-l -carboxylate (3.0 g, 17.4 mmol) in DMF (30 mL) at 0 °C. The reaction mixture was stirred under a nitrogen atmosphere at 0 °C for 0.5 h before 3 -bromoprop- l-ene (4.9 mL, 56.9 mmol) was added. The resulting mixture was stirred at room temperature for 2 h, quenched with saturated aqueous NH4CI solution (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (30 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product ethyl l-(allyloxy)cyclohexane-l -carboxylate (3.5 g, 16.5 mmol, 93% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) δ 6.02 - 5.88 (m, 1H), 5.26 - 5.22 (m, 1H), 5.18 - 5.13 (m, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.93 - 3.88 (m, 2H), 1.93 - 1.84 (m, 2H), 1.83 - 1.75 (m, 3H), 1.65 - 1.47 (m, 4H), 1.38 - 1.31 (m, 1H), 1.28 (t, J = 7.1 Hz, 3H).

[0227] Step 2: To a solution of ethyl l-(allyloxy)cyclohexane-l -carboxylate (3.5 g, 16.5 mmol) in THF (35 mL) was added dropwise LAH (2.5 in THF, 6.6 mL, 16.5 mmol) at -60 °C. The reaction mixture was stirred at -20 °C for 2 h under a nitrogen atmosphere, quenched with Na2SO4·10H2O at -20 °C and then stirred at room temperature for 30 min. The resulting reaction mixture was filtered through a Celite® pad and washed with DCM (50 mL). Thecombined filtrates was concentrated in vacuo and the remaining residue was purified by flash chromatography (silica gel, 10 - 40% EtOAc in PE) to obtain the product (1-(allyloxy)cyclohexyl)methanol (2.3 g, 13.5 mmol, 82% yield) as a colorless oil.1H NMR (400 MHz, CDCl₃) δ 6.05 - 5.87 (m, 1H), 5.39 - 5.27 (m, 1H), 5.22 - 5.10 (m, 1H), 3.95 -3.85 (m, 2H), 3.49 (s, 2H), 1.79 - 1.72 (m, 2H), 1.62 - 1.55 (m, 3H), 1.49 - 1.42 (m, 2H), 1.40 - 1.30 (m, 3H).

[0228] Step 3: To a mixture of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (2.3 g, 9.2 mmol) and (l-(allyloxy)cy cl ohexyl)m ethanol (2.4 g, 13.8 mmol) in chloroform (100 mL) was added BF3·Et2O (1.2 mL, 9.2 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h, concentrated, and the resulting residue was purified by flash chromatography (silica gel, 1 - 20% EtOAc in PE) to obtain the product methyl O-((l-(allyloxy)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3.4 g, 5.9 mmol, 64% yield) as a colorless oil. LC purity: 73% (UV at 254 nm); Mass calculated for C₂₃H₃₃NO₆ [M+1]+, 420.2, found 420.3; Retention time: 1.813 min.

[0229] Step 4: To a solution of methyl O-((l-(allyloxy)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4.6 g, 8.1 mmol) in THF (50 mL) was added dropwise BH₃·Me₂S (10 M, 4.1 mL, 40.5 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h under a nitrogen atmosphere. Then H2O (16 mL) and NaBO₃·4H₂O (6.2 g, 40.5 mmol) were slowly added. The resulting mixture was stirred at room temperature for 12 h, quenched with water (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 10 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-hydroxypropoxy)cyclohexyl)methyl)-L-threoninate (1.2 g, 2.8 mmol, 35% yield) as a colorless oil. LC purity: 73% (UV at 254 nm); Mass calculated for C₂₃H₃₅NO₇ [M+1]+, 438.2, found 438.2; Retention time: 1.570 min;1H NMR (400 MHz, CDCI3) δ 7.43 - 7.27 (m, 5H), 6.67 (d, J = 9.8 Hz, 1H), 5.15 (d, J = 5.2 Hz, 2H), 4.43 - 4.35 (m, 1H), 4.11 - 4.04 (m, 1H), 3.77 - 3.74 (m, 1H), 3.72 (s, 3H), 3.66 - 3.60 (m, 1H), 3.58 - 3.45 (m, 3H), 3.09 (d, J= 9.8 Hz, 1H), 1.88 - 1.79 (m, 1H), 1.76 - 1.69 (m, 1H), 1.68 - 1.61 (m, 4H), 1.57 - 1.47 (m, 3H), 1.44 - 1.34 (m, 2H), 1.23 - 1.20 (m, 4H).

[0230] Step 5: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-hydroxypropoxy)cyclohexyl)methyl)-L-threoninate (1.2 g, 2.8 mmol) in DCM (30 mL) were added TEA (0.8 mL, 5.7 mmol) and methanesulfonyl chloride (0.3 mL, 3.4 mmol) at 0 °Cunder a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 2 h, quenched with H2O (30 mL) and extracted with DCM (30 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to obtain the crude product methyl N-((benzyloxy)carbonyl)-O-((l-(3-((methylsulfonyl)oxy)propoxy)cyclohexyl)methyl)-L-threoninate (1.5 g, 2.9 mmol, 100% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 73% (UV at 254 nm); Mass calculated for C₂₄H₃₇NO₉S [M+1]+, 516.2, found 516.2; Retention time: 1.678 min.

[0231] Step 6: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-((methylsulfonyl)oxy)propoxy)cyclohexyl)methyl)-L-threoninate (1.5 g, 2.9 mmol) in DMF (30 mL) was added NaN3(473 mg, 7.3 mmol) at 0 °C. The reaction mixture was stirred at 60 °C under N2 for 12 h, cooled to room temperature, quenched with H2O (50 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were washed with brine (30 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 1 - 20% EtOAc in PE) to obtain methyl O-((l-(3-azidopropoxy)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.3 g, 2.8 mmol, 97% yield) as a colorless oil. LC purity: 81% (UV at 220 nm); Mass calculated for C₂₃H₃₄N₄O₆ [M+1]+, 463.3, found 463.2; Retention time: 1.957 min.

[0232] Step 7: To a solution of methyl O-((l-(3-azidopropoxy)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.3 g, 2.8 mmol) in THF (21 mL) and water (7 mL) was added Ph₃P (1.8 mg, 7.0 mmol). The reaction mixture was stirred at 60 °C for 1.5 h under N2 and cooled to room temperature. Boc2O (0.9 mL, 4.2 mmol) was added. The resulting mixture was stirred for 1 h, diluted with H2O (30 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were washed with brine (30 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 5 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propoxy)cyclohexyl)methyl)-L-threoninate (1.4 g, 2.8 mmol, 99% yield) as a colorless oil. LC purity: 88% (UV at 220 nm); Mass calculated for C₂₈H₄₄N₂O₈ [M+1]+, 537.3, found 537.3; Retention time: 1.933 min.

[0233] Step 8: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propoxy)cyclohexyl)methyl)-L-threoninate (1.5 g, 2.8 mmol) in THF (10 mL) and water (10 mL) was added LiOH·H2O (235 mg, 5.6 mmol). The reaction mixturewas stirred at room temperature for 2.5 h and the pH was adjusted to 4-5 with diluted aqueous HC1 solution (1.0 N). The solution was then diluted with H2O (30 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain the product N-((benzyloxy)carbonyl)-O-((l -(3-((tert-butoxycarbonyl)amino)propoxy)cyclohexyl)methyl)-L-threonine (1.4 g, 2.8 mmol, 100% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 78% (UV at 220 nm); Mass calculated for C₂₇H₄₂N₂O₈ [M+1]+, 523.3, found 523.3; Retention time: 1.735 min.

[0234] Step 9: To a solution of N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propoxy)cyclohexyl)methyl)-L-threonine (1.4 g, 2.7 mmol) in DCM (10 mL) was added HC1 solution (4 N in 1,4-dioxane, 5 mL, 20 mmol). The reaction mixture was stirred at room temperature for 2 h and concentrated in vacuo to obtain the product O-((l-(3-aminopropoxy)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.0 g, 2.3 mmol, 85% yield) as a light yellow oil, which was used directly in the next step without further purification. LC purity: 67% (UV at 220 nm); Mass calculated for C₂₂H₃₄N₂O₆ [M+1]+, 423.2, found 423.2; Retention time: 0.749 min.

[0235] Step 10: To a solution of O-((l-(3-aminopropoxy)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.0 g, 2.4 mmol) in DMF (200 mL) were added DIEA (2.1 mL, 11.8 mmol) and HATU (1.3 g, 3.6 mmol). The resulting mixture was stirred at room temperature for 0.5 h, quenched with saturated aqueous NaHCCl4solution (100 mL) and extracted with EtOAc (100 mL x 2). The combined organic layers were washed with brine (100 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 5 - 50% EtOAc in PE) to obtain the product benzyl ((13S,14R)-14-methyl-12-oxo-7,15-dioxa-l 1-azaspiro[5.10]hexadecan-13-yl)carbamate (260 mg, 0.6 mmol, 27% yield) as a colorless oil. LC purity: 60% (UV at 220 nm); Mass calculated for C₂₂H₃₂N₂O₅ [M+1]+, 405.2, found 405.2; Retention time: 1.536 min.

[0236] Step 11: To a solution of benzyl ((13S,14R)-14-methyl-12-oxo-7,15-dioxa-l 1-azaspiro[5.10]hexadecan-13-yl)carbamate (260 mg, 0.6 mmol) in THF (20 mL) were added 10% Pd / C (50 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at room temperature overnight, filtered through a Celite® pad and washed with MeOH. The combined filtrates were concentrated under reduced pressure to obtain the product (13S,14R)-13-amino-14-methyl-7,15-dioxa-ll-azaspiro[5.10]hexadecan-12-one (150 mg, 0.5 mmol, 86% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 65% (UV at 220 nm); Mass calculated for C₁₄H₂₆N₂O₃ [M+1]+, 271.2, found 271.0; Retention time: 0.274 min.

[0237] Step 12: To a mixture of (13S,14R)-13-amino-14-methyl-7,15-dioxa-l 1-azaspiro[5.10]hexadecan- 12-one (56 mg, 0.2 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (50 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (0.1 mL, 0.4 mmol) and HATU (78 mg, 0.2 mmol). The resulting mixture was stirred at room temperature for 1 h, quenched with saturated aqueous NaHCCl4solution (10 mL) and extracted with EtOAc (10 mL x 2). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The resulting residue was purified prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((13S,14R)-14-methyl-12-oxo-7,15-dioxa-ll-azaspiro[5.10]hexadecan-13-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (51 mg, 0.08 mmol, 60% yield) as a white solid. LC purity: 100% (UV at 220 nm); Mass calculated for C₃₁H₄₅N₅O₆S [M+1]+, 616.3, found 616.5; Retention time: 1.056 min;1H NMR (400 MHz, MeOD-d₄) δ 9.15 (s, 1H), 8.37 (d, J = 9.1 Hz, 1H), 4.44 - 4.21 (m, 4H), 4.18 - 3.85 (m, 8H), 3.60 - 3.54 (m, 2H), 3.53 - 3.37 (m, 3H), 1.92 - 1.77 (m, 3H), 1.69 - 1.58 (m, 2H), 1.51 - 1.35 (m, 7H), 1.20 - 1.14 (m, 6H), 1.11 (s, 3H), 1.06 - 0.98 (m, 2H), 0.82 - 0.74 (m, 1H).Example 11. (8S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((2lR,3lS)-2l-methyl-4l-oxo-ll-oxa-5l-azaspiro[bicvclo[2.2.2]octane-2,10l-cvcloundecan]-3l-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 1Example 12. (8S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((2lR,3lS)-2l-methyl-4l-oxo-ll-oxa-5l-azaspiro[bicvclo[2.2.2]octane-2,10l-cvcloundecan]-3l-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 2

[0238] Scheme 13Isomer 1 Isomer 2

[0239] Step 1: To methyl acrylate (6 mL, 69.7 mmol) in toluene (15 mL) was added cyclohexa-l,3-diene (10.5 mL, 116 mmol) at room temperature. The reaction was stirred at 100 °C under N2 for 48 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo and the resulting residue was purified by flash chromatography (silica gel, 0 - 15% EtOAc in PE) to obtain the product methyl bicyclo[2.2.2]oct-5-ene-2-carboxylate (4.1 g, 24.7 mmol, 35% yield) as a yellow oil. LC purity: 90% (UV at 220 nm);Mass calculated for C₁₀H₁₄O₂, [M+1]+, 167.1, found 167.1; Retention time: 1.544 min.

[0240] Step 2: To a solution of methyl bicyclo[2.2.2]oct-5-ene-2-carboxylate (3 g, 18.0 mmol) in MeOH (50 mL) was added 10% Pd / C (166 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at room temperature for 3 h, filtered through a Celite® pad, and washed with MeOH (50 mL). The filtrate was concentrated under reduced pressure to obtain the crude product methyl bicyclo[2.2.2]octane-2-carboxylate (2.8 g, 16.6 mmol, 92%yield) as a yellow oil. The crude product was used directly in the next step without further purification. LC purity: 90% (UV at 220 nm); Mass calculated for C₁₀H₁₆O₂, [M+1]+, 169.1, found 169.1; Retention time: 1.747 min.

[0241] Step 3: To a stirred suspension of methyl bicyclo[2.2.2]octane-2-carboxylate (2.8 g, 16.6 mmol) in THF (50 mL) was added dropwise LDA (2 in THF, 16.6 mL, 33.2 mmol) at -65 °C under N2. The resulting mixture was stirred at this temperature for 1 h under N2 before 3 -bromoprop- 1-ene (2.2 mL, 25 mmol) was added. The resulting mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous NH4CI solution (60 mL), and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 2-allylbicyclo[2.2.2]octane-2-carboxylate (2.9 g, 13.9 mmol, 84% yield) as a yellow oil. LC purity: 66% (UV at 220 nm); Mass calculated for C₁₃H₂₀O₂, [M+1]+, 209.1, found 209.1; Retention time: 1.972 min.

[0242] Step 4: To a stirred suspension of methyl 2-allylbicyclo[2.2.2]octane-2-carboxylate (2.9 g, 13.9 mmol) in THF (40 mL) was added dropwise LAH (1 in THF, 13.9 mL, 13.9 mmol) at -65 °C under N2 and the resulting mixture was stirred under N2 for 2 h. The reaction mixture was diluted with THF (50 mL), quenched with Na2SO4IOH2O, and stirred at room temperature for 30 min. The reaction mixture was then filtered, and the cake was washed with DCM (50 mL). The combined filtrates were concentrated in vacuo to obtain the product (2-allylbicyclo[2.2.2]octan-2-yl)methanol (2 g, 11.1 mmol, 79% yield) as a yellow oil. LC purity: 80% (UV at 220 nm); Mass calculated for C₁₂H₂₀O, [M+1]+, 181.1, found 181.1; Retention time: 1.972 min.

[0243] Step 5: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (1.3 g, 5.1 mmol) and (2-allyl-2-bicyclo[2.2.2]octanyl)methanol (2 g, 11.1 mmol) in chloroform (20 mL) was added dropwise BF3·Et2O (0.6 mL, 5.1 mmol) at 0 °C under N2 atmosphere. The resulting mixture was stirred at 25 °C for 2 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((2-allylbicyclo[2.2.2]octan-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (2.1 g, 4.8 mmol, 44% yield) as a yellow oil. LC purity: 90% (UV at 220 nm); Mass calculated for C₂₅H₃₅NO₅, [M+1]+, 430.3, found 430.2; Retention time: 2.433 min.

[0244] Step 6: To a solution of methyl O-((2-allylbicyclo[2.2.2]octan-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (2.1 g, 4.8 mmol) in THF (10 mL) and water (5 mL) was added LiOH·H2O (411 mg, 17.2 mmol). The resulting mixture was stirred at 25 °C for 2h, and the pH was adjusted to ~5 with diluted HC1 solution (1 N). The resulting mixture was extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 50% EtOAc in PE) to obtain the product O-((2-allylbicyclo[2.2.2]octan-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.9 g, 4.6 mmol, 93% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 91% (UV at 220 nm); Mass calculated for C₂₄H₃₃NO₅, [M+1]+, 416.2, found 416.1; Retention time: 2.085 min.

[0245] Step 7: To a solution of O-((2-allylbicyclo[2.2.2]octan-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (2 g, 4.8 mmol) and prop-2-en-l -amine (410 mg, 7.2 mmol) in DMF (30 mL) were added DIE A (1.7 mL, 9.6 mmol) and HATU (2.7 g, 7.2 mmol). The resulting mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with DCM (30 mL x 3). The combined organic layers were washed with brine (30 mL x 4), dried over by anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2S,3R)-l-(allylamino)-3-((2-allylbicyclo[2.2.2]octan-2-yl)methoxy)-l-oxobutan-2-yl)carbamate (1.5 g, 3.3 mmol, 69% yield) as a yellow oil. LC purity: 80% (UV at 220 nm); Mass calculated for C₂₇H₃₈N₂O₄, [M+1]+, 455.3, found 455.4; Retention time: 2.117 min.

[0246] Step 8: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((2-allylbicyclo[2.2.2]octan-2-yl)methoxy)-l-oxobutan-2-yl)carbamate (700 mg, 1.5 mmol) in DCM (700 mL) was added Grubbs 2nd generation catalyst (654 mg, 0.7 mmol). The resulting mixture was stirred under N2 atmosphere at 60 °C for 16 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((2'R,3'S, Z)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.2]octane-2,10'-cycloundecan]-7'-en-3'-yl)carbamate (250 mg, 0.6 mmol, 38% yield) as a black oil. LC purity: 57% (UV at 220 nm); Mass calculated for C₂₅H₃₄N₂O₄, [M+1]+, 427.3, found 427.2; Retention time: 1.853 min.

[0247] Step 9: To a solution of benzyl ((2'R,3'S, Z)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.2]octane-2,10'-cycloundecan]-7'-en-3'-yl)carbamate (250 mg, 0.6 mmol) in THF (30 mL) was added 10% Pd / C (100 mg). The reaction mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (15 mL). Thefiltrate was concentrated under reduced pressure to obtain the product (2'R,3'S)-3'-amino-2'-methyl-r-oxa-5'-azaspiro[bicyclo[2.2.2]octane-2,10'-cycloundecan]-4'-one (170 mg, 0.56 mmol, 98% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm), Mass calculated for C₁₇H₃₀N₂O₂, [M+1]+, 295.2, found 295.2; Retention time: 0.931 min.

[0248] Step 10: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (45 mg, 0.1 mmol) and (2'R,3'S)-3'-amino-2'-methyl-l'-oxa-5'-azaspiro[bicyclo[2.2.2]octane-2,10'-cycloundecan]-4'-one (54 mg, 0.3 mmol) in DMF (3 mL) were added DIEA (0.05 mL, 0.3 mmol) and HATU (38 mg, 0.1 mmol). The reaction mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4CI solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% NH4HCO3) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((2R,2'R,3'S)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.2]octane-2,10'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Example 11: Isomer 1, 7.1 mg, 0.01 mmol, 9% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C₃₄H₄₉N₅O₅S, [M+1]+, 640.1, found 639.9; Retention time: 1.673 min; ¹H NMR (400 MHz, MeOD-d₄) δ 9.15 (s, 1H), 8.37 (d, J = 9.4 Hz, 1H), 4.44 - 4.33 (m, 2H), 4.29 - 4.06 (m, 8H), 3.80 - 3.77 (m, 1H), 3.61 - 3.44 (m, 2H), 3.29 - 3.18 (m, 1H), 3.14 - 3.10 (m, 1H), 1.76 - 1.58 (m, 5H), 1.51 (d, J = 8.2 Hz, 4H), 1.43 - 1.30 (m, 6H), 1.27 - 1.15 (m, 8H), 1.13 - 0.98 (m, 6H), 0.80 - 0.75 (m, 1H); and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((2S,2'R,3'S)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.2]octane-2,10'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Example 12: Isomer 2, 8.5 mg, 0.01 mmol, 10% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C₃₄H₄₉N₅O₅S, [M+1]+, 640.1, found 639.9; Retention time: 1.695 min; ¹H NMR (400 MHz, MeOD-d₄) δ 9.15 (s, 1H), 8.36 (d, J = 14.0 Hz, 1H), 4.41 - 3.86 (m, 10H), 3.70 (d, J = 9.6 Hz, 1H), 3.56 - 3.39 (m, 3H), 3.00 - 2.92 (m, 1H), 1.91 - 1.75 (m, 2H), 1.55 - 1.37 (m, 12H), 1.31 - 1.14 (m, 10H), 1.10 - 1.02 (m, 4H), 0.80 - 0.72 (m, 2H).Example 13. (8S)-2-((S)-2,2-DimethylcvcloDroDane-l-carbonyl)-N-((2lR,3lS)-2l-methyl- 4l-oxo-ll-oxa-5l-azasDiro[bicyclo[2.2.1]heDtane-2,10l-cycloundecan]-3l-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0249] Scheme 14

[0250] Step 1: To a solution of methyl bicyclo[2.2.1]hept-5-ene-2-carboxylate (5 g, 32.9 mmol) in MeOH (100 mL) was added 10% Pd / C (500 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C overnight, filtered through a Celite® pad and washed with MeOH (50 mL). The filtrate was concentrated to obtain the product methyl bicyclo[2.2.1]heptane-2-carboxylate (4 g, 25.9 mmol, 78% yield) as a colorless oil. The crude product was used directly in the next step without further purification. LC purity: 90% (UV at 220 nm); Mass calculated for C9H14O2 [M+1]+, 155.1, found 154.9; Retention time: 1.433 mm.

[0251] Step 2: To a solution of methyl bicyclo[2.2. l]heptane-2-carboxylate (4 g, 25.9 mmol) in THF (50 mL) was added LDA (2 M in THF, 19 mL, 38.9 mmol) at -65 °C under N2 and the resulting mixture was stirred at this temperature for 0.5 h under N2 before 3-bromoprop-l-ene (3.4 mL, 38.9 mmol) was added. The reaction mixture was stirred at 25 °C overnight, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resultingresidue was purified by flash chromatography (silica gel, 0 - 5% EtOAc in PE) to obtain the product methyl 2-allylbicyclo[2.2.1]heptane-2-carboxylate (3.6 g, 18.5 mmol, 71% yield) as a colorless oil.1H NMR (400 MHz, CDCl3) 85.60 - 5.46 (m, 1H), 4.98 - 4.93 (m, 1H), 4.93 -4.89 (m, 1H), 3.60 (s, 3H), 2.50 (dd, J= 13.6, 7.2 Hz, 1H), 2.22 - 2.13 (m, 2H), 2.06 (dd, J = 13.6, 7.2 Hz, 1H), 1.92 (dd, J= 12.8, 2.5 Hz, 1H), 1.52 (d, J= 10.0 Hz, 1H), 1.46 - 1.34 (m, 2H), 1.32 - 1.26 (m, 1H), 1.20 - 1.10 (m, 2H), 1.10 - 1.02 (m, 1H).

[0252] Step 3: To a solution of methyl 2-allylbicyclo[2.2.1]heptane-2-carboxylate (3.6 g, 18.5 mmol) in THF (50 mL) was added dropwise LAH (1.0 AT in THF, 19 mL, 18.5 mmol) at -60 °C. The reaction mixture was stirred under a nitrogen atmosphere at -20 °C for 2 h, quenched with Na2SO4·10H2O at -20 °C and stirred at room temperature for 30 min. The resulting mixture was filtered through a Celite® pad and washed with DCM (50 mL). The combined filtrates were concentrated in vacuo and the resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product (2-allylbicyclo[2.2.1]heptan-2-yl)methanol (3 g, 17.9 mmol, 96% yield) as a light yellow oil.1H NMR (400 MHz, CDCI3) 65.87 - 5.73 (m, 1H), 5.08 - 4.96 (m, 2H), 3.50 (dd, J= 11.2, 5.8 Hz, 1H), 3.38 (dd, J= 11.2, 5.3 Hz, 1H), 2.26 (dd, J= 13.9, 7.4 Hz, 1H), 2.19 - 2.10 (m, 1H), 2.05 - 1.94 (m, 2H), 1.54 - 1.44 (m, 2H), 1.38 - 1.24 (m, 3H), 1.14 (d, J= 9.8 Hz, 1H), 1.06 -0.96 (m, 1H), 0.66 (dd, J= 12.4, 2.4 Hz, 1H).

[0253] Step 4: To a mixture of (2-allylbicyclo[2.2.1]heptan-2-yl)methanol (3 g, 18.1 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (3 g, 12.0 mmol) in chloroform (150 mL) was added BF3·Et2O (1.5 mL, 12.0 mmol) at 0 °C. The mixture was stirred at 0 °C for 2 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 1 - 10% EtOAc in PE) to obtain the product methyl (2S,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-2-(((benzyloxy)carbonyl)amino)butanoate (3.4 g, 5.9 mmol, 64% yield) as a colorless oil. LC purity: 70% (UV at 254 nm); Mass calculated for C24H33NO5 [M+1]+, 416.2, found 416.0; Retention time: 2.132 min.

[0254] Step 5: To a solution of methyl (2S,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-2-(((benzyloxy)carbonyl)amino)butanoate (4 g, 9.6 mmol) in THF (30 mL) and water (10 mL) was added LiOH H2O (808 mg, 19.3 mmol). The resulting mixture was stirred at room temperature for 2.5 h, and the pH was adjusted to 4-5 with diluted aqueous HCl solution (1.0 N). The resulting mixture was diluted with H2O (20 mL) and extracted withEtOAc (40 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 1 - 50% EtOAc in PE) to obtain the product O-((2-allylbicyclo[2.2.1]heptan-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (2.5 g, 6.2 mmol, 64% yield) as a white solid. LC purity: 82% (UV at 220 nm); Mass calculated for C23H31NO5 [M+1]+, 402.2, found 402.2; Retention time: 1.858 min.

[0255] Step 6: To a mixture of O-((2-allylbicyclo[2.2. l]heptan-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (2.5 g, 6.2 mmol) and prop-2-en-l -amine (758 mg, 8.1 mmol) in DMF (60 mL) were added DIEA (5.4 mL, 31.1 mmol) and HATU (3.5 g, 9.3 mmol). The resulting mixture was stirred at room temperature for 2 h, quenched with water (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 -20% EtOAc in PE) to obtain the product benzyl ((2S,3R)-l-(allylamino)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-l-oxobutan-2-yl)carbamate (2.20 g, 4.9 mmol, 80% yield) as a colorless oil. LC purity: 90% (UV at 220 nm); Mass calculated for C26H36N2O4 [M+1]+, 441.3, found 441.2; Retention time: 2.011 min.

[0256] Step 7: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-l-oxobutan-2-yl)carbamate (600 mg, 1.3 mmol) in DCM (600 mL) were added Grubbs 2ndgeneration catalyst (578 mg, 0.7 mmol). The reaction mixture was stirred under N2 at 70 °C for 12 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 25% EtOAc in PE) to obtain the product benzyl ((2'R,3'S, Z)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.1]heptane-2,10'-cycloundecan]-7'-en-3'-yl)carbamate (240 mg, 0.6 mmol, 42% yield) as a brown oil. LC purity: 81% (UV at 220 nm); Mass calculated for C24H32N2O4 [M+1]+, 413.2, found 413.1; Retention time: 1.694 min.

[0257] Step 8: To a solution of benzyl ((2'R,3'S, Z)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.1]heptane-2,10'-cycloundecan]-7'-en-3'-yl)carbamate (140 mg, 0.6 mmol) in THF (10 mL) was added 10% Pd / C (50 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C overnight, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated under reduced pressure to obtain the product (2'R,3'S)-3'-amino-2'-methyl-l'-oxa-5'-azaspiro[bicyclo[2.2.1]heptane-2,10'-cycloundecan]-4'-one (140 mg, 0.5 mmol, 85% yield) as a colorless oil. The crude product was used directly in the next step without further purification. LC purity: 50% (UV at 220 nm); Mass calculated for C16H28N2O2 [M+1]+, 281.2, found 281.4; Retention time: 0.739 min.

[0258] Step 9: To a mixture of (2'R,3'S)-3'-amino-2'-methyl-l'-oxa-5'-azaspiro[bicyclo[2.2.1]heptane-2,10'-cycloundecan]-4'-one (45 mg, 0.2 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (40 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (62 mg, 0.5 mmol) and HATU (91 mg, 0.2 mmol). The resulting mixture was stirred at room temperature for 1 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product ((8S)-2-((S)-2,2-dimethylcyclopropane-1-carbonyl)-N-((2'R,3'S)-2'-methyl-4'-oxo-l'-oxa-5'-azaspiro[bicyclo[2.2.1]heptane-2,10'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide) (24.7 mg, 0.03 mmol, 23% yield) as a white solid. LC purity: 100% (UV at 220 nm); Mass calculated for C33H47N5O5S [M+1]+, 626.3, found 626.6; Retention time: 1.455 min;1H NMR (400 MHz, MeOD-d4) δ 9.15 (s, 1H), 8.37 (d, J = 9.8 Hz, 1H), 4.44 - 4.20 (m, 4H), 4.18 - 4.04 (m, 4H), 4.03 - 3.86 (m, 3H), 3.54 - 3.45 (m, 2H), 3.28 - 3.17 (m, 2H), 2.14 - 2.09 (m, 1H), 1.93 - 1.87 (m, 1H), 1.81 - 1.73 (m, 1H), 1.71 - 1.60 (m, 2H), 1.58 - 1.49 (m, 2H), 1.46 - 1.31 (m, 5H), 1.27 - 1.14 (m, 9H), 1.10 (s, 3H), 1.06 -1.01 (m, 2H), 0.81 - 0.74 (m, 1H), 0.45 (d, J= 12.4 Hz, 1H).Example 14. (S)-2-((S)-2,2-Dimethylcyclopropane-1-carbonyl)-N-((8R,9S)-8-methyl-10-oxo-7-oxa-11-azaspiro[4.10]pentadecan-9-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide

[0259] Scheme 15

[0260] Step 1: To a stirred suspension of methyl cyclopentanecarboxylate (5 g, 39.1 mmol) in THF (50 mL) was added LDA (2 in THF, 29.3 mL, 58.6 mmol) at -65 °C and the resulting mixture was stirred for 0.5 h under N2 before 3 -bromoprop- 1-ene (5.1 mL, 58.6 mmol) was added at this temperature. The resulting mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 1-allylcyclopentane-1 -carboxylate (4.3 g, 25.6 mmol, 66% yield) as a yellow oil. LC purity:90% (UV at 220 nm); Mass calculated for C10H12O2, [M+1]+; 169.1, found 169.1; Retention time: 1.642 min.

[0261] Step 2: To a stirred suspension of methyl 1 -allylcyclopentane- 1 -carboxylate (2 g, 11.9 mmol) in THF (40 mL) was added LAH (1 in THF, 11.9 mL, 11.9 mmol) at -65 °C and the resulting mixture was stirred under N2 for 2 h. The reaction mixture was slowly quenched with diluted aqueous HC1 solution (1 A, 40 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product (1-allylcyclopentyl)methanol (1.7 g, 11.5 mmol, 97% yield) as a yellow oil.1H NMR (400 MHz,CDCl3) 65.84 - 5.71 (m, 1H), 5.03 - 4.96 (m, 2H), 3.32 (s, 2H), 2.09 (d, J= 7.4 Hz, 2H), 1.59 - 1.48 (m, 4H), 1.41 - 1.29 (m, 4H).

[0262] Step 3: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (4.6 mL, 4.6 mmol) and (l-allylcyclopentyl)methanol (1 g, 6.9 mmol) in chloroform (40 mL) was added dropwise BF3Et2O (0.6 mL, 4.6 mmol) under N2 atmosphere at 0 °C and the resulting mixture was stirred for 2 h at 0 °C. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l-allylcyclopentyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.7 g, 2.6 mmol, 57% yield) as a yellow oil. LC purity: 60% (UV at 254 nm); Mass calculated for C22H31NO5, [M+1]+; 390.2, found 390.1; Retention time: 2.057 min.

[0263] Step 4: To a solution of methyl O-((l-allylcyclopentyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.6 g, 4.1 mmol) in THF (10 mL) and water (10 mL) was added LiOH·H2O (196 mg, 8.2 mmol). The resulting mixture was stirred at 25 °C for 2 h, neutralized to pH 5 with aqueous HCl solution (1 N) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to obtain the product O-((l-allylcyclopentyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.5 g, 3.2 mmol, 79% yield) as a yellow oil. LC purity: 81% (UV at 254 nm); Mass calculated for C21H29NO5, [M+1]+, 376.2, found 376.2; Retention time: 1.867 min.

[0264] Step 5: To a solution of O-((l-allylcyclopentyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.5 g, 4.0 mmol) and prop-2-en-l -amine (374 mg, 4.8 mmol) in DMF (30 mL) were added DIEA (2.1 mL, 12.0 mmol) and HATU (2.3 g, 3.5 mmol). The resulting mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic phases were washed with brine (30 mL x 2), dried over by anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2S,3R)-l-(allylamino)-3-((l-allylcyclopentyl)methoxy)-l-oxobutan-2-yl)carbamate (900 mg, 1.6 mmol, 40% yield) as a yellow oil. LC purity: 74% (UV at 254 nm); Mass calculated for C24H34N2O4, [M+1]+, 415.3, found 415.1; Retention time: 1.906 min.

[0265] Step 6: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((l-allylcyclopentyl)methoxy)-l-oxobutan-2-yl)carbamate (900 mg, 2.2 mmol) in DCM (900mL) was added Grubbs 2ndgeneration catalyst (922 mg, 1.1 mmol). The resulting mixture was stirred under N2 atmosphere at 60 °C under N2 for 16 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((8R,9S, Z)-8-methyl-10-oxo-7-oxa-l l-azaspiro[4.10]pentadec-13-en-9-yl)carbamate (210 mg, 0.53 mmol, 24% yield) as a black oil. LC purity: 98% (UV at 220 nm); Mass calculated for C22H30N2O4, [M+1]+, 387.2, found 387.1; Retention time:1.599 min.

[0266] Step 7: To a solution of benzyl ((8R,9S, Z)-8-methyl-10-oxo-7-oxa-l 1-azaspiro[4.10]pentadec-13-en-9-yl)carbamate (210 mg, 0.55 mmol) in THF (10 mL) was added 10% Pd / C (100 mg). The reaction mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH. The filtrate was concentrated in vacuo to afford the product (8R,9S)-9-amino-8-methyl-7-oxa-ll-azaspiro[4.10]pentadecan-10-one (70 mg, 0.2 mmol, 41% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm); Mass calculated for C14H26N2O2, [M+1]+, 255.2, found 255.1; Retention time: 0.779 min.

[0267] Step 8: To a solution of (8R,9S)-9-amino-8-methyl-7-oxa-l 1-azaspiro[4.10]pentadecan-10-one (30 mg, 0.1 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (29 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (26 mg, 0.2 mmol) and HATU (45 mg, 0.1 mmol). The reaction mixture was stirred for 2 h at 25 °C, quenched with saturated aqueous NH4CI solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-((8R,9S)-8-methyl- 10-oxo-7-oxa- 11-azaspiro[4.10]pentadecan-9-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (6.1 mg, 0.01 mmol, 13% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C31H45N5O5S, [M+1]+, 600.3, found 600.2; Retention time: 1.391 min;1H NMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.37 (d, J= 8.4 Hz, 1H), 4.43 - 4.33 (m, 2H), 4.25 - 4.20 (m, 1H), 4.19 - 4.04 (m, 4H), 4.02 - 3.84 (m, 3H), 3.56 - 3.44 (m, 2H), 3.40 (d, J = 9.2 Hz, 2H), 3.20 - 3.13 (m, 1H), 1.85 - 1.75 (m, 1H), 1.70 - 1.66 (m, 2H), 1.61 - 1.51 (m, 4H), 1.47 - 1.33 (m, 6H), 1.26 - 1.20 (m, 4H), 1.16 - 1.14 (m, 3H), 1.10 (s, 2H), 1.05 -1.00 (m, 3H), 0.80 - 0.75 (m, 1H).Example 15. (8S)-2-((S)-2,2-Dimethylcyclopropane-1-carbonyl)-N-((9R,10S)-9-methyl-11-oxo-1,8-dioxa-12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide

[0268] Scheme 16

[0269] Step 1: To a solution of methyl tetrahydro-2H-pyran-2-carboxylate (9.5 g, 65.9 mmol) in THF (100 mL) was added NaHMDS (1 in THF, 99 mL, 98.8 mmol) at -75 °C under nitrogen. The mixture was stirred at -75 °C for 45 min before 3 -bromoprop- 1-ene (8.6 mL, 98.8 mmol) was added. The resulting solution was stirred at 25 °C for 1.5 h, quenched with saturated aqueous NH4CI solution (40 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl 2-allyltetrahydro-2H-pyran-2-carboxylate (6.7 g, 36.4 mmol, 55% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C10H16O3 [M+1]+, 185.1, found 185.2; Retention time: 1.325 min;1H NMR (400 MHz, DMSO-6) 65.77 - 5.61 (m, 1H), 5.07 - 4.95 (m, 2H), 3.75 - 3.68 (m, 1H), 3.64 (s, 3H), 3.54 - 3.45 (m, 1H), 2.37 - 2.25 (m, 2H), 2.05 - 1.96 (m, 1H), 1.70 - 1.61 (m, 1H), 1.46 - 1.22 (m, 4H).

[0270] Step 2: To a solution of methyl 2-allyltetrahydro-2H-pyran-2-carboxylate (6.7 g, 36.4 mmol) in THF (60 mL) was added LAH (1 M in THF, 44 mL, 43.6 mmol) at -78 °C under nitrogen. The mixture was stirred at -78 °C for 2 h, cautiously quenched with diluted HC1 solution (1 N, 50 mL) and extracted with EtOAc (50 mL x 3). The combined organiclayers were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product ((2-allyltetrahydro-2H-pyran-2-yl)methanol) (4.6 g, 29.4 mmol, 81% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C9H16O2 [M+1]+, 157.1, found 157.2; Retention time: 0.833 min;1H NMR (400 MHz, DMSO-6) 65.82 - 5.71 (m, 1H), 5.13 - 4.97 (m, 2H), 4.48 (t, J= 5.4 Hz, 1H), 3.60 - 3.52 (m, 2H), 3.36 - 3.31 (m, 1H), 3.27 - 3.20 (m, 1H), 2.42 - 2.35 (m, 1H), 2.25 - 2.17 (m, 1H), 1.62 - 1.52 (m, 2H), 1.45 - 1.31 (m, 4H).

[0271] Step 3: To a solution of (2-allyltetrahydro-2H-pyran-2-yl)methanol (2.1 g, 13.2 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (2.2 g, 8.83 mmol) in chloroform (30 mL) was added BF3·Et2O (1.1 mL, 8.83 mmol) at 0 °C under nitrogen. The mixture solution was stirred at 0 °C for 2 h, concentrated under reduced pressure and the remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((2-allyltetrahydro-2H-pyran-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3.4 g, 8.4 mmol, 95% yield) as a colorless oil. LC purity: 80% (UV at 220 nm); Mass calculated for C22H31NO6 [M+1]+, 406.2, found 406.2; Retention time: 1.715 min.

[0272] Step 4: To a solution of methyl O-((2-allyltetrahydro-2H-pyran-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3.4 g, 8.4 mmol) in THF (20 mL) and water (20 mL) was added LiOH H2O (706 mg, 16.8 mmol). The reaction mixture was stirred at 25 °C for 12 h, and the pH was adjusted to ~5 with aqueous HC1 solution (1 N). The resulting mixture was extracted with EtOAc (40 mL x 2). The combined organic layers were dried over anhydrous MgSO4, filtered and evaporated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 50% EtOAc in PE) to obtain the product O-((2-allyltetrahydro-2H-pyran-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.9 g, 4.9 mmol, 58% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C21H29NO6 [M+1]+, 392.2, found 392.2; Retention time: 1.445 min.

[0273] Step 5: To a solution of O-((2-allyltetrahydro-2H-pyran-2-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.9 g, 4.9 mmol) in DMF (20 mL) were added HATU (2 g, 5.3 mmol) and DIEA (2.5 mL, 14.6 mmol). The mixture solution was stirred at 25 °C for 30 min before prop-2-en-l -amine (276 mg, 4.9 mmol) was added. The resulting mixture was stirred at 25 °C for 1 h, quenched with saturated aqueous NH4CI solution (30 mL) andextracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((2S,3R)-l-(allylamino)-3-((2-allyltetrahydro-2H-pyran-2-yl)methoxy)-l-oxobutan-2-yl)carbamate (1.9 g, 4.4 mmol, 91% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C24H34N2O5 [M+1]+, 431.3, found 431.3; Retention time: 1.623 min.

[0274] Step 6: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((2-allyltetrahydro-2H-pyran-2-yl)methoxy)-l-oxobutan-2-yl)carbamate (500 mg, 1.2 mmol) in DCM (500 mL) was added Grubbs 2ndgeneration catalyst (394 mg, 0.47 mmol). The mixture was stirred at 70 °C for 24 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((9R,10S, Z)-9-methyl-ll-oxo-l,8-dioxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (70 mg, 0.17 mmol, 15% yield) as a black oil. LC purity: 50% (UV at 220 nm); Mass calculated for C22H30N2O5 [M+1]+, 403.2, found 403.2; Retention time: 1.347 min.

[0275] Step 7: To a solution of benzyl ((9R,10S, Z)-9-methyl-l l-oxo-l,8-dioxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (70 mg, 0.17 mmol) in THF (10 mL) was added 10% Pd / C (50 mg). The reaction mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (30 mL). The filtrate was concentrated in vacuo to afford the product (9R, 10 S)-10-amino-9-m ethyl- 1,8-di oxa- 12-azaspiro[5.10]hexadecan-ll-one (45 mg, 0.17 mmol, 96% yield) as a black solid, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm); Mass calculated for C₁₄H₂₆N₂O₃ [M+1]+, 271.2, found 271.3; Retention time: 0.544 min.

[0276] Step 8: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (60 mg, 0.17 mmol) in DMF (3 mL) were added HATU (76 mg, 0.2 mmol) and DIEA (0.09 mL, 0.5 mmol). The mixture solution was stirred at 25 °C for 25 min before (9R,10S)-10-amino-9-methyl-l,8-dioxa-12-azaspiro[5.10]hexadecan-ll-one (45 mg, 0.17 mmol) was added. The reaction mixture was stirred at 25 °C for 30 min, quenched with saturated aqueous NH4CI solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2Ocontaining 0.1% FA) to obtain the product (8S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R, 10S)-9-m ethyl- 11 -oxo- 1,8-dioxa- 12-azaspiro[5.10]hexadecan- 10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (5.0 mg, 0.008 mmol, 5.0% yield) as a yellow solid. LC purity: 97% (UV at 220 nm); Mass calculated for C₃₁H₄₅N₅O₆S [M+1]+, 616.3, found 616.3; Retention time: 1.114 min;1H NMR (400 MHz, MeOD-d4) δ 9.15 (s, 1H), 8.37 (d, J= 9.2 Hz, 1H), 4.45 - 4.30 (m, 2H), 4.27 - 4.22 (m, 1H), 4.20 - 4.03 (m, 4H), 4.02 - 3.82 (m, 3H), 3.63 - 3.33 (m, 6H), 3.24 - 3.18 (m, 1H), 1.85 - 1.56 (m, 5H), 1.52 - 1.36 (m, 7H), 1.24 - 1.18 (m, 4H), 1.18 - 1.08 (m, 6H), 1.06 - 1.01 (m, 1H), 0.81 - 0.74 (m, 1H).Example 16. (8S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-2, 8-dioxa-12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2, 6-diazaspiro[3.4]octane-8-carboxamide isomer 1Example 17. (8S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-2,8-dioxa-12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 2

[0277] Scheme 17

[0278] Step 1: To a stirred suspension of methyl tetrahydro-2H-pyran-3-carboxylate (10 g, 69.4 mmol) in THF (100 mL) was added LDA (2 in THF, 52 mL, 104.2 mmol) at -65 °C under N2 and the resulting mixture was stirred at this temperature for 1 h under N2 before 3-bromoprop- 1-ene (12.6 g, 104.2 mmol) was added. The reaction mixture was stirred at 25 °C for 12 h, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 3 -allyltetrahydro-2H-pyran-3 -carboxylate (1.8 g, 9.8 mmol, 14% yield) as a yellow oil. LC purity: 90% (UV at 220 nm); Mass calculated for C10H16O3 [M+1]+, 185.1, found 185.2; Retention time: 2.123 min.

[0279] Step 2: To a solution of methyl 3 -allyltetrahydro-2H-pyran-3 -carboxylate (1.8 g, 9.8 mmol) in THF (20 mL) was added dropwise LAH (1.0 in THF, 9.8 mL, 9.8 mmol) at -60 °C. The mixture was stirred at -60 °C for 2 h, diluted with THF (50 mL), quenched with Na2SO4·10H2O, and stirred at room temperature for 30 min. The resulting mixture was filtered, and the cake was washed with DCM (50 mL x 2). The combined filtrates were concentrated in vacuo to give the product (3-allyltetrahydro-2H-pyran-3-yl)methanol (1.2 g, 7.7 mmol, 79% yield) as a yellow oil. LC purity: 90% (UV at 220 nm); Mass calculated for C9H16O2 [M+1]+, 157.1, found 157.2; Retention time: 1.723 min.

[0280] Step 3: To a mixture of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (1.9 g, 7.7 mmol) and (3-allyltetrahydro-2H-pyran-3-yl)methanol (1.2 g, 7.7 mmol) in chloroform (25 mL) was added BF3·Et2O (1.1 mL, 8.3 mmol) at 0 °C. The reaction mixture was stirred under nitrogen at 0 °C for 2 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to afford the product methyl O-((3-allyltetrahydro-2H-pyran-3-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.5 g, 3.7 mmol, 48% yield) as a yellow oil. LC purity: 70% (UV at 220 nm); Mass calculated for C22H31NO6 [M+1]+, 406.2, found 406.3; Retention time: 1.809 min.

[0281] Step 4: To a solution of methyl O-((3-allyltetrahydro-2H-pyran-3-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.5 g, 3.7 mmol) in THF (20 mL) and water (10 mL) was added LiOH·H2O (546 mg, 13 mmol) at 0 °C and the resulting mixture was stirred at room temperature for 2 h. The pH of the reaction mixture was adjusted to 4-5 with diluted aqueous HC1 solution (1.0 N), diluted with H2O (20 mL), and extracted with EtOAc (40 mL x 3). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was concentrated in vacuo to obtain the product O-((3-allyltetrahydro-2H-pyran-3-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.4 g, 3.6 mmol, 97% yield) as a yellow oil. The crude product was used directly in the next step without further purification. LC purity: 50%; Mass calculated for C21H29NO6 [M+1]+, 392.2, found 392.2; Retention time: 1.624 min.

[0282] Step 5: To a solution of O-((3-allyltetrahydro-2H-pyran-3-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.4 g, 3.6 mmol) and DIEA (1.2 mL, 7.2 mmol) in DMF (20 mL) were added HATU (2 g, 5.4 mmol) and prop-2-en-l -amine (306 mg, 5.4 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (40 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to afford the product benzyl ((2S,3R)-l-(allylamino)-3-((3-allyltetrahydro-2H-pyran-3-yl)methoxy)-l-oxobutan-2-yl)carbamate (1 g, 2.3 mmol, 65% yield) as a yellow oil. LC purity: 80% (UV at 220 nm); Mass calculated for C24H34N2O5 [M+1]+, 431.3, found 431.3; Retention time: 1.674 min.

[0283] Step 6: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((3-allyltetrahydro-2H-pyran-3-yl)methoxy)-l-oxobutan-2-yl)carbamate (500 mg, 1.2 mmol) in DCM (500 mL) was added Grubbs 2ndgeneration catalyst (493 mg, 0.58 mmol). The reaction mixture was stirred under N2 at 60 °C for 24 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 25% EtOAc in PE) to afford the product benzyl ((9R, 10S, Z)-9-methyl-ll -oxo-2, 8-dioxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (80 mg, 0.2 mmol, 17% yield) as a brown solid. LC purity: 57% (UV at 220 nm); Mass calculated for C22H30N2O5 [M+1]+, 403.2, found 403.3; Retention time: 1.304 min.

[0284] Step 7: To a solution of benzyl ((9R,10S, Z)-9-methyl-l l-oxo-2,8-dioxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (80 mg, 0.2 mmol) in THF (10 mL) was added 10% Pd / C (50 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated under reduced pressure to give the product (9R,10S)-10-amino-9-methyl-2,8-dioxa-12-azaspiro[5.10]hexadecan-ll-one (50 mg, 0.2 mmol, 93% yield) as a colorless oil. The crude product was used directly in the next step without further purification. LC purity: 60% (UV at 220 nm); Mass calculated for C₁₄H₂₆N₂O₃ [M+1]+, 271.2, found 271.1;Retention time: 0.335 min.

[0285] Step 8: To a solution of (9R,10S)-10-amino-9-methyl-2,8-dioxa-12-azaspiro[5.10]hexadecan-l 1-one (54 mg, 0.2 mmol) and DIEA (0.02 mL, 0.12 mmol) in DMF (3 mL) were added HATU (60 mg, 0.16 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (58 mg, 0.16 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% NH4HCO3) to afford the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-((6R,9R, 10S)-9-m ethyl- 11 -oxo-2, 8-dioxa- 12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Isomer 1, 2.8 mg, 0.005 mmol, 4% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C₃₁H₄₅N₅O₆S [M+1]+, 615.3, found 616.2; Retention time: 1.018 min;1H NMR (400 MHz, MeOD-4) 69.15 (s, 1H), 8.37 (d, J= 92 Hz, 1H), 4.57 (s, 1H), 4.43 - 4.22 (m, 4H), 4.17 - 4.05 (m, 4H), 4.01 - 3.89 (m, 3H), 3.82 (d, J= 9.4 Hz, 1H), 3.68 - 3. 65 (m, 1H), 3.50 - 3.36 (m, 4H), 3.13 - 3.07 (m, 1H), 1.83 - 1.73 (m, 2H), 1.69 - 1.62 (m, 1H), 1.51 - 1.29 (m, 8H), 1.23 - 1.15 (m, 7H), 1.10 (s, 2H), 1.05 - 1.01 (m, 1H), 0.82 - 0.74 (m, 1H); and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((6S,9R, 10S)-9-m ethyl- 11 -oxo-2, 8-dioxa- 12-azaspiro[5.10]hexadecan- 10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide(Isomer 2, 2.9 mg, 0.005 mmol, 4% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C₃₁H₄₅N₅O₆S [M+1]+, 615.3, found 615.8; Retention time: 1.110 min;1H NMR (400 MHz, MeOD-4) 6 9.15 (s, 1H), 8.37 (d, J= 9.2 Hz, 1H), 4.58 (s, 1H), 4.43 - 4.33 (m, 2H), 4.26 - 4.22 (m, 2H), 4.19 - 3.89 (m, 7H), 3.77 - 3.69 (m, 1H), 3.60 - 3.49 (m, 4H), 3.43 - 3.36 (m, 1H), 3.05 - 2.97 (m, 1H), 1.87 - 1.78 (m, 1H), 1.53 - 1.28 (m, 9H), 1.23 - 1.15 (m, 8H), 1.10 (s, 2H), 1.07 -1.00 (m, 1H), 0.82 - 0.75 (m, 1H).Example 18. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((10R,llS)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.11]octadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0286] Scheme 18

[0287] Step 1: To a solution of methyl cycloheptanecarboxylate (1.8 g, 11.5 mmol) in THF (20 mL) was added LDA (2 M in THF, 8.6 mL, 17.3 mmol) at -78 °C and the resulting mixture was stirred for 1 h under N2 before 3 -bromoprop- 1-ene (1.5 mL, 17.3 mmol) was added at this temperature. The mixture was stirred at 25 °C for 12 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with EtOAc (20 mL x 3). The combined organic phases were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 1-allylcycloheptane-1 -carboxylate (2.1 g, 10.2 mmol, 88% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) δ 5.74 - 5.54 (m, 1H), 5.05 - 4.89 (m, 2H), 3.65 (s, 3H), 2.25 (d, J= 7.4 Hz, 2H), 2.09 - 2.00 (m, 2H), 1.55 - 1.43 (m, 10H).

[0288] Step 2: To a solution of methyl 1 -allylcycloheptane- 1 -carboxylate (2.2 g, 11.1 mmol) in THF (30 mL) was added dropwise LAH (1.0 M in THF, 16.7 mL, 16.7 mmol) at -60 °C. The mixture was stirred at -60 °C for 2 h, diluted with THF (50 mL), quenched with Na2SO4·10H2O, and stirred at room temperature for 30 min. The resulting mixture was filtered, and the cake was washed with DCM (50 mL x 2). The combined filtrates were concentrated in vacuo to obtain the product (l-allylcycloheptyl)methanol (1.6 g, 9.03 mmol, 81% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) 65.98 - 5.72 (m, 1H), 5.16 - 4.98 (m, 2H), 3.32 (s, 2H), 2.05 (d, J= 7.4 Hz, 2H), 1.53 - 1.44 (m, 8H), 1.41 - 1.30 (m, 4H).

[0289] Step 3: To a solution of (l-allylcycloheptyl)methanol (500 mg, 3.0 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (741 mg, 3.0 mmol) in chloroform (15 mL) was added BF3·Et2O (0.38 mL, 3.0 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. The volatiles were removed and the resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (600 mg, 1.0 mmol, 34% yield) as a yellow oil. LC purity:70% (UV at 254 nm); Mass calculated for C24H35NO5 [M+1]+, 418.3, found 418.2; Retention time: 2.387 min.

[0290] Step 4: To a solution of methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (500 mg, 1.2 mmol) in THF (10 mL) and water (5 mL) was added LiOH·H2O (151 mg, 3.6 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h, and the pH was adjusted to 4-5 with diluted aqueous HCl solution (1.0 N). The resulting mixture was diluted with H2O (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the product O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (390 mg, 0.97 mmol, 81% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 70% (UV at 200 nm); Mass calculated for C23H33NO5 [M+1]+, 404.2, found 404.3; Retention time: 1.269 min.

[0291] Step 5: To a solution of O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (390 mg, 1.0 mmol), DIEA (249 mg, 1.9 mmol), and HATU (404 mg, 1.1 mmol) in DMF (10 mL) was added but-3-en-l -amine (82 mg, 1.2 mmol). The reaction mixture was stirred at room temperature for 12 h, quenched with water (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 60% EtOAc in PE) to afford the product benzyl ((2S,3R)-3-((l-allylcycloheptyl)methoxy)-l-(but-3-en-l-ylamino)-l-oxobutan-2-yl)carbamate (380 mg, 0.8 mmol, 80% yield) as a yellow solid. LC purity: 80 % (UV at 200 nm); Mass calculated for C27H40N2O4 [M+1]+, 457.3, found 457.3; Retention time: 2.413 min.

[0292] Step 6: To a solution of benzyl ((2S,3R)-3-((l-allylcycloheptyl)methoxy)-l-(but-3-en-l-ylamino)-l-oxobutan-2-yl)carbamate (350 mg, 0.77 mmol) in DCM (350 mL) was added Grubbs 2ndgeneration catalyst (325 mg, 0.4 mmol) under N2 at room temperature. Themixture was stirred under N2 at 65 °C for 12 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to afford the product benzyl ((10R,l lS, Z)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.1 l]octadec-16-en-l 1-yl)carbamate (200 mg, 0.4 mmol, 52% yield) as a yellow oil. LC purity: 85% (UV at 254 nm); Mass calculated for C25H36N2O4 [M+1]+, 429.3, found 429.3; Retention time: 2.252 min.

[0293] Step 7: To a solution of benzyl ((10R,l lS, Z)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.1 l]octadec-16-en-l l-yl)carbamate (200 mg, 0.47 mmol) in TFE (20 mL) was added 10% Pd / C (40 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C for 10 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to give the product (10R,l 1 S)-l l-amino-10-methyl-9-oxa-13-azaspiro[6.1 l]octadecan- 12-one (140 mg, 0.33 mmol, 71% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 70% (UV at 200 nm); Mass calculated for C17H32N2O2 [M+1]+, 297.3, found 297.3; Retention time: 1.843 min.

[0294] Step 8: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (94 mg, 0.26 mmol), HATU (99 mg, 0.26 mmol), and DIEA (0.08 mL, 0.5 mmol) in DMF (3 mL) was added (10R,llS)-ll-amino-10-methyl-9-oxa-13-azaspiro[6.11]octadecan-12-one (70 mg, 0.24 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% NH4HCO3) to afford the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-(( 1 OR, 11 S)- 10-methyl- 12-oxo-9-oxa- 13-azaspiro[6.11]octadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (79 mg, 0.12 mmol, 51% yield). LC purity: 99% (UV at 254 nm); Mass calculated for C34H51N5O5S [M+1]+, 642.4, found 642.2; Retention time: 1.671 min;1H NMR (400 MHz, MeOD-d4) 89.15 (s, 1H), 8.37 (d, J= 9.4 Hz, 1H), 8.04 - 7.97 (m, 1H), 7.92 -7.84 (m, 1H), 4.42 - 4.36 (m, 1H), 4.35 - 4.30 (m, 1H), 4.28 - 4.24 (m, 1H), 4.19 - 4.03 (m, 3H), 4.02 - 3.85 (m, 4H), 3.57 - 3.46 (m, 1H), 3.42 - 3.38 (m, 2H), 3.28 - 3.20 (m, 1H), 2.74 (d, J= 8.6 Hz, 1H), 1.90 - 1.80 (m, 2H), 1.70 - 1.62 (m, 3H), 1.61 - 1.54 (m, 1H), 1.52 - 1.30 (m, 12H), 1.27 - 1.18 (m, 5H), 1.16 (s, 3H), 1.10 (s, 2H), 1.06 - 1.01 (m, 1H), 1.00 - 0.92 (m, 2H), 0.81 - 0.76 (m, 1H).Example 19. (S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((9R,10R)-ll-hvdroxy- 9-methyl-8-oxaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0295] Scheme 19

[0296] Step 1: To a solution of O-((l-allylcyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (4 g, 10.3 mmol), DIEA (5.4 mL, 30.9 mmol), and N, O-dimethylhydroxylamine hydrochloride (753 mg, 12.3 mmol) in DMF (40 mL) was added HATU (5.9 g, 15.4 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 2 h, quenched with saturated aqueous NH4CI solution (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with water (50 mL) and brine (50 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((2S,3R)-3-((l-allylcyclohexyl)methoxy)-l-(methoxy(methyl)amino)-l-oxobutan-2-yl)carbamate (2.6 g, 5.4 mmol, 53% yield) as a colorless oil. LC purity: 90 % (UV at 254 nm); Mass calculated for C24H36N2O5, [M+1]+, 433.3, found 433.2; Retention time: 2.172 min.

[0297] Step 2: To a solution of benzyl ((2S,3R)-3-((l-allylcyclohexyl)methoxy)-l-(methoxy(methyl)amino)-l-oxobutan-2-yl)carbamate (1 g, 2.3 mmol) in THF (15 mL) was added dropwise LAH (1.0 M in THF, 2.3 mL, 2.3 mmol) at 0 °C under N2. The resulting mixture was stirred at 0 °C for 1 h, cautiously quenched with diluted HCl solution (0.1 N) and extracted with DCM (50 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated at 0 °C to obtain the crude product benzyl ((2S,3R)-3-((l-allylcyclohexyl)methoxy)-l-oxobutan-2-yl)carbamate (800 mg, 1.5 mmol, 65% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80 % (UV at 254 nm); Mass calculated for C22H31NO4 [M+1]+, 374.2, found 374.2; Retention time: 2.233 min.

[0298] Step 3: To a solution of benzyl ((2S,3R)-3-((l-allylcyclohexyl)methoxy)-l-oxobutan-2-yl)carbamate (600 mg, 1.6 mmol) in THF (10 mL) was added dropwise but-3-en-1-ylmagnesium bromide (8 mL, 4.0 mmol) at 0 °C and the resulting mixture was stirred under N2 atmosphere at 0 °C for 1 h. The reaction mixture was poured into a stirred solution of diluted HCl solution (0.5 N) (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous MgSO4, filtered and evaporated in vacuo. The remaining residue was purified by reverse phase chromatography (gradient of MeCN in water containing 0.1% FA) to obtain the product benzyl ((2R,3R)-2-((l-allylcyclohexyl)methoxy)-4-hydroxyoct-7-en-3-yl)carbamate (260 mg, 0.5 mmol, 34% yield) as a colorless oil. LC purity: 90% (UV at 254 nm); Mass calculated for C26H39NO4 [M+1]+, 430.3, found 430.2; Retention time: 1.921 min.

[0299] Step 4: To a solution of benzyl ((2R,3R)-2-((l-allylcyclohexyl)methoxy)-4-hydroxyoct-7-en-3-yl)carbamate (260 mg, 0.5 mmol) in DCM (250 mL) was added Grubbs 2ndgeneration catalyst (138 mg, 0.16 mmol). The reaction mixture was stirred under N2 at 60 °C for 16 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((9R,10R, Z)-1 l-hydroxy-9-methyl-8-oxaspiro[5.10]hexadec-14-en-10-yl)carbamate (120 mg, 0.3 mmol, 49% yield) as a brown solid. LC purity: 90% (UV at 254 nm); Mass calculated for C24H35NO4 [M+1]+, 402.3, found 402.3; Retention time: 2.233 min.

[0300] Step 5: To a solution of benzyl ((9R,10R, Z)-ll-hydroxy-9-methyl-8-oxaspiro[5.10]hexadec-14-en-10-yl)carbamate (120 mg, 0.3 mmol) in TFE (5 mL) was added 10% Pd / C (40 mg) and the resulting mixture was stirred under H2atmosphere (1 atm) at 25°C for 10 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated under reduced pressure to obtain the product (9R,10R)-10-amino-9-methyl-8-oxaspiro[5.10]hexadecan-ll-ol (60 mg, 0.18 mmol, 60% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 80% (NO UV); Mass calculated for C16H31NO2 [M+1]+, 270.2, found 270.2; Retention time: 1.145 min.

[0301] Step 6: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (40 mg, 0.11 mmol), (9R,10R)-10-amino-9-methyl-8-oxaspiro[5.10]hexadecan-l l-ol (44 mg, 0.17 mmol), and DIEA (0.06 mL, 0.33 mmol) in DMF (3 mL) was added HATU (63 mg, 0.17 mmol) at 0 °C. The resulting mixture was stirred for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% NH4HCO3) to afford the product (8S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R,10R)-ll-hydroxy-9-methyl-8-oxaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (5.9 mg, 0.01 mmol, 9% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C33H50N4O5S [M+1]+, 615.4, found 615.2; Retention time: 1.645 min;1H NMR (400 MHz, MeOD-4) 89.16 (s, 1H), 8.37 (d, J= 13.6 Hz, 1H), 4.44 - 4.17 (m, 3H), 4.16 - 4.05 (m, 2H), 4.04 - 3.86 (m, 4H), 3.83 - 3.76 (m, 1H), 3.66 - 3.59 (m, 1H), 3.48 - 3.38 (m, 2H), 3.25 - 3.16 (m, 1H), 1.74 - 1.59 (m, 2H), 1.54 - 1.28 (m, 16H), 1.27 - 1.09 (m, 12H), 1.07 - 1.02 (m, 1H), 0.85 - 0.72 (m, 1H).Example 20. (S)-2-((S)-2.,2-DimethylcycloDroDane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxasDiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazasDiro[3.4]octane-8-carboxamide

[0302] Scheme 20

[0303] Step 1: To a solution of (8S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R,10R)-11-hydroxy-9-methyl-8-oxaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Example 19, 10 mg, 0.02 mmol) in DCM (2 mL) was added DMP (10 mg, 0.02 mmol) under N2 at 0 °C. The resulting mixture was stirred for 3 h, diluted with aqueous Na2S2C>3 solution (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% NH4HCO3) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-((9R, 10S)-9-methyl- 11 -oxo-8-oxaspiro[5.10]hexadecan- 10-yl)-6-(thiazole-5 -carbonyl)-2,6-diazaspiro[3,4]octane-8-carboxamide (2.7 mg, 0.004 mmol, 27% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C33H48N4O5S [M+1]+, 613.3, found 613.2; Retention time: 1.845 min;1H NMR (400 MHz, MeOD-4) 69.15 (s, 1H), 8.37 (d, J= 9.8 Hz, 1H), 4.57 - 4.49 (m, 1H), 4.42 - 4.25 (m, 2H), 4.24 - 4.03 (m, 4H), 4.03 - 3.80 (m, 3H), 3.60 - 3.47 (m, 2H), 2.99 -2.95 (m, 1H), 2.60 - 2.52 (m, 1H), 1.99 - 1.69 (m, 2H), 1.64 - 1.29 (m, 15H), 1.26 - 1.08 (m, 12H), 1.07 - 1.00 (m, 1H), 0.81 - 0.74 (m, 1H).Example 21. (S)-N-((9R,10S)-3,3-Difluoro-9-methyl-ll-oxo-8-oxa-12-azasDiro[5.11]heDtadecan-10-yl)-2-((S)-2,2-dimethylcycloDroDane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide

[0304] Scheme 21

[0305] Step 1: To a solution of methyl l-allyl-4,4-difluorocyclohexane-l -carboxylate (800 mg, 3.7 mmol) in THF (20 mL) was added LAH (1.0 in THF, 5.5 mL, 5.5 mmol) under N2 at 0 °C. The resulting mixture was stirred at 0 °C for 1 h, diluted with THF (50 mL), quenched with Na2SO4·10H2O, and stirred at room temperature for 30 min. The resulting mixture was filtered, and the cake was washed with DCM (50 mL x 2). The combined filtrates were concentrated in vacuo to obtain the product 1 -allyl-4,4-difluorocyclohexyl)methanol (500 mg, 2.4 mmol, 65% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) 65.91 - 5.76 (m, 1H), 5.16 - 5.09 (m, 2H), 3.46 (d, J= 5.3 Hz, 2H), 2.15 (d, J = 7.6 Hz, 2H), 1.95 - 1.84 (m, 4H), 1.58 - 1.53 (m, 4H).

[0306] Step 2: To a solution of (l-allyl-4,4-difluorocyclohexyl)methanol (350 mg, 1.8 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (459 mg, 1.8 mmol) in chloroform (10 mL) was added BF3·Et2O (0.23 mL, 1.8 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product methyl O-((l -allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (500 mg, 0.8 mmol, 43% yield) as a yellow oil. LC purity:70% (UV at 254 nm); Mass calculated for C23H31F2NO5 [M+1]+, 440.2, found 440.2; Retention time: 2.023 min.

[0307] Step 3: To a solution of methyl O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (500 mg, 0.8 mmol) in THF (10 mL) and water (5 mL) was added LiOH·H2O (100 mg, 2.4 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h, and the pH was adjusted to 4-5 with diluted HC1 solution (1 TV). The mixture was diluted with H2O (10 mL) and extracted with EA (10 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduce pressure to obtain the product O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (270 mg, 0.54 mmol, 68% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 85% (UV at 254 nm); Mass calculated for C22H29F2NO5 [M+1]+, 426.2, found 426.3; Retention time: 1.254 min.

[0308] Step 4: To a solution of O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (270 mg, 0.6 mmol), DIEA (0.2 mL, 1.3 mmol), and HATU (265 mg, 0.7 mmol) in DMF (5 mL) was added but-3-en-l -amine (50 mg, 0.7 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 12 h, quenched with saturated aqueous NH4CI solution (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over by anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((2S,3R)-3-((1 -allyl-4,4-difluorocyclohexyl)methoxy)- 1 -(but-3 -en- 1 -ylamino)- 1 -oxobutan-2-yl)carbamate (180 mg, 0.38 mmol, 59% yield) as a yellow solid. LC purity: 80% (UV at 254 nm); Mass calculated for C26H36F2N2O4 [M+1]+, 479.3, found 479.4; Retention time: 2.101 min.

[0309] Step 5: To a solution of benzyl ((2S,3R)-3-((l-allyl-4,4-difluorocy cl ohexyl)methoxy)-l -(but-3 -en-1 -ylamino)- l-oxobutan-2-yl)carbamate (180 mg, 0.38 mmol) in DCM (180 mL) was added Grubbs 2ndgeneration catalyst (160 mg, 0.2 mmol) at room temperature under N2. The resulting mixture was stirred under N2 at 65 °C for 12 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 50% EtOAc in PE) to obtain the product benzyl ((9R,10S, Z)-3,3-difluoro-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.11]heptadec-15-en-10-yl)carbamate (70 mg, 0.12 mmol, 33% yield) ) as a yellow oil. LC purity: 80% (UV at 254 nm); Mass calculated for C24H32F2N2O4 [M+1]+, 451.2, found 451.3; Retention time: 1.851 min.

[0310] Step 6: To a solution of benzyl ((9R,10S, Z)-3,3-difluoro-9-methyl-l l-oxo-8-oxa-12-azaspiro[5.11]heptadec-15-en-10-yl)carbamate (300 mg, 0.67 mmol) in TFE (10 mL) was added 10% Pd / C (40 mg) and the resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C for 3 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to obtain the product (9R,10S)-10-amino-3,3-difluoro-9-methyl-8-oxa-12-azaspiro[5.1 l]heptadecan-l l-one (200 mg, 0.56 mmol, 85% yield) as a pink solid, which was used directly in the next step without further purification. LC purity: 80% (UV at 200 nm); Mass calculated for C16H28F2N2O2 [M+1]+, 319.2, found 319.3; Retention time: 1.411 min.

[0311] Step 7: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (46 mg, 0.13 mmol), HATU (53 mg, 0.14 mmol), and DIEA (0.04 mL, 0.25 mmol) in DMF (3 mL) was added (9R,10S)-10-amino-3,3-difluoro-9-methyl-8-oxa-12-azaspiro[5.11]heptadecan-11-one (40 mg, 0.13 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to afford the product (S)-N-((9R,10S)-3,3 -difluoro-9-m ethyl- 11 -oxo-8-oxa- 12-azaspiro[5.11 ]heptadecan- 10-yl)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (69 mg, 0.1 mmol, 83% yield) as a white solid. LC purity: 100% (UV at 254 nm); Mass calculated for C33H47F2N5O5S [M+1]+, 664.3, found 664.3; Retention time: 1.456 min;1H NMR (400 MHz, MeOD-4) 89.16 (s, 1H), 8.37 (d, J= 8.8 Hz, 1H), 8.09 - 8.03 (m, 1H), 7.96 - 7.85 (m, 1H), 4.45 - 4.20 (m, 4H), 4.19 - 4.04 (m, 3H), 4.03 - 3.84 (m, 4H), 3.56 -3.47 (m, 2H), 3.29 - 3.21 (m, 1H), 2.99 (d, J= 9.0 Hz, 1H), 1.96 - 1.79 (m, 5H), 1.72 - 1.58 (m, 4H), 1.54 - 1.45 (m, 2H), 1.44 - 1.30 (m, 5H), 1.26 - 1.18 (m, 4H), 1.16 (s, 3H), 1.10 (s, 2H), 1.07 - 1.00 (m, 2H), 0.81 - 0.75 (m, 1H).Example 22. (R)-8-((S)-2,2-DimethylcvdoDropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxa-12-azasDiro[5.10]hexadecan-10-yl)-2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxamide isomer 1Example 23. (R)-8-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.10]hexadecan-10-yl)-2-(thiazole-5-carbonyl)-2,8-diazasDiro[4.5]decane-4-carboxamide isomer 2

[0312] Scheme 22

[0313] Step 1: To a solution of tert-butyl 4-(2-ethoxy-2-oxoethylidene)piperidine-l-carboxylate (5 g, 18.6 mmol) in toluene (50 mL) was added N-benzyl-1 -methoxy -N-((trimethylsilyl)methyl)methanamine (4.4 g, 18.6 mmol) at 0 °C under N2 and the resulting mixture was stirred at 0 °C for 10 min. Then a solution of TFA (0.14 mL, 1.9 mmol) in DCM (1.8 mL) was added and the resulting mixture was stirred at 25 °C for 16 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 -50% EtOAc in PE) to obtain the product 8-(tert-butyl) 4-ethyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (2.5 g, 6.0 mmol, 32% yield) as a yellow oil. LC purity: 97% (UV at 220 nm); Mass calculated for C23H34N2O4, [M+1]+; 403.3, found 403.4; Retention time: 0.640 min.

[0314] Step 2: To a solution of 8-(tert-butyl) 4-ethyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (2.4 g, 5.9 mmol) in EtOH (20 mL) was added 10% Pd / C (500 mg). The mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with EtOH (50 mL). The filtrate was concentrated in vacuo to obtain the product 8-(tert-butyl) 4-ethyl 2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (1.8 g, 4.6 mmol, 78% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm); Mass calculated for C16H28N2O4, [M+1]+; 313.2, found 313.2; Retention time: 0.329 min.

[0315] Step 3: To a mixture of 8-(tert-butyl) 4-ethyl 2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (1.8 g, 5.8 mmol) and thiazole-5-carboxylic acid (744 mg, 5.8 mmol) in DMF (20 mL) were added DIE A (3.0 mL, 17.3 mmol) and HATU (3.3 g, 8.6 mmol). The resulting mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over by anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 70% EtOAc in PE) to obtain the product 8-(tert-butyl) 4-ethyl 2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (2.0 g, 3.8 mmol, 66% yield) as a white solid. LC purity: 80% (UV at 254 nm); Mass calculated for C20H29N3O5S, [M+l-56]+, 368.2, found 368.0; Retention time: 1.204 min.

[0316] Step 4: To a mixture of 8-(tert-butyl) 4-ethyl 2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (2.0 g, 4.7 mmol) in DCM (20 mL) was added TFA (5 mL) and the mixture was stirred at 25 °C for 1 h, concentrated under reduced pressure to obtain the product ethyl 2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (1.3 g, 3.8 mmol, 80% yield) as a yellow oil. The crude product was used directly in the next step without further purification. LC purity: 94% (UV at 220 nm); Mass calculated for C15H21N3O3S, [M+1]+, 324.1, found 324.3; Retention time: 0.364 min.

[0317] Step 5: To a mixture of (S)-2,2-dimethylcyclopropane-l -carboxylic acid (459 mg, 4.0 mmol) and HATU (4.6 g, 12.1 mmol) in DMF (20 mL) was added DIEA (2.1 mL, 12.1 mmol). The resulting mixture was stirred at 25 °C for 1 h before ethyl 2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (1.3 g, 4.0 mmol) was added. The resulting mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over by anhydrous Na2SO4, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 100% EtOAc in PE) to obtain the product ethyl 8-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (2 g, 3.8 mmol,66% yield) as a yellow solid. LC purity: 94% (UV at 220 nm); Mass calculated for C21H29N3O4S, [M+1]+, 420.2, found 420.4; Retention time: 1.027 min.

[0318] Step 6: To a solution of ethyl 8-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (1.2 g, 2.9 mmol) in THF (20 mL) and water (10 mL) was added LiOH·H2O (137 mg, 5.7 mmol). The resulting mixture was stirred for 2 h at 25 °C, and the pH was adjusted to ~5 with diluted HC1 solution (1 N). The resulting mixture was extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 10% MeOH in DCM) to obtain the product 8-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxylic acid (760 mg, 1.8 mmol, 63% yield) as a yellow oil. LC purity: 93% (UV at 254 nm); Mass calculated for C19H25N3O4S, [M+1]+, 392.2, found 392.0; Retention time: 0.672 min.

[0319] Step 7: To a solution of 8-((S)-2,2-dimethylcyclopropane-l-carbonyl)-2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxylic acid (150 mg, 0.4 mmol) in MeCN (5 mL) were added 1 -methylimidazole (33 mg, 0.4 mmol), (9R,10S)-10-amino-9-methyl-8-oxa-12-azaspiro[5.10]hexadecan-ll-one (103 mg, 0.4 mmol), and chloro-N, N, N', N'-tetramethylformamidinium hexafluorophosphate (TCFH) (108 mg, 0.4 mmol) at 25 °C. The reaction mixture was stirred at 30 °C for 1 h, quenched with water (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated. The remaining residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to give the racemate (20 mg). The diastereomers were further separated by SFC (ChiralPak IB, 250x30 mm I. D., 5pm; Mobile phase: A for CO2 and B for MeOH + 0.1% NH3H2O) to obtain the product (R)-8-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-8-oxa-12-azaspiro[5.10]hexadecan-10-yl)-2-(thiazole-5-carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxamide (Example 22, Isomer 1, 9.2 mg, 0.01 mmol, 4% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C34H51N5O5S, [M+1]+, 642.4, found 642.3; Retention time: 1.535 min;1H NMR (400 MHz, MeOD-4) δ 9.14 (s, 1H), 8.42 - 8.33 (m, 1H), 7.78 - 7.65 (m, 1H), 4.62 - 4.55 (m, 1H), 4.02 - 3.88 (m, 4H), 3.86 - 3.74 (m, 3H), 3.67 -3.44 (m, 3H), 3.25 - 3.15 (m, 1H), 3.07 - 2.94 (m, 2H), 1.90 - 1.73 (m, 3H), 1.71 - 1.65 (m, 1H), 1.61 - 1.55 (m, 2H), 1.49 - 1.30 (m, 12H), 1.24 - 1.21 (m, 4H), 1.19 - 1.07 (m, 6H), 1.05 - 0.99 (m, 4H), 0.74 - 0.67 (m, 1H); and (S)-8-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R, 10S)-9-m ethyl- 11 -oxo-8-oxa- 12-azaspiro[5.10]hexadecan- 10-yl)-2-(thiazole-5 -carbonyl)-2,8-diazaspiro[4.5]decane-4-carboxamide (Example 23, Isomer 2, 8.0 mg, 0.01 mmol, 3% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C34H51N5O5S, [M+1]+, 642.4, found 642.3; Retention time: 1.507 min;1H NMR (400 MHz, MeOD-4) δ 9.14 (s, 1H), 8.38 (d, J= 12.8 Hz, 1H), 4.82 - 4.72 (m, 1H), 4.38 - 4.28 (m, 1H), 4.18 - 4.10 (m, 1H), 4.08 - 3.99 (m, 2H), 3.96 - 3.89 (m, 2H), 3.82 - 3.69 (m, 1H), 3.55 - 3.41 (m, 3H), 3.25 - 3.11 (m, 2H), 3.06 - 2.99 (m, 1H), 1.84 - 1.57 (m, 6H), 1.49 - 1.32 (m, 11H), 1.29 (s, 1H), 1.25 - 1.14 (m, 8H), 1.12 - 0.97 (m, 6H), 0.74 - 0.70 (m, 1H).Example 24. (S)-2-((S)-2.,2-DimethylcycloDroDane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-3-(trifluoromethyl)-8-oxa-12-azasDiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0320] Scheme 23carboxylate (4 g, 19.0 mmol) was added dropwise LDA (2 in THF, 14 mL, 28.5 mmol) at -65 °C under N2, and the resulting mixture was stirred under N2 for 1 h before 3 -bromoprop- 1-ene (2.5 mL, 28.5 mmol) was added at this temperature. The mixture was stirred at 25 °C for 12 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The remaining residuewas purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl l-allyl-4-(trifluoromethyl)cyclohexane-l -carboxylate (4.8 g, 17.3 mmol, 91% yield) as a yellow oil.1H NMR (400 MHz, CDCl3) 85.76 - 5.54 (m, 1H), 4.99 - 4.90 (m, 2H), 3.64 (s, 3H), 2.26 - 2.22 (m, 2H), 2.12 - 2.09 (m, 2H), 1.95 - 1.84 (m, 1H), 1.79 - 1.76 (m, 2H), 1.34 - 1.23 (m, 2H), 1.09 - 1.05 (m, 2H).

[0322] Step 2: To a solution of methyl l-allyl-4-(trifluoromethyl)cyclohexane-l-carboxylate (4.5 g, 18.0 mmol) in THF (40 mL) was added dropwise LAH (1.0 in THF, 18.0 mL, 18.0 mmol) under N2 at -60 °C. The resulting mixture was stirred at 0 °C for 1 h, diluted with THF (50 mL), quenched with Na2SO4·10H2O, and stirred at room temperature for 30 min. The resulting mixture was filtered, and the cake was washed with DCM (50 mL x 2). The combined filtrates were concentrated in vacuo to obtain the product (1 -allyl-4-(trifluoromethyl)cyclohexyl)methanol (3.8 g, 15.4 mmol, 86% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) 65.76 - 5.57 (m, 1H), 4.94 - 4.81 (m, 2H), 3.36 (s, 2H), 1.85 (d, J = 7.6 Hz, 2H), 1.81 - 1.71 (m, 1H), 1.56 (d, J= 11.4 Hz, 4H), 1.28 - 1.18 (m, 2H), 1.02 - 0.94 (m, 2H).

[0323] Step 3: To a solution of (l-allyl-4-(trifluoromethyl)cyclohexyl)methanol (3.8 g, 17.0 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (2.5 g, 10.1 mmol) in chloroform (80 mL) was added BF3·Et2O (1.3 mL, 10.1 mmol) at 0 °C under N2. The reaction mixture was stirred under nitrogen at 0 °C for 2 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l-allyl-4-(trifluoromethyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4.5 g, 9.5 mmol, 94% yield) as a yellow oil. LC purity: 90% (UV at 220 nm); Mass calculated for C24H32F3NO5 [M+1]+, 472.2, found 472.3; Retention time: 2.234 min.

[0324] Step 4: To a solution of methyl O-((l-allyl-4-(trifluoromethyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4.5 g, 9.5 mmol) in THF (30 mL) and water (30 mL) was added LiOH·H2O (456 mg, 19.0 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h, and the pH was adjusted to 4-5 with diluted HC1 solution (1 N). The mixture was diluted with H2O (20 mL) and extracted with DCM (40 mL x 3). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was concentrated under reduced pressure to obtain the product O-((l-allyl-4-(trifluoromethyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (4.3 g, 8.5 mmol,89% yield) as a white solid, which was used directly in the next step without further purification. LC purity: 90%; Mass calculated for C23H30F3NO5 [M+1]+, 458.2, found 458.3; Retention time: 2.043 min.

[0325] Step 5: To a solution of O-((l-allyl-4-(trifluoromethyl)cyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (4.3 g, 9.4 mmol) and DIEA (6.6 mL, 37.6 mmol) in DMF (40 mL) were added HATU (4.3 g, 11.3 mmol) and prop-2-en-l -amine (1.1 mL, 14.1 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 50% EtOAc in PE) to obtain the product benzyl ((2S,3R)-3-((l-allyl-4-(trifluoromethyl)cyclohexyl)methoxy)-l -(allylamino)- l-oxobutan-2-yl)carbamate (2.1 g, 3.4 mmol, 36% yield) as a brown solid. LC purity: 80% (UV at 220 nm); Mass calculated for C26H35F3N2O4 [M+1]+, 497.3, found 497.3; Retention time: 2.151 min.

[0326] Step 6: To a solution of benzyl ((2S,3R)-3-((l-allyl-4-(trifluoromethyl)cyclohexyl)methoxy)-l -(allylamino)- l-oxobutan-2-yl)carbamate (1.5 g, 3.0 mmol) in DCM (1.5 L) was added Grubbs 2ndgeneration catalyst (769 mg, 0.9 mmol). The reaction mixture was stirred under N2 at 60 °C for 24 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((9R,10S, Z)-9-methyl-l l-oxo-3-(trifluoromethyl)-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (300 mg, 0.58 mmol, 19% yield) as a brown solid. LC purity: 90% (UV at 220 nm); Mass calculated for C24H31F3N2O4 [M+1]+, 469.2, found 469.3; Retention time: 1.808 min.

[0327] Step 7: To a solution of benzyl ((9R,10S, Z)-9-methyl-ll-oxo-3-(trifluoromethyl)-8-oxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (150 mg, 0.32 mmol) in THF (10 mL) was added 10% Pd / C (50 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (50 mL). The filtrate was concentrated in vacuo to obtain the product (9R,10S)-10-amino-9-methyl-3-(trifluoromethyl)-8-oxa-12-azaspiro[5.10]hexadecan-ll-one (110 mg, 0.3 mmol, 92% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 90% (UV at 220 nm); Mass calculated for C16H27F3N2O2 [M+1]+, 337.2, found 337.2; Retention time: 1.008 min.

[0328] Step 8: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (60 mg, 0.17 mmol) and DIEA (0.12 mL, 0.7 mmol) in DMF (3 mL) were added HATU (75 mg, 0.2 mmol) and (9R, 1 OS)- 10-amino-9-methyl-3 -(trifluoromethyl)-8-oxa- 12-azaspiro[5.10]hexadecan- 11 -one (61 mg, 0.18 mmol) at 25 °C. The reaction mixture was stirred under N2 at 25 °C for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((9R,10S)-9-methyl-ll-oxo-3-(trifluoromethyl)-8-oxa-12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (50 mg, 0.07 mmol, 44% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C33H46F3N5O5S [M+1]+, 682.3, found 682.5; Retention time: 1.497 min;1H NMR (400 MHz, MeOD-d₄) δ 9.15 (s, 1H), 8.37 (d, J = 9.6 Hz, 1H), 7.79 (s, 1H), 4.45 - 4.33 (m, 2H), 4.32 - 4.17 (m, 2H), 4.13 - 4.06 (m, 3H), 4.02 - 3.85 (m, 4H), 3.57 - 3.37 (m, 3H), 3.05 - 3.00 (m, 1H), 2.08 - 1.95 (m, 2H), 1.85 - 1.76 (m, 1H), 1.70 - 1.60 (m, 4H), 1.54 - 1.36 (m, 4H), 1.32 - 1.28 (m, 1H), 1.25 - 1.22 (m, 2H), 1.21 - 1.08 (m, 9H), 1.04 - 0.96 (m, 2H), 0.83 - 0.74 (m, 2H).Example 25. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((10R,llR)-10-methyl-9,13-dioxaspiro[6.10]heptadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0329] Scheme 24Grubbs 2ndgeneration catalyst Pd(PPh3)4, toluene DCM

[0330] Step 1: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (6 g, 24.1 mmol) and (l-allylcycloheptyl)methanol (6 g, 36.1 mmol) in chloroform (50 mL) was added dropwise BFs Et2O (3.1 mL, 24.1 mmol) at 0 °C under N2 atmosphere. The resulting mixture was stirred at 25 °C for 2 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 30%EtOAc in PE) to obtain the product methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (7.5 g, 18 mmol, 75% yield) as a yellow oil. LC purity: 67% (UV at 220 nm); Mass calculated for C24H35NO5, [M+1]+, 418.3, found 418.2; Retention time: 2.377 min.

[0331] Step 2: To a solution of methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3 g, 7.2 mmol) in THF (40 mL) was added dropwise LAH (1 in THF, 7.2 mL, 7.2 mmol) at -78 °C under N2. The reaction mixture was stirred at -78 °C for 3 h, diluted with DCM (40 mL), quenched with Na2SO4IOH2O at -78 °C, and then stirred at room temperature for 30 min. The reaction mixture was filtered through a Celite® pad and the cake was washed with DCM (50 mL). The combined filtrateswere concentrated under reduced pressure and the remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((l-allylcycloheptyl)methoxy)-l-hydroxybutan-2-yl)carbamate (1 g, 2.6 mmol, 36% yield) asa yellow oil. LC purity: 90% (UV at 254 nm); Mass calculated for C22H35NO4 [M+1]+, 390.3, found 390.2; Retention time: 2.189 min.

[0332] Step 3: To a solution of benzyl ((2R, 3R)-3-((l -allylcy cl oheptyl)m ethoxy)- 1-hydroxybutan-2-yl)carbamate (1.0 g, 2.6 mmol) in toluene (20 mL) were added allyl tertbutyl carbonate (914 mg, 5.8 mmol) and Pd(PPh₃)4 (267 mg, 0.2 mmol). The resulting mixture was stirred under N2 at 110 °C for 12 h, cooled to room temperature and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 10 - 20% EtOAc in PE) to afford the product benzyl ((2R,3R)-3-((l-allylcycloheptyl)methoxy)-l -(allyloxy )butan-2-yl)carbamate (700 mg, 1.6 mmol, 71% yield) as a light yellow oil. LC purity: 52% (UV at 254 nm); Mass calculated for C26H39NO4 [M+1]+, 430.3, found 430.2; Retention time: 2.546 min.

[0333] Step 4: To a solution of benzyl ((2R, 3R)-3-((l -allylcy cl oheptyl)m ethoxy)- 1-(allyloxy)butan-2-yl)carbamate (700 mg, 1.6 mmol) in DCM (700 mL) was added Grubbs 2ndgeneration catalyst (692 mg, 0.8 mmol). The reaction mixture was stirred under N2 at 70 °C for 24 h. The volatiles were removed and the residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to afford the product benzyl ((10R,llR, Z)-10-methyl-9,13-dioxaspiro[6.10]heptadec-15-en-ll-yl)carbamate (200 mg, 0.5 mmol, 31% yield) as a brown oil. LC purity: 70% (UV at 254 nm); Mass calculated for C24H35NO4 [M+1]+, 402.2, found 402.3; Retention time: 2.464 min.

[0334] Step 5: To a solution of benzyl ((10R,l lR, Z)-10-methyl-9,13-dioxaspiro[6.10]heptadec-15-en-ll-yl)carbamate (200 mg, 0.5 mmol) in TFE (10 mL) was added 10% Pd / C (166 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at room temperature for 3 h, filtered through a Celite® pad and washed with MeOH (30 mL). The filtrate was concentrated under reduced pressure to give the crude product (10R,l 1R)-1O-methyl-9,13-dioxaspiro[6.10]heptadecan-ll-amine (106 mg, 0.4 mmol, 79% yield) as a brown oil. LC purity: 90% (UV at 254 nm); Mass calculated for C16H31NO2 [M+1]+, 270.2, found 270.1; Retention time: 1.349 min.

[0335] Step 6: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (27 mg, 0.07 mmol) and (10R,l lR)-10-methyl-9,13-dioxaspiro[6.10]heptadecan-l 1-amine (30 mg, 0.3 mmol) in DMF (3 mL) were added DIEA (19 mg, 0.03 mmol) and HATU (42 mg, 0.1 mmol). The reaction mixture was stirred at 25 °C for 1 h, quenched with water (10 mL) and extractedwith EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((10R,llR)-10-methyl-9,13-dioxaspiro[6.10]heptadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (15 mg, 0.02 mmol, 32% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C33H50N4O5S [M+1]+, 615.4, found 615.4; Retention time: 1.966 min;1H NMR (400 MHz, MeOD-d₄) δ 9.15 (s, 1H), 8.36 (d, J = 14.6 Hz, 1H), 4.46 - 4.20 (m, 3H), 4.17 - 4.05 (m, 3H), 4.01 - 3.90 (m, 2H), 3.87 - 3.82 (m, 2H), 3.78 - 3.69 (m, 1H), 3.62 - 3.56 (m, 1H), 3.52 - 3.41 (m, 3H), 3.39 - 3.33 (m, 1H), 2.94 (d, J= 9.6 Hz, 1H), 1.92 -1.82 (m, 1H), 1.80 - 1.76 (m, 1H), 1.72 - 1.61 (m, 2H), 1.55 - 1.48 (m, 4H), 1.42 - 1.28 (m, 9H), 1.23 - 1.11 (m, 10H), 1.07 - 1.01 (m, 1H), 0.98 - 0.90 (m, 1H), 0.80 - 0.74 (m, 1H).Example 26. (S)-N-((9R,10R)-3.,3-Difluoro-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-yl)-2-((S)-2,2-dimethylcycloDroDane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0336] Scheme 25

[0337] Step 1: To a stirred suspension of methyl 4,4-difluorocyclohexane-l -carboxylate (5 g, 28.1 mmol) in THF (100 mL) was added dropwise LDA (2 in THF, 27.4 mL, 54.8 mmol) at -65 °C. The resulting mixture was stirred at this temperature for 0.5 h under N2before 3 -bromoprop- 1-ene (3.6 mL, 42.1 mmol) was added. The resulting mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (60 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl l-allyl-4,4-difluorocyclohexane-l -carboxylate (5 g, 20.6 mmol, 74% yield) as a yellow oil.1H NMR (400 MHz, CDCI3) 65.72 - 5.61 (m, 1H), 5.10 - 5.00 (m, 2H), 3.70 (s, 3H), 2.27 (d, J= 7.6 Hz, 2H), 2.22 - 2.17 (m, 2H), 2.04 - 1.95 (m, 2H), 1.89 - 1.69 (m, 2H), 1.57 - 1.49 (m, 2H).

[0338] Step 2: To a stirred suspension of methyl l-allyl-4,4-difluorocyclohexane-l-carboxylate (5 g, 22.9 mmol) in THF (50 mL) was added dropwise LAH (1 in THF, 23 mL, 22.9 mmol) at -65 °C under N2. The resulting mixture was stirred under N2 for 2 h, quenched with diluted HC1 solution (1 N, 40 mL) and extracted with EtOAc (40 mL x 3). The combined organic phases were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product (l-allyl-4,4-difluorocyclohexyl)methanol (4.1 g, 19.4 mmol, 85% yield) as a yellow oil.1H NMR (400 MHz, CDCI3) 85.89 - 5.78 (m, 1H), 5.18 - 5.05 (m, 2H), 3.46 (s, 2H), 2.16 - 2.14 (m, 2H), 1.96 - 1.84 (m, 4H), 1.60 - 1.49 (m, 4H).

[0339] Step 3: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (3.6 g, 14.4 mmol) and (l-allyl-4,4-difluorocyclohexyl)methanol (4.1 g, 21.6 mmol) in chloroform (10 mL) was added dropwise BF3·Et2O (1.8 mL, 14.4 mmol) at 0 °C under N2 atmosphere. The resulting mixture was stirred at 25 °C for 2 h. The volatiles were removed and the residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4.5 g, 7.5 mmol, 52% yield) as a yellow oil. LC purity: 78% (UV at 220 nm); Mass calculated for C23H31F2NO5, [M+1]+, 440.2, found 440.2;Retention time: 1.919 min.

[0340] Step 4: To a stirred suspension of methyl O-((l-allyl-4,4-difluorocyclohexyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4.5 g, 10.2 mmol) in THF (100 mL) was added LAH (1 in THF, 10 mL, 10.2 mmol) at -65 °C under N2. The resulting mixture was stirred under N2 for 2 h, quenched with diluted HC1 solution (1 N) (40mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((l-allyl-4,4-difluorocyclohexyl)methoxy)-l-hydroxybutan-2-yl)carbamate (2.1 g, 3.8 mmol, 37% yield) as a yellow oil. LC purity: 75% (UV at 254 nm); Mass calculated for C22H31F2NO4, [M+1]+, 412.2, found 412.1; Retention time: 1.799 min.

[0341] Step 5: To a solution of benzyl ((2R,3R)-3-((l-allyl-4,4-difluorocyclohexyl)methoxy)-l-hydroxybutan-2-yl)carbamate (1.0 g, 2.4 mmol) in toluene (20 mL) were added allyl tert-butyl carbonate (961 mg, 6.1 mmol) and Pd(PPh₃)4 (281 mg, 0.3 mmol). The resulting mixture was stirred under N2 at 110 °C for 12 h, cooled to room temperature and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((l-allyl-4,4-difluorocy cl ohexyl)methoxy)-l -(allyloxy )butan-2-yl)carbamate (600 mg, 0.7 mmol, 27% yield) as a light yellow oil. LC purity: 50% (UV at 254 nm); Mass calculated for C25H35F2NO4, [M+1]+, 452.3, found 452.2; Retention time: 2.088 min.

[0342] Step 6: To a solution of benzyl ((2R,3R)-3-((l-allyl-4,4-difluorocyclohexyl)methoxy)-l -(allyloxy )butan-2-yl)carbamate (600 mg, 1.3 mmol) in DCM (600 mL) was added Grubbs 2ndgeneration catalyst (564 mg, 0.7 mmol). The resulting mixture was stirred under N2 atmosphere at 60 °C for 16 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product benzyl ((9R,10R, Z)-3,3-difluoro-9-methyl-8,12-dioxaspiro[5.10]hexadec-14-en-10-yl)carbamate (170 mg, 0.4 mmol, 28% yield) as a black oil. LC purity: 92% (UV at 220 nm); Mass calculated for C23H31F2NO4, [M+1]+, 424.2, found 424.4; Retention time: 1.836 min.

[0343] Step 7: To a solution of benzyl ((9R,10R, Z)-3,3-difluoro-9-methyl-8,12-dioxaspiro[5.10]hexadec-14-en-10-yl)carbamate (50 mg, 0.1 mmol) in TFE (10 mL) was added 10% Pd / C (50 mg). The resulting mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to afford the product (9R,10R)-3,3-difluoro-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-amine (37 mg, 0.1 mmol, 86% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220nm); Mass calculated for C15H27F2NO2, [M+1]+, 292.2, found 292.2; Retention time: 0.868 min.

[0344] Step 8: To a solution of (9R,10R)-3,3-difluoro-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-amine (37 mg, 0.1 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (30 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (0.04 mL, 0.2 mmol) and HATU (45 mg, 0.1 mmol). The reaction mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4Cl solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The remaining residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-N-((9R,10R)-3,3-difluoro-9-methyl-8,12-dioxaspiro[5.10]hexadecan-10-yl)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (21 mg, 0.03 mmol, 40% yield) as a gray solid. LC purity: 99% (UV at 254 nm); Mass calculated for C32H46F2N4O5S, [M+1]+, 637.3, found 637.2; Retention time: 1.609 min;1H NMR (400 MHz, MeOD-4) δ 9.16 (s, 1H), 8.37 (d, J = 14.4 Hz, 1H), 4.41 - 4.32 (m, 1H), 4.30 - 4.18 (m, 2H), 4.17 - 4.11 (m, 1H), 4.10 - 4.03 (m, 2H), 4.01 - 3.89 (m, 2H), 3.87 -3.78 (m, 2H), 3.74 - 3.65 (m, 1H), 3.63 - 3.56 (m, 1H), 3.55 - 3.45 (m, 3H), 3.43 - 3.33 (m, 1H), 3.22 (d, J= 9.6 Hz, 1H), 1.93 - 1.76 (m, 5H), 1.71 - 1.59 (m, 3H), 1.47 - 1.39 (m, 4H), 1.37 - 1.26 (m, 3H), 1.19 (s, 1H), 1.17 - 1.13 (m, 4H), 1.11 (s, 4H), 1.06 - 1.01 (m, 1H), 0.82 -0.74 (m, 1H).Example 27. (S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((2R,3lR,4lR)-4l-methyl-ll,5l-dioxasDiro[bicyclo[2.2.2]octane-2,7l-cycloundecan]-3l-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 1Example 28. (S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((2R,3lR,4lR)-4l-methyl-ll,5l-dioxaspiro[bicvclo[2.2.2]octane-2,7l-cvcloundecan]-3l-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 2

[0345] Scheme 26

[0346] Step 1: To a solution of methyl O-((2-allylbicyclo[2.2.2]octan-2-yl)methyl)-N- ((benzyloxy)carbonyl)-L-threoninate (1.2 g, 2.8 mmol) in THF (20 mL) was added dropwise LAH (1 in THF, 2.8 mL, 2.8 mmol) at -78 °C under N2. The resulting mixture was stirred at 0 °C for 1 h, quenched with Na₂SO₄·10H₂O, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.2]octan-2-yl)methoxy)-l-hydroxybutan-2-yl)carbamate (900 mg, 2.2 mmol, 78% yield) as a yellow oil. LC purity: 90 % (UV at 254 nm); Mass calculated for C24H35NO4, [M+1]+, 402.3, found 402.1; Retention time: 2.196 min.

[0347] Step 2: To a solution of benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.2]octan-2-yl)methoxy)-l-hydroxybutan-2-yl)carbamate (900 mg, 2.2 mmol) in toluene (20 mL) were added allyl tert-butyl carbonate (886 mg, 5.6 mmol) and Pd(PPh₃)4 (259 mg, 0.22 mmol). The resulting mixture was stirred under N2 at 110 °C for 12 h, cooled to room temperature and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.2]octan-2-yl)methoxy)-l -(allyloxy )butan-2-yl)carbamate (200 mg, 0.45 mmol, 21% yield) as a yellow oil. LC purity: 65 % (UV at 220 nm); Mass calculated for C27H39NO4 [M+1]+, 442.3, found 442.2; Retention time: 2.234 min.

[0348] Step 3: To a solution of benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.2]octan-2-yl)methoxy)-l -(allyloxy )butan -2 -yl)carbamate (200 mg, 0.45 mmol) in DCM (200 mL) was added Grubbs 2ndgeneration catalyst (115 mg, 0.136 mmol). The reaction mixture was stirred under N2 at 60 °C for 16 h. The volatiles were removed and the residue was purified by flash chromatography (silica gel, 0 - 25% EtOAc in PE) to obtain the product benzyl ((3'R,4'R, Z)-4'-methyl-r,5'-dioxaspiro[bicyclo[2.2.2]octane-2,7'-cycloundecan]-9'-en-3'-yl)carbamate (60 mg, 0.12 mmol, 26% yield) as a brown solid. LC purity: 80% (UV at 254 nm); Mass calculated for C25H35NO4 [M+1]+, 414.3, found 414.2; Retention time: 1.882 min.

[0349] Step 4: To a solution of benzyl ((3'R,4'R, Z)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.2]octane-2,7'-cycloundecan]-9'-en-3'-yl)carbamate (60 mg, 0.15 mmol) in TFE (5 mL) was added 10% Pd / C (20 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at 25 °C for 16 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to afford the product (3'R,4'R)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.2]octane-2,7'-cycloundecan]-3'-amine (40 mg, 0.14 mmol, 93% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 80 % (UV at 200 nm); Mass calculated for C17H31NO2 [M+1]+, 282.2, found 282.2; Retention time: 1.345 min.

[0350] Step 5: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (40 mg, 0.11 mmol), (3'R,4'R)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.2]octane-2,7'-cycloundecan]-3'-amine (40 mg, 0.14 mmol), and DIEA (0.06 mL, 0.33 mmol) in DMF (3 mL) was added HATU (53 mg, 0.14 mmol) at 0 °C. The resulting mixture was stirred for 2 h, quenched with saturated aqueous NH4CI solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((2R,3'R,4'R)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.2]octane-2,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Example 27, Isomer 1, 5.0 mg, 0.008 mmol, 7% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C34H50N4O5S [M+1]+, 627.4, found 627.2; Retention time: 1.914 min;1H NMR (400 MHz, MeOD-4) δ 9.15 (s, 1H), 8.36 (d, J = 14.4 Hz, 1H), 7.94 - 7.59 (m, 1H), 4.40 - 4.22 (m, 2H), 4.15 - 3.96 (m, 5H), 3.86 - 3.64 (m, 5H), 3.59 - 3.45 (m, 4H), 3.06 (d, J= 9.6 Hz, 1H), 1.94 - 1.82 (m, 2H), 1.59 -1.35 (m, 17H), 1.18 - 1.10 (m, 9H), 1.08 - 1.00 (m, 1H), 0.81 - 0.77 (m, 1H); and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((2S,3'R,4'R)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.2]octane-2,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Example 28, Isomer 2, 5.1 mg, 0.008 mmol, 7% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C34H50N4O5S [M+1]+, 627.4, found 627.2; Retention time: 1.940 min;1H NMR (400 MHz, MeOD-4) δ 9.15 (s, 1H), 8.36 (d, J = 14.0 Hz, 1H), 8.02 - 7.47 (m, 1H), 4.37 - 4.22 (m, 2H), 4.18 - 3.94 (m, 5H), 3.87 - 3.65 (m, 5H), 3.61 - 3.42 (m, 4H), 3.06 (d, J= 9.6 Hz, 1H), 1.92 - 1.83 (m, 2H), 1.61 -1.35 (m, 17H), 1.19 - 1.10 (m, 9H), 1.07 - 1.00 (m, 1H), 0.81 - 0.77 (m, 1H).Example 29. 2.,2-Dimethylcvclopropane-l-carbonyl)-N-((2R.,3lR.,4lR)-4l-methyl-r.,5l-dioxaspiro[bicvclo[2.2.1]heptane-2.,7l-cvcloundecan]-3l-yr)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 1Example 30. -2-((S)-2.,2-Dimethylcvclopropane-l-carbonyl)-N-((2R.,3lR.,4lR)-4l-methyl-r.,5l-dioxasDiro[bicyclo[2.2.1]heDtane-2.,7l-cycloundecan]-3l-yr)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide isomer 2

[0351] Scheme 27

[0352] Step 1: To a solution of methyl (2S,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-2-(((benzyloxy)carbonyl)amino)butanoate (2.5 g, 6.0 mmol) in THF (30 mL) was added dropwise LAH (1 in THF, 2.8 mL, 2.8 mmol) at -65 °C. The resulting mixture was stirred at 0 °C for 1 h, quenched with Na₂SO₄·10H₂O, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 -30% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-l-hydroxybutan-2-yl)carbamate (1.1 g, 2.6 mmol, 42% yield) as colorless oil. LC purity: 70% (UV at 200 nm); Mass calculated for C23H33NO4 [M+1]+, 388.2, found 388.2; Retention time: 2.074 min.

[0353] Step 2: To a solution of benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-l-hydroxybutan-2-yl)carbamate (1.1 g, 2.8 mmol) in toluene (15 mL) were added allyl tert-butyl carbonate (449 mg, 2.8 mmol) and Pd(PPh3)4 (328 mg, 0.28 mmol) at room temperature. The resulting mixture was stirred under N2 at 110 °C for 12 h, cooled to room temperature, quenched with H₂O (20 mL), and extracted with EtOAc (20 mL x 2). The combined organic layers were washed with brine (15 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-l-(allyloxy)butan-2-yl)carbamate (700 mg, 1.5 mmol, 52% yield) as a yellow oil. LC purity: 90% (UV at 254 nm); Mass calculated for C26H37NO4 [M+1]+, 428.3, found 428.3; Retention time: 2.722 min.

[0354] Step 3: To a solution of benzyl ((2R,3R)-3-((2-allylbicyclo[2.2.1]heptan-2-yl)methoxy)-l -(allyloxy )butan -2 -yl)carbamate (500 mg, 1.2 mmol) in DCM (500 mL) was added Grubbs 2ndgeneration catalyst (497 mg, 0.6 mmol) at room temperature under N2. The resulting mixture was stirred under N2 at 65 °C for 12 h, cooled to room temperature and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((3'R,4'R, Z)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.1]heptane-2,7'-cycloundecan]-9'-en-3'-yl)carbamate (136 mg, 0.31 mmol, 26% yield) as a yellow oil. LC purity: 80% (UV at 220 nm); Mass calculated for C24H33NO4 [M+1]+, 400.2, found 400.3; Retention time: 2.538 min.

[0355] Step 4: To a solution of benzyl ((3'R,4'R, Z)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.1]heptane-2,7'-cycloundecan]-9'-en-3'-yl)carbamate (80 mg, 0.2 mmol) in TFE (5 mL) was added 10% Pd / C (20 mg). The resulting mixture was stirred underH2atmosphere (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to afford the product (3'R,4'R)-4'-methyl-T,5'-dioxaspiro[bicyclo[2.2.1]heptane-2,7'-cycloundecan]-3'-amine (50 mg, 0.15 mmol, 75% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 70% (UV at 200 nm); Mass calculated for C16H29NO2 [M+1]+, 268.2, found 268.1; Retention time: 2.462 min.

[0356] Step 5: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (61 mg, 0.17 mmol) and HATU (70 mg, 0.19 mmol) in DMF (3 mL) were added DIE A (43 mg, 0.34 mmol) and (3'R,4'R)-4'-methyl-T,5'-dioxaspiro[bicyclo[2.2.1]heptane-2,7'-cycloundecan]-3'-amine (45 mg, 0.17 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature, stirred for 12 h, quenched with saturated aqueous NH4CI solution (5 mL) and extracted with EtOAc (5 mL x 3). The combined organic phases were washed with brine (10 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (8S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((2R,3'R,4'R)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.1]heptane-2,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Isomer 1, 6.7 mg, 0.01 mmol, 6% yield) as a white solid. LC purity: 98.74% (UV at 254 nm); Mass calculated for C33H48N4O5S [M+1]+, 613.3, found 613.3; Retention time: 1.934 min;1H NMR (400 MHz, MeOD-4) 89.16 (s, 1H), 8.36 (d, J= 14.2 Hz, 1H), 8.05 - 7.85 (m, 1H), 4.44 - 4.18 (m, 3H), 4.16 - 3.93 (m, 5H), 3.86 -3.78 (m, 2H), 3.75 - 3.67 (m, 1H), 3.59 - 3.46 (m, 4H), 3.38 - 3.32 (m, 2H), 2.20 - 2.16 (m, 1H), 2.09 - 2.04 (m, 1H), 1.78 - 1.71 (m, 1H), 1.66 - 1.58 (m, 2H), 1.56 - 1.50 (m, 1H), 1.47 -1.36 (m, 5H), 1.34 - 1.26 (m, 3H), 1.18 - 1.10 (m, 12H), 1.06 - 1.02 (m, 1H), 0.82 - 0.75 (m, 1H); and the product (8S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((2S,3'R,4'R)-4'-methyl-l',5'-dioxaspiro[bicyclo[2.2.1]heptane-2,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (Isomer 2, 17 mg, 0.03 mmol, 16% yield) as a white solid. LC purity: 99.71% (UV at 220 nm); Mass calculated for C33H48N4O5S [M+1]+, 613.3, found 613.3; Retention time: 1.972 min;1H NMR (400 MHz, MeOD-4) 6 9.16 (s, 1H), 8.37 (d, J= 13.4 Hz, 1H), 7.98 - 7.69 (m, 1H), 4.45 - 4.18 (m, 3H), 4.17 - 3.92 (m, 5H), 3.88 - 3.83 (m, 1H), 3.79 - 3.71 (m, 1H), 3.66 - 3.58 (m, 1H), 3.56 - 3.46 (m, 4H), 3.44 - 3.32 (m, 2H), 2.16 - 2.10 (m, 1H), 1.98 - 1.92 (m, 1H), 1.87 - 1.76 (m, 1H), 1.74 - 1.65(m, 2H), 1.61 (d, J= 9.6 Hz, 1H), 1.54 - 1.42 (m, 3H), 1.41 - 1.29 (m, 4H), 1.27 - 1.19 (m, 2H), 1.18 - 1.10 (m, 9H), 1.07 - 1.00 (m, 2H), 0.81 - 0.75 (m, 1H), 0.54 (d, J= 12.2 Hz, 1H).Example 31. (8S)-2-((S)-2,2-Dimethylcyclopropane-1-carbonyl)-N-((3'R,4'R)-4'-methyl-1',5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide

[0357] Scheme 28

[0358] Step 1: To a solution of 8-oxabicyclo[3.2. l]octan-3-one (8 g, 63.4 mmol) and p-toluenesulfonylmethyl isocyanide (TosMIC) (16.1 g, 82.4 mmol) in DME (240 mL) and ethanol (6.3 mL) was added t-BuOK (17.1 mg, 152 mmol) at 0 °C. The resulting mixture was stirred under N2 at room temperature for 12 h, diluted with H2O (100 mL) and extracted with MTBE (100 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to afford the crude product 8-oxabicyclo[3.2.1]octane-3-carbonitrile (8.7 g, 63.4 mmol, 77% yield), which was used directly in the next step without further purification.

[0359] Step 2: To a mixture of 8-oxabicyclo[3.2. l]octane-3-carbonitrile (8.7 g, 63.4 mmol) and acetic acid (40 mL) was added concentrated HC1 (40 mL) at 0 °C. After being stirred atroom temperature for 30 min, the reaction mixture was stirred at 100 °C for 4 h and cooled to room temperature. The mixture was poured into ice water (100 mL) and extracted with DCM (100 mL x 3). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product 8-oxabicyclo[3.2.1]octane-3-carboxylic acid (4 g, 25.6 mmol, 40% yield) as a black oil. The crude product was used directly in the next step without further purification.1H NMR (400 MHz, DMSO-6) 64.34 - 4.25 (m, 2H), 2.74 - 2.59 (m, 1H), 1.83 - 1.75 (m, 2H), 1.74 - 1.68 (m, 2H), 1.68 - 1.55 (m, 4H).

[0360] Step 3: To a solution of 8-oxabicyclo[3.2.1]octane-3-carboxylic acid (4.0 g, 25.6 mmol) in methanol (50 mL) was added concentrated H2SO4 (0.2 mL, 3.7 mmol). The reaction mixture was stirred at 60 °C under N2 for 8 h, cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 15% EtOAc in PE) to obtain the product methyl 8-oxabicyclo[3.2.1]octane-3-carboxylate (2.5 g, 14.7 mmol, 57% yield) as a yellow oil.1H NMR (400 MHz, DMSO-d₆) 6 4.35 - 4.22 (m, 2H), 3.57 (s, 3H), 2.82 - 2.71 (m, 1H), 1.84 - 1.74 (m, 2H), 1.74 - 1.67 (m, 2H), 1.67 - 1.55 (m, 4H).

[0361] Step 4: To a solution of methyl 8-oxabicyclo[3.2.1]octane-3-carboxylate (2.5 g, 14.7 mmol) in THF (25 mL) was added dropwise LDA (2.0 M in THF, 12 mL, 22.0 mmol) at -65 °C under N2. The resulting mixture was stirred at this temperature for 0.5 h under N2 before 3 -bromoprop- 1-ene (1.9 mL, 22.0 mmol) was added. The resulting mixture was stirred at 25 °C for 3 h, quenched with saturated aqueous NH4CI solution (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 3-allyl-8-oxabicyclo[3.2.1]octane-3-carboxylate (1.9 g, 9.4 mmol, 63% yield) as a yellow oil.1H NMR (400 MHz, DMSO-6) 65.69 - 5.48 (m, 1H), 5.07 - 4.90 (m, 2H), 4.31 - 4.15 (m, 2H), 3.63 (d, J= 0.8 Hz, 3H), 2.21 (d, J= 13.8 Hz, 2H), 2.13 (d, J= 7.4 Hz, 2H), 1.75 - 1.65 (m, 2H), 1.63 - 1.53 (m, 4H).

[0362] Step 5: To a solution of methyl 3-allyl-8-oxabicyclo[3.2.1]octane-3-carboxylate (1.9 g, 9.4 mmol) in THF (20 mL) was added dropwise LAH (1 M in THF, 9.4 mL, 9.4 mmol) at -65 °C under N2. The resulting mixture was stirred under N2 for 2 h, quenched with diluted HC1 solution (1 A, 15 mL) and extracted with EtOAc (20 mL x 3). The combined organiclayers were washed with brine (20 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product (3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methanol (1.7 g, 9.3 mmol, 99% yield) as a yellow oil.1H NMR (400 MHz, DMSO-6) 65.82 - 5.68 (m, 1H), 5.09 - 4.94 (m, 2H), 4.66 (t, J= 5.2 Hz, 1H), 4.29 - 4.14 (m, 2H), 3.28 (d, J= 5.2 Hz, 2H), 1.91 (d, J= 7.4 Hz, 2H), 1.80 - 1.72 (m, 2H), 1.68 (dd, J= 14.2, 6.4 Hz, 2H), 1.50 (dd, J = 14.2, 4.8 Hz, 2H), 1.41 (d, J= 14.2 Hz, 2H).

[0363] Step 6: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (1.6 g, 6.2 mmol) and (3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methanol (1.7 g, 9.3 mmol) in chloroform (60 mL) was added dropwise BF3·Et2O (0.8 mL, 6.2 mmol) under N2 at 0 °C atmosphere. The resulting mixture was stirred at 0 °C for 2 h, quenched with saturated aqueous NaHCCl4solution (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 -30% EtOAc in PE) to obtain the product methyl (2S,3R)-3-((3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methoxy)-2-(((benzyloxy)carbonyl)amino)butanoate (1.6 g, 3.7 mmol, 59% yield) as a light yellow oil. LC purity: 70% (UV at 220 nm); Mass calculated for C24H33NO6 [M+1]+, 432.2, found 432.2; Retention time: 1.705 min.

[0364] Step 7: To a solution of methyl (2S,3R)-3-((3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methoxy)-2-(((benzyloxy)carbonyl)amino)butanoate (1.6 g, 3.8 mmol) in THF (20 mL) was added dropwise LAH (1 M in THF, 3.8 mL, 3.8 mmol) at -65 °C under N2. The resulting mixture was stirred under N2 for 2 h, quenched with diluted HC1 solution (1 N, 10 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 50% EtOAc in PE) to obtain the product benzyl ((2R,3R)-3-((3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methoxy)-l-hydroxybutan-2-yl)carbamate (550 mg, 1.3 mmol, 36% yield) as a colorless oil. LC purity: 92% (UV at 220 nm); Mass calculated for C23H33NO5 [M+1]+, 404.2, found 404.2; Retention time: 1.440 min.

[0365] Step 8: To a mixture of benzyl ((2R,3R)-3-((3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methoxy)-l-hydroxybutan-2-yl)carbamate (550 mg, 1.3 mmol) and allyl tert-butyl carbonate (539 mg, 3.4 mmol) in toluene (20 mL) was added Pd(PPh3)4 (158 mg, 0.1 mmol).The reaction mixture was stirred under N2 at 110 °C for 4 h. The volatiles were removed and the residue was purified by flash chromatography (silica gel, 10 - 30% EtOAc in PE) to afford the product benzyl ((2R,3R)-3-((3-allyl-8-oxabicyclo[3.2. l]octan-3-yl)m ethoxy)- 1-(allyloxy)butan-2-yl)carbamate (100 mg, 0.2 mmol, 16% yield) as a colorless oil. LC purity: 78% (UV at 220 nm); Mass calculated for C26H37NO5 [M+1]+, 444.3, found 444.2; Retention time: 1.894 min.

[0366] Step 9: To a solution of benzyl ((2R,3R)-3-((3-allyl-8-oxabicyclo[3.2.1]octan-3-yl)methoxy)-l -(allyloxy )butan -2 -yl)carbamate (100 mg, 0.2 mmol) in DCM (100 mL) was added Grubbs 2ndgeneration catalyst (96 mg, 0.1 mmol). The reaction mixture was stirred under N2 at 60 °C for 12 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 10 - 30% EtOAc in PE) to afford the product benzyl ((3'R,4'R, Z)-4'-methyl-l',5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-9'-en-3'-yl)carbamate (45 mg, 0.1 mmol, 48% yield) as a brown oil. LC purity: 77% (UV at 220 nm); Mass calculated for C24H33NO5 [M+1]+, 416.2, found 416.2; Retention time: 1.609 min.

[0367] Step 10: To a solution of benzyl ((3'R,4'R, Z)-4'-methyl-l',5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-9'-en-3'-yl)carbamate (45 mg, 0.1 mmol) in 2,2,2-trifluoroethanol (5 mL) was added 10% Pd / C (20 mg). The reaction mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to obtain the product (3'R,4'R)-4'-methyl-l',5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-3'-amine (29 mg, 0.1 mmol, 94% yield) as a light yellow oil. The crude product was used directly in the next step without further purification. LC purity: 50% (UV at 220 nm); Mass calculated for C16H29NO3 [M+1]+, 284.2, found 284.1; Retention time: 0.448 min.

[0368] Step 11: To a mixture of (3'R,4'R)-4'-methyl-l',5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-3'-amine (29 mg, 0.1 mmol) and (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (30 mg, 0.1 mmol) in DMF (3 mL) were added DIEA (40 mg, 0.3 mmol) and HATU (47 mg, 0.2 mmol). The resulting mixture was stirred at room temperature for 1 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (8S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-N-((3'R,4'R)-4'-methyl-T,5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (3.5 mg, 0.01 mmol, 5% yield) as a white solid. LC purity: 100% (UV at 220 nm); Mass calculated for C33H48N4O6S [M+1]+, 629.3, found 629.5; Retention time: 1.211 min;1H NMR (400 MHz, MeOD-d4) δ 9.16 (s, 1H), 8.37 (d, J = 14.6 Hz, 1H), 4.41 - 4.04 (m, 8H), 4.02 - 3.89 (m, 3H), 3.88 - 3.81 (m, 2H), 3.69 - 3.61 (m, 1H), 3.60 - 3.53 (m, 1H), 3.51 - 3.34 (m, 3H), 3.18 - 3.12 (m, 1H), 2.11 - 2.03 (m, 1H), 1.97 - 1.86 (m, 3H), 1.86 - 1.66 (m, 4H), 1.47 - 1.29 (m, 6H), 1.19 - 1.09 (m, 9H), 1.07 - 1.00 (m, 1H), 0.91 - 0.76 (m, 2H).Example 32. (S)-2-((S)-2,2-Dimethylcyclopropane-1-carbonyl)-N-((9R,10S)-9-methyl-11-oxo-3,8-dioxa-12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide

[0369] Scheme 29

[0370] Step 1: To a solution of methyl tetrahydro-2H-pyran-4-carboxylate (6 g, 41.6 mmol) in THF (50 mL) was added dropwise NaHMDS (1 M in THF, 62 mL, 62.4 mmol) at -50 °C under nitrogen. The mixture solution was stirred at 50 °C for 30 min under nitrogen before 3 -bromoprop- 1-ene (4.5 mL, 52.0 mmol) was added. The resulting mixture was stirred at 50 °C for 2 h, cooled to room temperature, quenched with saturated aqueous NH4CI solution (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were dried over anhydrous MgSO4, filtered and evaporated in vacuo. The resulting residue waspurified by flash chromatography (silica gel, 10 - 20% EtOAc in PE) to obtain the product methyl 4-allyltetrahydro-2H-pyran-4-carboxylate (5.5 g, 29.9 mmol, 72% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C10H16O3, [M+1]+, 185.1, found 185.2; Retention time: 1.339 min;1H NMR. (400 MHz, DMSO-ifc) 85.70 - 5.57 (m, 1H), 5.08 - 4.99 (m, 2H), 3.75 - 3.67 (m, 2H), 3.62 (s, 3H), 3.31 - 3.25 (m, 2H), 2.25 (d, J = 7.6 Hz, 2H), 1.93 - 1.86 (m, 2H), 1.50 - 1.40 (m, 2H).

[0371] Step 2: To a solution of methyl 4-allyltetrahydro-2H-pyran-4-carboxylate (5.5 g, 29.9 mmol) in THF (60 mL) was added dropwise LAH (1 in THF, 36 mL, 35.8 mmol) at -78 °C under nitrogen. The mixture solution was stirred at -78 °C for 2 h, quenched with diluted HC1 solution (1 TV, 30 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product (4-allyltetrahydro-2H-pyran-4-yl)m ethanol (4.2 g, 26.9 mmol, 90% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C9H16O2, [M+1]+, 157.3, found 157.3; Retention time: 0.830 min;1H NMR (400 MHz, DMSO-6) 65.88 - 5.72 (m, 1H), 5.08 - 5.00 (m, 2H), 4.51 (t, J= 5.4 Hz, 1H), 3.54 (t, J= 5.8 Hz, 4H), 3.25 (d, J= 5.4 Hz, 2H), 2.12 (d, J= 7.6 Hz, 2H), 1.44 - 1.33 (m, 2H), 1.31 - 1.23 (m, 2H).

[0372] Step 3: To a solution of 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (2.2 g, 8.8 mmol) and (4-allyltetrahydro-2H-pyran-4-yl)methanol (2.1 g, 13.2 mmol) in chloroform (40 mL) was added BF3·Et2O (1.1 mL, 8.8 mmol) undernitrogen at 0 °C. The mixture solution was stirred at 0 °C for 2 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((4-allyltetrahydro-2H-pyran-4-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3.1 g, 7.7 mmol, 87% yield) as a colorless oil. LC purity: 97% (UV at 220 nm); Mass calculated for C22H31NO6, [M+1]+, 406.2, found 406.2; Retention time: 1.678 min.

[0373] Step 4: To a solution of methyl O-((4-allyltetrahydro-2H-pyran-4-yl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (3.1 g, 7.7 mmol) in THF (15 mL) and water (15 mL) was added LiOH·H2O (643 mg, 15.3 mmol). The mixture solution was stirred at 25 °C for 5 h. The pH of the mixture was adjusted to ~3 with HC1 solution (1 TV, 15 mL), and the mixture was extracted with EtOAc (30 mL x 2). The combined organic layers were dried overanhydrous MgSO4and filtered. The filtrate was evaporated in vacuo to obtain the product O-((4-allyltetrahydro-2H-pyran-4-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.2 g, 3.1 mmol, 40% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 95% (UV at 220 nm); Mass calculated for C21H29NO6, [M+1]+, 392.2, found 392.4; Retention time: 1.531 min.

[0374] Step 5: To a solution of O-((4-allyltetrahydro-2H-pyran-4-yl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (1.2 g, 3.1 mmol) in DMF (15 mL) were added HATU (1.4 g, 3.7 mmol) and DIEA (1.6 mL, 9.2 mmol). The mixture was stirred at 25 °C for 5 min before prop-2-en-l -amine (0.23 mL, 3.1 mmol) was added. The resulting mixture was stirred at 25 °C for 1 h, diluted with water (20 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were dried over anhydrous MgSO4, filtered, and evaporated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((2S,3R)-l-(allylamino)-3-((4-allyltetrahydro-2H-pyran-4-yl)methoxy)-l-oxobutan-2-yl)carbamate (1.2 g, 2.8 mmol, 91% yield) as a colorless oil. LC purity: 95% (UV at 220 nm); Mass calculated for C24H34N2O5, [M+1]+, 431.3, found 431.3; Retention time: 1.520 min.

[0375] Step 6: To a solution of benzyl ((2S,3R)-l-(allylamino)-3-((4-allyltetrahydro-2H-pyran-4-yl)methoxy)-l-oxobutan-2-yl)carbamate (1.2 g, 2.8 mmol) in DCM (1.2 L) was added Grubbs 2ndgeneration catalyst (0.95 g, 1.1 mmol). The mixture solution was stirred under nitrogen at 70 °C for 12 h. The volatiles were removed and the remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product benzyl ((9R,10S, Z)-9-methyl-ll-oxo-3,8-dioxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (300 mg, 0.77 mmol, 27% yield) as a black oil. LC purity: 90% (UV at 220 nm); Mass calculated for C22H30N2O5, [M+1]+, 403.2, found 403.3; Retention time: 1.256 min.

[0376] Step 7: To a solution of benzyl ((9R,10S, Z)-9-methyl-l l-oxo-3,8-dioxa-12-azaspiro[5.10]hexadec-14-en-10-yl)carbamate (300 mg, 0.75 mmol) in TFE (20 mL) was added 10% Pd / C (50 mg). The mixture was stirred under H2 (1 atm) at 25 °C for 12 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to obtain the product (9R,10S)-10-amino-9-methyl-3,8-dioxa-12-azaspiro[5.10]hexadecan-l l-one (200 mg, 0.74 mmol, 99% yield) as a black solid, which wasused directly in the next step without further purification. LC purity: 90% (UV at 220 nm);Mass calculated for C14H26N2O3, [M+1]+, 271.2, found 271.2; Retention time: 0.429 min.

[0377] Step 8: To a solution of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6- (thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (54 mg, 0.15 mmol) in DMF (3 mL) were added HATU (68 mg, 0.18 mmol) and DIEA (0.078 mL, 0.44 mmol).The mixture solution was stirred at 25 °C for 5 min before (9R,10S)-10-amino-9-methyl-3,8- dioxa-12-azaspiro[5.10]hexadecan-ll-one (40 mg, 0.15 mmol) was added. The resulting mixture was stirred at 25 °C for 30 min, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-((9R, 10S)-9-m ethyl- 11 -oxo-3, 8-dioxa- 12-azaspiro[5.10]hexadecan-10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8- carboxamide (11 mg, 0.02 mmol, 12% yield) as a yellow solid. LC purity: 97% (UV at 220 nm); Mass calculated for C31H45N5O6S, [M+1]+, 616.3, found 616.3; Retention time: 1.037 min;1H NMR (400 MHz, MeOD-4) 69.15 (s, 1H), 8.43 - 8.31 (m, 1H), 4.67 - 4.55 (m, 1H), 4.50 - 4.43 (m, 1H), 4.33 - 4.12 (m, 3H), 4.07 - 3.82 (m, 5H), 3.69 - 3.47 (m, 7H), 3.29 - 3.23 (m, 1H), 3.15 - 3.03 (m, 1H), 1.89 - 1.77 (m, 1H), 1.57 - 1.04 (m, 20H), 0.81 - 0.73 (m, 1H).Example 33. (8S)-2-((S)-2,2-Dimethylcyclopropane-1-carbonyl)-N-((3'R,4'R)-4'-methyl-1',5',8-trioxaspiro[bicyclo[3.2.1]octane-3,7'-cycloundecan]-3'-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide

[0378] Scheme 30

[0379] Step 1: To a solution of benzyl ((9R,10R, Z)-l l-hydroxy-9-methyl-8- oxaspiro[5.10]hexadec-14-en-10-yl)carbamate (intermediate of Example 19, Step 4, 30 mg, 0.07 mmol) in DCM (2 mL) was added BCl3(1 M in DCM, 0.7 mL, 0.7 mmol) at 0 °C under N2. The resulting mixture was stirred at room temperature for 16 h, poured into a stirred solution of aqueous NaHCCl4solution (3 mL) and extracted with DCM (5 mL x 3). Thecombined organic layers were dried over anhydrous MgSO4, filtered and evaporated to obtain the product (9R,10R, Z)-10-amino-9-methyl-8-oxaspiro[5.10]hexadec-14-en-ll-ol (15 mg, 0.03 mmol, 45% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 60% (UV at 200 nm); Mass calculated for C16H29NO2, [M+1]+, 268.2, found 268.2; Retention time: 1.069 min.

[0380] Step 2: To a solution of (9R,10R, Z)-10-amino-9-methyl-8-oxaspiro[5.10]hexadec-14-en-ll-ol (15 mg, 0.03 mmol), (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (11 mg, 0.03 mmol), and DIEA (73 mg, 0.1 mmol) in DMF (3 mL) was added HATU (17 mg, 0.05 mmol) at 0 °C. The resulting mixture was stirred for 2 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (8 S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-((9R, 10R, Z)- 11 -hydroxy-9-methyl-8-oxaspiro[5.10]hexadec- 14-en- 10-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (1.5 mg, 0.002 mmol, 8% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C33H48N4O5S [M+1]+, 613.3, found 612.9; Retention time: 1.633 min;1H NMR (400 MHz, MeOD-4) δ 9.16 (s, 1H), 8.37 (d, J= 8.8 Hz, 1H), 5.65 - 5.26 (m, 2H), 4.40 - 4.21 (m, 3H), 4.17 - 4.06 (m, 2H), 3.99 - 3.80 (m, 4H), 3.79 - 3.54 (m, 1H), 3.49 - 3.35 (m, 2H), 3.27 - 3.18 (m, 1H), 3.13 - 2.84 (m, 1H), 2.70 - 2.33 (m, 1H), 2.28 - 1.80 (m, 3H), 1.70 - 1.30 (m, 13H), 1.18 - 1.05 (m, 9H), 1.03 - 0.98 (m, 1H), 0.84 - 0.71 (m, 1H).Example 34. (8S)-2-((S)-2,2-DimethylcvdoDroDane-l-carbonyl)-N-((3lR,4lR)-4l-methyl-1,8-trioxaspiro [bicyclo [3.2. l]octane-3,7l-cvcloundecan]-3l-yl)-6-(thiazole-5-carbonyl)-2.,6-diazasDiro [3.4] octane-8-carboxamide

[0381] Scheme 31

[0382] Step 1: To a solution of methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (4 g, 9.6 mmol) in THF (40 mL) was added dropwise BH3·Me2S (10 in THF, 4.8 mL, 47.9 mmol) at 0 °C and the mixture was stirred under nitrogen atmosphere at 0 °C for 2 h before water (5 mL) and NaBO3·4H2O (7.4 g, 47.9 mmol) were added. The resulting reaction mixture was stirred at 25 °C for 25 h, poured into water (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-hydroxypropyl)cycloheptyl)methyl)-L-threoninate (2.2 g, 4.6 mmol, 47% yield) as a colorless oil. LC purity: 90% (UV at 254 nm); Mass calculated for C24H37NO6 [M+1]+, 436.3, found 436.3; Retention time: 1.992 min.

[0383] Step 2: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-hydroxypropyl)cycloheptyl)methyl)-L-threoninate (2.2 g, 5.1 mmol) and TEA (1.4 mL, 10.1 mmol) in DCM (30 mL) was added methanesulfonyl chloride (0.4 mL, 5.6 mmol) under N2 at 0 °C and the resulting mixture was stirred at 25 °C for 2 h, quenched with saturated aqueous NH4CI solution (20 mL) and extracted with DCM (20 mL x 2). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was evaporated under reduced pressure to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-((methylsulfonyl)oxy)propyl)cycloheptyl)methyl)-L-threoninate (2.5 g, 3.9 mmol, 77% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 254 nm); Mass calculated for C25H39NO8S [M+1]+, 514.2, found 514.3; Retention time: 2.088 min.

[0384] Step 3: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(3-((methylsulfonyl)oxy)propyl)cycloheptyl)methyl)-L-threoninate (2.2 g, 4.3 mmol) in DMF (30 mL) was added NaN3(0.7 g, 10.7 mmol) at 25 °C. The resulting mixture was stirred at 60 °C under N2 for 12 h, cooled to room temperature, quenched with H2O (40 mL), and extracted with EtOAc (40 mL x 2). The combined organic layers were washed with brine (40 mL x 2), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 40% EtOAc in PE) to obtain the product methyl O-((l -(3-azidopropyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.8 g, 3.1 mmol, 73% yield) as a yellow oil. LC purity: 80% (UV at 254 nm); Mass calculated for C24H36N4O5 [M+1]+, 461.3, found 461.2; Retention time: 2.354 min.

[0385] Step 4: To a stirred mixture of methyl O-((l-(3-azidopropyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.7 g, 3.7 mmol) in THF (20 mL) and water (20 mL) was added PPh3(2.9 g, 11.1 mmol) and the resulting mixture was stirred at 60 °C under N2 for 2 h before Boc2O (1.2 g, 5.5 mmol) was added. The reaction mixture was stirred at room temperature for another 1 h, diluted with H2O (40 mL) and extracted with EtOAc (40 mL x 2). The combined organic layers were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cycloheptyl)methyl)-L-threoninate (1.5 g, 2.8 mmol, 81% yield) as a yellow oil. LC purity: 80% (UV at 254 nm); Mass calculated for C29H46N2O7 [M+1]+, 535.3, found 535.3; Retention time: 2.252 min.

[0386] Step 5: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l -(3 -((tertbutoxy carbonyl)amino)propyl)cy cl oheptyl)methyl)-L-threoninate (1.5 g, 2.8 mmol) in THF (20 mL) and water (7 mL) was added LiOH·H2O (0.35 g, 8.4 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 h, and the pH was adjusted to 4-5 with aqueous HC1 solution (1.0 N). The mixture was then diluted with H2O (30 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4and filtered. The filtrate was concentrated under reduced pressure to obtain the product N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cycloheptyl)methyl)-L-threonine (1.2 g, 2.3 mmol, 74% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 90% (UV at 220 nm); Mass calculated for C28H44N2O7 [M+1]+, 521.3, found 521.4; Retention time: 1.537 min.

[0387] Step 6: To a mixture of N-((benzyloxy)carbonyl)-O-((l-(3-((tert-butoxycarbonyl)amino)propyl)cycloheptyl)methyl)-L-threonine (800 mg, 1.54 mmol) in DCM (5 mL) was added HCl-di oxane (4 N in dioxane, 4 mL, 16 mmol) at room temperature. The reaction was stirred at room temperature for 1 h. The mixture was evaporated in vacuo to obtain the product O-((l-(3-aminopropyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine hydrochloride (450 mg, 0. 96 mmol, 63% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 90% (UV at 254 nm); Mass calculated for C23H36N2O5 [M+1]+, 421.3, found 421.1; Retention time: 1.396 min.

[0388] Step 7: To a solution of O-((l-(3-aminopropyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (450 mg, 1.1 mmol) in DMF (30 mL) were added DIEA (0.6 mL, 3.3 mmol) and HATU (448 mg, 1.2 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 4 h, quenched with saturated aqueous NH4CI solution (60 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with brine (50 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product benzyl ((10R,l IS)- 10-m ethyl- 12-oxo-9-oxa-13-azaspiro[6.9]hexadecan-ll-yl)carbamate (160 mg, 0.36 mmol, 33% yield) as a yellow solid. LC purity: 90% (UV at 254 nm); Mass calculated for C23H34N2O4 [M+1]+, 403.3, found 403.3; Retention time: 1.962 min.

[0389] Step 8: To a solution of benzyl ((10R,llS)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.9]hexadecan-l l-yl)carbamate (80 mg, 0.2 mmol) in TFE (5 mL) was added 10% Pd / C (30 mg). The resulting mixture was stirred under H2atmosphere (1 atm) for 2 h, filtered through a Celite® pad and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to obtain the product (10R,l 1 S)-l l-amino-10-methyl-9-oxa-13-azaspiro[6.9]hexadecan-12-one (50 mg, 0.17 mmol, 84% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 90% (UV at 200 nm); Mass calculated for C15H28N2O2 [M+1]+, 269.2, found 269.3; Retention time: 1.418 min.

[0390] Step 9: To a solution of (5)-2-((5)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (68 mg, 0.19 mmol), DIEA (48 mg, 0.37 mmol), and HATU (78 mg, 0.2 mmol) in DMF (3 mL) was added (10R,l 1S)-1 l-amino-10-methyl-9-oxa-13-azaspiro[6.9]hexadecan-12-one (50 mg, 0.19 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 1 h, quenched with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-(( 1 OR, 11 S)- 10-methyl- 12-oxo-9-oxa- 13-azaspiro[6.9]hexadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (49 mg, 0.08 mmol, 43% yield) as a white solid. LC purity: 99% (UV at 254 nm); Mass calculated for C32H47N5O5S [M+1]+, 614.3, found 614.2; Retention time: 1.490 min;1H NMR (400 MHz, MeOD-4) 69.15 (s, 1H), 8.36 (d, J= 10.8 Hz, 1H), 7.85 - 7.61 (m, 1H), 4.38 - 4.31 (m, 1H), 4.29 - 4.21 (m, 2H), 4.19 - 4.03 (m, 3H), 4.02 - 3.85 (m, 4H), 3.50 (d, = 9.4 Hz, 1H), 3.45 - 3.33 (m, 2H), 3.15 - 3.00 (m, 1H), 2.90 (d, J= 8.8 Hz, 1H), 1.87 -1.78 (m, 1H), 1.68 - 1.60 (m, 2H), 1.49 - 1.29 (m, 12H), 1.26 - 1.13 (m, 8H), 1.10 (s, 2H), 1.06 - 1.01 (m, 1H), 0.94 - 0.86 (m, 1H), 0.81 - 0.74 (m, 1H).Example 35. (S)-2-((S)-2,2-Dimethylcvdopropane-l-carbonyl)-N-((10R,llS)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.8]pentadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0391] Scheme 32

[0392] Step 1: To a solution of methyl O-((l-allylcycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (5 g, 12.0 mmol) and 2,6-lutidine (5.6 mL, 47.9 mmol) in 1,4-dioxane (60 mL) and water (20 mL) was added K2OsO4(373 mg, 1.2 mmol) under N2. After stirring for 10 mins, NalCh (10.2 g, 47.9 mmol) was added, and the resulting mixture was stirred at 25 °C for 30 min under N2 atmosphere. After filtration, the filtrate was poured into saturated aqueous NH4CI (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (40 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(2-oxoethyl)cycloheptyl)methyl)-L-threoninate (3.5 g, 8.3 mmol, 70% yield) as a colorless oil. LC purity: 58% (UV at 254 nm); Mass calculated for C₂₃H₃₃NO₆ [M+1]+, 420.2, found 420.3; Retention time: 1.936 min.

[0393] Step 2: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(2-oxoethyl)cycloheptyl)methyl)-L-threoninate (3.5 g, 8.3 mmol) in MeOH (20 mL) wasadded NaBH4(221 mg, 5.8 mmol) in portions at 0 °C. The mixture was stirred at 0 °C for 10 min, quenched with H₂O (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silicagel, 0 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(2-hydroxyethyl)cycloheptyl)methyl)-L-threoninate (2.7 g, 6.4 mmol, 77% yield) as a colorless oil. LC purity: 69% (UV at 254 nm); Mass calculated for C23H35NO6 [M+1]+, 422.3, found 422.4; Retention time: 1.841 min.

[0394] Step 3: To a solution methyl N-((benzyloxy)carbonyl)-O-((l-(2-hydroxyethyl)cycloheptyl)methyl)-L-threoninate (2.7 g, 6.4 mmol) in DCM (20 mL) were added TEA (2.7 mL, 19.2 mmol) and MsCl (0.75 mL, 9.6 mmol) at 0 °C under N2. The reaction mixture was stirred under nitrogen atmosphere at room temperature for 2 h, quenched with H2O (10 mL) and extracted with DCM (10 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(2-((methylsulfonyl)oxy)ethyl)cycloheptyl)methyl)-L-threoninate (3 g, 6 mmol, 94% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 90% (UV at 254 nm); Mass calculated for C24H37NO8S [M+1]+, 500.2, found 500.2; Retention time: 2.066 min.

[0395] Step 4: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(2-((methylsulfonyl)oxy)ethyl)cycloheptyl)methyl)-L-threoninate (1 g, 2.0 mmol) in DMF (20 mL) was added NaN3(260 mg, 4 mmol) at 0 °C. The reaction mixture was stirred at 60 °C under nitrogen atmosphere for 12 h, cooled to room temperature, diluted with H2O (20 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl O-((l-(2-azidoethyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (670 mg, 1.5 mmol, 75% yield) as a colorless oil. LC purity: 65% (UV at 220 nm); Mass calculated for C23H34N4O5 [M+1]+, 447.3, found 447.2; Retention time: 2.308 min.

[0396] Step 5: To a solution of methyl O-((l-(2-azidoethyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threoninate (670 mg, 1.5 mmol) in THF (6 mL) and water (2 mL) was added Ph3P (590 mg, 2.25 mmol). The reaction mixture was stirred under N2 at 60 °C for 1.5 h. After cooled to room temperature, Boc2O (491 mg, 2.25 mmol) was added and the resulting mixture was stirred for 1 h, diluted with H2O (20 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified byflash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-((l-(2-((tert-butoxycarbonyl)amino)ethyl)cycloheptyl)methyl)-L-threoninate (325 mg, 0.6 mmol, 42% yield) as a colorless oil. LC purity: 78% (UV at 220 nm); Mass calculated for C28H44N2O7 [M+1]+, 521.3, found 521.4; Retention time: 2.343 min.

[0397] Step 6: To a solution of methyl N-((benzyloxy)carbonyl)-O-((l-(2-((tert-butoxycarbonyl)amino)ethyl)cycloheptyl)methyl)-L-threoninate (325 mg, 0.6 mmol) in THF (5 mL) and water (1 mL) was added LiOH·H2O (122 mg, 2.9 mmol), and stirred at room temperature for 12 h. The reaction mixture was adjusted to pH 4 with aqueous HCl solution (1 N), diluted with water (20 mL), and extracted with EtOAc (20 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo to obtain the product N-((benzyloxy)carbonyl)-O-((l-(2-((tert-butoxycarbonyl)amino)ethyl)cycloheptyl)methyl)-L-threonine (310 mg, 0.6 mmol, 98% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 71% (UV at 220 nm); Mass calculated for C27H42N2O7 [M+1]+, 507.3, found 507.3; Retention time: 2.134 min.

[0398] Step 7: To a solution of N-((benzyloxy)carbonyl)-O-((l-(2-((tert-butoxycarbonyl)amino)ethyl)cycloheptyl)methyl)-L-threonine (310 mg, 0.6 mmol) in DCM (5 mL) was added HCl-dioxane (4 TV in 1,4-dioxane, 4 mL, 0.9 mmol). The mixture was stirred at room temperature for 2 h and concentrated in vacuo to obtain the product O-((l-(2-aminoethyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (240 mg, 0.59 mmol, 99% yield) as a yellow solid, which was used directly in the next step without further purification. LC purity: 70% (UV at 220 nm); Mass calculated for C22H34N2O5 [M+1]+, 407.3, found 407.2; Retention time: 1.147 min.

[0399] Step 8: To a solution of O-((l-(2-aminoethyl)cycloheptyl)methyl)-N-((benzyloxy)carbonyl)-L-threonine (240 mg, 0.59 mmol) in DMF (150 mL) were added DIEA (0.3 mL, 1.8 mmol) and HATU (337 mg, 0.9 mmol). The resulting mixture was stirred at room temperature for 0.5 h, quenched with saturated aqueous NaHCCl4solution (100 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with brine (50 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 20 - 50% EtOAc in PE) to obtain the product benzyl ((10R,l lS)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.8]pentadecan-l 1-yl)carbamate (150 mg, 0.4 mmol, 66% yield) as a colorless oil. LC purity: 83% (UV at 220nm); Mass calculated for C22H32N2O4 [M+1]+, 389.2, found 389.3; Retention time: 1.764 min.

[0400] Step 9: To a solution of benzyl ((10R,l lS)-10-methyl-12-oxo-9-oxa-13-azaspiro[6.8]pentadecan-l l-yl)carbamate (150 mg, 0.4 mmol) in TFE (10 mL) was added 10% Pd / C (60 mg). The resulting mixture was stirred under H2atmosphere (1 atm) at room temperature for 12 h, filtered and concentrated under reduced pressure to obtain the product (10R,llS)-ll-amino-10-methyl-9-oxa-13-azaspiro[6.8]pentadecan-12-one (94 mg, 0.4 mmol, 96% yield) as a colorless oil, which was used directly in the next step without further purification. LC purity: 50% (UV at 220 nm); Mass calculated for C14H26N2O2 [M+1]+, 255.2, found 255.1; Retention time: 0.657 min.

[0401] Step 10: To a mixture of (S)-2-((S)-2,2-dimethylcyclopropane-l-carbonyl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxylic acid (90 mg, 0.2 mmol) and (10R,llS)-ll-amino-10-methyl-9-oxa-13-azaspiro[6.8]pentadecan-12-one (94 mg, 0.4 mmol) in DMF (3 mL) were added DIEA (0.1 mL, 0.5 mmol) and HATU (141 mg, 0.4 mmol). The resulting mixture was stirred at room temperature for 1 h, quenched with saturated aqueous NaHCCl4solution (10 mL) and extracted with EtOAc (10 mL x 2). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by prep-HPLC (gradient of CH3CN / H2O containing 0.1% FA) to obtain the product (S)-2-((S)-2,2-dimethylcyclopropane- 1 -carbonyl)-N-(( 1 OR, 11 S)- 10-methyl- 12-oxo-9-oxa- 13-azaspiro[6.8]pentadecan-ll-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro[3.4]octane-8-carboxamide (20 mg, 0.03 mmol, 13% yield) as a white solid. LC purity: 100% (UV at 220 nm); Mass calculated for C31H45N5O5S [M+1]+, 600.3, found 599.9; Retention time: 1.513 min;1H NMR (400 MHz, MeOD-4) 89.15 (s, 1H), 8.36 (d, J= 14.6 Hz, 1H), 4.39 - 4.24 (m, 2H), 4.20 - 3.86 (m, 6H), 3.73 - 3.66 (m, 1H), 3.50 - 3.36 (m, 3H), 3.14 - 2.74 (m, 2H), 2.66 - 2.34 (m, 1H), 1.90 - 1.78 (m, 1H), 1.77 - 1.72 (m, 2H), 1.52 - 1.40 (m, 4H), 1.38 - 1.27 (m, 4H), 1.21 - 1.14 (m, 8H), 1.10 - 0.94 (m, 6H), 0.80 - 0.74 (m, 1H).Example 36. (S)-2-((S)-2,2-Dimethylcvclopropane-l-carbonyl)-N-((13S,14R)-14-methyl-12-oxo-7,15-dioxa-ll-azaspiro[5.11]heptadecan-13-yl)-6-(thiazole-5-carbonyl)-2,6-diazaspiro [3.4] octane-8-carboxamide

[0402] Scheme 33

[0403] Step 1: A mixture of Zn (11.9 g, 183.4 mmol) and trimethyl silyl chloride (2 mL, 15.2 mmol) in THF (250 mL) was stirred at 70 °C for 1 h. Then cyclohexanone (10 g, 101.9 mmol) and methyl 2-bromoacetate (11 mL, 122.2 mmol) was added to above mixture at70 °C and the resulting mixture was stirred at 70 °C for 2 h. After cooled to room temperature, the reaction mixture was quenched with diluted aqueous HC1 solution (2 N, 100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with saturated aqueous NaHCCl4solution (50 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 2-(l-hydroxycyclohexyl)acetate (14.8 g, 85.9 mmol, 84% yield) as a colorless oil.1H NMR (400 MHz, DMSO-6) 64.31 (s, 1H), 3.56 (s, 3H), 2.37 (s, 2H), 1.60 - 1.33 (m, 9H), 1.23 - 1.12 (m, 1H).

[0404] Step 2: To a solution of methyl 2-(l-hydroxycyclohexyl)acetate (16 g, 92.9 mmol) in toluene (200 mL) were added allyl tert-butyl carbonate (29.4 g, 185.8 mmol) and Pd(PPh₃)4 (10.7 g, 9.3 mmol). The resulting mixture was stirred under N2 at 100 °C for 12 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The remaining residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product methyl 2-(l-(allyloxy)cyclohexyl)acetate (2.9 g, 13.7 mmol, 15% yield) as a light yellow oil. LC purity: 86% (UV at 220 nm); Mass calculated for C12H20O3 [M+1]+, 213.1, found 213.0; Retention time: 1.564 min.

[0405] Step 3: To a stirred suspension of methyl 2-(l-(allyloxy)cyclohexyl)acetate (2.9 g, 13.7 mmol) in THF (40 mL) was added dropwise LAH (1 in THF, 13.7 mL, 13.7 mmol) at -65 °C under N2. The resulting mixture was stirred under N2 for 2 h, quenched with diluted aqueous HCl solution (1 N, 20 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 10% EtOAc in PE) to obtain the product 2-(l-(allyloxy)cyclohexyl)ethan-l-ol (1.9 g, 10.2 mmol, 73% yield) as a yellow oil.1H NMR (400 MHz, DMSO-6) 85.96 - 5.82 (m, 1H), 5.24 (dd, J= 17.2, 1.8 Hz, 1H), 5.06 (dd, J= 10.4, 1.6 Hz, 1H), 4.24 (t, J= 5.0 Hz, 1H), 3.79 (d, J= 5.0 Hz, 2H), 3.48 - 3.40 (m, 2H), 1.71 - 1.60 (m, 4H), 1.53 - 1.42 (m, 3H), 1.40 -1.32 (m, 2H), 1.31 - 1.15 (m, 3H).

[0406] Step 4: To a solution of 2-(l-(allyloxy)cyclohexyl)ethan-l-ol (1.9 g, 10.2 mmol) and 1-benzyl 2-methyl (2S,3S)-3-methylaziridine-l,2-dicarboxylate (1.7 g, 6.8 mmol) in chloroform (120 mL) was added dropwise BF3·Et2O (0.9 mL, 6.8 mmol) at 0 °C under N2 atmosphere. The resulting mixture was stirred at 0 °C for 2 h, quenched with saturated aqueous NaHCCl4solution (20 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 0 - 20% EtOAc in PE) to obtain the product methyl O-(2-(l-(allyloxy)cyclohexyl)ethyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.1 g, 2.5 mmol, 37% yield) as a yellow oil. LC purity: 53% (UV at 254 nm); Mass calculated for C24H35NO6, [M+1]+, 434.3, found 434.2; Retention time: 1.978 min.

[0407] Step 5: To a solution of methyl O-(2-(l-(allyloxy)cyclohexyl)ethyl)-N-((benzyloxy)carbonyl)-L-threoninate (1.1 g, 2.5 mmol) in THF (40 mL) was added dropwise BH3·Me2S (10 M, 1.2 mL, 12.7 mmol) at 0 °C. The reaction mixture was stirred undernitrogen atmosphere at 0 °C for 2 h. Then H2O (5 mL) and NaBO3·4H2O (1.9 g, 12.7 mmol) were slowly added. The resulting mixture was stirred at room temperature for 12 h, quenched with brine (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc in PE) to obtain the product methyl N-((benzyloxy)carbonyl)-O-(2-(l-(3-hydroxypropoxy)cyclohexyl)ethyl)-L-threoninate (440 mg, 0.9 mmol, 38% yield) as a colorless oil. LC purity: 92% (UV at 220 nm); Mass calculated for C24H37NO7 [M+1]+, 452.3, found 452.2; Retention time: 1.609 min;1H NMR (400 MHz, DMSO-6) 67.41 - 7.24 (m, 6H), 5.05 (s, 2H), 4.32 (t, J= 5.0 Hz, 1H), 4.19 (dd, J= 8.8, 4.0 Hz, 1H), 3.88 - 3.79 (m, 1H), 3.64 (s, 3H), 3.50 - 3.43 (m, 3H), 3.31 -3.26 (m, 1H), 3.23 (t, J= 6.2 Hz, 2H), 1.64 - 1.55 (m, 6H), 1.50 - 1.39 (m, 3H), 1.38 - 1.29 (m, 2H), 1.24 - 1.15 (m, 3H), 1.08 (d, J = 6.2 Hz, 3H).

[0408] Step 6: To a solution of methyl N-((benzyloxy)carbonyl)-O-(2-(l-(3-hydroxypropoxy)cyclohexyl)ethyl)-L-threoninate (440 g, 0.9 mmol) in DCM (10 mL) were added TEA (0.3 mL, 1.9 mmol) and methanesulfonyl chloride (0.1 mL, 1.2 mmol) under nitrogen atmosphere at 0 °C. The reaction mixture was stirred at room temperature for 2 h, quenched with H₂O (20 mL) and extracted with DCM (20 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to afford the crude product methyl N-((benzyloxy)carbonyl)-O-(2-(l-(3-((methylsulfonyl)oxy)propoxy)cyclohexyl)ethyl)-L-threoninate (570 mg, 1.1 mmol, 100% yield) as a yellow oil, which was used directly in the next step without further purification. LC purity: 80% (UV at 220 nm); Mass calculated for C25H39NO9S [M+1]+, 530.2, found 530.3; Retention time: 1.768 min.

[0409] Step 7: To a solution of methyl N-((benzyloxy)carbonyl)-O-(2-(l-(3-((methylsulfonyl)oxy)propoxy)cyclohexyl)ethyl)-L-threoninate (570 mg, 1.1 mmol) in DMF (15 mL) was added NaN3(140 mg, 2.2 mmol) at 0 °C. The reaction mixture was stirred under N2 at 60 °C for 4 h, cooled to room temperature, quenched with H2O (50 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The remaining residue was purified by flash chromatography (silica gel, 1 - 30% EtOAc in PE) to obtain the product methyl O-(2-(l-(3-azidopropoxy)cyclohexyl)ethyl)-N-((benzyloxy)carbonyl)-L-threoninate (420 mg, 0.9 mmol, 82% yield) as a colorless oil. LC purity: 92% (UV at 220 nm); Mass calculated for C24H36N4O6 [M+1]+, 477.3, found 477.3; Retention time: 2.030 min;1H NMR(400 MHz, DMSO-6) 67.47 - 7.21 (m, 6H), 5.05 (s, 2H), 4.19 (dd, J= 8.6, 4.0 Hz, 1H), 3.89 - 3.77 (m, 1H), 3.62 (d, J= 19.0 Hz, 3H), 3.47 (dd, J= 15.6, 8.2 Hz, 1H), 3.40 (t, J= 6.8 Hz, 2H), 3.31 - 3.21 (m, 3H), 1.75 - 1.66 (m, 2H), 1.65 - 1.54 (m, 4H), 1.51 - 1.32 (m, 5H), 1.25 -1.15 (m, 3H), 1.09 (d, J = 6.2 Hz, 3H).

[0410] Step 8: To a solution of methyl O-(2-(l-(3-azidopropoxy)cyclohexyl)ethyl)-N-((benzyloxy)carbonyl)-L-threoninate (420 mg, 0.9 mmol) in THF (9 mL) and water (3 mL) was added Ph3P (462.3 mg, 1.8 mmol). The reaction mixture was stirred under N2 at 60 °C for 1.5 h. After cooled to room temperature, Boc2O (0.3 mL, 1.3 mmol) was added and the resulting mixture was stirred for 1 h, diluted wi...

Claims

1. Attorney Docket No.: 064105-502001WO2.WHAT IS CLAIMED IS:

1. A compound of Formula I:

5. 7.or pharmaceutically acceptable salt thereof,8.wherein9.each X1, X2, X3is -C(Xla)2-, -NXlb-, -O-, or -S(0)o-2-;10.X4is -C(Xla)2-, -NXlb-, -O-, -S(0)o-2-, or is absent;11.provided that when one of X1and X2is -NXlb-, -O-, or -S(0)o-2-, then the other is - C(Xla)2-, and when X4is present, one of X3and X4is -NXlb-, -O-, or -S(0)o-2-, then the other is -C(Xla)2-;12.each Xlais independently H, C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, Ci-Ce alkoxy, Ci-C6haloalkyl, Ci-C6haloalkoxy, halogen, -ORla, -NRlbRlc, -CN, -C(O)Rla, -C(O)NRlbRlc, -OC(O)NRlbRlc, -N(Rla)C(O)NRlbRlc, or -S(O)2NRlaRlb, or alternatively, two Xlaattached to the same carbon combine to form C3-C6 cycloalkyl, 4- to 10-membered heterocyclyl, or oxo;13.Xlbis H, C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, or Ci-Ce haloalkyl;14.'ll represents a single or a double bond;15.L is -C(=O)-NH-, -NH-C(=O)-, -CH(RL)-NH-, -CH(RL)-N(CH3)-, -NH-CH(RL)-, - CH(RL)-O-, -O-CH(RL)-, or a 5-membered heteroaryl;16.RLis H or C1-C6alkyl;17.R1is C1-C6alkyl, C2-Ce alkenyl, C2-Ce alkynyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, halogen, -ORla, -NRlbRlc, -CN, -C(O)Rla,18.-C(O)NRlbRlc, -OC(O)NRlbRlc, -N(Rla)C(O)NRlbRlc, or -S(O)2NRlaRlb, or alternatively, two R1attached on the same carbon combine to form C3-C6 cycloalkyl; each Rla, Rlb, and Rlcis independently H, C1-C6alkyl, or Ci-Ce haloalkyl; Attorney Docket No.: 064105-502001WO19.variable m is 1 or 2;20.variable n is 0, 1, 2, or 3, provided that if variable n is 0 or 1, then at least one of X2and X3is -C(Xla)2-;21.variable p is 0, 1, 2, 3, or 4;22.variable q is 0 or 1;23.Ring A is a C5-C8cycloalkyl or 5- to 8-membered heterocyclyl, wherein the cycloalkyl or heterocyclyl is substituted with 0, 1, 2, 3, or 4 R2;24.each R2is independently C1-C6alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce alkoxy, Ci- Ce haloalkyl, Ci-Ce haloalkoxy, or halogen, or two R2attached to the same carbon combine to form oxo;25.R3is a 8- to 12-membered spiro heterocyclyl, which is substituted by two L3a; each L3ais independently R3a, R3a-C(=O)-, (R3a)NH-C(=O)-, (R3a)O-C(=O)-, - CH(R3a)-, or a 5-membered heteroaryl substituted by R3a;26.each R3ais independently Ci-Ce haloalkyl, C3-C8 cycloalkyl, 4- to 10-membered heterocyclyl, Ce-Cio aryl, or 5- to 10-membered heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, or 3 C1-C6alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, or halogen; and27.R4is C1-C6alkyl or Ci-Ce haloalkyl.

2. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein29.X1is -C(=O); and30.X2is -NH-.

3. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein32.X1is -CH2-; and33.X2is -O-.

4. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein35.X1is -CH(OH)-; and36.X2is -CH2-. Attorney Docket No.: 064105-502001WO5. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 4, wherein X3is -CH2-.

6. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 5, wherein X4is -CH2-.

7. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 5, wherein X4is -O-.

8. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, wherein L is -C(=O)-NH-, -CH(RL)-NH-, or a 5-membered heteroaryl.

9. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 8, wherein RLis H.

10. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 9, wherein R1is C1-C6alkyl, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, halogen, -ORla, -NRlbRlc, or -CN.

11. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 10, wherein variable m is 1.

12. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 11, wherein variable n is 1 or 2.

13. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 12, wherein variable p is 0.

14. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 13, having the structure of Formula la:Attorney Docket No.: 064105-502001WO48.

15. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 11, having the structure of Formula Ib:

52.

16. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 15, wherein Ring A is cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, or tetrahydropyranyl, which are each substituted with 0, 1, 2, or 3 R2.

17. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 16, wherein each R2is independently halogen.

18. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 17, wherein R3isAttorney Docket No.: 064105-502001WO58. 60.wherein61.variable t is 1 or 2; and62.variable w is 1 or 2.

19. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 18, wherein R4is methyl.

20. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 19, having the structure of Formula (II):

66.

21. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 20, wherein each R3ais independently C3-C8 cycloalkyl or 5- to 10-membered heteroaryl, wherein the cycloalkyl and heteroaryl is independently substituted with 0, 1, 2, or 3 C1-C6alkyl or halogen.

22. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 21, wherein the compound is one selected from Table 1.

23. A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 22, and a pharmaceutically acceptable excipient.Attorney Docket No.: 064105-502001WO24. A method of inhibiting activity of a cyclin-dependent kinase (CDK) in a cell, comprising administering to the cell an effective amount of a compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 22, or a pharmaceutical composition of claim 23.

25. A method of treating a disease or disorder associated with CDK2 activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 22, or a pharmaceutical composition of claim 23.

26. The method of claim 25, wherein the disease or disorder associated with CDK2 activity is a cancer, a myeloproliferative disorder, an autoimmune disorder, an inflammatory disorder, a viral infection, or a fibrotic disorder.

27. The method of claim 25, the disease or disorder associated with CDK2 activity is a cancer.

28. The method of claim 25, the disease or disorder associated with CDK2 activity is breast cancer, ovarian cancer, endometrial cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, esophagogastric cancer, head and neck cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, melanoma, thyroid cancer, and glioblastoma.

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