Macrocyclic immunomodulators

Abstract

In accordance with the present disclosure, macrocyclic compounds have been discovered that can bind to PD-1 and inhibit the interaction of PD-1 with PD-L1, and these macrocyclic compounds demonstrate in vitro immunomodulatory efficacy and thus represent therapeutic candidates for the treatment of a variety of diseases, including cancer and infectious diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 324,433, filed March 28, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure provides macrocyclic compounds that can bind to PD-1 and inhibit the interaction between PD-1 and PD-L1. These macrocyclic compounds exhibit in vitro immunomodulatory efficacy and are therefore therapeutic candidates for the treatment of various diseases, including cancer. [Background technology]

[0003] Human cancers possess numerous genetic and epigenetic alterations, generating neoantigens potentially recognizable by the immune system (Non-Patent Document 1). The adaptive immune system, composed of T and B lymphocytes, possesses broad capabilities and excellent specificity in responding to diverse tumor antigens, resulting in potent anticancer potential. Furthermore, the immune system exhibits considerable plasticity and memory components. By exploiting all these attributes of the adaptive immune system, immunotherapy is unique among all cancer treatments.

[0004] The protein programmed death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4).

[0005] The PD-1 protein is a 55-kDa type I transmembrane protein that is part of the Ig gene superfamily (Non-Patent Document 2). PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM) (Non-Patent Document 5; Non-Patent Document 6). PD-1 is structurally similar to CTLA-4 but lacks the MYPPY motif, which is important for CD80 CD86 (B7-2) binding. Two ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (b7-DC), have been identified. It has been shown that activation of PD-1-expressing T cells is downregulated upon interaction with cells expressing PD-L1 or PD-L2 (Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9). Both PD-L1 and PD-L2 are B7 protein family members that bind to PD-1 but not other CD28 family members. PD-L1 ligands are abundant in various human cancers (Non-Patent Document 10). Interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancerous cells (Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13). Immune suppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked (Non-Patent Document 14; Non-Patent Document 15).

[0006] When PD-1-expressing T cells come into contact with cells expressing its ligand, their functional activities in response to antigenic stimulation, including proliferation, cytokine secretion, and cytotoxicity, are reduced. PD-1 / PD-L1 or PD-L2 interactions downregulate immune responses during infection or tumor resolution, or during the development of self-tolerance (Non-Patent Document 16). Chronic antigenic stimulation, such as that occurring during tumor disease or chronic infection, results in T cells that express elevated levels of PD-1 and are dysfunctional with respect to activity against chronic antigens (reviewed in Non-Patent Document 17). This is called "T cell exhaustion." B cells also exhibit PD-1 / PD-ligand suppression and "exhaustion."

[0007] In addition to enhancing immunological responses to chronic antigens, blockade of the PD-1 / PD-L1 pathway has also been shown to enhance responses to vaccination, including therapeutic vaccination in the setting of chronic infection (Non-Patent Document 18; Non-Patent Document 19; Non-Patent Document 20).

[0008] The PD-1 pathway is a key inhibitory molecule in T cell exhaustion resulting from chronic antigen stimulation during tumor disease, and therefore, agents that block the interaction between PD-1 and PD-L1 are desirable. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Sjoblom et al., 2006 [Non-patent document 2] Agata et al., Int. Immunol., 8:765-772(1996) [Non-patent document 3] Okazaki et al.,Curr.Opin.Immunol.,14:779-782(2002) [Non-patent document 4] Bennett et al., J. Immunol., 170:711-718 (2003) [Non-patent document 5] Thomas, ML, J. Exp. Med., 181:1953-1956 (1995) [Non-patent document 6] Vivier, E. et al., Immunol.Today, 18:286-291(1997) [Non-Patent Document 7] Freeman et al., J. Exp. Med., 192:1027-1034 (2000) [Non-patent document 8] Latchman et al., Nat. Immunol., 2:261-268 (2001) [Non-Patent Document 9] Carter et al., Eur.J.Immunol.,32:634-643(2002)

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

[0010] The present disclosure provides macrocyclic compounds that inhibit the PD-1 / PD-L1 protein / protein interaction and are therefore useful in ameliorating various diseases, including cancer.

[0011] In one aspect, the present disclosure provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 is C1-C6 alkyl, amino C1-C6 alkyl, aryl C1-C6 alkyl, heteroaryl C1-C6 alkyl, hydroxy C1-C6 alkyl, -XR 31 , -(CH2) z -O-(CH2) z -Triazolyl-XR 35 and NHC(X'')NHC1-C6 alkyl, where X'' is O or NH, and the aryl moiety of the arylC1-C6 alkyl and the heteroaryl moiety of the heteroarylC1-C6 alkyl are selected from carboxy, carboxyC1-C6 alkoxy, -XR 31 , and -O-(CH2) z -Triazolyl-XR35 and optionally substituted with one or two groups independently selected from R 2 is selected from aryl C1-C6 alkyl and heteroaryl C1-C6 alkyl, and the aryl moiety of the aryl C1-C6 alkyl and the heteroaryl moiety of the heteroaryl C1-C6 alkyl are carboxy, carboxy C1-C6 alkoxy, -XR 31 , and -O-(CH2) z -Triazolyl-XR 35 and optionally substituted with one or two groups independently selected from R 3 is carboxy C1-C3 alkyl, R 4 is selected from aryl C1-C6 alkyl and heteroaryl C1-C6 alkyl, wherein the aryl portion of the aryl C1-C6 alkyl and the heteroaryl portion of the heteroaryl C1-C6 alkyl are optionally substituted with one or two C1-C6 alkyl groups; R 5 is C1-C6 alkyl, aryl C1-C6 alkyl, -XR 31 , and -(CH2) z -O-(CH2) z -Triazolyl-XR 35 The aryl moiety of the aryl C1-C6 alkyl is selected from carboxy, carboxy C1-C6 alkoxy, hydroxy, -XR 31 , and -O-(CH2) z -Triazolyl-XR 35 and optionally substituted with one or two groups independently selected from R 6 is aryl-aryl C1-C3 alkyl, R 7 is C1-C6 alkyl, aryl C1-C6 alkyl, -XR 31 , and -(CH2) z -O-(CH2) z -Triazolyl-XR 35The aryl moiety of the aryl C1-C6 alkyl is selected from carboxy, carboxy C1-C6 alkoxy, -XR 31 , and -O-(CH2) z -Triazolyl-XR 35 and optionally substituted with one or two groups independently selected from R 8 is C1-C6 alkyl, amino C1-C6 alkyl, -XR 31 and -O-(CH2) z -Triazolyl-XR 35 is selected from R 9 is a C1-C6 alkyl, R 10 is amide C1-C6 alkyl, amino C1-C6 alkyl, -XR 31 , and -(CH2) z -O-(CH2) z -Triazolyl-XR 35 is selected from R 11 is (C3-C8 cycloalkyl)C1-C6 alkyl, R 12 is selected from C1-C6 alkyl, R 13 is aryl C1-C6 alkyl, carboxy C1-C6 alkyl, hydroxy C1-C6 alkyl, -XR 31 and -(CH2) z -O-(CH2) z -Triazolyl-XR 35 The aryl moiety of the aryl C1-C6 alkyl is selected from carboxy, carboxy C1-C6 alkoxy, -XR 31 and -O-(CH2) z -Triazolyl-XR 35 and optionally substituted with one or two groups independently selected from R 14 is -C(O)NH2 or -C(O)NHCHR 15 C(O)NHR 50 and R 15is selected from hydrogen, C1-C6 alkyl, and amino C1-C6 alkyl; R 50 is selected from hydrogen and NHC(O)CH(OCHCH)-; R 31 -CO2H, -C(O)NR w R x , -CH3, alexa-5-SDP, and biotin; each z is independently 1, 2, 3, 4, 5, or 6; R 35 -CO2H, -C(O)NR w R x , CH3, biotin, 2-fluoropyridine, —C(O)—(CH2)2—C(O)-vitamin E, and —C(O)-vitamin E; R x and R w are independently selected from hydrogen and C1-C6 alkyl; X is a chain of 1 to 172 atoms, the atoms being selected from carbon and oxygen, the chain optionally containing embedded therein 1, 2, 3, or 4 groups selected from -NHC(O)NH- and -C(O)NH-, the chain being optionally substituted with 1 to 6 groups independently selected from -COH, -C(O)NH, -CHC(O)NH, and -(CH)COH; However, R 1 , R 2 , R 5 , R 7 , R 8 , R 10 , and R 13 At least one of the following is -XR 31 , -(CH2) z -O-(CH2) z -Triazolyl-XR 35 , and -O-(CH2) z -Triazolyl-XR 35 or is substituted with a group selected from:

[0012] In some embodiments, vitamin E is [ka] refers to, [ka] indicates the point of attachment to the carbonyl group.

[0013] In some embodiments, alexa-5-SDP is [ka] wherein W is O or NH; [ka] indicates the point of attachment to the parent molecular moiety.

[0014] In some embodiments, biotin is [ka] wherein W is O or NH; [ka] indicates the point of attachment to the parent molecular moiety.

[0015] In some embodiments, R 31 is —COH. In some embodiments, R 31 is -NH2.

[0016] In some embodiments, R 35 is —COH. In some embodiments, R 35 is -NH2.

[0017] In some aspects, the disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein: R 1 is C1-C6 alkyl, amino C1-C6 alkyl, aryl C1-C6 alkyl, heteroaryl C1-C6 alkyl, hydroxy C1-C6 alkyl, HO2C(CH2) 10 C(O)NH(CH2)z - and NHC(X'')NHC1-C6 alkyl, where z is 1, 2, 3, or 4, X'' is O or NH, and the aryl portion of the arylC1-C6 alkyl and the heteroaryl portion of the heteroarylC1-C6 alkyl are selected from carboxy, carboxyC1-C6 alkoxy, and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH)triazolylCHO—; R 2 is selected from aryl C1-C6 alkyl and heteroaryl C1-C6 alkyl, and the aryl moiety of the aryl C1-C6 alkyl and the heteroaryl moiety of the heteroaryl C1-C6 alkyl are selected from carboxy, carboxy C1-C6 alkoxy, HO2C(CH2) 10 C(O)NH(CH2) z - and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH)triazolyl optionally substituted with one or two groups independently selected from CHO—, where z is 1, 2, 3, or 4; R 5 is C1-C6 alkyl, aryl C1-C6 alkyl, and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolylCH2OCH2-, and the aryl portion of the aryl C1-C6 alkyl is selected from carboxy, carboxy C1-C6 alkoxy, hydroxy, and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH)triazolylCHO—; R 7 is C1-C6 alkyl, aryl C1-C6 alkyl, HO2C(CH2) 10 C(O)NH(CH2)z -, HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolylCH2OCH2-, where z is 1, 2, 3, or 4; aryl C1-C6 ア The aryl portion of alkyl is carboxy, carboxy C1-C6 alkoxy, and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH)triazolylCHO—; R 8 is C1-C6 alkyl, amino C1-C6 alkyl, HO2C(CH2) 10 C(O)NH(CH2) z -, HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2triazolylCH2OCH2-, where z is 1, 2, 3, or 4; R 10 is amide C1-C6 alkyl, amino C1-C6 alkyl, and HO2C(CH2) 10 C(O)NH(CH2) z and z is 1, 2, 3, or 4; R 13 is aryl C1-C6 alkyl, carboxy C1-C6 alkyl, hydroxy C1-C6 alkyl, HO2C(CH2) 10 C(O)NH(CH2) z - and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2triazolylCH2OCH2-, where z is 1, 2, 3, or 4; However, R 1 , R 2 , R 5 , R 7 , R 8 , R 10 and R 13At least one of the following is HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 TriazolylCH2O-, HO2C(CH2) 10 C(O)NH(CH2) z , and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolylCH2OCH2- is or is substituted with a group selected from the group:

[0018] In some embodiments, R 1 , R 7 , R 8 , R 10 , and R 13 At least one of the following is HO2C(CH2) 10 C(O)NH(CH2) z - and z is 1, 2, 3, or 4.

[0019] In some embodiments, R 1 , R 2 , R 5 , and R 7 At least one of the groups is an aryl C1-C6 alkyl or a heteroaryl C1-C6 alkyl, and the aryl portion of the aryl C1-C6 alkyl and the heteroaryl portion of the heteroaryl C1-C6 alkyl are HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolylCH2O- or HO2C(CH2) 10 C(O)NH(CH2) z - is replaced by

[0020] In some embodiments, R 5 , R 7 , R 8 , and R 13 At least one of the following is HO2C(CH2) 16C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolyl CH2OCH2-.

[0021] In some embodiments, R 2 is aryl C1-C6 alkyl, and the aryl portion of the aryl C1-C6 alkyl is carboxy, carboxy C1-C6 alkoxy, HO2C(CH2) 10 C(O)NHCH2- and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolyl optionally substituted with one or two groups independently selected from CH2O-.

[0022] In some embodiments, R 4 is aryl C1-C6 alkyl, the aryl portion of the aryl C1-C6 alkyl being optionally substituted with one or two C1-C6 alkyl groups.

[0023] In some embodiments, R 5 is aryl C1-C6 alkyl, and the aryl portion of the aryl C1-C6 alkyl is carboxy, carboxy C1-C6 alkoxy, hydroxy, and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolyl optionally substituted with one or two groups independently selected from CH2O-.

[0024] In some embodiments, R 7 is aryl C1-C6 alkyl, and the aryl portion of the aryl C1-C6 alkyl is carboxy, carboxy C1-C6 alkoxy, and HO2C(CH2) 16 C(O)NHCH(CO2H)(CH2)2C(O)NH(CH2CH2O) 11 (CH2)2 triazolyl optionally substituted with one or two groups independently selected from CH2O-.

[0025] In some embodiments, R 8 is amino C1-C6 alkyl.

[0026] In some embodiments, R 10 is amino C1-C6 alkyl.

[0027] In some aspects, the present disclosure provides a pharmaceutical composition comprising a compound of any of the above aspects, or a pharmaceutically acceptable salt thereof.

[0028] In some aspects, the present disclosure provides a method of enhancing, stimulating, and / or increasing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any of the above aspects, or a pharmaceutically acceptable salt thereof.

[0029] In some aspects, the disclosure provides methods of blocking the interaction of PD-1 and PD-L1 in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of any of the above aspects, or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE INVENTION

[0030] Unless otherwise indicated, any atom with unsatisfied valences is assumed to have enough hydrogen atoms to satisfy the valences.

[0031] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise.

[0032] As used herein, the term "or" is a logical or (i.e., and / or) and does not indicate an exclusive or unless expressly indicated by words such as "either," "otherwise," "alternatively," and words of similar effect.

[0033] As used herein, the phrase "or a pharmaceutically acceptable salt thereof" refers to at least one compound, or at least one salt of a compound, or a combination thereof. For example, "a compound of Formula (I) or a pharmaceutically acceptable salt thereof" includes, but is not limited to, a compound of Formula (I), two compounds of Formula (I), a pharmaceutically acceptable salt of a compound of Formula (I), one or more pharmaceutically acceptable salts of a compound of Formula (I) and a compound of Formula (I), and two or more pharmaceutically acceptable salts of a compound of Formula (I).

[0034] The term "C1-C6 alkoxy," as used herein, refers to a C1-C6 alkyl group attached to the parent molecular moiety through an oxygen atom.

[0035] The term "C1-C6 alkyl" as used herein refers to a group derived from a straight or branched chain saturated hydrocarbon containing from 1 to 6 carbon atoms.

[0036] The term "amide" as used herein refers to -C(O)NH2.

[0037] The term "amido C1-C6 alkyl," as used herein, refers to an amido group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0038] The term "amino" as used herein refers to --NH.sub.2.

[0039] The term "amino C1-C6 alkyl," as used herein, refers to an amino group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0040] The term "aryl," as used herein, refers to a phenyl group or a bicyclic fused ring system in which one or both rings are phenyl groups. Bicyclic fused ring systems consist of a phenyl group fused to a 4- to 6-membered aromatic or non-aromatic carbocyclic ring. The aryl groups of the present disclosure can be attached to the parent molecular moiety through any substitutable carbon atom of the group. Representative examples of aryl groups include, but are not limited to, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl.

[0041] The term "aryl C1-C6 alkyl," as used herein, refers to an aryl group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0042] The term "carboxy" as used herein refers to -CO2H.

[0043] The term "carboxy C1-C6 alkoxy," as used herein, refers to a carboxy C1-C6 alkyl group attached to the parent molecular moiety through an oxygen atom.

[0044] The term "carboxy C1-C6 alkyl," as used herein, refers to a carboxy group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0045] The term "C3-C8 cycloalkyl" as used herein refers to a saturated monocyclic or bicyclic hydrocarbon ring system having 3 to 8 carbon atoms and 0 heteroatoms. Bicyclic rings can be fused, spirocyclic, or bridged. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, octahydropentalene, and bicyclo[3.1.1]heptyl.

[0046] The term "(C3-C8 cycloalkyl)C1-C6 alkyl," as used herein, refers to a C3-C8 cycloalkyl group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0047] The terms "halo" and "halogen" as used herein refer to F, Cl, Br, or I.

[0048] The term "heteroaryl," as used herein, refers to an aromatic 5- or 6-membered ring in which at least one atom is selected from N, O, and S, and the remaining atoms are carbon. The term "heteroaryl" also includes bicyclic systems in which a heteroaryl ring is fused to a 4- to 6-membered aromatic or non-aromatic ring containing 0, 1, or 2 additional heteroatoms selected from N, O, and S; and tricyclic systems in which a bicyclic system is fused to a 4- to 6-membered aromatic or non-aromatic ring containing 0, 1, or 2 additional heteroatoms selected from N, O, and S. A heteroaryl group is attached to the parent molecular moiety through any substitutable carbon or nitrogen atom in the group. Representative examples of heteroaryl groups include, but are not limited to, alloxazine, benzo[1,2-d:4,5-d']bisthiazole, benzoxadiazolyl, benzoxazolyl, benzofuranyl, benzothienyl, furanyl, imidazolyl, indazolyl, indolyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, purine, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, quinolinyl, thiazolyl, thienopyridinyl, thienyl, triazolyl, thiadiazolyl, and triazinyl.

[0049] The term "heteroaryl C1-C6 alkyl," as used herein, refers to a heteroaryl group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0050] As used herein, the term "hydroxy" refers to --OH.

[0051] The term "hydroxy C1-C6 alkyl," as used herein, refers to a hydroxy group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0052] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules, which result in the selective damage, destruction, or elimination from the body of invading pathogens, pathogen-infected cells or tissues, cancerous cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0053] The terms "programmed death-ligand 1," "programmed cell death-ligand 1," "PD-L1," "PDL1," "hPD-L1," "hPD-LI," and "B7-H1" are used interchangeably and include variants, isoforms, species homologs of human PD-L1, and analogs that share at least one epitope with PD-L1. The complete PD-L1 sequence can be found in GENBANK® Accession No. NP_054862.

[0054] The terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "hPD-1," and "hPD-1" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one epitope with PD-1. The complete PD-L1 sequence can be found in GENBANK® Accession No. U64863.

[0055] The term "treating" refers to i) inhibiting the disease, disorder, or condition, i.e., arresting its development, and / or ii) alleviating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and / or condition and / or symptoms associated with the disease, disorder, and / or condition.

[0056] The present disclosure is intended to include all isotopes of atoms present in the present compounds. Isotopes include atoms having the same atomic number but different mass numbers. Common examples include, but are not limited to, isotopes of hydrogen, such as deuterium and tritium. Isotopes of carbon include, but are not limited to, 13 C and 14C. Isotopically labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described herein, using an appropriate isotope-labeled reagent in place of an unlabeled reagent used in other methods. Such compounds may have a variety of potential uses, for example, as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds may have the potential to advantageously modify biological, pharmacological, or pharmacokinetic properties.

[0057] An additional aspect of the subject matter described herein is the use of the disclosed compounds as radiolabeled ligands for the development of ligand binding assays or for monitoring in vivo adsorption, metabolism, distribution, receptor binding or occupancy, or compound disposition. For example, the macrocyclic compounds described herein can be prepared using radioisotopes, and the resulting radiolabeled compounds can be used to develop binding assays or for metabolic studies. Alternatively, and for the same purpose, the macrocyclic compounds described herein can be converted to radiolabeled forms by catalytic tritiation using methods known to those skilled in the art.

[0058] The macrocyclic compounds of the present disclosure may also be used as PET imaging agents by adding a radioactive tracer using methods known to those skilled in the art.

[0059] Those skilled in the art will recognize that amino acids have the general structure: [ka] where R and R' are as discussed herein. Unless otherwise indicated, the term "amino acid" as used herein, alone or as part of another group, includes, but is not limited to, an amino group and a carboxyl group attached to the same carbon, referred to as the "α" carbon, and R and / or R' may be natural or unnatural side chains containing hydrogen. The absolute "S" configuration at the "α" carbon is commonly referred to as the "L" or "natural" configuration. When both the "R" and "R'" (prime) substituents are equal to hydrogen, the amino acid is glycine and is not chiral.

[0060] Unless specifically specified, amino acids described herein may be of D- or L-stereochemistry and may be substituted as described elsewhere in this disclosure. Where stereochemistry is not specified, it is understood that the disclosure encompasses all stereochemical isomers, or mixtures thereof, that have the ability to inhibit the interaction between PD-1 and PD-L1. Individual stereoisomers of a compound can be prepared synthetically from commercially available starting materials that contain chiral centers, or by preparation of a mixture of enantiomeric products followed by separation, e.g., conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, or direct separation of enantiomers on a chiral chromatographic column. Starting compounds of particular stereochemistry are commercially available or can be made and resolved by techniques known in the art.

[0061] Certain compounds of the present disclosure may exist in different stable conformational forms that may be separable. Torsional asymmetry resulting from restricted rotation about an asymmetric single bond, for example due to steric hindrance or ring strain, may allow for the separation of different conformers. The present disclosure includes each conformational isomer of these compounds and mixtures thereof.

[0062] Certain compounds of the present disclosure can exist as tautomers, which are compounds that are generated by the phenomenon that the protons of a molecule are shifted to different atoms within the molecule.The term "tautomer" also refers to one of two or more structural isomers that exist in equilibrium and can be easily converted from one isomer to another isomer.All tautomers of the compounds described herein are included in the present disclosure.

[0063] The pharmaceutical compounds of the present disclosure may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM et al., J. Pharm. Sci., 66:1-19 (1977)). Salts can be obtained during the final isolation and purification of the compounds described herein, or by separately reacting a free base functional group of the compound with a suitable acid, or by reacting an acidic group of the compound with a suitable base. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, and from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0064] Administration of a therapeutic agent described herein includes, but is not limited to, administration of a therapeutically effective amount of the therapeutic agent. As used herein, the term "therapeutically effective amount" refers to, but is not limited to, the amount of a therapeutic agent to treat a condition treatable by administration of a composition comprising a PD-1 / PD-L1 binding inhibitor described herein. The amount is an amount sufficient to exhibit a detectable therapeutic or ameliorative effect. The effect can include, for example, but is not limited to, treatment of the conditions listed herein. The precise effective amount for a subject will depend on the subject's size and health, the nature and extent of the condition being treated, the recommendations of the treating physician, and the therapeutic agent or combination of therapeutic agents selected for administration.

[0065] For administration of the macrocyclic peptides described herein, dosages range from about 0.0001 to 100 mg / kg, more typically 0.01 to 40 mg / kg, of the host body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or within the range of 10 to 40 mg / kg. Exemplary treatment regimens entail administration once daily, once every other week, once every three weeks, once every week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferred dosing regimens for the macrocyclic peptides of the present disclosure include 1 mg / kg or 3 mg / kg body weight by intravenous administration, with the macrocyclic peptide being administered using one of the following dosing schedules: (i) six doses every four weeks, followed by a dose every three months; (ii) a dose every three weeks; or (iii) one dose of 3 mg / kg body weight, followed by a dose of 1 mg / kg body weight every three weeks.

[0066] In another aspect, the present disclosure relates to methods for inhibiting tumor cell growth in a subject using the macrocyclic compounds of the present disclosure. In certain embodiments, the compounds of the present disclosure can bind to PD-1, disrupt the interaction between PD-1 and PD-L1, compete with PD-1 binding to certain anti-PD-1 monoclonal antibodies known to block PD-L1 interaction, and enhance CMV-specific T cell IFNγ secretion. As a result, the compounds of the present disclosure can be useful for modifying immune responses, treating diseases such as cancer, stimulating protective autoimmune responses, or stimulating antigen-specific immune responses (e.g., by coadministering a PD-L1 blocking compound with an antigen of interest). For example, the compounds of the present disclosure can be used to treat cancers selected from melanoma, renal cell carcinoma, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, gastrointestinal cancer, and breast cancer, as well as hematological malignancies.

[0067] The compounds of the present disclosure can also be used to treat infectious diseases, such as those caused by viruses, including, but not limited to, HIV, hepatitis A, hepatitis B, hepatitis C, herpes viruses, and influenza.

[0068] The compounds of the present disclosure can also be used to treat septic shock.

[0069] Pharmaceutical Composition In another aspect, the present disclosure provides compositions, e.g., pharmaceutical compositions, containing one or a combination of the compounds described within this disclosure formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present disclosure can also be administered in combination therapy, i.e., in combination with other drugs. For example, the combination therapy can include a macrocyclic compound combined with at least one other anti-inflammatory or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the compounds of the present disclosure.

[0070] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0071] The pharmaceutical compositions of the present disclosure may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0072] Pharmaceutical compositions of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired results. In some embodiments, routes of administration of macrocyclic compounds of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0073] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the above-listed ingredients as needed, and then sterilizing by microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from the above-listed ingredients.For the preparation of sterile injectable solution, some preparation methods are vacuum drying and lyophilization, thereby obtaining the powder of active ingredient plus any additional desired ingredients from its solution that has been previously sterilized and filtered.

[0074] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0075] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0076] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is well known in the art.Except where any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated.Auxiliary active compounds can also be incorporated into the composition.

[0077] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0078] Alternatively, the compounds of the present disclosure can be administered parenterally, including topical, epidermal, or mucosal routes of administration, such as intranasally, orally, intravaginally, rectally, sublingually, or topically.

[0079] Any pharmaceutical composition contemplated herein can be orally delivered, for example, via any acceptable and suitable oral preparation. Exemplary oral preparations include, but are not limited to, tablets, troches, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups, and elixirs. Pharmaceutical compositions intended for oral administration can be prepared according to any method known in the art for producing pharmaceutical compositions intended for oral administration. To provide a pharmaceutically palatable preparation, pharmaceutical compositions according to the present disclosure can contain at least one agent selected from sweeteners, flavoring agents, coloring agents, demulcents, antioxidants, and preservatives.

[0080] Tablets can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for tablet manufacture. Exemplary excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents such as microcrystalline cellulose, croscarmellose sodium, cornstarch, and alginic acid; binders such as starch, gelatin, polyvinylpyrrolidone, and acacia; and lubricants such as magnesium stearate, stearic acid, and talc. Additionally, tablets can be uncoated or coated by known techniques to mask the unpleasant taste of unpleasant-tasting drugs or delay disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby prolonging the effect of the active ingredient. Exemplary water-soluble taste-masking substances include, but are not limited to, hydroxypropylmethylcellulose and hydroxypropylcellulose. Exemplary time delay materials include, but are not limited to, ethyl cellulose and cellulose acetate butyrate.

[0081] Hard gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one salt thereof with at least one inert solid diluent, such as calcium carbonate; calcium phosphate; and kaolin.

[0082] Soft gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one water-soluble carrier, such as polyethylene glycol; and at least one oil medium, such as peanut oil, liquid paraffin, and olive oil.

[0083] Aqueous suspensions, for example, comprise at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof in a suspension containing, for example, a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, gum tragacanth, and gum arabic; a dispersing or wetting agent, such as a naturally occurring phosphatide, for example, lecithin; a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; a condensation product of an ethylene oxide with a long chain aliphatic alcohol, for example, a soluble solid; The aqueous suspension may be prepared by mixing the aqueous suspension with at least one excipient suitable for the manufacture of an aqueous suspension, including, but not limited to, condensation products of ethylene oxide with a fatty acid and a partial ester derived from a hexitol, such as, for example, heptadecathylene-oxycetanol; condensation products of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as, for example, polyoxyethylene sorbitol monooleate; condensation products of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as, for example, polyethylene sorbitan monooleate. The aqueous suspension may also contain at least one preservative, such as, for example, ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate; at least one coloring agent; at least one flavoring agent; and / or at least one sweetening agent, including, but not limited to, sucrose, saccharin, and aspartame.

[0084] Oily suspensions can be prepared by suspending at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof in vegetable oils, such as peanut oil, sesame oil, and coconut oil; or mineral oils, such as liquid paraffin.Oily suspensions can also contain at least one thickening agent, such as beeswax, hard paraffin, and cetyl alcohol.To provide a smooth oily suspension, at least one sweetener and / or at least one flavoring agent described herein above can be added to the oily suspension.Oily suspensions can further contain at least one preservative, including but not limited to, for example, antioxidants, such as butylated hydroxyanisole and alpha-tocopherol.

[0085] Dispersible powders and granules can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one dispersing agent and / or wetting agent, at least one suspending agent and / or at least one preservative. Suitable dispersing agents, wetting agents, and suspending agents have already been described above. Exemplary preservatives include, but are not limited to, antioxidants, such as ascorbic acid. Furthermore, dispersible powders and granules can contain at least one excipient, including, but not limited to, sweeteners, flavoring agents, and coloring agents.

[0086] An emulsion of at least one compound of Formula (I) and / or at least one pharmaceutically acceptable salt thereof can be prepared, for example, as an oil-in-water emulsion. The oily phase of an emulsion containing a compound of Formula (I) can be composed of known ingredients in a known manner. The oily phase can be provided by, for example, but not limited to, vegetable oils such as olive oil and peanut oil; mineral oils such as liquid paraffin; and mixtures thereof. The phase can simply contain an emulsifier, but can also contain a mixture of at least one emulsifier with a fat or oil, or both a fat and an oil. Suitable emulsifiers include, for example, but are not limited to, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. In some embodiments, a hydrophilic emulsifier is included together with a lipophilic emulsifier, which acts as a stabilizer. In some cases, it may be desirable to include both an oil and a fat. The emulsifier, with or without a stabilizer, also constitutes a so-called emulsifying wax, which, together with the fat or oil, forms the oily dispersed phase of the cream formulation, forming a so-called emulsifying ointment base. The emulsion may also contain sweeteners, flavorings, preservatives, and / or antioxidants. Suitable emulsifiers and emulsion stabilizers for use in the formulations of the present disclosure include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with wax, or other materials known in the art.

[0087] Active compounds can be prepared with carriers that protect compounds from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Many methods for preparing such formulations are patented or generally known to those skilled in the art.For example, see Robinson, JR, ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York (1978).

[0088] Therapeutic compositions can be administered using medical devices known in the art.For example, in one embodiment, the therapeutic compositions of the present disclosure can be administered using needleless hypodermic injection devices, such as those disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Examples of well-known implants and modules useful in the present disclosure include U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates; U.S. Pat. No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0089] In certain embodiments, the compounds of the present disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can contain one or more moieties that are selectively transported to specific cells or organs, thus enhancing targeted drug delivery (see, for example, Ranade, VV, J. Clin. Pharmacol., 29:685 (1989)). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun., 153:1038 (1988)); macrocycles (Bloeman, P. Get al., FEBS Lett., 357:140 (1995); Owais, M. et al., Antimicrob. Agents Chemother., 39:180 (1995)); surfactant protein A receptor (Briscoe et al., Am. J. Physiol., 1233:134 (1995)); p120 (Schreier et al., J. Biol. Chem., 269:9090 (1994)); Keinanen, K. et al., FEBS Lett., 357:140 (1995); Owais, M. et al., Antimicrob. Agents Chemother., 39:180 (1995)); surfactant protein A receptor (Briscoe et al., Am. J. Physiol., 1233:134 (1995)); p120 (Schreier et al., J. Biol. Chem., 269:9090 (1994)); Lett., 346:123 (1994); see also Killion, JJ et al., Immunomethods 4:273 (1994).

[0090] In certain embodiments, the compounds of the present disclosure can be administered parenterally, i.e., by injection, including but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, epidural, and intrasternal injection and / or infusion.

[0091] In some embodiments, the compounds of the present disclosure can be administered orally, i.e., via gelatin capsules, tablets, hard or soft capsules, or liquid capsules. The compounds can be made by methods known in the art, including those described below and including variations within the skill of one of ordinary skill in the art. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using readily available materials. Any variables used to describe the synthesis of the compounds (e.g., numbered "R" substituents) are intended only to illustrate how the compounds are made and should not be confused with variables used in the claims or other sections of this specification. The following methods are for illustrative purposes and are not intended to limit the scope of the disclosure. [Example]

[0092] The following examples are included to demonstrate various aspects of the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made to the specific compounds disclosed and still obtain like or similar results without departing from the spirit and scope of the present disclosure.

[0093] The compounds can be made by methods known in the art, including those described below and including variations within the skill of one in the art. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using readily available materials. Any variables used to describe the synthesis of the compounds (e.g., numbered "R" substituents) are intended only to illustrate how the compounds are made and should not be confused with variables used in the claims or other sections of this specification. The following methods are for illustrative purposes and are not intended to limit the scope of this disclosure.

[0094] Abbreviations used in the schemes generally follow conventions used in the art. Chemical abbreviations used in this specification and in the compounds are defined as follows: Ph = phenyl; Bn = benzyl; i-Bu = isobutyl; i-Pr = isopropyl; Me = methyl; Et = ethyl; Pr = n-propyl; Bu = n-butyl; t-Bu = tert-butyl; Trt = trityl; TMS = trimethylsilyl; TIS = triisopropylsilane; EtO = diethyl ether; HOAc or AcOH = acetic acid; MeCN or AcCN = acetonitrile; DMF = N,N-dimethylformamide; EtOAc = ethyl acetate. ;THF = tetrahydrofuran; TFA = trifluoroacetic acid; TFE = α,α,α-trifluoroethanol; Et2NH = diethylamine; NMM = N-methylmorpholine; NMP = N-methylpyrrolidone; DCM = dichloromethane; TEA = trimethylamine; min. = minute; h or hr = hour; L = liter; mL or mL = milliliter; μL = microliter; g = gram; mg = milligram; mol = mole; mmol = millimole; meq = milliequivalent; rt or RT = room temperature; sat or sat'd = saturated; aq.= aqueous; mp = melting point; BOP Reagent = benzotriazol-1-yloxy-tris-dimethylamino-phosphonium hexafluorophosphate (Castro's Reagent); PyBOP Reagent = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate; HBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HATU = O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HCTU = 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium Hexafluorophosphate; T3P = 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide; DMAP = 4-(dimethylamino)pyridine; DIEA = diisopropylethylamine; Fmoc or FMOC = fluorenylmethyloxycarbonyl; Boc or BOC = tert-butyloxycarbonyl; HOBT or HOBT·HO = 1-hydroxybenzotriazole hydrate; Cl-HOBt = 6-chlorobenzotriazole; HOAT = 1-hydroxy-7-azabenzotriazole; HPLC = high-performance liquid chromatography; LC / MS = high-performance liquid chromatography / mass spectrometry; MS or Mass Spec = mass spectrometry; NMR = nuclear magnetic resonance; Sc or SC or SQ = subcutaneous; IP or ip = intraperitoneal.

[0095] Example 1: General synthetic procedures and analytical methods The macrocyclic compounds of the present disclosure can be produced by methods known in the art, such as by chemical synthesis, recombinant synthesis in a cell-free system, recombinant synthesis in a cell, or by isolation from a biological source. Chemical synthesis of the macrocyclic compounds of the present disclosure can be performed using a variety of art-recognized methods, including stepwise solid-phase synthesis, semisynthesis by conformation-assisted religation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. A preferred method of synthesizing the macrocycles and analogs thereof described herein is chemical synthesis using various solid-phase techniques, such as those described in Chan, W C et al, eds., Fmoc Solid Phase Synthesis, Oxford University Press, Oxford (2000); Barany, G. et al, The Peptides: Analysis, Synthesis, Biology, Vol. 2: "Special Methods in Peptide Synthesis, Part A", pp. 3-284, Gross, E. et al, eds., Academic Press, New York (1980); Atherton, E., Sheppard, R C Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, England (1989); and Stewart, J M Young, J D Solid-Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co., Rockford, IL (1984).A preferred strategy is based on the combination of the (9-fluorenylmethyloxycarbonyl) group (Fmoc) for temporary protection of the α-amino group and the tert-butyl group (tBu) for temporary protection of the amino acid side chain (see, e.g., Atherton, E. et al., "The Fluorenylmethoxycarbonyl Amino Protecting Group," in The Peptides: Analysis, Synthesis, Biology, Vol. 9: "Special Methods in Peptide Synthesis, Part C," pp. 1-38, Undenfriend, S. et al., eds., Academic Press, San Diego (1987)).

[0096] Compounds can be synthesized stepwise on an insoluble polymeric support (also called a "resin"), starting from the C-terminus of the peptide. Synthesis begins by adding the C-terminal amino acid of the peptide to the resin via the formation of an amide or ester bond, allowing for the eventual release of the resulting peptide as a C-terminal amide or carboxylic acid, respectively.

[0097] The C-terminal amino acid and all other amino acids used in the synthesis must have their α-amino groups and side chain functional groups (if present) separately protected so that the α-amino protecting groups can be selectively removed during synthesis. Coupling of an amino acid is carried out by activating its carboxyl group as an active ester and reacting it with the unblocked α-amino group of the N-terminal amino acid attached to the resin. This series of deprotection and coupling of the α-amino groups is repeated until the entire peptide sequence is assembled. The peptide is then released from the resin with simultaneous deprotection of the side chain functional groups, usually in the presence of appropriate scavengers to limit side reactions. The resulting peptide is finally purified by reverse-phase HPLC.

[0098] Synthesis of the peptidyl resins required as precursors to the final peptides utilizes commercially available cross-linked polystyrene polymer resins (Novabiochem, San Diego, CA; Applied Biosystems, Foster City, CA). Preferred solid supports are: 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin); 9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin); and, for C-terminal carboxamides, 4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy)valerylaminomethyl-Merrifield resin (PAL resin). Coupling of the first and subsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt, or HOAt active esters generated from DIC / HOBt, HBTU / HOBt, BOP, PyBOP, or DIC / 6-Cl-HOBt, HCTU, DIC / HOAt, or HATU, respectively. Preferred solid supports are: 2-chlorotrityl chloride resin and 9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin) for protected peptide fragments. Loading of the first amino acid onto the 2-chlorotrityl chloride resin is optimally accomplished by reacting the Fmoc-protected amino acid with the resin in dichloromethane and DIEA. If necessary, a small amount of DMF may be added to solubilize the amino acid.

[0099] Synthesis of the compound analogs described herein can be performed using a single or multi-channel peptide synthesizer such as a CEM Liberty Microwave synthesizer, or a Protein Technologies, Inc. Prelude (6 channel) or Symphony (12 channel) or Symphony X (24 channel) synthesizer.

[0100] Useful Fmoc amino acid derivatives are listed in Table 1.

[0101] [Table 1]

[0102] The peptidyl resin precursor for each compound can be cleaved and deprotected using any standard procedure. (For compounds, see King, D.S. et al., Int. J. Peptide Protein Res., 36:255-266 (1990)). A preferred method is the use of TFA in the presence of TIS as a scavenger and DTT or TCEP as a disulfide reducing agent. Typically, the peptidyl resin is stirred in TFA / TIS / DTT (95:5:1 to 97:3:1, v:v:w; 1-3 mL / 100 mg of peptidyl resin) for 1.5-3 hours at room temperature. The spent resin is then filtered off, the TFA solution is cooled, and EtO solution is added. The precipitate is recovered by centrifuging and decanting the ether layer (three times). The resulting crude peptide is either directly redissolved in DMF or DMSO or CH3CN / H2O for purification by preparative HPLC or used directly in the next step.

[0103] Compounds of the desired purity can be obtained by preparative HPLC purification on a Waters Model 4000 or Shimadzu Model LC-8A liquid chromatograph. The crude compound solution is injected onto a YMC S5 ODS (20 x 100 mm) column and eluted with a linear gradient of MeCN in water, both buffered with 0.1% TFA, using a flow rate of 14-20 mL / min, with monitoring of the eluate by UV absorbance at 217 or 220 nm. The structure of the purified compound can be confirmed by electrospray MS analysis.

[0104] A list of the unnatural amino acids referred to herein is provided in Table 2.

[0105] [Table 2]

[0106]

Table 3

[0107]

Table 4

[0108]

Table 5

[0109]

Table 6

[0110]

Table 7

[0111]

Table 8

[0112]

Table 9

[0113]

Table 10

[0114]

Table 11

[0115]

Table 12

[0116]

Table 13

[0117] [Table 14]

[0118] [Table 15]

[0119] Solid phase peptide synthesis and cyclization of compounds The procedures described in this example were used in whole or in part, where noted, to synthesize the macrocyclic peptides described herein.

[0120] Scheme 1 - General synthetic methodology used for thioether macrocyclic peptides [ka] [ka]

[0121] General Protocol for Solid Phase Peptide Synthesis (SPPS) and Macrocyclization On a Symphony Peptide Synthesizer (Protein Technology Inc., Tucson, AZ), Prelude Peptide Synthesizer (Protein Technology Inc., Tucson, AZ), or Symphony X Peptide Synthesizer (Protein Technology Inc., Tucson, AZ), chlorotrityl resin preloaded with Fmoc-Pra-OH (0.100 mmol) was swollen with CHCl and then DMF under a gentle N2 stream and mixing. The solvent was drained, and the first amino acid was coupled using the following method: The Fmoc group was removed from the resin-supported component by washing the resin twice with 20% piperidine in DMF, mixing every 30 seconds with a gentle N2 stream. The resin was washed 5–6 times with DMF. Fmoc-Ala-OH (0.2 M solution in DMF) was then added, followed by the addition of a coupling activator (i.e., HATU (Chem-Impex International, 0.4 M solution in DMF) and a base (i.e., N-methylmorpholine (Aldrich, 0.8 M in DMF)). The reaction mixture was agitated with a gentle stream of nitrogen for 1-2 hours. The reagents were drained from the reaction vessel, and the resin was washed 5-6 times with DMF. The resulting resin-bound Fmoc-protected dipeptide was then sequentially deprotected and iteratively coupled with a third amino acid, etc., to give the desired resin-bound product.

[0122] The Fmoc group was removed from the N-terminus by washing the resin twice with a 20% piperidine solution in DMF under a gentle stream of nitrogen. The resin was washed with DMF (5-6 times). The peptide-resin was treated with chloroacetic anhydride (0.2 M in DMF) followed by NMM (0.8 M in DMF). This reaction was repeated. After draining all reagents and solvents, the resin was washed with DMF and DCM and then dried.

[0123] LCMS analysis confirmed the formation of the desired linear sequence by treating an aliquot of the peptide cleaved from the resin (analytical volume) with a small amount of TFA / TIS / DTT (96:4:1) solution at room temperature.

[0124] The peptide was globally deprotected and cleaved from the resin upon treatment with a TFA / TIS / DTT (96:4:1) solution for 1.5 hours. The resin was removed by filtration, washed with a small amount of cleavage cocktail, and the combined filtrates were added to cold EtO. The solution was cooled to 0 °C to precipitate the peptide from solution. The slurry was centrifuged to pellet the solid, and the supernatant was decanted. Fresh EtO was added, and the process was repeated three times to wash the solid. To the air-dried solid, a solution of DIEA / DMF (1–3 mL of DIEA in 40–45 mL of DMF) or 0.1 M NH4HCO3 / acetonitrile (1 / 1–3 / 1 (v / v)) was added until the solution pH exceeded 8. The solution was stirred for 16–72 hours and monitored by LCMS. The reaction solution was purified by preparative reverse-phase HPLC to yield the desired product.

[0125] General analytical protocols and synthetic methods Analysis data: Mass spectrometry: "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode; "ESI-MS(-)" refers to electrospray ionization mass spectrometry performed in negative ion mode; "ESI-HRMS(+)" refers to high-resolution electrospray ionization mass spectrometry performed in positive ion mode; "ESI-HRMS(-)" refers to high-resolution electrospray ionization mass spectrometry performed in negative ion mode. Detected masses are reported according to the "m / z" unit designation. Compounds with exact masses greater than 1000 are often detected as doubly or triply charged ions.

[0126] The crude material was purified by preparative LC / MS. Fractions containing the desired product were combined and dried via centrifugal evaporation.

[0127] Analytical LC / MS conditions A: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50 °C; Gradient: 0 to 100% B over 3 min, then hold at 100% B for 0.75 min; Flow rate: 1.0 mL / min; Detection: UV at 220 nm.

[0128] Analytical LC / MS conditions B: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50 °C; Gradient: 0 to 100% B over 3 min, then hold at 100% B for 0.75 min; Flow rate: 1.0 mL / min; Detection: UV at 220 nm.

[0129] Analytical LC / MS conditions C: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 70 °C; Gradient: 0 to 100% B over 3 min, then hold at 100% B for 2.0 min; Flow rate: 0.75 mL / min; Detection: UV at 220 nm.

[0130] Analytical LC / MS conditions D: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 70 °C; Gradient: 0 to 100% B over 3 min, then hold at 100% B for 2.0 min; Flow rate: 0.75 mL / min; Detection: UV at 220 nm.

[0131] Analytical LC / MS conditions E: Column: Kinetex XB C18, 3.0 × 75 mm, 2.6 μm particles; Mobile phase A: 10 mM ammonium formate in water:acetonitrile (98:2); Mobile phase B: 10 mM ammonium formate in water:acetonitrile (02:98); Gradient: 20 to 100% B over 4 min, then hold at 100% B for 0.6 min; Flow rate: 1.0 mL / min; Detection: UV at 254 nm.

[0132] Analytical LC / MS conditions F: Column: Ascentis Express C18, 2.1 x 50 mm, 2.7 μm particles; Mobile phase A: 10 mM ammonium acetate in water:acetonitrile (95:5); Mobile phase B: 10 mM ammonium acetate in water:acetonitrile (05:95); Temperature: 50 °C; Gradient: 0 to 100% B over 3 min; Flow rate: 1.0 mL / min; Detection: UV at 220 nm.

[0133] Analytical LC / MS conditions G: Column: X Bridge C18, 4.6 x 50 mm, 5 μm particles; Mobile phase A: 0.1% TFA in water; Mobile phase B: acetonitrile, Temperature: 35 °C; Gradient: 5 to 95% B over 4 min; Flow rate: 4.0 mL / min; Detection: UV at 220 nm.

[0134] Analytical LC / MS conditions H: Column: X Bridge C18, 4.6 x 50 mm, 5 μm particles; Mobile phase A: 10 mM NH4OAc; Mobile phase B: Methanol, Temperature: 35 °C; Gradient: 5 to 95% B over 4 min; Flow rate: 4.0 mL / min; Detection: UV at 220 nm.

[0135] Analytical LC / MS conditions I: Column: X Bridge C18, 4.6 x 50 mm, 5 μm particles; Mobile phase A: 10 mM NH4OAc; Mobile phase B: acetonitrile, Temperature: 35 °C; Gradient: 5 to 95% B over 4 min; Flow rate: 4.0 mL / min; Detection: UV at 220 nm.

[0136] Analytical LC / MS conditions J: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; Temperature: 70 °C; Gradient: 0 to 100% B over 1.5 min, then hold at 100% B for 2.0 min; Flow rate: 0.75 mL / min; Detection: UV at 254 nm.

[0137] Analytical LC / MS conditions K: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 100% water with 0.05% trifluoroacetic acid; Mobile phase B: 100% acetonitrile with 0.05% trifluoroacetic acid; Temperature: 50 °C; Gradient: 2 to 98% B over 1.0 min, then hold at 98% B from 1.0 to 1.5 min; Flow rate: 0.80 mL / min; Detection: UV at 220 nm.

[0138] Analytical LC / MS conditions L: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Buffer: 10 mM ammonium acetate. Mobile phase A: Buffer "CH3CN (95 / 5)"; Mobile phase B: Buffer: ACN (5:95); Temperature: 50 °C; Gradient: 20 to 98% B over 2.0 min, then hold at 100% B for 0.2 min; Flow rate: 0.70 mL / min; Detection: UV at 220 nm.

[0139] Analytical LC / MS conditions M: Column: Waters Acquity UPLC BEH C18, 3.0 x 50 mm, 1.7 μm particles; Mobile phase A: 95% water and 5% water with 0.1% trifluoroacetic acid; Mobile phase B: 95% acetonitrile and 5% water with 0.1% trifluoroacetic acid; Temperature: 50°C; Gradient: 20 to 100% B over 2.0 min, then hold at 100% B from 2.0 to 2.3 min; Flow rate: 0.7 mL / min; Detection: UV at 220 nm.

[0140] Analytical LC / MS conditions N: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 100% water with 0.05% trifluoroacetic acid; Mobile phase B: 100% acetonitrile with 0.05% trifluoroacetic acid; Temperature: 50 °C; Gradient: 2 to 98% B over 5.0 min, then hold at 98% B from 5.0 to 5.5 min; Flow rate: 0.80 mL / min; Detection: UV at 220 nm.

[0141] Analytical LC / MS conditions O: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; Temperature: 50 °C; Gradient: 2% to 98% B over 2 min, then hold at 98% B for 0.5 min; Flow rate: 0.8 mL / min; Detection: UV at 220 nm.

[0142] Analytical LC / MS conditions P: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; Temperature: 50 °C; Gradient: 0% to 100% B over 3 min, then hold at 100% B for 0.5 min; Flow rate: 1.0 mL / min; Detection: UV at 220 nm.

[0143] Analytical LC / MS conditions Q: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; Temperature: 50 °C; Gradient: 0% to 100% B over 1 min, then hold at 100% B for 0.5 min; Flow rate: 1.0 mL / min; Detection: UV at 220 nm.

[0144] Analytical LC / MS conditions R: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; Buffer: 10 mM ammonium acetate. Mobile phase A: Buffer "CH3CN (95 / 5)"; Mobile phase B: Buffer: ACN (5:95); Temperature: 50 °C; Gradient: 0% to 100% B over 1 min, then hold at 100% B for 0.5 min; Flow rate: 1.0 mL / min; Detection: UV at 220 nm.

[0145] General steps: Prelude Act: All operations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). Unless otherwise specified, all procedures were performed in a 45 mL polypropylene reaction vessel equipped with a bottom frit. The reaction vessel was connected to the Prelude peptide synthesizer through both the bottom and top of the vessel. DMF and DCM could be added through the top of the vessel, which evenly washed down the sides of the vessel. The remaining reagents were added through the bottom of the reaction vessel, passing through the frit and contacting the resin. All solution was removed through the bottom of the reaction vessel. "Periodic agitation" refers to short pulses of N2 gas through the bottom frit; pulses lasted approximately 5 seconds and occurred every 30 seconds. Amino acid solutions were generally not used more than two weeks after preparation. HATU solutions were used within 7–14 days of preparation.

[0146] Sieber amide resin = 9-Fmoc-aminoxanthen-3-yloxy polystyrene resin ("3-yloxy" refers to the position and type of linkage to the polystyrene resin). The resin used is polystyrene with a Sieber linker (Fmoc-protected at the nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol / g loading.

[0147] Rink = (2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine ("4-alkoxy" refers to the position and type of connectivity to the polystyrene resin). The resin used is Merrifield polymer (polystyrene) with a Rink linker (Fmoc-protected on the nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol / g loading.

[0148] 2-Chlorotrityl chloride resin (2-chlorotriphenylmethyl chloride resin), 50-150 mesh, 1% DVB, 1.54 mmol / g loading. Fmoc-glycine-2-chlorotrityl chloride resin, 200-400 mesh, 1% DVB, 0.63 mmol / g loading.

[0149] PL-FMP resin: (4-formyl-3-methoxyphenoxymethyl) polystyrene.

[0150] Commonly used amino acids are listed below with the side chain protecting groups shown in parentheses: Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(tBu)-OH; Fmoc-Bip-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-L Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Orn(Boc)-OH, Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH and their corresponding D-amino acids.

[0151] The "Prelude Method" procedures describe experiments performed on a 0.100 mmol scale, determined by the amount of Sieber, Rink, 2-chlorotrityl, or PL-FMP resin. This scale corresponds to approximately 140 mg of Sieber amide resin, as described above. All procedures can be scaled down or up from the 0.100 mmol scale by adjusting the listed volumes by a factor of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin swelling procedure, described below as the "Resin Swelling Procedure." Coupling of amino acids to the primary amine N-terminus was performed using the "Single Coupling Procedure," described below. Coupling of amino acids to the secondary amine N-terminus or the N-terminus of Arg(Pbf)- and D-Arg(Pbf)- was performed using the "Double Coupling Procedure," described below.

[0152] Resin swelling procedure: To a 45 mL polypropylene solid-phase reaction vessel was added Sieber amide resin (140 mg, 0.100 mmol). The resin was washed (swollen) twice as follows: DMF (5.0 mL) was added to the reaction vessel through the top of the vessel (DMF topwash), and the mixture was periodically stirred for 10 minutes, after which the solvent was drained through a frit.

[0153] Single Coupling Procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 5.0 mL) was added. The mixture was periodically stirred for 5.0 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 5.0 mL) was added. The mixture was periodically stirred for 5.0 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (6.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 1.0 minutes, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 5.0 mL, 10 equiv.) was added, followed by HATU (0.4 M in DMF, 2.5 mL, 10 equiv.), and finally NMM (0.8 M in DMF, 2.5 mL, 20 equiv.). The mixture was periodically stirred for 60–120 minutes, then the reaction solution was drained through a frit. The resin was washed four times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, the resulting mixture was periodically stirred for 1.0 min, and then the solution was drained through a frit. The resulting resin was used directly in the next step.

[0154] Double coupling procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 5.0 mL) was added. The mixture was periodically stirred for 5.0 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 5.0 mL) was added. The mixture was periodically stirred for 5.0 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (6.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 1.0 minutes, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 5.0 mL, 10 equiv.) was added, followed by HATU (0.4 M in DMF, 2.5 mL, 10 equiv.), and finally NMM (0.8 M in DMF, 2.5 mL, 20 equiv.). The mixture was periodically stirred for 1-1.5 hours, then the reaction solution was drained through a frit. The resin was washed twice in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 1.0 min before the solution was drained through the frit. To the reaction vessel was added amino acid (0.2 M in DMF, 5.0 mL, 10 equiv.), followed by HATU (0.4 M in DMF, 2.5 mL, 10 equiv.), and finally NMM (0.8 M in DMF, 2.5 mL, 20 equiv.). The mixture was periodically stirred for 1–1.5 h, and then the reaction solution was drained through the frit. The resin was washed four times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 1.0 min before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0155] Manually Adding a Single Coupling Procedure A: Piperidine:DMF (20:80 v / v, 5.0 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 5.0 mL) was added to the reaction vessel. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The reaction was paused. The reaction vessel was opened, and with the bottom of the vessel still attached to the instrument, the unnatural amino acid (2–4 equivalents) in DMF (1–2 mL) was manually added through the top of the vessel using a pipette. The vessel was then closed. The automated program was resumed, and HATU (0.4 M in DMF, 1.3 mL, 4 equiv.) and NMM (1.3 M in DMF, 1.0 mL, 8 equiv.) were added sequentially. The mixture was periodically stirred for 2–3 h, and then the reaction solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0156] Single Coupling Manual Addition Procedure B: Piperidine:DMF (20:80 v / v, 5.0 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 5.0 mL) was added to the reaction vessel. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The reaction was paused. The reaction vessel was opened, and with the bottom of the vessel still attached to the instrument, the unnatural amino acid (2–4 equivalents) in DMF (1–1.5 mL) was manually added through the top of the vessel using a pipette, followed by the manual addition of HATU (2–4 equivalents, the same equivalent as the unnatural amino acid), and then the vessel was closed. The automated program was resumed, and NMM (1.3 M in DMF, 1.0 mL, 8 equiv.) was added sequentially. The mixture was periodically stirred for 2-3 h, and then the reaction solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0157] Peptoid installation (50 μmol) procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 5.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 60 seconds, after which the solution was drained through a frit. To the reaction vessel, bromoacetic acid (0.4 M in DMF, 2.0 mL, 16 equivalents) was added, followed by DIC (0.4 M in DMF, 2.0 mL, 16 equivalents). The mixture was periodically stirred for 1 hour, then the reaction solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. An amine (0.4 M in DMF, 2.0 mL, 16 equivalents) was added to the reaction vessel. The mixture was periodically stirred for 1 hour, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0158] Chloroacetic anhydride coupling: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 5.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times in succession as follows: for each wash, DMF (6.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 1 minute, after which the solution was drained through a frit. To the reaction vessel, chloroacetic anhydride solution (0.4 M in DMF, 5.0 mL, 20 equivalents) was added, followed by N-methylmorpholine (0.8 M in DMF, 5.0 mL, 40 equivalents). The mixture was periodically stirred for 15 minutes, then the reaction solution was drained through a frit. The resin was washed twice as follows: for each wash, DMF (6.0 mL) was added through the top of the vessel, and the resulting mixture was stirred periodically for 1 minute before draining the solution through the frit. A chloroacetic anhydride solution (0.4 M in DMF, 5.0 mL, 20 equivalents) was added to the reaction vessel, followed by N-methylmorpholine (0.8 M in DMF, 5.0 mL, 40 equivalents). The mixture was stirred periodically for 15 minutes, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (6.0 mL) was added through the top of the vessel, and the resulting mixture was stirred periodically for 1 minute before draining the solution through the frit. The resin was washed four times in succession as follows: for each wash, DCM (6.0 mL) was added through the top of the vessel, and the resulting mixture was stirred periodically for 1 minute before draining the solution through the frit. The resin was then dried with a stream of nitrogen for 10 minutes. The resulting resin was used directly in the next step.

[0159] Symphony method: All operations were performed under automation on a 12-channel Symphony peptide synthesizer (Protein Technologies). Unless otherwise specified, all procedures were performed in a 25 mL polypropylene reaction vessel equipped with a bottom frit. The reaction vessel was connected to the Symphony peptide synthesizer through both the bottom and top of the vessel. DMF and DCM could be added through the top of the vessel, which evenly washed down the sides of the vessel. The remaining reagents were added through the bottom of the reaction vessel, passing through the frit and contacting the resin. All solution was removed through the bottom of the reaction vessel. "Periodic stirring" refers to short pulses of N2 gas through the bottom frit; pulses lasted approximately 5 seconds and occurred every 30 seconds. Amino acid solutions were generally not used more than two weeks after preparation. HATU solutions were used within 7–14 days of preparation.

[0160] Sieber amide resin = 9-Fmoc-aminoxanthen-3-yloxy polystyrene resin ("3-yloxy" refers to the position and type of linkage to the polystyrene resin). The resin used is polystyrene with a Sieber linker (Fmoc-protected at the nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol / g loading.

[0161] Rink = (2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine ("4-alkoxy" refers to the position and type of connectivity to the polystyrene resin). The resin used is Merrifield polymer (polystyrene) with a Rink linker (Fmoc-protected on the nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol / g loading.

[0162] 2-Chlorotrityl chloride resin (2-chlorotriphenylmethyl chloride resin), 50–150 mesh, 1% DVB, 1.54 mmol / g loading.

[0163] PL-FMP resin: (4-formyl-3-methoxyphenoxymethyl) polystyrene.

[0164] Fmoc-glycine-2-chlorotrityl chloride resin, 200–400 mesh, 1% DVB, 0.63 mmol / g loading.

[0165] Commonly used amino acids are listed below with the side chain protecting groups shown in parentheses: Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(tBu)-OH; Fmoc-Bip-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH Fmoc-Gly-OH;Fmoc-His(Trt)-OH;Fmoc-Hyp(tBu)-OH;Fmoc-Ile-OH;Fmoc-Leu-OH;Fmoc-Lys(Boc)-OH;Fmoc-Nle-OH;Fmoc-Met-OH;Fmoc-[N-Me]Ala-OH;Fmoc-[N-Me]Nle-OH;F moc-Orn(Boc)-OH, Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH and their corresponding D-amino acids.

[0166] The "Symphony Method" procedures describe experiments performed on a 0.05 mmol scale, which is determined by the amount of Sieber, Rink, chlorotrityl linker, or PL-FMP attached to the resin. This corresponds to approximately 70 mg of the Sieber resin described above. All procedures can be scaled up from the 0.05 mmol scale by adjusting the volumes listed by a factor of the scale.

[0167] Prior to amino acid coupling, all peptide synthesis sequences began with a resin swelling procedure, described below as the "resin swelling procedure." Coupling of amino acids to the primary amine N-terminus used the "single coupling procedure," described below. Coupling of amino acids to the secondary amine N-terminus or the N-terminus of Arg(Pbf)- and D-Arg(Pbf)- used the "double coupling procedure," described below.

[0168] Resin swelling procedure: Resin (0.05 mmol) was added to a 25 mL polypropylene solid-phase reaction vessel. The resin was washed (swelled) as follows: DMF (2.0–3.0 mL, 1–2 times) was added to the reaction vessel, and the mixture was periodically stirred for 10 minutes, after which the solvent was drained through a frit. Sometimes, the resin was washed (swelled) as follows: CHCl (3–5 mL, 2 times) was added to the reaction vessel, and the mixture was periodically stirred for 30 minutes, after which the solvent was drained through a frit. Then, DMF (2.0–3.0 mL, 1–6 times) was added, and the mixture was periodically stirred for 2–10 minutes, after which the solvent was drained through a frit.

[0169] Single Coupling Procedure: DMF (2.5–3.75 mL) was added three times to the reaction vessel containing the resin from the previous step; the mixture was stirred for 30 seconds, and the solvent was drained through the frit each time. Piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added to the resin; the mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added to the reaction vessel; the mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Occasionally, the deprotection step was performed a third time. The resin was washed six times consecutively as follows: for each wash, DMF (2.5–3.75 mL) was added to the vessel; the resulting mixture was periodically stirred for 30 seconds, and then the solution was drained through the frit. To the reaction vessel, amino acid (0.2 M in DMF, 2.0–2.5 mL, 8–10 equiv.) was added, followed by HATU (0.4 M in DMF, 1.0–1.25 mL, 8–10 equiv.), and finally NMM (0.8 M in DMF, 1.0–1.25 mL, 20 equiv.). The mixture was periodically stirred for 30–120 min, and then the reaction solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (2.5–3.0 mL) was added, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0170] Single Coupling Manual Addition Procedure: To the reaction vessel containing the resin from the previous step, DMF (3.0–3.75 mL) was added three times; the mixture was stirred for 30 seconds, and the solvent was drained through the frit each time. Piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added to the resin; the mixture was periodically stirred for 5.0 minutes, and then the solution was drained through the frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added. The mixture was periodically stirred for 5.0 minutes, and then the solution was drained through the frit. The mixture was periodically stirred for 5.0 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (3.0–3.75 mL) was added to the vessel, and the resulting mixture was periodically stirred for 30 seconds, and then the solution was drained through the frit. Premixed amino acids (2.0–5.0 equiv.) and HATU (0.4 M in DMF, 2.0–5.0 equiv.) were added to the reaction vessel, followed by NMM (0.8 M in DMF, 4.0–10.0 equiv.), with a molar ratio of 1:1:2 between amino acids, HATU, and NMM. The mixture was periodically stirred for 2–6 h, and then the reaction solution was drained through a frit. The resin was washed four times consecutively as follows: for each wash, DMF (3.75 mL) was added to the vessel, the resulting mixture was periodically stirred for 30 s, and then the solution was drained through a frit. The resulting resin was used directly in the next step.

[0171] Double coupling procedure: To the reaction vessel containing the resin from the previous step, DMF (2.5–3.75 mL) was added three times. The mixture was stirred for 30 seconds, and then the solvent was drained through the frit each time. Piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added to the reaction vessel. The mixture was stirred periodically for 5 minutes, and then the solution was drained through the frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added. The mixture was stirred periodically for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (3.0–3.75 mL) was added to the vessel, and the resulting mixture was stirred periodically for 30 seconds, and then the solution was drained through the frit. To the reaction vessel, amino acid (0.2 M in DMF, 2.0–2.5 mL, 8–10 equiv.) was added, followed by HATU (0.4 M in DMF, 1.0–1.25 mL, 10 equiv.), and finally NMM (0.8 M in DMF, 1.0–1.25 mL, 16–20 equiv.). The mixture was periodically stirred for 1 h, and then the reaction solution was drained through a frit. The resin was washed twice with DMF (3.0–3.75 mL), and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through a frit each time. To the reaction vessel, amino acid (0.2 M in DMF, 2.0–2.5 mL, 8–10 equiv.) was added, followed by HATU (0.4 M in DMF, 1.0–1.25 mL, 8–10 equiv.), and finally NMM (0.8 M in DMF, 1.0–1.25 mL, 16–20 equiv.). The mixture was periodically stirred for 1–2 h, and then the reaction solution was drained through a frit. The resin was washed six successive times as follows: for each wash, DMF (3.0–3.75 mL) was added to the vessel, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0172] Peptoid installation (50 μmol) procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 3.75 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 3.75 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times in succession as follows: for each wash, DMF (3.75 mL) was added to the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through a frit. To the reaction vessel, bromoacetic acid (0.4 M in DMF, 2.5 mL, 10 equivalents) was added, followed by DIC (0.4 M in DMF, 2.5 mL, 10 equivalents). The mixture was periodically stirred for 60 minutes, then the reaction solution was drained through a frit. The resin was washed two times in succession as follows: for each wash, DMF (3.75 mL) was added to the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. An amine (0.4 M in DMF, 2.5 mL, 10 equivalents) was added to the reaction vessel, and the mixture was periodically agitated for 60 minutes, after which the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (3.75 mL) was added to the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0173] Chloroacetic anhydride coupling: To the reaction vessel containing the resin from the previous step, DMF (3.0–3.75 mL) was added three times. The mixture was stirred for 30 seconds, and then the solvent was drained through the frit each time. To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added. The mixture was stirred periodically for 5 minutes, and then the solution was drained through the frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 3.0–3.75 mL) was added. The mixture was stirred periodically for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (3.0–3.75 mL) was added through the top of the vessel, and the resulting mixture was stirred periodically for 30 seconds, and then the solution was drained through the frit. To the reaction vessel was added chloroacetic anhydride solution (0.4 M in DMF, 3.0–3.75 mL, 30 equiv.), followed by NMM (0.8 M in DMF, 2.5 mL, 40 equiv.). The mixture was periodically stirred for 15 min, then the reaction solution was drained through a frit. The resin was washed once as follows: DMF (5.0–6.25 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through the frit. To the reaction vessel was added chloroacetic anhydride solution (0.4 M in DMF, 3.75 mL, 30 equiv.), followed by NMM (0.8 M in DMF, 2.5 mL, 40 equiv.). The mixture was periodically stirred for 15 min, then the reaction solution was drained through a frit. The resin was washed six times in succession as follows: for each wash, DMF (2.5 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resin was washed four times in succession as follows: for each wash, DCM (2.5 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was dried using a nitrogen stream for 10 minutes before being used directly in the next step.

[0174] Symphony X method: All operations were performed under automation on a Symphony X peptide synthesizer (Protein Technologies). Unless otherwise specified, all procedures were performed in a 45 mL polypropylene reaction vessel equipped with a bottom frit. The reaction vessel was connected to the Symphony X peptide synthesizer through both the bottom and top of the vessel. DMF and DCM could be added through the top of the vessel, evenly washing down the sides of the vessel. The remaining reagents were added through the bottom of the reaction vessel, passing through the frit and contacting the resin. All solution was removed through the bottom of the reaction vessel. "Periodic agitation" refers to short pulses of N2 gas through the bottom frit; pulses lasted approximately 5 seconds and occurred every 30 seconds. "Single-shot" addition mode refers to the addition of all solution contained in a single-shot Falcon tube, typically of any volume less than 5 mL. Amino acid solutions were generally not used for more than two weeks after preparation. HATU solutions were used within 14 days of preparation.

[0175] Sieber amide resin = 9-Fmoc-aminoxanthen-3-yloxy polystyrene resin ("3-yloxy" refers to the position and type of linkage to the polystyrene resin). The resin used is polystyrene with a Sieber linker (Fmoc-protected at the nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol / g loading.

[0176] Rink = (2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine, where "4-alkoxy" represents the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Rink linker (Fmoc-protected on the nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol / g loading.

[0177] 2-Chlorotrityl chloride resin (2-chlorotriphenylmethyl chloride resin), 50-150 mesh, 1% DVB, 1.54 mmol / g loading. Fmoc-glycine-2-chlorotrityl chloride resin, 200-400 mesh, 1% DVB, 0.63 mmol / g loading.

[0178] PL-FMPMP resin: (4-formyl-3-methoxyphenoxymethyl) polystyrene.

[0179] Commonly used amino acids are listed below with the side chain protecting groups shown in parentheses: Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(tBu)-OH; Fmoc-Bip-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-L Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Orn(Boc)-OH, Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH and their corresponding D-amino acids.

[0180] The "Symphony X" procedure describes experiments performed on a 0.050 mmol scale, determined by the amount of Sieber, Rink, 2-chlorotrityl, or PL-FMP bound to the resin. This scale corresponds to approximately 70 mg of Sieber amide resin, as described above. All procedures can be scaled above or below the 0.050 mmol scale by adjusting the listed volumes by multiples of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin swelling procedure, described below as the "Resin Swelling Procedure." Coupling of amino acids to the primary amine N-terminus was performed using the "Single Coupling Procedure," described below. Coupling of amino acids to the secondary amine N-terminus or N-terminus of Arg(Pbf)- and D-Arg(Pbf)- or D-Leu was performed using the "Double Coupling Procedure" or "Single Coupling 2-Hour Procedure," described below. Unless otherwise noted, the final step in the automated synthesis is the installation of an acetyl group, described below as the "Installation of Chloroacetyl Anhydride." All syntheses are concluded with a final rinse and dry step described as "Standard Final Rinse and Dry Procedure."

[0181] Resin swelling procedure: To a 45 mL polypropylene solid-phase reaction vessel was added Sieber amide resin (70 mg, 0.050 mmol). The resin was washed (swollen) three times as follows: DMF (5.0 mL) was added to the reaction vessel through the top of the vessel (DMF topwash), and the mixture was periodically stirred for 3 minutes, after which the solvent was drained through a frit.

[0182] Single Coupling Procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 2.0 mL, 8 equiv.) was added, followed by HATU (0.4 M in DMF, 1.0 mL, 8 equiv.), and finally NMM (0.8 M in DMF, 1.0 mL, 16 equiv.). The mixture was periodically stirred for 1-2 hours, then the reaction solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, the resulting mixture was periodically agitated for 30 seconds, and then the solution was drained through the frit. The resulting resin was used directly in the next step.

[0183] Single Coupling 4 Equivalent Procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, and then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, and then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 1.0 mL, 4 equiv.) was added, followed by HATU (0.2 M in DMF, 1.0 mL, 4 equiv.), and finally NMM (0.8 M in DMF, 1.0 mL, 16 equiv.). The mixture was periodically stirred for 1-2 hours, and then the reaction solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, the resulting mixture was periodically agitated for 30 seconds, and then the solution was drained through the frit. The resulting resin was used directly in the next step.

[0184] Double coupling procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. To the reaction vessel, amino acid (0.2 M in DMF, 2.0 mL, 8 eq.) was added, followed by HATU (0.4 M in DMF, 1.0 mL, 8 eq.), and finally NMM (0.8 M in DMF, 1.0 mL, 16 eq.). The mixture was periodically stirred for 1 hour, then the reaction solution was drained through a frit. The resin was washed twice in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added amino acid (2.0 mL, 8 equiv., 0.2 M in DMF), followed by HATU (1.0 mL, 8 equiv., 0.4 M in DMF), and finally NMM (1.0 mL, 16 equiv., 0.8 M in DMF). The mixture was periodically agitated for 1-2 hours, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0185] Double Coupling 4 Equivalent Procedures: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 4.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. To the reaction vessel, amino acid (0.2 M in DMF, 1.0 mL, 4 eq.) was added, followed by HATU (0.2 M in DMF, 1.0 mL, 4 eq.), and finally NMM (0.8 M in DMF, 1.0 mL, 16 eq.). The mixture was periodically stirred for 1 hour, then the reaction solution was drained through a frit. The resin was washed twice in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added amino acid (0.2 M in DMF, 1.0 mL, 4 equiv.), followed by HATU (0.2 M in DMF, 1.0 mL, 4 equiv.), and finally NMM (0.8 M in DMF, 1.0 mL, 16 equiv.). The mixture was periodically agitated for 1-2 hours, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0186] Manually Adding a Single Coupling Procedure A: Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The reaction was paused. The reaction vessel was opened, and with the bottom of the vessel still attached to the instrument, the unnatural amino acid (2–4 equivalents) in DMF (1–1.5 mL) was manually added through the top of the vessel using a pipette. The vessel was then closed. The automated program was resumed, and HATU (0.4 M in DMF, 1.0 mL, 8 equiv.) and NMM (0.8 M in DMF, 1.0 mL, 16 equiv.) were added sequentially. The mixture was periodically stirred for 2–3 h, and then the reaction solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0187] Single Coupling Manual Addition Procedure B: Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The reaction was paused. The reaction vessel was opened, and with the bottom of the vessel still attached to the instrument, the unnatural amino acid (2–4 equivalents) in DMF (1–1.5 mL) was manually added through the top of the vessel using a pipette, followed by HATU (2–4 equivalents, the same equivalent as the unnatural amino acid), and then the vessel was closed. The automated program was resumed, and NMM (0.8 M in DMF, 1.0 mL, 16 equiv.) was added sequentially. The mixture was periodically stirred for 2-3 h, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 s, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0188] Single Coupling Manual Addition Procedure C: Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel. The mixture was periodically stirred for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The reaction was paused. The reaction vessel was opened, and while the bottom of the vessel was still attached to the instrument, unnatural amino acids (2–4 equivalents) and NMM (4–8 equivalents) in DMF (1–1.5 mL) containing HATU (equimolar amount relative to the unnatural amino acid) were manually added through the top of the vessel using a pipette. The automated program was resumed, and the mixture was periodically stirred for 2–3 hours, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, the resulting mixture was periodically agitated for 30 seconds, and then the solution was drained through the frit. The resulting resin was used directly in the next step.

[0189] Single Coupling Manual Addition Procedure D: Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was periodically agitated for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel. The mixture was periodically agitated for 5 minutes, and then the solution was drained through the frit. The resin was washed six times consecutively as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. The reaction was paused. The reaction vessel was opened, and while the bottom of the vessel was still attached to the instrument, the unnatural amino acid (2–4 equivalents) in DMF (1–1.5 mL) containing DIC (equimolar amount relative to the unnatural amino acid) and HOAt (equimolar amount relative to the unnatural amino acid) was manually added through the top of the vessel using a pipette. The automated program was restarted, and the mixture was periodically stirred for 2-3 hours, after which the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0190] Peptoid installation (50 μmol) procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 3.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 3.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (3.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. To the reaction vessel, bromoacetic acid (0.4 M in DMF, 1.0 mL, 8 equivalents) was added, followed by DIC (0.4 M in DMF, 1.0 mL, 8 equivalents). The mixture was periodically stirred for 1 hour, then the reaction solution was drained through a frit. The resin was washed two times in succession as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. An amine (0.4 M in DMF, 2.0 mL, 16 equivalents) was added to the reaction vessel. The mixture was periodically agitated for 1 hour, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (3.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0191] Chloroacetic anhydride coupling: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 3.0 mL) was added. The mixture was periodically stirred for 3.5 or 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 3.0 mL) was added. The mixture was periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (3.0 mL) was added through the top of the vessel, and the resulting mixture was periodically stirred for 30 seconds, after which the solution was drained through the frit. To the reaction vessel, chloroacetic anhydride solution (0.4 M in DMF, 2.5 mL, 20 equivalents) was added, followed by N-methylmorpholine (0.8 M in DMF, 2.0 mL, 32 equivalents). The mixture was periodically stirred for 15 minutes, then the reaction solution was drained through a frit. The resin was washed twice as follows: for each wash, DMF (3.0 mL) was added through the top of the vessel, and the resulting mixture was stirred periodically for 1.0 min before the solution was drained through the frit. To the reaction vessel was added chloroacetic anhydride solution (0.4 M in DMF, 2.5 mL, 20 equivalents), followed by N-methylmorpholine (0.8 M in DMF, 2.0 mL, 32 equivalents). The mixture was stirred periodically for 15 min, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (3.0 mL) was added through the top of the vessel, and the resulting mixture was stirred periodically for 1.0 min before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0192] Final rinsing and drying procedure: The resin from the previous step was washed six times in succession as follows: for each wash, DCM (5.0 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resin was then dried using a stream of nitrogen for 10 minutes. The resulting resin was used directly in the next step.

[0193] Sonata method: Unless otherwise noted, all procedures were performed under automation on a Sonata peptide synthesizer (Protein Technologies). Unless otherwise noted, all procedures were performed in a 200 mL glass reaction vessel connected to the Sonata peptide synthesizer via both the bottom and top of the vessel. All reagents were added through the bottom of the vessel, and washing was performed with one top wash followed by five bottom washes. All solutions were removed through the bottom of the vessel. "Periodic stirring" refers to brief pulses of N2 gas through the bottom frit; pulses last approximately 5 seconds and occur every 30 seconds. Mechanical stirring was performed during addition and throughout the wash and / or reaction cycles. The chloroacetic acid / DIC solution in DMF was used within 0.25 hours of preparation. The amino acid solutions were not used more than 3 days after preparation. The HATU solution was used within 5 days of preparation. DMF = N,N-dimethylformamide; NMM = N-methylmorpholine.

[0194] The "Prelude Method" procedures describe experiments performed on a 2 mmol scale, with the scale determined by the amount of Sieber or Rink, or 2-chlorotrityl, or PL-FMP resin. The procedures described below can be scaled up beyond the 2 mmol scale by simply adjusting the volumes described by multiples of the scale. The general synthesis outline is as follows: Prior to amino acid coupling, all peptide synthesis sequences began with a resin swelling procedure, described below as the "Resin Swelling Procedure." Coupling of amino acids to the primary amine N-terminus used the "Single Coupling Procedure," described below. Coupling of amino acids to the secondary amine N-terminus or the N-terminus of Arg(Pbf)- and D-Arg(Pbf)- used the "Double Coupling Procedure," described below.

[0195] Resin swelling (automated) Amino Acid Coupling Step (Repeat of General Procedures A and B; Automated) Capping with chloroacetic acid (automated) Global deprotection (manual) Cyclization (manual)

[0196] Resin swelling and first coupling procedure: NOTE: This procedure includes a swelling step and is used as the first coupling cycle.

[0197] The resin was added to a 200 mL glass reaction vessel. The resin was washed four times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 10 seconds, approximately 50 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was mechanically and periodically stirred for 5 minutes, after which the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 10 seconds, approximately 50 mL) was added to the reaction vessel. The mixture was mechanically and periodically stirred for 5 minutes, after which the solution was drained through the frit. The resin was washed six times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added to the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. To the reaction vessel, amino acid (0.2 M in DMF, 20 mL, 2 equivalents) was added, followed by HATU (0.4 M in DMF, 10 mL, 2 equivalents), and finally NMM (0.8 M in DMF, 10 mL, 4 equivalents). The mixture was mechanically and periodically stirred for 30 minutes, and then the reaction solution was drained through a frit. The resin was washed five times consecutively as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through a frit. To the reaction vessel, approximately 45 mL of 10% (v / v) acetic anhydride and 10% (v / v) d-IPEA in DMF was added over 9 seconds. The mixture was mechanically and periodically stirred for 10 minutes, and then the solution was drained through a frit. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 90 seconds, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0198] Single coupling procedure with capping: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, approximately 50 mL, 10-second delivery) was added. The mixture was mechanically and periodically stirred for 5 minutes, and then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, approximately 50 mL, 10-second delivery) was added. The mixture was mechanically and periodically stirred for 5 minutes, and then the solution was drained through a frit. The resin was washed six times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 20 mL, 2 equivalents) was added, followed by HATU (0.4 M in DMF, 10 mL, 2 equivalents), and finally NMM (0.8 M in DMF, 10 mL, 4 equivalents). The mixture was mechanically and periodically stirred for 30 minutes, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. Approximately 45 mL of 10% (v / v) acetic anhydride and 10% (v / v) DIEA in DMF was added to the reaction vessel over 9 seconds. The mixture was mechanically and periodically stirred for 10 minutes, and then the solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 90 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0199] Double coupling procedure with capping: Note: There are two exposures to the amino acid and coupling reagent ("double coupling"). This procedure is typically used when the reactive terminal amine is secondary rather than primary.

[0200] To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added. The mixture was mechanically and periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added. The mixture was mechanically and periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (9 s delivery, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 20 mL, 2 eq.) was added, followed by HATU (0.4 M in DMF, 10 mL, 2 eq.), and finally NMM (0.8 M in DMF, 10 mL, 4 eq.). The mixture was mechanically and periodically stirred for 30 minutes, and then the reaction solution was drained through the frit. The resin was washed twice as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. To the reaction vessel, amino acid (0.2 M in DMF, 20 mL, 2 equivalents) was added, followed by HATU (0.4 M in DMF, 10 mL, 2 equivalents), and finally NMM (0.8 M in DMF, 10 mL, 4 equivalents). The resin was washed five times consecutively as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. To the reaction vessel, approximately 45 mL of 10% (v / v) acetic anhydride and 10% (v / v) d-IPEA in DMF was added over 9 seconds. The mixture was mechanically and periodically stirred for 10 minutes, and then the solution was drained through a frit. The resin was washed five times consecutively as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 90 seconds, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0201] Single coupling, extended time procedure: Note: Coupling time is extended to 2 hours.

[0202] To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added. The mixture was mechanically and periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added. The mixture was mechanically and periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (9 s delivery, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through a frit. To the reaction vessel, amino acid (0.2 M in DMF, 20 mL, 2 eq.) was added, followed by HATU (0.4 M in DMF, 10 mL, 2 eq.), and finally NMM (0.8 M in DMF, 10 mL, 4 eq.). The mixture was mechanically and periodically stirred for 120 minutes, and then the reaction solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. Approximately 45 mL of 10% (v / v) acetic anhydride and 10% (v / v) d-IPEA in DMF was added to the reaction vessel over 9 seconds. The mixture was mechanically and periodically stirred for 10 minutes, and then the solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 90 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0203] Final coupling steps: Note: This condition differs from general procedure B in the fact that it includes a final DCM wash of the peptide to prevent any loss of the terminal FMOC from the linear peptide.

[0204] Piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added to the reaction vessel containing the resin from the previous step. The mixture was mechanically and periodically stirred for 5 minutes, and then the solution was drained through the frit. Piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added to the reaction vessel. The mixture was mechanically and periodically stirred for 5 minutes, and then the solution was drained through the frit. The resin was washed once in succession as follows: DMF (9 s, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (9 s, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2 M in DMF, 20 mL, 2 equivalents), followed by HATU (0.4 M in DMF, 10 mL, 2 equivalents), and finally NMM (0.8 M in DMF, 10 mL, 4 equivalents). The mixture was mechanically and periodically stirred for 30 minutes, and then the reaction solution was drained through a frit. The resin was washed twice as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through a frit. To the reaction vessel was added the amino acid (0.2 M in DMF, 20 mL, 2 equivalents), followed by HATU (0.4 M in DMF, 10 mL, 2 equivalents), and finally NMM (0.8 M in DMF, 10 mL, 4 equivalents). The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added to the top of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through the frit. Approximately 45 mL of 10% (v / v) acetic anhydride and 10% (v / v) DIEA in DMF was added to the reaction vessel over 9 seconds. The mixture was mechanically and periodically stirred for 10 minutes, and then the solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 90 seconds, after which the solution was drained through the frit.The resin was washed seven times in succession as follows: for each wash, DCM (10 seconds, approximately 50 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 90 seconds, after which the solution was drained through a frit. The resulting resin was used directly in the next step.

[0205] Manual mode chloroacetic acid capping procedure: To the reaction vessel containing the resin from the previous step, piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added. The mixture was mechanically and periodically stirred for 5 minutes, then the solution was drained through a frit. To the reaction vessel, piperidine:DMF (20:80 v / v, 10 s delivery, approximately 50 mL) was added. The mixture was mechanically and periodically stirred for 5 minutes, then the solution was drained through a frit. The resin was washed six times consecutively as follows: for each wash, DMF (9 s delivery, approximately 45 mL) was added through the bottom of the vessel, and the resulting mixture was mechanically and periodically stirred for 1 minute, after which the solution was drained through a frit. To an Erlenmeyer flask, 1.9 g (10 eq.) of chloroacetic acid and 4.7 mL (15 eq.) of DIC in 40 mL of DMF were added. This prepared solution was added to a 200 mL glass reactor containing the resin using manual mode for 2 seconds, followed by the addition of approximately 10 mL of DMF through the bottom of the frit, and the resulting mixture was mechanically and periodically stirred for 2.5 days. The reaction time is likely to be much shorter and can be monitored using the Kaiser ninhydrin test. The resin was washed five times in succession as follows: for each wash, DMF (9 seconds, approximately 45 mL) was added through the bottom of the vessel, the resulting mixture was mechanically and periodically stirred for 1 minute, and then the solution was drained through the frit. The resin was washed seven times in succession as follows: for each wash, DCM (10 seconds, approximately 50 mL) was added through the bottom of the vessel, the resulting mixture was mechanically and periodically stirred for 1 minute, and then the solution was drained through the frit.

[0206] Alternatively, when 4 equivalents of amino acid were used, acetic anhydride capping was not performed (no capping) in the single and double coupling steps above.

[0207] Manual operation and operation of all the following procedures: All procedures were performed as follows: A "deprotection solution" was prepared by combining 1% (wt%) dithiothreitol (75 mg), 2.5% (v / v) triisopropylsilane (1.875 mL), and trifluoroacetic acid (75 mL) in a 200 mL Erlenmeyer flask. The deprotection solution was cooled to 5 °C in an ice-water bath before adding to the resin. 0.8 mmol ~ 4 g of resin was placed in a 100 mL peptide synthesis vessel, and the cold "deprotection solution" was added all at once. The mixture was capped and shaken on a shaker for 1.5 h. The filtrate was collected equally into 8 x 50 mL polypropylene Falcon tubes. 30 mL of ether was added to each tube, capped, and shaken to obtain a white precipitate. The tubes were cooled in a refrigerator for 1 h before centrifugation. Each tube was centrifuged (3 min, 2500 rpm), and the ether layer was discarded. The precipitate was washed with ether (3 x 20 mL), and the centrifugation was repeated to obtain the crude linear chloroacylated peptide.

[0208] 7.5 mL of acetonitrile was added to each tube, which was then diluted to 35 mL with 0.1 M aqueous NH4HCO3, and the resulting mixture was shaken to dissolve all solids. Note: CO2 results from quenching excess TFA with ammonium bicarbonate. All solutions were transferred to a lyophilization flask, and each Falcon tube was washed with 5 mL of a 1:1 mixture of acetonitrile and 0.1 M aqueous NH4HCO3. The resulting solution was carefully adjusted to pH 8 using 1.0 M NaOH. The solution was allowed to stand overnight (18 hours). Completion of the reaction was confirmed by HPLC MS. The mixture was then frozen in a dry ice / acetone bath and lyophilized to yield a white solid for purification.

[0209] General deprotection, cyclization, N-methylation, Click, and Suzuki procedures: Global Deprotection Method A: Unless otherwise specified, all operations were performed manually. The "Global Deprotection Method" procedure describes experiments performed at a 0.050 mmol scale, determined by the amount of Sieber, Rink, Wang, chlorotrityl resin, or PL-FMP resin. The procedure can be scaled beyond 0.05 mmol by adjusting the listed volumes by a factor of 1. To a 50 mL Falcon tube, resin and 2.0–5.0 mL of cleavage cocktail (TFA:TIS:DTT, v / v / w = 95:5:1) were added. The volume of cleavage cocktail used for each individual linear peptide can be varied. Generally, the greater the number of protecting groups present on the peptide side chains, the larger the volume of cleavage cocktail required. The mixture was shaken at room temperature for 1–2 hours, usually approximately 1.5 hours. 35–50 mL of cold diethyl ether was added to the suspension. Vigorous mixing of the mixture precipitated a significant amount of white solid. The mixture was centrifuged for 3–5 minutes, and then the solution was decanted from the solid and discarded. The solid was suspended in EtO (30-40 mL); the mixture was then centrifuged for 3-5 min; the solution was decanted from the solid and discarded. Finally, the solid was suspended in EtO (30-40 mL); the mixture was centrifuged for 3-5 min; the solution was decanted from the solid and discarded. After drying under a stream of nitrogen and / or house vacuum, the crude peptide, along with the cleaved resin, was obtained as a white to off-white solid. The crude material was used for the cyclization step the same day.

[0210] Global Deprotection Method B: Unless otherwise stated, all operations were performed manually. The "Global Deprotection Method" procedure describes experiments performed at a 0.050 mmol scale, determined by the amount of Sieber, Rink, Wang, chlorotrityl resin, or PL-FMP resin. The procedure can be scaled beyond 0.05 mmol by adjusting the stated volumes by a factor of 1. To a 30 mL Bio-Rad poly-prep chromatography column, the resin and 2.0–5.0 mL of cleavage cocktail (TFA:TIS:HO:DTT, v / v / w = 94:3:3:1) were added. The volume of cleavage cocktail used for each individual linear peptide can be varied. Generally, the greater the number of protecting groups present on the peptide side chains, the larger the volume of cleavage cocktail required. The mixture was shaken at room temperature for 1–2 h, typically approximately 1.5 h. The acidic solution was drained into 40 mL of cold diethyl ether, and the resin was washed twice with 0.5 mL of TFA solution. The mixture was centrifuged for 3-5 minutes, and then the solution was decanted from the solid and discarded. The solid was suspended in EtO (35 mL); the mixture was then centrifuged for 3-5 minutes; the solution was decanted from the solid and discarded. Finally, the solid was suspended in EtO (35 mL); the mixture was centrifuged for 3-5 minutes; the solution was decanted from the solid and discarded. After drying under a stream of nitrogen and / or house vacuum, the crude peptide was obtained as a white to off-white solid. The crude was used for the cyclization step the same day.

[0211] Cyclization method A: Unless otherwise noted, all operations were performed manually. The procedure for "Cyclization Method A" describes experiments performed at a 0.05 mmol scale, determined by the amount of Sieber, Rink, Wang, or PL-FMP resin used to generate the peptide. This scale is not based on a direct determination of the amount of peptide used in the procedure. The procedure can be scaled beyond the 0.05 mmol scale by adjusting the listed volumes by a factor of 1. The crude peptide solid from global deprotection was dissolved in DMF (30-45 mL) in a 50 mL centrifuge tube at room temperature, and DIEA (1.0-2.0 mL) was added to the solution. The pH of the reaction mixture was adjusted to 8. The solution was then shaken at room temperature for several hours, overnight, or 2-3 days. The reaction solution was concentrated to dryness using a speedvac or genevac EZ-2, and the crude residue was then dissolved in DMF or DMF / DMSO (2 mL). After filtration, the solution was subjected to single-compound reverse-phase HPLC purification to obtain the desired cyclic peptide.

[0212] Cyclization method B: Unless otherwise noted, all operations were performed manually. The procedure for "Cyclization Method B" describes experiments performed at a 0.05 mmol scale, determined by the amount of Sieber, Rink, Wang, or PL-FMP resin used to generate the peptide. This scale is not based on a direct determination of the amount of peptide used in the procedure. The procedure can be scaled beyond the 0.05 mmol scale by adjusting the stated volumes by a factor of 1. The crude peptide solid in a 50 mL centrifuge tube was dissolved in a 0.1 M aqueous solution of ammonium bicarbonate in CH3CN (1:1, v / v, 30-45 mL). The solution was then shaken at room temperature for several hours. The reaction solution was checked by pH paper and LCMS, and the pH can be adjusted to above 8 by adding 0.1 M aqueous ammonium bicarbonate (5-10 mL). After completion of the reaction based on the disappearance of the linear peptide by LCMS, the reaction was concentrated to dryness in a speedvac or genevac EZ-2. The resulting residue was charged with CHCN:HO (2:3, v / v, 30 mL) and concentrated to dryness in a speedvac or genevac EZ-2. This procedure was repeated (usually twice). The resulting crude solid was then dissolved in DMF, DMF / DMSO, or CHCN / HO / formic acid. After filtration, the solution was subjected to single-compound reverse-phase HPLC purification to yield the desired cyclic peptide.

[0213] N-Methylation on-resin method A. To the resin (50 μmol) in a Bio-Rad tube was added CHCl (2 mL) and shaken at room temperature for 5 minutes. 2-Nitrobenzene-1-sulfonyl chloride (44.3 mg, 200 μmol, 4 equiv.) was added, followed by 2,4,6-trimethylpyridine (0.040 mL, 300 μmol, 6 equiv.). The reaction was shaken at room temperature for 2 hours. The solvent was drained, and the resin was rinsed with CHCl (3 x 5 mL), DMF (3 x 5 mL), and THF (3 x 5 mL). THF (1 mL) was added to the resin. Triphenylphosphine (65.6 mg, 250 μmol, 5 equiv.), methanol (0.020 mL, 500 μmol, 10 equiv.), and diethyl azodicarboxylate or DIAD (0.040 mL, 250 μmol, 5 equiv.) were added. The mixture was shaken at room temperature for 2-16 hours. The reaction was repeated. Triphenylphosphine (65.6 mg, 250 μmol, 5 equiv.), methanol (0.020 mL, 500 μmol, 10 equiv.), and diethyl azodicarboxylate or DIAD (0.040 mL, 250 μmol, 5 equiv.) were added. The mixture was shaken at room temperature for 1-16 hours. The solvent was drained, and the resin was washed with THF (5 mL × 3) and CHCl3 (5 mL × 3). The resin was air-dried and used directly in the next step. The resin was shaken in DMF (2 mL). 2-Mercaptoethanol (39.1 mg, 500 μmol) was added, followed by DBU (0.038 mL, 250 μmol, 5 equiv.). The reaction was shaken for 1.5 hours. The solvent was drained. The resin was washed with DMF (4 times). It was air-dried and used directly in the next step.

[0214] N-Methylation on-resin Method B (Turner, RA et al., Org. Lett., 15(19):5012-5015 (2013)). All operations were performed manually unless otherwise noted. The procedure for "N-Methylation on-resin Method A" describes experiments performed at a 0.100 mmol scale, which is determined by the amount of Sieber or Rink linker attached to the resin used to generate the peptide. This scale is not based on a direct determination of the amount of peptide used in the procedure. The procedure can be scaled beyond the 0.10 mmol scale by adjusting the stated volumes by a factor of the scale. The resin was transferred to a 25 mL fritted syringe. Piperidine:DMF (20:80 v / v, 5.0 mL) was added to the resin; the mixture was shaken for 3 minutes, and then the solution was drained through the frit. The resin was washed three times with DMF (4.0 mL). Piperidine:DMF (20:80 v / v, 4.0 mL) was added to the reaction vessel. The mixture was shaken for 3 minutes, and then the solution was drained through a frit. The resin was washed successively with three portions of DMF (4.0 mL) and three portions of DCM (4.0 mL). The resin was suspended in DMF (2.0 mL), ethyl trifluoroacetate (0.119 mL, 1.00 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.181 mL, 1.20 mmol). The mixture was placed on a shaker for 60 minutes. The solution was drained through a frit. The resin was washed successively with three portions of DMF (4.0 mL) and three portions of DCM (4.0 mL). The resin was washed three times with dry THF (2.0 mL) to remove residual water. To an oven-dried 4.0 mL vial was added THF (1.0 mL) and triphenylphosphine (131 mg, 0.500 mmol) on dry 4 Å molecular sieves (20 mg). The solution was transferred to the resin, and diisopropyl azodicarboxylate (0.097 mL, 0.5 mmol) was slowly added. The resin was agitated for 15 min. The solution was drained through a frit, and the resin was washed three times with dry THF (2.0 mL) to remove residual water. To an oven-dried 4.0 mL vial was added THF (1.0 mL), triphenylphosphine (131 mg, 0.50 mmol) on dry 4 Å molecular sieves (20 mg).The solution was transferred to the resin, and diisopropyl azodicarboxylate (0.097 mL, 0.5 mmol) was added slowly. The resin was stirred for 15 minutes. The solution was drained through a frit. The resin was washed successively with three portions of DMF (4.0 mL) and three portions of DCM (4.0 mL). The resin was suspended in ethanol (1.0 mL) and THF (1.0 mL), and sodium borohydride (37.8 mg, 1.000 mmol) was added. The mixture was stirred for 30 minutes and drained. The resin was washed successively with three portions of DMF (4.0 mL) and three portions of DCM (4.0 mL).

[0215] N-Alkylated On-Resin Procedure A: To the nosylated resin (0.186 g, 0.100 mmol), a solution of the alcohol corresponding to the alkylating group (0.046 g, 1.000 mmol), triphenylphosphine (0.131 g, 0.500 mmol), and DIAD (0.097 mL, 0.500 mmol) in 3 mL of THF was added, and the reaction mixture was stirred at room temperature for 16 h. The resin was washed three times with THF (5 mL), and the above procedure was repeated one to three times. The progress of the reaction was monitored by TFA microcleavage of a small resin sample treated with a solution of 50 μL of TIS in 1 mL of TFA for 1.5 h.

[0216] N-Alkylated On-Resin Procedure B: The nosylated resin (0.100 mmol) was washed three times with N-methylpyrrolidone (NMP) (3 mL). A solution of NMP (3 mL), alkyl bromide (20 equiv., 2.000 mmol), and DBU (20 equiv., 0.301 mL, 2.000 mmol) was added to the resin, and the reaction mixture was stirred at room temperature for 16 h. The resin was washed with NMP (3 mL), and the above procedure was repeated once more. The progress of the reaction was monitored by TFA microcleavage of a small resin sample treated with a solution of 50 μL of TIS in 1 mL of TFA for 1.5 h.

[0217] N-Nosylate Formation Procedure: A solution of collidine (10 equiv.) in DCM (2 mL) was added to the resin, followed by a solution of Nos-Cl (8 equiv.) in DCM (1 mL). The reaction mixture was stirred at room temperature for 16 h. The resin was washed three times with DCM (4 mL) and three times with DMF (4 mL). Alternating washes of DCM and DMF were repeated three times, followed by one final set of four DCM washes (4 mL).

[0218] N-Nosylate Removal Procedure: The resin (0.100 mmol) was swelled using three washes with DMF (3 mL) and three washes with NMP (3 mL). A solution of NMP (3 mL), DBU (0.075 mL, 0.500 mmol), and 2-mercaptoethanol (0.071 mL, 1.000 mmol) was added to the resin, and the reaction mixture was stirred at room temperature for 5 min. After filtration and washing with NMP (3 mL), the resin was re-treated with a solution of NMP (3 mL), DBU (0.075 mL, 0.500 mmol), and 2-mercaptoethanol (0.071 mL, 1.000 mmol) for 5 min at room temperature. The resin was washed three times with NMP (3 mL), four times with DMF (4 mL), and four times with DCM (4 mL) and returned to the Symphony reaction vessel for completion of sequence assembly on the Symphony peptide synthesizer.

[0219] General procedure for preloading PL-FMP resin with amines: PL-FMP resin (Novabiochem, 1.00 mmol / g substitution) was swollen in DMF (20 mL / mmol) at room temperature. The solvent was drained, and 10 mL of DMF was added, followed by the amine (2.5 mmol) and acetic acid (0.3 mL). After stirring for 10 minutes, sodium triacetoxyborohydride (2.5 mmol) was added. The reaction was stirred overnight. The resin was washed with DMF (1×), THF / HO / AcOH (6:3:1) (2×), DMF (2×), and DCM (3×) and dried. The resulting amine-preloaded PL-FMP resin was confirmed by the following method: 100 mg of the resin was reacted with benzoyl chloride (5 equiv.) and DIEA (10 equiv.) in DCM (2 mL) at room temperature for 0.5 hours. The resin was washed with DMF (2x), MeOH (1x), and DCM (3x). The sample was then cleaved with 40% TFA / DCM (1 h). The product was collected and analyzed by HPLC and MS. The collected sample was dried and weighed to calculate the resin loading.

[0220] General procedure for preloading Fmoc-amino acids onto Cl-trityl resin: 2-Chloro-chlorotrityl resin mesh 50-150 (1.54 meq / gram, 1.94 grams, 3.0 mmol) was added to a glass reaction vessel equipped with a frit and allowed to swell in DCM (5 mL) for 5 minutes. A solution of the acid (3.00 mmol, 1.0 equiv.) in DCM (5 mL) was added to the resin, followed by DIPEA (2.61 mL, 15.00 mmol, 5.0 equiv.). The reaction was shaken at room temperature for 60 minutes. DIEA (0.5 mL) and methanol (3 mL) were added and shaken for an additional 15 minutes. The reaction solution was filtered through a frit, and the resin was rinsed with DCM (4 × 5 mL), DMF (4 × 5 mL), DCM (4 × 5 mL), diethyl ether (4 × 5 mL), and dried using a stream of nitrogen. The resin loading can be determined as follows:

[0221] A resin sample (13.1 mg) was treated with 20% piperidine / DMF (v / v, 2.0 mL) by shaking for 10 minutes. 1 mL of this solution was transferred to a 25.0 mL volumetric flask and diluted with methanol to a total volume of 25.0 mL. A blank solution of 20% piperidine / DMF (v / v, 1.0 mL) was diluted to 25.0 mL with methanol in a volumetric flask. The UV was set to 301 nm and 0 on the blank solution, and the solution was subsequently read, resulting in an absorbance of 1.9411. (1.9411 / 20 mg) * 6.94 = 0.6736. The resin loading was determined to be 0.6736 mmol / g.

[0222] Click reaction on resin method A: This procedure describes experiments performed at the 0.050 mmol scale. It can be scaled above or below the 0.050 mmol scale by adjusting the volumes listed. The alkyne-containing resins (50 μmol each) were transferred to Bio-Rad tubes and swelled with DCM (2 × 5 mL × 5 min) followed by DMF (2 × 5 mL × 5 min). A 200 mL bottle was charged with 30 portions of the following: ascorbic acid (vitamin C, 0.026 g, 0.150 mmol), bis(2,2,6,6-tetramethyl-3,5-heptanedionato)copper(II) (10.75 mg, 0.025 mmol), DMF (1.5 mL), 2,6-lutidine (0.058 mL, 0.50 mmol), and THF (1.5 mL), followed by DIEA (0.087 mL, 0.50 mmol) and the corresponding azide used in the examples (1.0–2.0 equiv.). The mixture was stirred until everything was dissolved. The DMF in the Bio-Rad tubes was drained, and the click solution (3 mL each) was added to each Bio-Rad tube. The tubes were shaken overnight on an orbital shaker. The solution was drained through the frit. The resin was washed with DMF (3 x 2 mL) and DCM (3 x 2 mL).

[0223] Click reaction on resin method B: This procedure describes experiments performed at a 0.050 mmol scale. It can be scaled above or below the 0.050 mmol scale by adjusting the volumes listed. The alkyne-containing resins (50 μmol each) were transferred to Bio-Rad tubes and swollen with DCM (2 × 5 mL × 5 min) followed by DMF (2 × 5 mL × 5 min). In a separate bottle, nitrogen was bubbled through 4.0 mL of DMSO for 15 min. To the DMSO was added copper iodide (9.52 mg, 0.050 mmol, 1.0 equiv.) (sonicated), lutidine (58 μL, 0.500 mmol, 10.0 equiv.), and DIEA (87 μL, 0.050 mmol, 10.0 equiv.). The solution was again purged with nitrogen. The DCM was drained through a frit. In a separate vial, ascorbic acid (8.8 mg, 0.050 mmol, 1.0 equiv.) was dissolved in water (600 μL). This solution was sparged with nitrogen for 10 minutes. The coupling partner was dispensed into the tube (0.050–0.10 mmol, 1.0–2.0 equiv.), followed by the DMSO copper and base solutions, and finally the aqueous ascorbic acid solution. The solution was blanketed with nitrogen and capped. The tube was placed on a rotating mixer for 16 hours. The solution was drained through a frit. The resin was washed with DMF (3 × 2 mL) and DCM (3 × 2 mL).

[0224] Suzuki reaction-on-resin procedure: A BioRad tube was charged with 50 μmol of dried Rink resin, an N-terminal Fmoc-protected linear polypeptide containing a 4-bromophenylalanine side chain. The resin was swollen in DMF (2 × 5 mL). To this was added a DMF solution (2 mL) of p-tolylboronic acid (0.017 g, 0.125 mmol), potassium phosphate (0.2 mL, 0.400 mmol), followed by the catalyst [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) [PdCl2(dtbpf)] (3.26 mg, 5.00 μmol). The tube was shaken overnight at room temperature. The solution was drained, and the resin was washed with DMF (5 × 3 mL), followed by alternating washes with DCM (2 × 3 mL), then DMF (2 × 3 mL), then DCM (5 × 3 mL). A small sample of the resin was microcleaved using 235 μL of TIS in 1 mL of TFA for 1 h at room temperature. The remaining resin was used in the next step of N-terminal peptide coupling or chloroacetic acid capping.

[0225] Solution-phase click reaction method A: To a 20 mL scintillation vial was added 100x sodium ascorbate (sodium (R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate) and copper(II) sulfate pentahydrate (CuSO4:sodium ascorbate molar ratio: 1:3 to 1:5). The reaction was diluted with water. The solution was shaken at room temperature for 1 to 10 minutes. The resulting yellowish slurry was added to the reaction mixture.

[0226] To a vial containing the alkyne and azide (1.0–2.0 equiv.), an aliquot of the above copper solution (CuSO: 0.3–1.0 equiv. alkyne) was added. The mixture was shaken at room temperature for 1–3 h, and progress was monitored by LC / MS. If necessary, additional amounts of azide or copper solution could be added to promote triazole formation. Upon completion, the mixture was diluted with CHCN:NHCO solution (1:1 v / v), filtered, and purified the same day by reverse-phase HPLC purification.

[0227] Solution-phase click reaction method B: Stock solutions of CuSO4 and sodium ascorbate were prepared by diluting a 1:2–1:3 molar ratio of dried copper(II) sulfate pentahydrate and sodium ascorbate to a concentration of 0.1–0.3 M relative to copper sulfate pentahydrate. To a solution of the peptide alkyne in DMF (0.05–0.1 M), the corresponding azide (1.0–2.0 equiv.) used in the examples was added, followed by the freshly prepared aqueous copper solution (0.03–1.0 equiv.) described above. The mixture was stirred at room temperature and monitored by LCMS. Additional amounts of azide or copper solution could be added to promote triazole formation, if necessary. Upon complete conversion, the mixture was diluted, filtered, and purified by reverse-phase HPLC the same day.

[0228] Symphony Dde / ivDde Deprotection Procedure: The reaction vessel containing the resin from the previous step was washed twice in succession as follows: for each wash, DMF (2.5 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. The resin was washed five times in succession as follows: for each wash, a solution of hydrazine in DMF (2% v / v, 2.5 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 5 minutes, after which the solution was drained through the frit. The resin was washed six times in succession as follows: for each wash, DMF (2.5 mL) was added through the top of the vessel, and the resulting mixture was periodically agitated for 30 seconds, after which the solution was drained through the frit. The resulting resin was used directly in the next step.

[0229] General purification procedure: The crude material was purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm × 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; Gradient: Hold at a specific percentage of B for 0 min, then linearly increase from this percentage to a higher percentage of B over 20–30 min, followed by a hold at 100% B for 0 min; Flow rate: 20–40 mL / min; Column temperature: 25 °C. Fraction collection was initiated by MS and UV signals. Fractions containing the desired product were combined and dried via centrifugal evaporation. If the material was not pure based on orthogonal analytical data, it was further purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 30 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with ammonium acetate; Gradient: Hold at a specific percentage of B for 0 min, then linear increase from the starting percentage of B over 20-30 min, then hold at 100% B for 0 min; Flow rate: 20-40 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. Fractions containing the desired product were combined and dried via centrifugal evaporation. Product yield and purity were determined.

[0230] Alternatively, based on the initial analytical data, the crude material was purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm × 30 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with ammonium acetate; Gradient: Hold at a specific percentage of B for 0 min, then linear increase from the starting percentage of B over 20 min, then hold at 100% B for 0 min; Flow rate: 40 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. Fractions containing the desired product were combined and dried via centrifugal evaporation. If the material was not pure based on orthogonal analytical data, it was further purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm × 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; Gradient: Hold at a specific percentage of B for 0 min, then linearly increase from this percentage to a higher percentage of B over 20 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was initiated by MS and UV signals. Fractions containing the desired product were combined and dried via centrifugal evaporation. Product yield and purity were determined.

[0231] Fatty Acid Chain Coupling Procedure A: To the synthesized peptide, DMF (2.0 mL), fatty acid activated ester (0.077–0.205 mmol, 1.5–4.0 equiv.), and DIEA (0.036–0.072 mL, 0.205 mmol, 4.0–8.0 equiv.) were added. The reaction mixture was shaken for 1 h. The reaction mixture was neutralized with a few drops of acetic acid and subjected to purification.

[0232] Fatty acid chain coupling procedure B: To the synthesized peptide, DMF (2.0 mL), fatty activated ester (0.077–0.205 mmol, 1.5–4.0 equiv.), and DIEA (0.036–0.072 mL, 0.205 mmol, 4.0–8.0 equiv.) were added. The reaction mixture was shaken for 1 h. The reaction mixture was concentrated to dryness using a Biotage V10 apparatus. 2.0 mL of TFA / water (90:10, v:v) solution was added to the crude PDTS and shaken for 20 min. The reaction mixture was then concentrated to dryness and redissolved in 2.0 mL of DMF for purification.

[0233] Unnatural Amino Acid Synthesis: Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)-3-(1-(2-(tert-butoxy)-2-oxoethyl)-1H-indol-3-yl)propanoic acid Scheme: [ka]

[0234] Step 1: To a solution of (S)-benzyl 2-((((benzyloxy)carbonyl)amino)-3-(1H-indol-3-yl)propanoate (25.0 g, 58.3 mmol) and cesium carbonate (20.9 g, 64.2 mmol) in DMF (200 mL) at 0° C. was added tert-butyl 2-bromoacetate (9.36 mL, 64.2 mmol). The solution was allowed to warm slowly to room temperature with stirring for 18 hours. The reaction mixture was cooled to ice-water:aqueous 1N The resulting solid was poured into HCl (1:1) and extracted with EtOAc. The organic layer was washed with brine, collected, dried over MgSO, filtered, and concentrated in vacuo. The resulting solid was subjected to flash chromatography (330 g column, 0-50% EtOAc:Hex over 20 column volumes) to afford (S)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(1-(2-(tert-butoxy)-2-oxoethyl)-1H-indol-3-yl)propanoate as a white solid (29.6 g, 93%).

[0235] Step 2: H2 was slowly bubbled through a mixture of (S)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(1-(2-(tert-butoxy)-2-oxoethyl)-1H-indol-3-yl)propanoate (29.6 g, 54.5 mmol) and Pd—C (1.45 g, 1.36 mmol) in MeOH (200 mL) at room temperature for 10 min. The mixture was then stirred under a positive pressure of H2, and the conversion was monitored by LCMS. After 48 h, the reaction mixture was filtered through diatomaceous earth and evaporated to give crude (S)-2-amino-3-(1-(2-(tert-butoxy)-2-oxoethyl)-1H-indol-3-yl)propanoic acid (17.0 g), which was used in Step 3 without further purification.

[0236] Step 3: To a solution of (S)-2-amino-3-(1-(2-(tert-butoxy)-2-oxoethyl)-1H-indol-3-yl)propanoic acid (5.17 g, 16.2 mmol) and sodium bicarbonate (6.8 g, 81 mmol) in acetone:water (50.0 mL:100 mL) was added (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl)carbonate (5.48 g, 16.2 mmol). The mixture was stirred overnight, at which point LCMS analysis indicated complete conversion. The vigorously stirred mixture was acidified by the slow addition of aqueous 1 N HCl. Once acidified, the mixture was diluted with DCM (150 mL), and the isolated organic phase was then washed with water followed by brine. The organic layer was collected, dried over sodium sulfate, and concentrated in vacuo to give the crude product. The crude material was purified by silica gel chromatography (330 g column, 20-80% EtOAc:Hex over 20 columns, 25 volumes) to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(2-(tertbutoxy)-2-oxoethyl)-1H-indol-3-yl)propanoic acid as a white foam (7.26 g, 83%). 1H NMR(500MHz, methanol-d4)δ 7.80(d,J=7.6Hz,2H),7.67-7.60(m,2H),7.39(t,J=7.5Hz,2H),7.32-7.22(m,3H),7.18(td,J=7.6,0.9Hz,1H) ,7.08(td,J=7.5,0.9Hz,1H),7.04(s,1H),4.54(dd,J=8.4,4.9Hz,1H),4.36-4.23(m,2H),4.23-4.14(m,1H),30 3.43-3.35(m, 2H), 3.25-3.09(m, 1H), 1.55-1.38(m, 9H). ESI-MS (+) m / z = 541.3 (M+H).

[0237] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(2-(tert-butoxy)-2-oxoethoxy)phenyl)propanoic acid Scheme: [ka]

[0238] Step 1: To a cooled, stirred solution of (S)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoate (70 g, 173 mmol) and K2CO3 (35.8 g, 259 mmol) in DMF (350 mL) was added tert-butyl-2-bromoacetate (30.6 mL, 207 mmol) dropwise, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with 10% brine solution (1000 mL) and extracted with ethyl acetate (2 x 250 mL). The combined organic layers were washed with water (500 mL), saturated brine solution (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a colorless gum. The crude compound was purified by flash column chromatography using 20% ​​ethyl acetate in petroleum ether as eluent to give a white solid (78 g, 85%).

[0239] Step 2: (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(2-(tert-butoxy)-2-oxoethoxy)phenyl)propanoate (73 g, 140 mmol) was dissolved in MeOH (3000 mL) and purged with nitrogen for 5 minutes. To the above purged mixture was added Pd / C (18 g, 16.91 mmol) and stirred at 3 kg hydrogen pressure for 15 hours. The reaction mixture was filtered through a bed of diatomaceous earth (Celite®) and washed with methanol (1000 mL). The filtrate was concentrated in vacuo to give a white solid (36 g, 87%).

[0240] Step 3: To a stirred solution of (S)-2-amino-3-(4-(2-(tert-butoxy)-2-oxoethoxy)phenyl)propanoic acid (38 g, 129 mmol) and sodium bicarbonate (43.2 g, 515 mmol) in water (440 mL) was added Fmoc-OSu (43.4 g, 129 mmol) dissolved in dioxane (440 mL) dropwise, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with 1.5 N HCl (200 mL) and water (500 mL) and extracted with ethyl acetate (2 × 250 mL). The combined organic layers were washed with water (250 mL), saturated brine solution (250 mL), dried over NaSO, filtered, and concentrated to give a pale yellow gum. The crude compound was purified by column chromatography using 6% MeOH in chloroform as eluent to give a pale green gum. The gum was further triturated with petroleum ether to give an off-white solid (45g, 67%). 1 H NMR(400MHz,DMSO-d6)δ 12.86-12.58(m,1H),7.88(d,J=7.5Hz,2H),7.73-7.61(m,3H),7.58-7.47(m,1H),7.44-7.27 (m,4H),7.18(d,J=8.5Hz,2H),6.79(d,J=8.5Hz,2H),4.57(s,2H),4.25-4.10(m,4H),3.34(br s,3H),3.02(dd,J=13.8,4.3Hz,1H),2.81(dd,J=14.1,10.5Hz,1H),1.41(s,9H).

[0241] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxycarbonyl)phenyl)propanoic acid Scheme: [ka]

[0242] Step 1. (S) Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoate (10 g, 24.66 mmol) was placed in DCM (100 mL) in a 250 mL multi-necked round-bottom flask with magnetic stirring and a N outlet. The reaction mixture was cooled to -40°C, pyridine (5.49 mL, 67.8 mmol) was added slowly, and then stirred at the same temperature for 20 minutes. Subsequently, trifluoromethanesulfonic anhydride (11.46 mL, 67.8 mmol) was added slowly at -40°C, and the mixture was stirred at -40°C for 2 hours. The reaction mixture was quenched with water at -10°C, and then citric acid solution (50 mL) was added. The organic layer was extracted into DCM and the separated organic layer was dried over anhydrous NaSO, filtered and then evaporated to give (S)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(((trifluoromethyl)sulfonyl)oxy)phenyl)propanoate (11.93 g, 22.20 mmol, 90% yield) as a pale yellow solid.

[0243] Step 2. The DMF solution (1500 mL) was purged with nitrogen for 10 minutes. To this was added sodium formate (114 g, 1676 mmol) and acetic anhydride (106 mL, 1123 mmol). Purging was continued and the mixture was cooled to 0° C. DIPEA (194 mL, 1111 mmol) was added and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour.

[0244] DMF (3200 mL) was added to a 10-liter autoclave, and the system was purged with nitrogen. Under nitrogen purging conditions, (S)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-(((trifluoromethyl)sulfonyl)oxy)phenyl)propanoate (300 g, 558 mmol), lithium chloride (71 g, 1675 mmol), 1,3-bis(diphenylphosphino)propane (24.17 g, 58.6 mmol) were added, followed by palladium(II) acetate (12.9 g, 57.5 mmol). The solution prepared above was added to this reaction mixture, which was then heated to 80°C for 16 hours.

[0245] The reaction mass was diluted with ethyl acetate and water. The phases were separated, and the ethyl acetate layer was washed with water and brine solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude material was loaded onto a Trent column and eluted with petroleum ether and ethyl acetate. The fraction from 30% to 65% ethyl acetate in petroleum ether was concentrated to give a cream-colored solid (300 g), which was dissolved in ethyl acetate (700 mL), and petroleum ether was added slowly. At approximately 20% ethyl acetate in petroleum ether, a white solid precipitated, which was filtered and washed with 20% ethyl acetate in petroleum ether to give a white solid (180 g, 74% yield).

[0246] Step 3. A 2000 mL multi-necked round-bottom flask was charged with (S)-4-(3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropyl)benzoic acid (130 g, 300 mmol), dichloromethane (260 mL), and cyclohexane (130 mL). To the slurry reaction mixture was added BF3.OEt2 (3.80 mL, 30.0 mmol) at room temperature, followed by the slow addition of tert-butyl 2,2,2-trichloroacetylimidate (262 g, 1200 mmol) over 30 minutes at room temperature. Upon addition, the slurry began to slowly dissolve and was completely dissolved by the end of the addition. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with DCM, and the remaining solids were removed by filtration. The filtrate was concentrated and purified by flash chromatography. The crude material was purified by Torrent using a 1.5 kg silicon cycle column. The product spot was eluted with a 15% ethyl acetate / petroleum ether mixture, and the collected fractions were concentrated to give a colorless liquid (120 g, 82% yield).

[0247] Step 4. (S)-tert-Butyl 4-(3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropyl)benzoate (200 g, 409 mmol) was dissolved in MeOH (4000 mL) and purged with N for 10 minutes. Pd / C (27.4 g, 25.7 mmol) was added. The reaction was shaken under H at room temperature for 16 hours. The reaction mass was filtered through a bed of Celite and the bed was washed with methanol. The resulting filtrate was concentrated to give a pale yellow solid. The resulting solid was stirred with a 5% methanol:diethyl ether mixture for 15 minutes, then filtered and dried under vacuum to give a pale yellow solid. This was slurried in 5% methanol in diethyl ether, stirred for 15 minutes, filtered, and dried to give (S)-2-amino-3-(4-(tert-butoxycarbonyl)phenyl)propanoic acid as a white solid (105 g, 97% yield). Analytical Condition E: Retention time = 0.971 min; ESI-MS (+) m / z [M+H] + :266.2.

[0248] Step 5. (S) 2-Amino-3-(4-(tert-butoxycarbonyl)phenyl)propanoic acid (122 g, 460 mmol) was dissolved in acetone (1000 mL), followed by the addition of water (260 mL) and sodium bicarbonate (116 g, 1380 mmol). This was cooled to 0 °C, and Fmoc-OSu (155 g, 460 mmol) was added portionwise to the reaction mixture. After the addition was complete, this was stirred at room temperature for 16 h. The reaction mixture was diluted with dichloromethane (2 L), and then water was added (1.5 L). The organic layer was washed with saturated citric acid solution, extracted, and the aqueous layer was extracted again with DCM. The combined organic layers were washed with 10% citric acid solution, brine solution, dried over Na2SO4, and evaporated to dryness. The resulting white solid was slurried with diethyl ether, filtered, and dried to give the desired product as a white solid (80 g, 35% yield). 1 H NMR(400MHz,DMSO-d6)δ 7.87(d,J=7.5Hz,2H),7.83-7.73(m,3H),7.60(t,J=8.5Hz,2H),7.51-7.24(m,7H),4.26-4.11(m,4H),3.45-3.27(m,4H),3.17(br dd,J=13.8,4.3Hz,1H),2.94(dd,J=13.5,11.0Hz,1H),2.52-2.48(m,4H),1.51(s,9H).

[0249] Preparation of tert-butyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate Scheme: [ka]

[0250] Step 1. To a solution of (R)-2-amino-3-chloropropanoic acid hydrochloride (125 g, 781 mmol) in a 1:1 mixture of acetone (1 L) and water (1 L) was added Na2CO3 (182 g, 1719 mmol), followed by Fmoc-OSu (250 g, 742 mmol). The reaction was stirred overnight at room temperature. It was then added to ethyl acetate (2 × 500 mL) and the layers were acidified with 5 N HCl. The HCl solution was extracted with ethyl acetate (1500 mL, then 2 × 500 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated to give the crude product (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-chloropropanoic acid. The product (220 g) was used directly in the next step.

[0251] Step 2. A solution of (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-chloropropanoic acid (220 g, 636 mmol) in DCM (2 L) was cooled to −20° C. 2-Methylpropene (200 mL, 636 mmol) was bubbled through the solution for 15 min, then HSO (57.7 mL, 1082 mmol) was added and the mixture was stirred at room temperature overnight. Water (500 mL) was added to the reaction mixture. The layers were separated and the aqueous layer was extracted with DCM (2×500 mL). The combined organic layers were dried over anhydrous MgSO, filtered and evaporated. The crude product was purified by flash chromatography using petroleum ether and ethyl acetate as eluents. The desired fractions were combined and concentrated to give the product (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-chloropropanoate (83 g, 182 mmol, 29% yield).

[0252] Step 3. To a solution of (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-chloropropanoate (80 g, 199 mmol) in acetone (1000 mL) was added sodium iodide (119 g, 796 mmol) and the reaction was heated to reflux for 40 h. The acetone was removed by rotary evaporation and the crude product was diluted with water (1000 mL) and DCM (1000 mL). The layers were separated and the organic layer was washed with saturated aqueous sodium sulfite (1000 mL) and brine (1000 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude material was purified by flash chromatography using 7-9% ethyl acetate in petroleum ether. The desired product fractions were combined and concentrated to give the product (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (83 g, 156 mmol, 79%). 1 H NMR(400MHz,CDCl3)δ 7.77(d,J=7.5Hz,2H),7.62(d,J=7.5Hz,2H),7.45-7.30(m,4H),5.67(br d,J=7.0Hz,1H),4.54-4.32(m,3H),4.30-4.21(m,1H),3.71-3.50(m,2H),1.56-1.48(m,9H).

[0253] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methyl-1H-indol-3-yl)propanoic acid [ka]

[0254] Step 1. In a 100 mL three-neck flame-dried, nitrogen-purged round-bottom flask, zinc (2.319 g, 35.5 mmol) was added under an argon atmosphere, and the flask was heated to 150 °C using a hot gun and purged with argon. DMF (50 mL) was added to the reaction flask, followed by 1,2-dibromoethane (0.017 mL, 0.20 mmol) and TMS-Cl (0.026 mL, 0.20 mmol) under an argon atmosphere, and then stirred for 10 minutes. To the reaction mixture, (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (5 g, 10.14 mmol) was added, and the reaction was stirred for 1 hour. The progress of the reaction was monitored by TLC and LCMS until the starting iodide was completely converted to the Zn complex. The solution of organozinc reagent was cooled to room temperature, and then tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (0.23 g, 0.25 mmol), dicyclohexyl(2',6'-dimethoxy-[1,1'-biphenyl]-2-yl)phosphine (SPhos) (0.21 g, 0.51 mmol), and tert-butyl 3-bromo-2-methyl-1H-indole-1-carboxylate (3.77 g, 12.16 mmol) were added. The reaction mixture was stirred under a positive pressure of nitrogen at room temperature for 1 hour and then heated to 50 °C for 6 hours. The progress of the reaction was monitored by LCMS. The mixture was diluted with EtOAc (700 mL) and filtered through Celite. The organic phase was washed with saturated NH4Cl (250 mL), water (2 × 200 mL), and saturated NaCl (aq) (250 mL), dried over anhydrous Na2SO4, concentrated, and dried under vacuum to give the crude compound (19 g). This was purified by ISCO flash chromatography using a 330 g Redisep column, and the product was eluted with 7–9% ethyl acetate in petroleum ether. The above reaction and purification were repeated. Pure fractions were concentrated to give tert-butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)-2-methyl-1H-indole-1-carboxylate as a brownish solid (10.2 g, 95% purity, approximately 80% yield).Analysis condition G: retention time = 4.23 min; ESI-MS(+)m / z[M+2H][M-Boc-tBu+H]. + :441.2.

[0255] Step 2. To a 25 mL multi-necked round-bottom flask was added DCM (65 mL), followed by (S)-tert-butyl 3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)-2-methyl-1H-indole-1-carboxylate (6.5 g, 10.89 mmol) at room temperature under a nitrogen atmosphere. The reaction mixture was cooled to 0° C., and triethylsilane (4.18 mL, 26.1 mmol) was added, followed by the dropwise addition of TFA (5.87 mL, 76 mmol) at 0° C. The temperature of the reaction mixture was slowly brought to room temperature and stirred at room temperature for 4 hours. The progress of the reaction was monitored by TLC. To the reaction mixture was added TFA (5.87 mL, 76 mmol). The reaction mixture was stirred at room temperature overnight and concentrated under reduced pressure. The crude material was triturated with hexane and stored in the refrigerator to give a brown solid (crude weight: 6.5 g), which was purified by reverse-phase flash chromatography and the pure fractions were concentrated to give the desired final product as an off-white powder (2.3 g, 46%). 1 H NMR(DMSO-d6):δ ppm:10.65(s,1H),7.84(d,J=9.12Hz,2H),7.65(d,J=9.12Hz,2H),7.42-7.49(m,1H),7.30-7.38(m,2H),7.26-7.29(m,2H),7.17-7.19(m,2H) ),6.91-6.95(m,1H),6.85-6.88(t,J=7.85Hz,1H),4-16-4.18(m,2H), 4.01-4.06(m,1H),3.09-3.14(m,1H),2.96-2.99(m,1H),2.50(s,3H). Analysis conditions F: retention time = 1.37 minutes; ESI-MS(+)m / z[M+2H][M+H] + :441.2.

[0256] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(7-methyl-1H-indol-3-yl)propanoic acid [ka]

[0257] Step 1. A 50 mL round-bottom flask was charged with dry zinc (0.928 g, 14.19 mmol) and flushed with argon three times. The flask was then heated to 150° C. for 5 minutes, then cooled to room temperature and flushed with argon three times. DMF (20 mL) was added, followed by 1,2-dibromoethane (6.99 μl, 0.081 mmol) and TMS-Cl (0.013 mL, 0.10 mmol). Successful zinc insertion was accompanied by a noticeable exotherm. After 5 minutes, (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (2.0 g, 4.05 mmol) was added, and the reaction was stirred for 30 minutes. In a 50 mL round-bottom flask charged with argon, the above alkylzinc reagent, tert-butyl 3-bromo-7-methyl-1H-indole-1-carboxylate (1.26 g, 4.05 mmol), was added, followed by 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos) (0.083 g, 0.20 mmol) and Pd2(dba)3 (0.093 g, 0.101 mmol). After the addition, the reaction mixture was heated to 50 °C overnight. Another equivalent of SPhos and Pd2(dba)3 was added, and heating was continued for an additional 16 h. The reaction mixture was diluted with EtOAc (100 mL) and filtered through Celite. The organic phase was washed with saturated aqueous NH4Cl (100 mL), water (50 mL), and saturated NaCl (100 mL), dried over anhydrous Na2SO4, concentrated, and dried under vacuum. After purification by flash chromatography, the desired tert-butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)-2-methyl-1H-indole-1-carboxylate was obtained in 58% yield.

[0258] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methyl-1H-indol-3-yl)propanoic acid. TFA hydrolysis with triethylsilane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(7-methyl-1H-indol-3-yl)propanoic acid as an off-white solid in 64% yield after purification by reverse-phase flash chromatography. Analytical condition E: retention time = 2.16 min; ESI-MS (+) m / z [M+H] + :441.1. 1 H NMR(300MHz,DMSO-d6) shift 12.70(br s,1H),10.81(br s,1H),7.88(d,J=7.6Hz,2H),7.76-7.56(m,2H),7.49-7.21(m,5H),7.17(d,J=2.3Hz,1H),6.94-6.84(m,2H),4.29-4.13(m,3H),4.07(br s,1H),3.19(br dd,J=14.7,4.5Hz,1H),3.01(br dd,J=14.5,9.6Hz,1H),2.47-2.40(m,3H),0.02--0.06(m,1H).

[0259] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(quinolin-6-yl)propanoic acid [ka]

[0260] Step 1. A 25 mL round-bottom flask was charged with dry zinc (2.32 g, 35.5 mmol) and flushed with argon three times. The flask was heated to 150 °C for 5 minutes, then cooled to room temperature and flushed with argon three times. DMF (50 mL) was added, followed by 1,2-dibromoethane (0.017 mL, 0.20 mmol) and TMS-Cl (0.032 mL, 0.25 mmol). Successful zinc insertion was accompanied by a noticeable exotherm. After 5 minutes, (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (5.0 g, 10.14 mmol) was added, and the reaction was stirred for 30 minutes.

[0261] Into an argon-purged 250 mL round-bottom flask was added DMF (50 mL), a previously prepared solution of the alkylzinc reagent, 6-bromoquinoline (2.53 g, 12.16 mmol), (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (5.0 g, 10.14 mmol), followed by 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (RuPhos) (0.24 g, 0.51 mmol) and Pd2(dba)3 (0.23 g, 0.25 mmol). The reaction mixture was stirred at room temperature for 5 h and then heated to 50 °C for 16 h. It was cooled to room temperature, filtered through Celite, and rinsed with ethyl acetate. The solution was concentrated on a rotary evaporator. Purification by flash chromatography gave the desired compound as a viscous brown liquid in quantitative yield. Analytical Condition E: Retention time = 3.47 min; ESI-MS (+) m / z [M+H] + :495.2.

[0262] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methyl-1H-indol-3-yl)propanoic acid. TFA hydrolysis with triethylsilane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(quinolin-6-yl)propanoic acid as a beige solid in 40% yield after solid-liquid extraction with diethyl ether and water. 1 H NMR(300MHz,DMSO-d6)δ 8.94(br d,J=4.5Hz,1H),8.49(d,J=8.7Hz,1H),8.01-7.92(m,2H),7.85-7.79(m,3H),7.65(dd,J=8.3,4.5Hz,1H),7.55(dd,J=7.2,4.2 Hz,2H),7.36(t,J=7.4Hz,2H),7.26-7.14(m,2H),4.32(dd,J=10.6,4.5Hz,1H),4.18-4.08(m,3H),3.38-3.29(m,2H),3.11(br d,J=10.6Hz,1H),2.72(s,1H),1.07(t,J=7.0Hz,1H),-0.02(s,1H). Analysis condition E: retention time = 1.54 minutes; ESI-MS(+)m / z[M+H] + :439.0.

[0263] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-6-yl)propanoic acid [ka]

[0264] Step 1. To a 50 mL, three-neck, flame-dried round-bottom flask under an argon atmosphere was added zinc (1.392 g, 21.28 mmol), and the flask was heated to 150 °C using a hot gun and purged with argon. To the reaction was added DMF (30 mL), followed by 1,2-dibromoethane (10.48 μl, 0.12 mmol) and TMS-Cl (0.016 mL, 0.12 mmol) under argon. The reaction was stirred for 10 minutes. To the reaction mixture was added (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (3.0 g, 6.08 mmol), and the reaction was stirred for 1 hour. To the reaction mixture was added 6-bromoisoquinoline (1.52 g, 7.30 mmol) and bis-(triphenylphosphino)-palladium chloride (0.20 g, 0.30 mmol), and the reaction was stirred for 16 hours. The reaction mixture was diluted with ethyl acetate (50 mL), filtered through Celite, and washed with ethyl acetate (50 mL). The filtrate was concentrated under reduced pressure to give the crude product as a red, viscous gum. The crude product was purified by flash chromatography using 40-42% EtOAc in petroleum ether. After concentration on a rotary evaporator, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-6-yl)propanoate (2.0 g, 66%) was obtained as a yellow gum. Analysis condition B: retention time = 2.46 minutes; ESI-MS(+)m / z[[M+H] + :495.3.

[0265] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methyl-1H-indol-3-yl)propanoic acid. TFA hydrolysis with triethylsilane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-6-yl)propanoic acid as a gray solid in 90% yield after recrystallization in EtOAc and hexanes. 1H NMR(400MHz, methanol-d4)δ 9.55(s,1H),8.46(d,J=6.5Hz,1H),8.33(d,J=8.5Hz,1H),8.17(d,J=6.0Hz,1H),8 .08(s,1H),7.99-7.86(m,1H),7.78(dd,J=7.5,4.0Hz,2H),7.66-7.48(m,2H),7.4 3-7.30(m,2H),7.30-7.17(m,2H),4.68(dd,J=10.0,4.5Hz,1H),4.32-4.13(m,2H) ,4.12-3.84(m,1H),3.61(dd,J=13.8,4.8Hz,1H),3.32-3.26(m,1H),1.46(s,1H). Analysis condition B: retention time = 2.77 minutes; ESI-MS(+)m / z[M+H] + :439.2.

[0266] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-4-yl)propanoic acid [ka]

[0267] Step 1. To a stirred mixture of zinc (2.319 g, 35.5 mmol) in DMF (50 mL) was added dibromomethane (0.071 mL, 1.014 mmol) and TMS-Cl (0.130 mL, 1.014 mmol). An exotherm was observed. The reaction mixture was stirred for 10 minutes. (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (5 g, 10.14 mmol) was added and again an exotherm was observed. The reaction was stirred at room temperature for 1 hour. 2-Dicyclohexylphosphino-2',6'-dimethoxybiphenyl (0.21 g, 0.51 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.23 g, 0.25 mmol), and 4-bromoisoquinoline (2.11 g, 10.14 mmol) were added sequentially, and the reaction was heated to 50 °C for 16 h. The reaction mixture was cooled to room temperature and treated with saturated ammonium chloride solution (200 mL). The crude material was diluted with ethyl acetate (300 mL). The layers were separated, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography eluting with 30% ethyl acetate in petroleum ether to give tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-4-yl)propanoate (2.5 g, 50%). Analytical condition E: retention time = 3.44 min; ESI-MS (+) m / z [M+H] + :495.2.

[0268] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methyl-1H-indol-3-yl)propanoic acid. TFA hydrolysis afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-4-yl)propanoic acid as an off-white solid in quantitative yield after purification and diethyl ether trituration. 1H NMR(400MHz,DMSO-d6)δ 9.55(s,1H),8.52(s,1H),8.44-8.24(m,2H),8.18-8.00(m,1H),7.95-7.80(m,4H),7.59(br d,J=7.5Hz,1H),7.56(br d,J=7.5Hz,1H),7.47-7.34(m,2H),7.34-7.24(m,2H),4.46-4.30(m,1H),4.25-4.02( m,3H),3.69(dd,J=14.1,4.5Hz,1H),3.37(dd,J=14.1,10.5Hz,1H),0.10-0.11(m,1H). Analysis condition E: retention time = 1.57 minutes; ESI-MS(+)m / z[M+H] + :441.2.

[0269] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3,5-difluorophenyl)propanoic acid [ka]

[0270] Step 1. The compound was prepared following the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-4-yl)propanoate. Initial Negishi coupling with methyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate at 50 °C gave the desired methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-2,6-difluorophenyl)propanoate (5.5 g, 48.5% yield) after purification by flash chromatography. Analytical condition E: retention time = 3.99 min; ESI-MS (+) m / z [M+NH4] + :527.2.

[0271] Step 2. In a multi-necked round-bottom flask, methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3,5-difluorophenyl)propanoate (11 g, 21.59 mmol) was added, followed by tetrahydrofuran (132 mL) at room temperature under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and a solution of LiOH (1.09 g, 45.3 mmol) in water (132 mL) was added. The reaction was stirred for 3 h. It was concentrated under reduced pressure below 38 °C to remove the solvent. The crude compound was cooled to 0 °C. Citric acid solution was added to adjust the pH to 4-5. It was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (200 mL) followed by brine (200 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product (12 g) as a colorless, viscous mass. The crude compound was purified by ISCO using a 120 g redisep column, and the product was eluted with 20% ethyl acetate in petroleum ether. The reaction was concentrated to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3,5-difluorophenyl)propanoic acid (9.0 g, 82%, HPLC purity 97%) as a white, fluffy solid. Analytical condition E: retention time = 3.62 min; ESI-MS (+) m / z [M+H] + :513.2. 1 H NMR(CDCl3,400MHz)d 7.75(d,J=7.6Hz,2H),7.60(m,2H),7.39(t,J=7.6Hz,2H),7.30(m,2H),6.71(d,J=7.6Hz,2H ),5.26(m,1H),4.65(m,1H),4.48-4.38(m,2H),4.20(m,1H),3.14-2.99(m,1H),1.35(s,9H).

[0272] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-8-yl)propanoic acid [ka]

[0273] Step 1. Zinc (0.79 g, 12.00 mmol) was added to a flame-dried, nitrogen-purged, side-arm round-bottom flask. DMF (5 mL) was added via syringe, followed by a catalytic amount of iodine (0.16 g, 0.63 mmol). A color change in the DMF was observed from colorless to yellow and back again. Protected (R)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (1.97 g, 4.00 mmol) was added immediately, followed by a catalytic amount of iodine (0.16 g, 0.63 mmol). The solution was stirred at room temperature. Successful insertion of the zinc was accompanied by a noticeable exotherm. The solution of organozinc reagent was cooled to room temperature, followed by the sequential addition of Pd(dba) (0.088 g, 0.096 mmol), dicyclohexyl(2',6'-dimethoxy-[1,1'-biphenyl]-2-yl)phosphine (0.082 g, 0.200 mmol), and 8-bromoisoquinoline (1.082 g, 5.20 mmol). The reaction mixture was stirred at 50 °C under a positive nitrogen pressure for 4 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (200 mL), and passed through Celite. The organic phase was washed with saturated aqueous NHCl (200 mL), water (150 mL), and saturated aqueous NaCl (200 mL), dried over NaSO, concentrated, and dried under vacuum to give the crude compound. This was purified using ISCO Combiflash column chromatography (24 g silica gel column, hexane / ethyl acetate as eluent) to give (S)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-8-yl)propanoate (380 mg, 0.768 mmol, 19.21% yield). Analysis condition G: retention time = 2.59 min; ESI-MS (+) m / z [M+H] + :495.3.

[0274] Step 2. (S)-tert-Butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-8-yl)propanoate (380 mg, 0.768 mmol) was placed in a 50 mL round-bottom flask and dissolved in DCM (8 mL). Triethylsilane (0.31 mL, 1.92 mmol) was added, followed by trifluoroacetic acid (2.66 mL, 34.6 mmol). The reaction mixture was stirred at room temperature for 5 hours. The solvent was evaporated, and the residue was dissolved in diethyl ether. The product was precipitated by the addition of petroleum ether. The resulting powder was then triturated with petroleum ether to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-8-yl)propanoic acid (320 mg, 0.712 mmol, 93% yield) as an off-white solid. 1 H-NMR:(400MHz,DMSO-d6)δ ppm:12.98(bs,1H),9.79(s,1H),8.62(d,J=9.42Hz,1H),8.22(d,J=9.42Hz,1H),8.06(d,J=9.42Hz,1H),7.84-7.93(m,4H),7.74-7.76(m,1 H),7.56-7.58(m,1H),7.38-7.42(m,2H),(m,3H),7.26-7.30(m,2H),4 .41(m,1H),4.10-4.15(m,3H),3.731-3.66(m,1H),3.47-3.50(m,1H). Analysis condition G: retention time = 2.012 minutes; ESI-MS(+)m / z[M+H] + :439.2, purity 97.5%.

[0275] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(7-fluoro-1H-indol-3-yl)propanoic acid [ka]

[0276] Step 1. Synthesis of tert-butyl 6-fluoro-3-iodo-1H-indole-1-carboxylate from 6-fluoro-1H-indole: A solution of iodine (3.76 g, 14.80 mmol) in DMF (15 mL) was added dropwise to a solution of 6-fluoro-1H-indole (2 g, 14.80 mmol) and potassium hydroxide (2.076 g, 37.0 mmol) in DMF (15 mL) at room temperature, and the mixture was stirred for 45 minutes. The reaction mixture was then poured into 200 mL of ice water containing 0.5% ammonia and 0.1% sodium disulfite. The mixture was placed in a refrigerator to ensure complete precipitation. The precipitate was filtered, washed with 100 mL of ice water, and dried in vacuo to give 3.80 g. The solid was suspended in 25 mL of dichloromethane. 4-Dimethylaminopyridine (160 mg, 10 mol%) and di-tert-butyl dicarbonate (4.84 g, 22.20 mmol) were dissolved in dichloromethane (15 mL) and added to the reaction. The resulting mixture was stirred at room temperature for 30 minutes, washed with 0.1 N HCl (25 mL), and the aqueous phase was extracted with dichloromethane (3 × 35 mL, monitored by TLC). The combined organic layers were dried over sodium sulfate, and the solvent was removed under reduced pressure to give tert-butyl 6-fluoro-3-iodo-1H-indole-1-carboxylate (4.16 g, 11.52 mmol, 78% yield) as an orange solid. 1 H-NMR(CDCl3)δ ppm:7.82(d,J=8.23Hz,1H),7.68(s 1H),7.30-7.34(m,1H),7.03-7.08(m,1H),1.66(s,9H).

[0277] Step 2. The compound was prepared according to the same procedure as (S)-tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-8-yl)propanoate. An initial Negishi coupling at 50 °C gave the desired tert-butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)-7-fluoro-1H-indole-1-carboxylate (690 mg, 1.149 mmol, 57.4% yield) after purification by flash chromatography. Analytical condition H: retention time = 3.885 min; ESI-MS (+) m / z [M-Boc-tBu+H] + :445.2.

[0278] Step 3 The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(isoquinolin-8-yl)propanoic acid. TFA hydrolysis afforded the product via reversed-phase preparative HPLC (96 mg, 0.191 mmol, 16.63% yield) (column 80 g size, Silisep C18, 19x150 mm, 5 μm, Mobile phase: A = 10 mM ammonium acetate in water, B = MeOH. 15 mL / min flow gradient: 0-20 min, 5-30% B, 20-55 min, 30-80% B, 55-60 min, 80-100% B, 5 min hold at 100% B. The compound was eluted at 75% B. Purification by HPLC followed by lyophilization afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(7-fluoro-1H-indol-3-yl)propanoic acid as an off-white powder. Analytical conditions: F: retention time = 1.367 min; ESI-MS (+) m / z [M+H] + :445.3. 1H-NMR(400MHz,DMSO-d6)δ ppm:11.22(s,1H),7.86(d,J=8.72Hz,2H),7.62-7.65(m,1H),7.52-7.55(m,3H),7.40-7.42(m,2H ),7.26-7.38(m,2H),6.78-6.83(m,2H),4.12-4.21(m,4H),3.15-3.18(m,1H),2.97-3.03(m,1H).

[0279] Preparation of (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)butanoic acid [ka] The compound (2S,3S)-2-azido-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)butanoic acid was prepared according to the procedure reported in Tetrahedron Letters 2001, 42, 4601-4603. The azide reduction step used different conditions as detailed below.

[0280] Step 1. To a solution of (2S,3S)-2-azido-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)butanoic acid (1000 mg, 2.90 mmol) in THF (58 mL) was added platinum(IV) oxide (132 mg, 0.58 mmol). The reaction mixture was evacuated and filled with hydrogen. The reaction mixture was stirred at room temperature with a hydrogen balloon for 2 hours. The reaction mixture was evacuated and filled with nitrogen three times. The solution was filtered through Celite®. The solvent was removed in vacuo and the crude residue was redissolved in EtOH. The solution was filtered through Celite® to give a clear solution, which was concentrated in vacuo (0.89 g, 96% yield). 1H NMR(400MHz, methanol-d4)δ 8.13(br d,J=8.0Hz,1H),7.75(d,J=7.8Hz,1H),7.61(s,1H),7.46-7.18(m,2H),4.89(s,2H) ),3.80(d,J=6.5Hz,1H),3.58(t,J=7.2Hz,1H),1.68(s,9H),1.53(d,J=7.3Hz,3H). Analysis condition B: retention time = 0.93 minutes; ESI-MS(+)m / z[M+H] + :319.1.

[0281] Step 2. To a solution of (2S,3S)-2-amino-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)butanoic acid (3.96 g, 12.44 mmol) in MeOH (25 mL) was added (9H-fluoren-9-yl)methyl 2,5-dioxopyrrolidine-1-carboxylate (888 mg, 2.76 mmol), followed by EtN (0.385 mL, 2.76 mmol). The reaction was stirred at room temperature for 2 hours. The solvent was removed in vacuo, and the residue was redissolved in EtOAc and washed with 1N aqueous HCl, followed by brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo to give the desired product (1.3 g, 89% yield), which was not further purified. 1 H NMR(500MHz,DMSO-d6)δ 12.78(br s,1H),8.07-7.80(m,2H),7.76-7.48(m,4H),7.46-7.15(m,6H),5.75(s,1H),4.44(t ,J=8.2Hz,1H),4.33-4.22(m,1H),4.19-4.07(m,2H),1.56(s,9H),1.39-1.27(m,3H).

[0282] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-(o-tolyl)pyridin-3-yl)propanoic acid [ka]

[0283] Step 1. To a stirred solution of tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-bromopyridin-3-yl)propanoate (1750 mg, 3.35 mmol) in toluene / iPrOH (1:1, v:v, 50 mL) was added o-tolylboronic acid (911.6 mg, 6.7 mmol) and 2 M aqueous NaCO solution (25.0 mL). The mixture was purged with argon three times. Dichlorobis(tricyclohexylphosphine)palladium(II) (123.6 mg, 0.167 mmol) was added, and the reaction mixture was purged with argon twice. The reaction was heated to 80 °C for 20 h. The reaction was cooled to room temperature, and iPrOH was removed by rotary evaporation. The crude material was partitioned between water and EtOAc. The aqueous phase was extracted with EtOAc. The organic phases were combined and dried over anhydrous MgSO. After filtration and concentration, the crude product was obtained as a brown oil. Purification by flash chromatography using EtOAc:DCM (1:9) as the eluent afforded tert-butyl (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-(o-tolyl)pyridin-3-yl)propanoate (1.81 g, 3.39 mmol, 90%) as a colorless oil.

[0284] Step 2. (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-(o-tolyl)pyridin-3-yl)propanoate (1750 mg, 3.19 mmol) was dissolved in trifluoroacetic acid (5.00 mL) and the reaction was stirred at room temperature for 2 hours. The reaction was dried on a rotary evaporator and the crude product was dissolved in diethyl ether and 1 M HCl in diethyl ether. The mixture was sonicated for 2 hours to give a white solid. The product was isolated by filtration and washed with water to give (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-(o-tolyl)pyridin-3-yl)propanoic acid (1.91 g, 3.99 mmol, 100%) as a white solid. 1H NMR(499MHz,DMSO-d6)δ 8.90(s,1H),8.48(br d,J=8.0Hz,1H),7.96(t,J=6.9Hz,2H),7.89(d,J=7.5Hz,2H),7.64(dd,J=7.2,4.8Hz,2H),7.52-7.45(m,1H),7.43-7.29(m,7H),4 .46(ddd,J=10.7,8.9,4.5Hz,1H),4.25-4.15(m,3H),3.45-3.34(m,1H),3.18-3.10(m,1H),3.08-3.00(m,1H),2.27-2.20(m,3H).

[0285] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-acetamido-[1,1'-biphenyl]-4-yl)propanoic acid [ka]

[0286] Step 1. A 5.0 L multi-necked round-bottom flask was charged in one portion with a solution of (S)-2-amino-3-(4-bromophenyl)propanoic acid (150.0 g, 615 mmol), Fmoc-OSu (207 g, 615 mmol), and sodium bicarbonate (258 g, 3073 mmol) in water (3000 mL) in acetone (1500 mL) and stirred at room temperature for 16 hours. The reaction mixture was slowly acidified to pH 1 with 10 N HCl solution and stirred for 15 minutes. The slurry was filtered, dried under vacuum, and the cake was washed with water (3.0 L). The solid was dried for 16 hours. The desired product was obtained as a white solid (280 g, 98%) and was used in the next step. Analytical condition E: retention time = 2.17 minutes; ESI-MS (+) m / z [M+H] + :466.2.

[0287] Step 2. A stirred solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-bromophenyl)propanoic acid (1.0 g, 2.144 mmol) and (4-acetamidophenyl)boronic acid (0.576 g, 3.22 mmol) in 150 mL of THF (50 mL) in a pressure tube was purged with argon for 5 minutes. Tribasic potassium phosphate (1.366 g, 6.43 mmol) was then added and purging continued for another 5 minutes. 1,1′-bis(di-tert-butylphosphino)ferrocenepalladium dichloride (0.140 g, 0.214 mmol) was then added and purging continued for another 5 minutes. The reaction mixture was heated to 65° C. for 26 hours. The reaction mass was diluted with EtOAc (25 mL) and washed with 10% aqueous citric acid (10 mL) followed by brine solution to give the crude product. This was triturated with 20% DCM, stirred for 10 minutes, filtered through a Buchner funnel, and then dried for 10 minutes. The crude material was purified by flash chromatography to give 0.7 g (57%) of the desired product as a brown solid. Analytical condition E: retention time = 1.79 minutes; ESI-MS (+) m / z [M+H] + :519.0. 1 H NMR(400MHz,DMSO-d6)δ 12.75(br s,1H),9.99(s,1H),7.87(d,J=7.5Hz,2H),7.77-7.49(m,9H),7.47-7.22(m,7H),4.26-4.13(m,4H),3.11(br dd,J=13.8,4.3Hz,1H),2.91(dd,J=13.8,10.8Hz,1H),2.12-2.01(m,4H). [ka]

[0288] General procedure for Suzuki-Miyaura coupling (SMC) reaction in Scheme 1. To a N2-flushed 20 mL scintillation vial equipped with a magnetic stir bar was added Fmoc-halo-Phe-OH (0.5 mmol), boronic acid (1.5–2.5 equiv.), and anhydrous THF (6 mL). The suspension was degassed by bubbling N2 through the vial for several minutes. Palladium(II) acetate (4.5 mol%), DtBuPF (5 mol%), and then anhydrous K3PO4 (2.5 equiv.) were added. The suspension was degassed for several minutes, and then the vial was capped with a septum. The reaction mixture was stirred at 50 °C for 16 h. After cooling, the reaction was acidified by adding 20% ​​aqueous citric acid. The organic layer was separated, and the aqueous layer was extracted with EtOAc (2x). Silica gel was added to the combined organic layers, and the mixture was concentrated to dryness. The residue was dry-loaded onto a silica gel column (ISCO system) and eluted with hexane / EtOAc to give the desired product. Occasionally, further elution with MeOH / CH2Cl2 is necessary for compounds that tail in the hexane / EtOAc system.

[0289] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-(tert-butoxycarbonyl)-[1,1'-biphenyl]-4-yl)propanoic acid [ka] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-(tert-butoxycarbonyl)-[1,1'-biphenyl]-4-yl)propanoic acid was prepared according to SMC general procedure. Yield: 78% (439 mg); colorless solid. 1H NMR(400MHz, methanol-d4)δ 7.94(d,J=8.3Hz,2H),7.74(d,J=7.6Hz,2H),7.56(d,J=8.4Hz,4H),7.51(d ,J=8.1Hz,2H),7.38-7.28(m,4H),7.28-7.17(m,2H),4.56-4.38(m,1H),4.2 9(dd,J=10.5,7.0Hz,1H),4.17(dd,J=10.5,7.1Hz,1H),4.08(t,J=7.0Hz,1 H), 3.29-3.21(m, 1H), 2.98&2.80(dd, J=13.8, 9.6Hz, total 1H), 1.59(s, 9H). ESI-HRMS:C 35 H 34 NO6[M+H] + The calculated value was 564.23806, the measured value was 564.23896, and the mass difference was 1.588 ppm.

[0290] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3'-(tert-butoxycarbonyl)-[1,1'-biphenyl]-4-yl)propanoic acid [ka] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-(tert-butoxycarbonyl)-[1,1'-biphenyl]-4-yl)propanoic acid was prepared according to the SMC general procedure. Yield: 85% (240 mg); off-white solid. 1H NMR(500MHz,DMSO-d6)δ 8.08(t,J=1.8Hz,1H),7.86(dd,J=7.7,1.4Hz,3H),7.83(d,J=8.1Hz,1H),7.64(d,J= 7.7Hz,1H),7.63(d,J=7.5Hz,1H),7.58-7.48(m,3H),7.41-7.35(m,2H),731(d,J=7. 8Hz, 2H), 7.30-7.23 (m, 2H), 4.31-4.10 (m, 4H), 4.05 (td, J=8.2, 4.5Hz, 1H), 3.13 & 2.9 (dd, J=13.6, 4.5Hz, combined 1H), 2.94 & 2.76 (dd, J=13.6, 8.7Hz, combined 1H), 1.56 (s, 9H). ESI-HRMS: C 35 H 37 N2O6[M+NH4] + The calculated value was 581.26461, the measured value was 581.26474, and the mass difference was 0.218 ppm.

[0291] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-boronophenyl)propanoic acid [ka] A 75 mL pressure bottle was charged with (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-bromophenyl)propanoic acid (6.0 g, 12.87 mmol) and 2-methyl-THF (250 mL), and the solution was purged with argon for 5 minutes. Tri-o-tolylphosphine (0.31 g, 1.03 mmol), tetrahydroxydiboron (2.31 g, 25.7 mmol), and potassium acetate (3.79 g, 38.6 mmol) were added over 10 minute intervals, followed by MeOH (100 mL) and Pd(OAc) (0.12 g, 0.52 mmol), and the solution was purged with argon for 10 minutes. The reaction was heated at 50 °C overnight. The reaction mixture was transferred to a 1 L separatory funnel, diluted with 2-methyl-THF, and acidified to pH = 2 with 1.5 N HCl. The organic layer was washed with brine, dried (sodium sulfate), passed through Celite, and concentrated to give a crude black material. The crude was treated with petroleum ether to give a solid (10 g), which was dissolved in 2-methyl-THF, and charcoal (2 g) was added. The mixture was heated on a rotary evaporator at 50° C. without vacuum. After filtration, the filtrate was passed through Celite and concentrated. The resulting solid was treated with 30% ethyl acetate in petroleum ether and filtered to give 8 g of crude product as a fine off-white solid, which was further purified by flash chromatography and then triturated with petroleum ether to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-boronophenyl)propanoic acid (4.0 g, 9.28 mmol, 72.1% yield) as a white solid. LCMS: 432.1 (M+H), tr=0.82 min. 1 H NMR(500MHz,DMSO-d6)δ 7.88(d,J=7.6Hz,2H),7.85-7.77(m,1H),7.71(br d,J=7.9Hz,3H),7.68-7.60(m,2H),7.41(br d,J=6.6Hz,2H),7.35-7.20(m,4H),4.30-4.11(m,5H),3.16-3.03(m,1H),2.95-2.83(m,1H).

[0292] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid [ka] To a stirred solution of (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-boronophenyl)propanoic acid (217.5 mg, 0.504 mmol), 1-bromo-4-fluorobenzene (0.083 mL, 0.757 mmol), and XPhos Pd G2 (9.7 mg, 0.012 mmol) in THF (1 mL) was added 0.5 M aqueous K3PO4 (2 mL, 1.000 mmol) at room temperature. N2 was purged three times in vacuo, and the mixture was stirred at 80 °C for 16 h. The mixture was cooled to room temperature. To the reaction was added 10% citric acid until the pH was <6. It was partitioned between EtOAc and HO, and the organic phase was separated, washed with brine, and dried over sodium sulfate. The mixture was filtered, SiO2 (5 g) was added, and concentrated. The material was then purified by flash chromatography (Teledyne ISCO CombiFlash® f The resulting mixture was purified by RediSep SiO (40 g) with a gradient of 0% to 20% MeOH / CHCl over 15 column volumes. Fractions containing the desired product were collected and concentrated to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (206.1 mg, 0.43 mmol, 85% yield) as a cream-colored solid: HPLC: RT = 1.04 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CHCN / H2O / 0.05% TFA, 1 min, gradient, wavelength = 254 nm); MS (ES): m / z = 482 [M+H] + . 1H NMR(499MHz,DMSO-d6)δ 12.78(br s,1H),7.88(d,J=7.5Hz,3H),7.71-7.61(m,5H),7.53(d,J=8.1Hz,2H),7.39(q,J=7.3Hz,3H),7. 36-7.23(m,8H),4.24-4.13(m,5H),3.12(dd,J=14.0,4.5Hz,1H),2.91(dd,J=13.6,10.3Hz,1H).

[0293] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3',5'-difluoro-[1,1'-biphenyl]-4-yl)propanoic acid [ka] The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid. The Suzuki coupling reaction afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3',5'-difluoro-[1,1'-biphenyl]-4-yl)propanoic acid (197.1 mg, 0.40 mmol, 78% yield) as a colorless solid after purification by flash chromatography. HPLC: RT = 1.06 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 500 [M+H] + . 1 H NMR(499MHz,DMSO-d6)δ 12.90-12.67(m,1H),7.87(d,J=7.5Hz,2H),7.69-7.61(m,4H),7.45-7.35(m,6H),7.33-7.27(m,2H),7.22-7. 16(m,1H),4.25-4.18(m,3H),4.17-4.12(m,1H),3.14(dd,J=13.8,4.4Hz,1H),2.92(dd,J=13.7,10.6Hz,1H).

[0294] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)propanoic acid [ka] The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid. Suzuki coupling gave the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)propanoic acid (218.5 mg, 0.422 mmol, 84% yield) as a colorless solid after purification by flash chromatography. HPLC: RT = 1.466 min (Waters Acquity BEH C18 1.7 um 2.1 × 50 mm column, Shimadzu UPLC with CHCN / HO / 0.1% TFA, 3 min gradient, wavelength = 254 nm); MS (ES): m / z = 556. 1 H NMR(499MHz,DMSO-d6)δ 12.79(br s,1H),7.87(d,J=7.6Hz,2H),7.75(d,J=8.6Hz,1H),7.69-7.58(m,6H),7.44-7.35(m,4H),7.33-7.25(m ,2H),4.27-4.17(m,3H),4.17-4.10(m,1H),3.14(dd,J=13.8,4.4Hz,1H),2.92(dd,J=13.7,10.7Hz,1H).

[0295] Scheme. General procedure for photoredox reactions. [ka] Ir[dF(CF)ppy](dtbbpy)PF (0.018 g, 0.016 mmol, 1 mol%), tert-butyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (1.181 g, 2.393 mmol, 1.5 equiv.), bromo-pyridine derivative (1.596 mmol, 1.00 equiv.), powdered NaCO (0.338 g, 3.19 mmol, 2.00 equiv.), and tris(trimethylsilane)silane (0.278 g, 1.596 mmol, 1.00 equiv.) were placed in an oven-dried 40 mL pressure-relief screw-cap vial. The vial was capped, purged with nitrogen, diluted with THF (45.0 mL), and then sonicated. A separate vial was charged with NiCl2-glyme (18 mg, 0.080 mmol, 5 mol%) and di-tert-butylbipyridine (18 mg, 0.096 mmol, 6 mol%) in 1 mL of dioxane. The vial was purged with nitrogen for 10 minutes. The nickel-ligand complex solution was transferred to the main reaction vial, and the mixture was degassed with a gentle stream of nitrogen for 20 minutes. The reactor was sealed with parafilm, placed between two 34 W blue LED Kessel lamps (approximately 7 cm apart), and vigorously stirred. After 16 hours, the reaction was monitored by LCMS analysis. The resulting oil was dissolved in 4 M HCl in dioxane (15 mL). After 16 hours, the reaction mixture was dried on a rotary evaporator. The crude product was dissolved in a minimal amount of methanol and dry-loaded onto a silica gel column for purification.

[0296] Preparation of (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(2-methoxypyridin-4-yl)propanoic acid [ka] The mixture was rotary evaporated onto silica gel and purified by ISCO using 10% to 80% EtOAc / Hexanes. Fractions were pooled and concentrated to give the desired product as a clear oil (237 mg, 100%). Analytical condition D: Retention time 1.74 min; ES+ 475.1.

[0297] Preparation of ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid [ka]

[0298] Step 1 Four separate 40 mL vials were charged with Ir(dF(CF)ppy)(dtbbpy)PF (5.6 mg, 4.99 μmol) and NaCO (249 mg, 2.35 mmol) in dioxane (18 mL) and equipped with Teflon screw caps and stir bars. 1-Iodo-4-(trifluoromethoxy)benzene (0.16 mL, 1.02 mmol) was added to the mixture, stirred briefly, and then tris(trimethylsilyl)silane (0.23 mL, 0.75 mmol) was added via syringe. The suspension was degassed with nitrogen for 5 minutes (cap on). To separate 40 mL vials, nickel(II) chloride ethylene glycol dimethyl ether complex (22 mg, 0.10 mmol) and 4,4'-di-tert-butyl-2,2'-bipyridine (33 mg, 0.12 mmol) were added. Dioxane (10 mL) was added, and the solution was degassed with nitrogen gas (capped) for 10 minutes and stirred. To the Ir mixture, 2.5 mL of Ni solution and 5 mL of a solution of iodoalanine, tert-butyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (987 mg, 2.0 mmol) in dioxane (20 mL) were added, and the mixture was then degassed with nitrogen gas (capped) for an additional 5 minutes. The vials were sealed with parafilm, placed in a circular photoredox reactor with the light and fan on, and stirred for 40 hours. The reactions were removed from the light / reactor. The dark reaction mixture from each vial was poured into a 500 mL Erlenmeyer flask, and EtOAc (200 mL) was added. The mixture was filtered through Celite, washed with EtOAc, and concentrated. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% gradient using solvent A / B=CH 2 Cl 2 / EtOAc over 10 column volumes, RediSep SiO 2 80 g, loaded as a solution in DCM).Fractions containing the desired product were collected and concentrated to give the product tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate (865.2 mg, 1.64 mmol, 82% yield, only about 73% HPLC purity was obtained as a colorless oil and used directly in the deprotection step): HPLC: RT = 1.62 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 550 [M+23]+.

[0299] Step 2. To a stirred solution of tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate (865.2 mg, 1.64 mmol) in dichloromethane (8.2 mL) at room temperature was added HCl (4 M in dioxane, 8.20 mL, 32.8 mmol). The mixture was stirred at room temperature for 18 hours. The mixture was concentrated in vacuo and then dried under vacuum. The residue was dissolved in DMF (4 mL) and purified in two injections on an ISCO ACCQ Prep. Fractions containing the desired product were combined and partially concentrated on a rotary evaporator, and then air was bubbled through the mixture over the weekend. The residue was dissolved in CH3CN, diluted with water, frozen, and lyophilized. The product, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid (344.1 mg, 0.73 mmol, 44.5% yield), was obtained as a colorless solid. HPLC: RT = 1.38 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CH3CN / HO / 0.05% TFA, 1.5 min gradient, wavelength = 254 nm); MS (ES): m / z = 472 [M+1]+. 1H NMR(499MHz,DMSO-d6)ppm δ 7.88(d,J=7.5Hz,2H),7.63(d,J=7.4Hz,2H),7.44-7.37(m,2H),7.35-7.25(m,4H),7.19(br d,J=7.6Hz,3H),4.30-4.20(m,1H),4.21-4.13(m,2H),4.04(br d,J=3.5Hz,1H),3.11(br dd,J=13.6,4.4Hz,1H),2.91(br dd,J=13.6,9.1Hz,1H).

[0300] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,5-dimethylphenyl)propanoic acid [ka]

[0301] Step 1. The compound was prepared according to the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,5-dimethylphenyl)propanoate (140.5 mg, 0.298 mmol, 61.1% yield), after purification by flash chromatography. HPLC: RT = 1.21 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); analytical condition F: retention time = 1.21 min; ESI-MS (+) m / z [M-tBu+H] + :416. 1H NMR(499MHz,chloroform-d)δ 7.78(d,J=7.5Hz,2H),7.63-7.56(m,2H),7.42(t,J=7.4Hz,2H),7.37-7.3 0(m,2H),7.07(d,J=7.7Hz,1H),6.98(d,J=7.7Hz,1H),6.96(s,1H),4.58-4 .51(m,1H),4.39(dd,J=10.5,7.3Hz,1H),4.34(dd,J=10.5,7.2Hz,1H),4. 24-4.19(m,1H),3.10-3.01(m,2H),2.34(s,3H),2.28(s,3H),1.40(s,8H).

[0302] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,5-dimethylphenyl)propanoic acid (115.2 mg, 0.277 mmol, 93% yield) as a cream-colored solid after purification by reverse-phase flash chromatography. HPLC: RT = 1.03 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 416 [M+H] + . 1 H NMR(499MHz,chloroform-d)δ 7.88(d,J=7.4Hz,2H),7.79(br d,J=8.6Hz,1H),7.67(d,J=7.4Hz,1H),7.64(d,J=7.5Hz,1H),7.41(td,J=7.3,4.2Hz,3H),7.35-7.29(m,2H),7.29-7.25(m,1H),7.02(br d,J=8.9Hz,2H),6.91(br d,J=7.4Hz,1H),4.21-4.10(m,5H),3.07(dd,J=14.1,4.4Hz,1H),2.80(dd,J=14.1,10.3Hz,1H),2.24(s,3H),2.18(s,3H).

[0303] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-fluoro-3-methylphenyl)propanoic acid [ka]

[0304] Step 1. The compound was prepared according to the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-fluoro-3-(trifluoromethyl)phenyl)propanoate (66.3 mg, 0.13 mmol, 24.9% yield), as a colorless solid after purification by flash chromatography. HPLC: RT = 1.19 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CH3CN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 474 [M-tBu] + . 1 H NMR(499MHz,chloroform-d)δ 7.80(d,J=7.5Hz,2H),7.60(dd,J=7.6,3.3Hz,2H),7.47-7.39(m,3H),7.38-7.32(m,2H),7.16-7.09(m,1H),5.34(br d,J=7.7Hz,1H),4.57-4.47(m,2H),4.40(dd,J=10.3,6.9Hz,1H),4.26-4.21(m,1H),3.14(br d,J=4.9Hz,2H),1.44(s,9H).

[0305] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-fluoro-3-methylphenyl)propanoic acid (58.3 mg, 0.139 mmol, 85% yield) as a cream-colored solid after purification by reverse-phase flash chromatography. HPLC: RT=1.02 min (Waters Acquity UPLC BEH C18 1.7 um 2.1×50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength=254 nm); MS (ES): m / z=420 [M+H] + . 1 H NMR(499MHz,DMSO-d6)δ 12.86-12.66(m,1H),7.89(d,J=7.5Hz,2H),7.73(d,J=8.3Hz,1H),7.65(t,J=7.5Hz,2H),7.42(t,J=7.5Hz,2H),7.35-7.26(m,2H),7.17(br d,J=7.5Hz,1H),7.14-7.08(m,1H),7.06-6.99(m,1H),4.24-4.11(m,4H), 3.03(dd,J=13.7,4.3Hz,1H),2.82(dd,J=13.6,10.6Hz,1H),2.17(s,3H).

[0306] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,4-difluoro-5-methoxyphenyl)propanoic acid [ka]

[0307] Step 1. The compound was prepared according to the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,4-difluoro-5-methoxyphenyl)propanoate (77.1 mg, 0.151 mmol, 29.1% yield), as a colorless solid after purification by flash chromatography. HPLC: RT = 1.15 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 454 [Mt-Bu]+. 1 H NMR(499MHz,chloroform-d)δ 7.79(d,J=7.4Hz,2H),7.59(t,J=6.4Hz,2H),7.43(t,J=7.3Hz,2H),7.33(td ,J=7.5,1.1Hz,3H),6.85(dd,J=10.8,9.3Hz,1H),6.83-6.79(m,1H),5.40(br d,J=8.1Hz,1H),4.58-4.51(m,1H),4.38(dd,J=7.0,4.5Hz,2H),4.25-4.20(m,1H),3.82(s,3H),3.18-3.05(m,2H),1.45(s,9H).

[0308] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,4-difluoro-5-methoxyphenyl)propanoic acid (45.9 mg, 0.101 mmol, 66.9% yield) as a cream-colored solid after purification by reverse-phase flash chromatography. HPLC: RT=0.99 min (Waters Acquity UPLC BEH C18 1.7 um 2.1×50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength=254 nm); MS (ES): m / z=454 [M+1]+. 1 H NMR(499MHz,DMSO-d6)δ 12.92(br s,1H),7.89(d,J=7.5Hz,2H),7.71-7.65(m,1H),7.63(d,J=7.5Hz,2H),7.41(t,J=7.5 Hz,2H),7.34-7.25(m,2H),7.24-7.15(m,2H),4.24-4.12(m,4H),3.77(s,3H),3.16(br dd,J=13.8,4.6Hz,1H),2.82(dd,J=13.6,10.7Hz,1H).

[0309] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,3-dimethylphenyl)propanoic acid [ka]

[0310] Step 1. The compound was prepared according to the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,3-dimethylphenyl)propanoate (107.5 mg, 0.228 mmol, 55.5% yield), as a tan viscous oil after purification by flash chromatography. HPLC: RT = 1.21 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CH3CN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 416 [Mt-Bu]+. 1 H NMR (499 MHz, chloroform-d) δ 7.79 (d, J = 7.5 Hz, 2H), 7.61-7.56 (m, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.35-7.31 (m, 2H), 7.09-7.06 (m, 1H), 7.02 (t, J = 7.5 Hz, 1H), 7.00-6.96 (m, 1H), 5.30 (br d,J=8.3Hz,1H),4.53(q,J=7.4Hz,1H),4.39(dd,J=10.6,7.3Hz,1H),4.34(dd,J=10.4,7.0Hz,1H),4.21(t,J= 7.2Hz,1H), 3.15(dd,J=14.2,7.0Hz,1H),3.08(dd,J=14.1,7.3Hz,1H),2.29(s,3H),2.28(s,3H),1.40(s,9H).

[0311] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane gave the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,3-dimethylphenyl)propanoic acid (72.9 mg, 0.175 mmol, 77% yield) as a cream-colored solid after purification by reverse-phase flash chromatography. HPLC: RT=1.03 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength=254 nm); MS (ES): m / z=416 [M+H]+. 1 H NMR(499MHz,DMSO-d6)δ 12.76(br d,J=1.8Hz,1H),7.89(d,J=7.5Hz,2H),7.79-7.71(m,1H),7.66(dd,J=13.6,7.6 Hz,2H),7.42(td,J=7.2,4.1Hz,2H),7.35-7.27(m,2H),7.07(d,J=7.3Hz,1H),7. 04-6.99(m,1H),6.99-6.94(m,1H),4.24-4.14(m,3H),4.13-4.05(m,1H),3.15( dd,J=14.1,4.1Hz,1H),2.85(dd,J=13.9,10.4Hz,1H),2.22(s,3H),2.19(s,3H).

[0312] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-3-methylphenyl)propanoic acid [ka]

[0313] Step 1 The compound was prepared according to the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-3-methylphenyl)propanoate (136.9 mg, LCMS showed 77% product and 23% impurity) as a viscous oil after purification by flash chromatography. Used as is and purified after tBu hydrolysis.

[0314] Step 2 The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-3-methylphenyl)propanoic acid (79.7 mg, 0.190 mmol, 66.0% yield) as a cream-colored solid after purification by reverse-phase flash chromatography. HPLC: RT = 1.02 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 420 [M+1] + . 1 H NMR(499MHz,DMSO-d6)δ 12.79(br s,1H),7.89(d,J=7.7Hz,2H),7.78(d,J=8.6Hz,1H),7.65(dd,J=11.6,7.5Hz,2H),7.44-7.39(m,3H),7.37-7.25(m,3H),7.14(br t,J=7.4Hz,2H),7.01-6.96(m,1H),4.24-4.12(m,4H),3.17(dd,J=13.8,4.8Hz,1H),2.86(dd,J=13.6,10.8Hz,1H),2.21(s,3H). 11 H NMR and LCMS showed 14% impurity.

[0315] Preparation of ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-5-methylphenyl)propanoic acid [ka] The compound was prepared following the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-5-methylphenyl)propanoate (148.1 mg, 0.311 mmol, 65.4% yield), as a colorless gum after purification by flash chromatography. HPLC: RT = 1.19 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CH3CN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 420 [Mt-Bu] + . 1 H NMR (499 MHz, chloroform-d) δ 7.79 (d, J = 7.6 Hz, 2H), 7.60 (t, J = 7.2 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.37-7.30 (m, 2H), 7.06-6.99 (m, 2H), 6.97-6.90 (m, 1H), 5.41 (br d,J=8.1Hz,1H),4.60-4.54(m,1H),4.43(dd,J=10.4,7.2Hz,1H),4.30(dd,J=10.1,7.5Hz,1H),4.26 -4.21(m,1H),3.16(dd,J=13.9,6.7Hz,1H),3.10(dd,J=13.9,6.4Hz,1H),2.28(s,3H),1.44(s,9H).

[0316] Step 2 The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-5-methylphenyl)propanoic acid (98.1 mg, 0.23 mmol, 75% yield) as a colorless solid after purification by reverse-phase flash chromatography. HPLC: RT=1.01 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength=254 nm); MS (ES): m / z=420 [M+1] + . 1 H NMR(499MHz,DMSO-d6)δ 12.82(br s,1H),7.89(d,J=7.5Hz,2H),7.78(d,J=8.6Hz,1H),7.67(d,J=7.4Hz,1H),7.64(d,J=7.4Hz,1H),7.42(td,J=7.4,3.0Hz,2H),7.34-7.27( m,2H),7.16-7.11(m,1H),7.08-6.97(m,2H),4.26-4.12(m,5H),3.15(dd,J=13.8,4.9Hz,1H),2.83(dd,J=13.8,10.3Hz,1H),2.20(s,3H).

[0317] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-5-methoxyphenyl)propanoic acid [ka]

[0318] Step 1 The compound was prepared following the same procedure as above: tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-5-methoxyphenyl)propanoate (117.7 mg, 0.24 mmol, 50.4% yield) as a colorless solid after purification by flash chromatography. HPLC: RT = 1.15 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CH3CN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 436 [Mt-Bu] + . 1 H NMR (499 MHz, chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.63-7.56 (m, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.37-7.30 (m, 2H), 7.01-6.93 (m, 1H), 6.79-6.72 (m, 2H), 5.41 (br d,J=8.2Hz,1H),4.62-4.55(m,1H),4.41(dd,J=10.4,7.3Hz,1H),4.31(dd,J=10.5,7.4Hz,1H),4.26 -4.20(m,1H),3.75(s,3H),3.17(dd,J=13.9,6.7Hz,1H),3.11(dd,J=14.4,6.6Hz,1H),1.45(s,9H).

[0319] Step 2 The final product was obtained following the same procedure as for (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-fluoro-5-methoxyphenyl)propanoic acid (79.5 mg, 0.183 mmol, 76% yield) as a colorless solid after purification by flash chromatography. HPLC: RT = 0.98 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 436 [M+1] + . A base peak at 214 = fully deprotected amino acid fragment was also observed. 1 H NMR(499MHz,DMSO-d6)δ 12.84(br s,1H),7.89(d,J=7.5Hz,2H),7.79(d,J=8.6Hz,1H),7.64(t,J=8.4Hz,2 H),7.45-7.38(m,2H),7.34-7.25(m,2H),7.07(t,J=9.2Hz,1H),6.94(d d,J=6.1,3.2Hz,1H),6.80(dt,J=8.9,3.6Hz,1H),4.25-4.13(m,4H),3.69(s,3H),3.17(dd,J=13.9,4.6Hz,1H),2.83(dd,J=13.7,10.7Hz,1H).

[0320] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methoxy-5-methylphenyl)propanoic acid [ka]

[0321] Step 1. The compound was prepared following the same procedure as tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoate. Photoredox coupling afforded the desired product, tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methoxy-5-methylphenyl)propanoate (73.9 mg, 0.15 mmol, 31.3% yield), as a colorless gum after purification by flash chromatography. HPLC: RT = 1.20 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 488 [M-tBu+H] + . 1 H NMR(499MHz,chloroform-d)δ 7.78(d,J=7.6Hz,2H),7.61-7.54(m,2H),7.41(t,J=7.4Hz,2H),7.34-7.30(m,2H) ,7.05(dd,J=8.1,1.5Hz,1H),6.98(d,J=1.4Hz,1H),6.79(d,J=8.3Hz,1H),5.70(br d,J=7.7Hz,1H),4.49(q,J=7.4Hz,1H),4.33(d,J=7.4Hz,2H),4.25-4.18(m,1H),3.82(s,3H),3.10-3.02(m,2H),2.26(s,3H),1.43(s,9H).

[0322] Step 2. The final product was obtained following the same procedure as for (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(trifluoromethoxy)phenyl)propanoic acid. Removal of the tBu ester using HCl / dioxane afforded the desired (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-methoxy-5-methylphenyl)propanoic acid (44.7 mg, 0.104 mmol, 68.4% yield) as a colorless solid after purification by flash chromatography. HPLC: RT = 1.02 min (Waters Acquity UPLC BEH C18 1.7 um 2.1 x 50 mm, CHCN / HO / 0.05% TFA, 1 min gradient, wavelength = 254 nm); MS (ES): m / z = 432 [M+H] + . 1 H NMR(499MHz,DMSO-d6)δ 12.61(br s,1H),7.89(d,J=7.5Hz,2H),7.67(d,J=7.5Hz,1H),7.63(d,J=7.5Hz,1H),7.60(br d,J=8.1Hz,1H),7.42(td,J=7.2,3.5Hz,2H),7.32(td,J=7.5,1.0Hz,1H),7.30-7.26(m,1H),7.02-6.97(m,2H),6.84(d, J=8.9Hz,1H),4.26-4.10(m,4H),3.75(s,3H),3.12(dd,J=13.5,4.8Hz,1H),2.72(dd,J=13.4,10.2Hz,1H),2.16(s,3H).

[0323] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-3-methylbutanoic acid Scheme: [ka]

[0324] Step 1. A 10-L multi-neck round-bottom flask was charged with methyl (tert-butoxycarbonyl)-D-serinate (50 g, 228 mmol) and diethyl ether (4200 mL). The mixture was cooled to -78 °C, and methylmagnesium bromide (456 mL, 1368 mmol) was added dropwise over 30 minutes. The reaction was stirred at room temperature for 1 hour. It was then cooled to 0 °C, and saturated NH4Cl solution (1500 mL) was added dropwise and stirred for 10 minutes. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 2000 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated at 40 °C to give a colorless viscous liquid. The crude material was purified by I2PAC. The desired fractions were eluted with 50% EtOAc:petroleum ether mixture, which was collected and concentrated at 40° C. to give tert-butyl (R)-(1,3-dihydroxy-3-methylbutan-2-yl)carbamate (43.5 g, 87%) as a white solid. 1 H NMR (MeOD, 300MHz) δ 3.70 (m, 1H), 3.48 (m, 1H), 3.21 (m, 1H), 1.35 (s, 9H), 1.13 (s, 3H), 1.05 (s, 3H).

[0325] Step 2. A 50 mL single-neck round-bottom flask was charged with tert-butyl (R)-(1,3-dihydroxy-3-methylbutan-2-yl)carbamate (43.0 g, 196 mmol) and acetonitrile (650 mL) and stirred until the solution became clear. Sodium phosphate buffer (460 mL, 196 mmol) (pH = 6.7, 0.67 M), (diacetoxyiodo)benzene (4.48 g, 13.92 mmol), and TEMPO (2.206 g, 14.12 mmol) were added sequentially. The reaction was then cooled to 0 °C, and sodium chlorite (19.95 g, 221 mmol) was added. The color of the reaction changed to black. The reaction was stirred at 0 °C for 2 hours and then at room temperature overnight. The orange reaction was quenched with saturated ammonium chloride solution (1000 mL) and adjusted to pH = 2 using 1.5 N HCl (330 mL) using a pH meter. The aqueous solution was saturated with solid NaCl and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over NaSO, and concentrated to give crude (S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid (34.0 g, 74.3% yield) as an off-white solid, which was used directly in the next step. 1 H NMR (MeOD, 300MHz) δ 3.98 (s, 1H), 1.35 (s, 9H), 1.19 (s, 3H), 1.16 (9s, 3H).

[0326] Step 3. A 2000 mL single-neck flask was charged with (S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid (90 g, 386 mmol) in dioxane (450 mL) and cooled to 0° C. 4 N HCl in dioxane (450 mL, 1800 mmol) was added dropwise over 10 minutes. The reaction was stirred at room temperature for 3 hours. It was concentrated, azeotroped with toluene (2×), and then stirred with ethyl acetate for 10 minutes. It was filtered and dried under vacuum to give crude (S)-2-amino-3-hydroxy-3-methylbutanoic acid, HCl (70 g, 107% yield) as a white solid, which was used directly in the next step.

[0327] Step 4 A 3000 mL multi-necked round-bottom flask was charged with (S)-2-amino-3-hydroxy-3-methylbutanoic acid, HCl (70 g, 413 mmol), dioxane (1160 mL), and water (540 mL). The stirred solution became clear, and a solution of sodium bicarbonate (104 g, 1238 mmol) in water (1160 mL) was added in one portion at room temperature. The reaction mass was stirred at room temperature for 30 minutes. A solution of Fmoc-OSu (139 g, 413 mmol) in 1,4-dioxane (1460 mL) was added in one portion at room temperature. The reaction was stirred at room temperature for 16 hours. The reaction was concentrated to remove dioxane. Water was added to the resulting solution and washed with ethyl acetate (3 x 1000 mL). The aqueous solution was acidified to pH 1-2 and extracted with ethyl acetate. The combined organic layers were washed with water, followed by brine, and finally dried over NaSO and concentrated to give an off-white solid (135.7 g). To remove trapped dioxane and ethyl acetate, the following procedure was followed: the solid was dissolved in ethyl acetate (1200 mL) and stripped with n-hexane (3000 mL). The resulting slurry was stirred for 10 minutes, filtered, and dried under vacuum to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-3-methylbutanoic acid (112.0 g, 74.8% yield over two steps) as a white solid.

[0328] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3,4,5-trifluorophenyl)propanoic acid [ka]

[0329] Step 1. To a stirred solution of 2-((diphenylmethylene)amino)acetonitrile (100 g, 454 mmol) in DCM (1000 mL) was added 5-(bromomethyl)-1,2,3-trifluorobenzene (66.5 mL, 499 mmol) and benzyltrimethylammonium chloride (16.86 g, 91 mmol). To this was added 10 M NaOH (136 mL, 1362 mmol) solution and stirred at room temperature overnight. After 26 h, the reaction mixture was diluted with water (500 mL) and the DCM layer was separated. The aqueous layer was further extracted with DCM (2 × 250 mL). The organic layers were combined, washed with water and brine solution, dried over NaSO, filtered, and concentrated in vacuo. The crude compound was purified by flash column chromatography (1.5 kg, silica gel, 0-10% ethyl acetate / petroleum ether mixture). The desired fractions were collected and concentrated to give 2-((diphenylmethylene)amino)-3-(3,4,5-trifluorophenyl)propanenitrile (140 g, 384 mmol, 85% yield) as a yellow solid. Analytical condition E: retention time = 3.78 min; ESI-MS (+) m / z [M+H] + :365.2.

[0330] Step 2. To a stirred solution of 2-((diphenylmethylene)amino)-3-(3,4,5-trifluorophenyl)propanenitrile (80 g, 220 mmol) in 1,4-dioxane (240 mL) was added concentrated aqueous HCl (270 mL, 3293 mmol) and the mixture was stirred at 90° C. for 16 h. The reaction mixture was used directly in the next step.

[0331] Step 3. To the crude dioxane solution, 10 N NaOH solution was added until the solution became neutral. Then, Na2CO3 (438 mL, 438 mmol) was added, followed by Fmoc-OSu (81 g, 241 mmol). The mixture was stirred at room temperature overnight. The aqueous solution was acidified with 1.5 N HCl to pH = 2, and the formed solid was filtered and dried to obtain the crude compound. This was first slurried in 5% EtOAc / petroleum ether for 30 min and filtered. Racemic 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3,4,5-trifluorophenyl)propanoic acid (90 g, 204 mmol, 93% yield) was obtained as an off-white solid. This racemic compound was separated into two isomers by SFC purification (column: Chiralcel OD-H, MeOH as co-solvent) to give the desired isomer. After concentration of the desired isomer, it was slurried in 5% EtOAc / petroleum ether and filtered to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3,4,5-trifluorophenyl)propanoic acid (43 g, 95 mmol, 43.3% yield) as an off-white solid. 1 H NMR(MeOD,400MHz)δ 7.78(d,J=7.2Hz,2H),7.60(t,J=8.0Hz,2H),7.38(t,J=8.0Hz,2H),7.28(t,J=7.6Hz, 2H),7.01(t,J=7.8Hz,2H),4.48-4.26(m,3H),4.18(m,1H),3.18(m,1H),2.91(m,1H). 19 F(MeOD,376MHz)δ -137.56(d,J=19.6Hz,2F),-166.67(t,J=19.6Hz,1F). Analysis condition E: retention time = 3.15 minutes; ESI-MS(+)m / z[M+H] + :442.2.

[0332] The other fractions were concentrated to give (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3,4,5 trifluorophenyl)propanoic acid (40 g, 91 mmol, 41.4% yield) as an off-white solid.

[0333] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-3,3-dimethyl-4-oxobutanoic acid [ka]

[0334] Step 1. To a stirred solution of 4-(tert-butyl) 1-methyl L-aspartate, HCl salt (34 g, 142 mmol) in acetonitrile (550 mL) under a nitrogen atmosphere, lead(II) nitrate (47.0 g, 142 mmol), potassium phosphate (66.2 g, 312 mmol), and triethanolamine (19.77 mL, 142 mmol) were added. The mixture was cooled to 0 °C, and then a solution of 9-bromo-9-phenylfluorene (43.3 g, 135 mmol) in acetonitrile (100 mL) was added. The reaction mixture was stirred at room temperature for 48 h, and the progress of the reaction was monitored by TLC (50% EA in PE) and LCMS. The reaction mixture was filtered through Celite, washed with chloroform, and evaporated to give a viscous pale yellow liquid, to which ethyl acetate (3500 mL) was added. The EtOAc layer was washed with 5% citric acid solution (500 mL) followed by brine solution. The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give a pale yellow viscous liquid, which was scraped with petroleum ether and filtered to give 4-(tert-butyl) 1-methyl(9-phenyl-9H-fluoren-9-yl)-L-aspartate (55 g, 124 mmol, 87% yield) as a white solid. Analytical conditions: Retention time = 1.73 min; ESI-MS (+) m / z [M+Na] + :466.40.

[0335] Step 2. A solution of 4-(tert-butyl)1-methyl(9-phenyl-9H-fluoren-9-yl)-L-aspartic acid (22.5 g, 50.7 mmol) was cooled to −78° C. under Ar, and a solution of KHMDS (127 mL, 127 mmol, 1 M in THF) was added with stirring over 30 min. The reaction was warmed to −40° C., and methyl iodide (9.52 mL, 152 mmol) was added dropwise. The reaction was stirred at −40° C. for 5 h. The reaction was monitored by TLC and LCMS. Saturated NH4Cl (400 mL) was added, followed by HO (100 mL). The resulting mixture was extracted with EtOAc (3×), and the combined organic extracts were washed with 2% citric acid (200 mL), aqueous NaHCO3 (200 mL), and brine. The organic layer was dried over anhydrous Na2SO4, evaporated in vacuo, and recrystallized from hexane to give 1-(tert-butyl) 4-methyl(S)-2,2-dimethyl-3-((9-phenyl-9H-fluoren-9-yl)amino)succinate (18.5 g, 39.2 mmol, 77% yield) as a white solid, which was used in the next step. Analytical conditions: L: retention time = 2.04 min; ESI-MS (+) m / z [M+Na] + :494.34.

[0336] Step 3. A stirred solution of 1-(tert-butyl) 4-methyl(S)-2,2-dimethyl-3-((9-phenyl-9H-fluoren-9-yl)amino)succinate (24 g, 50.9 mmol) in methanol (270 mL) and ethyl acetate (100 mL) was degassed with nitrogen. Pd—C (2.71 g, 2.54 mmol) (10 wt %) was added, the mixture was flushed with hydrogen gas, and then stirred overnight at room temperature in a 1-liter autoclave containing 50 psi. The reaction mixture was filtered through a Celite pad and washed with a mixture of methanol and ethyl acetate. The combined solvents were evaporated to dryness, and the precipitated white solid was removed by filtration to give 1-(tert-butyl) 4-methyl(S)-3-amino-2,2-dimethylsuccinate (11.7 g) as a pale yellow liquid, which was used directly in the next step.

[0337] Step 4. To a stirred solution of 1-(tert-butyl) 4-methyl(S)-3-amino-2,2-dimethylsuccinate (11.0 g, 47.6 mmol) cooled in an ice bath, lithium hydroxide (428 mL, 86 mmol, 0.2 M solution in water) was added, and the reaction was slowly brought to room temperature. The reaction was monitored by TLC and LCMS. The reaction mixture was evaporated and used directly in the next step. To a stirred solution of (S)-2-amino-4-(tert-butoxy)-3,3-dimethyl-4-oxobutanoic acid (15 g, 69.0 mmol) (which was in water from a previous batch) in acetonitrile (200 mL) cooled to 0 °C, sodium bicarbonate (5.80 g, 69.0 mmol) and Fmoc-OSu (46.6 g, 138 mmol) were added. The reaction mixture was stirred at room temperature overnight. This was acidified to pH=4 with 2N HCl, then extracted with ethyl acetate (3×500 mL), and the combined organic layers were washed with brine, dried over sodium sulfate, and evaporated to give an off-white solid, which was purified by ISCO flash chromatography using 20% ​​EA in petroleum ether to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-3,3-dimethyl-4-oxobutanoic acid (12.2 g, 26.9 mmol, 39.0% yield) as a white solid. 1 HNMR(CDCl3,400MHz)δ 7.77(d,J=7.6Hz,2H),7.60(m,2H),7.42(t,J=8.0Hz,2H),7.33(t,J=7.6Hz, 2H),4.65(m,2H),4.34(m,1H),4.25(m,1H),3.18(m,1H),1.40-1.27(m,6H). Analysis condition E: retention time = 1.90 minutes; ESI-MS(+)m / z[M+H] + :440.2.

[0338] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(tert-butoxycarbonyl)phenyl)propanoic acid [ka]

[0339] Step 1. To a solution of (S)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (80 g, 365 mmol), O-methylhydroxylamine hydrochloride (36.6 g, 438 mmol) in CHCl (2000 mL) was added TEA (153 mL, 1095 mmol) at room temperature. The reaction was cooled to 0 °C, and 1-propanephosphonic anhydride (326 mL, 547 mmol) was added dropwise. The reaction was stirred at room temperature for 2 hours. It was quenched with saturated ammonium chloride (500 mL) and extracted with EtOAc (3 × 300 mL). The combined organic layers were washed with saturated brine, dried over NaSO, and concentrated under reduced pressure. The crude product was purified by Combiflash using a 120 g silica column with 38–45% EtOAc in petroleum ether to give (S)-2-(1,3-dioxoisoindolin-2-yl)-N-methoxypropanamide (80 g, 322 mmol, 88% yield). 1 H NMR (DMSO-d6,400MHz)δ 11.36(s,1H),7.91-7.85(m,4H),4.75-4.69(m,1H),3.56(s,3H),1.51(d,J=7.6Hz,3H).

[0340] Step 2. To a solution of (S)-2-(1,3-dioxoisoindolin-2-yl)-N-methoxypropanamide (20 g, 81 mmol), palladium(II) acetate (1.809 g, 8.06 mmol), and silver acetate (26.9 g, 161 mmol) in a 1000 mL sealed tube was added tert-butyl 3-iodobenzoate (36.8 g, 121 mmol), 2,6-lutidine (2.395 mL, 24.17 mmol), and HFIP (300 mL) at 25 °C under a N atmosphere. The reaction was stirred at 25 °C under N for 15 min and then heated to 80 °C with vigorous stirring for 24 h. The reaction mixture was filtered through CELITE and washed with DCM (200 mL). The combined organic layers were concentrated under reduced pressure. The crude product was purified by Combiflash using a 220 g silica column eluting with 25-30% EtOAc:CHCl to give the desired product, tert-butyl (S)-3-(2-(1,3-dioxoisoindolin-2-yl)-3-(methoxyamino)-3-oxopropyl)benzoate (11 g, 25.9 mmol, 32.2% yield). Analytical condition E: retention time = 2.52 min; ESI-MS (+) m / z [M−H] + :423.2. 1 H NMR(DMSO-d6,400MHz)δ 11.46(s,1H),7.82(m,4H),7.63(d,J=7.6Hz,1H),7.54(s,1H),7.40(d,J=7.6Hz,1H),7.30(t,J =7.6Hz,1H),4.93-4.89(m,1H),3.59(s,3H),3.56-3.49(m,1H),3.36-3.27(m,1H),1.40(s,9H).

[0341] Step 3. To a solution of tert-butyl (S)-3-(2-(1,3-dioxoisoindolin-2-yl)-3-(methoxyamino)-3-oxopropyl)benzoate (15 g, 35.3 mmol) in methanol (200 mL) was added (diacetoxyiodo)benzene (12.52 g, 38.9 mmol) at room temperature. The temperature was slowly raised to 80 °C and stirred at 80 °C for 3 h. The reaction was concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography (100-200 mesh eluted with 20% EA:hexane) to give the desired compound, tert-butyl (S)-3-(2-(1,3-dioxoisoindolin-2-yl)-3-methoxy-3-oxopropyl)benzoate (10 g, 24.42 mmol, 69.1% yield). 1 H NMR(CDCl3,400MHz)δ 7.80-7.76(m,4H),7.72-7.68(m,2H),7.34-7.26(m,1H),7.25-7.23(m,1H) ,5.14(dd,J=10.8,5.6Hz,1H),3.76(s,3H),3.65-3.49(m,2H),1.50(s,9H).

[0342] Step 4. To a solution of tert-butyl (S)-3-(2-(1,3-dioxoisoindolin-2-yl)-3-methoxy-3-oxopropyl)benzoate (15 g, 36.6 mmol) in methanol (25 mL) was added ethylenediamine (12.25 mL, 183 mmol) at room temperature. The reaction temperature was slowly raised to 40 °C and stirred at 40 °C for 3 h. The mixture was concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography (100-200 mesh eluted with 20% EA:hexane) to give the desired compound (S)-3-(2-amino-3-methoxy-3-oxopropyl)benzoate (8.3 g, 29.7 mmol, 81% yield). 1 H NMR(DMSO-d6,400MHz)δ 8.32(s,1H),7.77-7.72(m,2H),7.46-7.38(m,1H),3.61-3.57(m,4H),2.96-2.91(m,1H),2.85-2.82(m,1H),1.79(br.s,2H),1.55(s,9H).

[0343] Step 5. To a solution of tert-butyl (S)-3-(2-amino-3-methoxy-3-oxopropyl)benzoate (10 g, 35.8 mmol) in dioxane (150 mL) was added sodium bicarbonate (6.01 g, 71.6 mmol), followed by 9-fluorenylmethyl chloroformate (13.89 g, 53.7 mmol) at room temperature. The reaction was stirred at room temperature for 12 hours. It was diluted with water and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure to give the crude product. This was purified via silica gel chromatography (100-200 mesh eluted with 20% EA:hexane) to give the desired compound, tert-butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)benzoate (15 g, 29.9 mmol, 84% yield).

[0344] Step 6. To a solution of tert-butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)benzoate (18.00 g, 35.9 mmol) in THF (150 mL) and HO (150 mL) at room temperature was added lithium hydroxide monohydrate (1.66 g, 39.5 mmol). The reaction was stirred at room temperature for 2 hours. The reaction was concentrated under reduced pressure to remove THF. In basic medium, the mixture was extracted with diethyl ether to remove non-polar impurities. The aqueous layer was acidified with aqueous citric acid and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give the desired compound as a gummy solid, which was further lyophilized to give an off-white solid. Desired Compound Lot 1: (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(tert-butoxycarbonyl)phenyl)propanoic acid (11 g, 22.56 mmol, 62.9% yield). Lot 2: (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(tert-butoxycarbonyl)phenyl)propanoic acid (5 g, 10.26 mmol, 28.6% yield). 7.86(t,J=7.6Hz,2H),7.75(d,J=7.6Hz,1H),7.66-7.59(m,2H),7.52(m,2H),7.41-7.37 (m,3H),7.31-7.24(m,2H),4.21-4.16(m,4H),3.17(m,1H),2.96(m,1H),1.53(br,s.9H). Analysis condition E: retention time = 3.865 minutes; ESI-MS(+)m / z[MH] + :486.2.

[0345] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(m-tolyl)propanoic acid [ka] The compound was synthesized according to the same procedure as (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(tert-butoxycarbonyl)phenyl)propanoic acid. Analysis condition E: Retention time = 3.147 min; ESI-MS (+) m / z [M+H] + :402.0. 1 H NMR(DMSO-d6,300MHz)δ 7.88(d,J=7.5Hz,2H),7.64(t,J=6.8Hz,2H),7.44(t,J=7.5Hz,2H),7.36-7.28(m,2H),7.18(t,J=7.5 Hz,1H),7.09-7.02(m,3H),4.24-4.17(m,4H),3.21-3.04(m,1H),2.89-2.81(m,1H),2.26(s,3H)ppm.

[0346] Preparation of ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate [ka]

[0347] Step 1: The compound was synthesized using a procedure similar to that described in the reference: To a 1000 mL flask equipped with a septum inlet and a magnetic stir bar, bismuth(III) chloride (5.25 g, 16.64 mmol) was added. The flask was connected to an argon line, and thionyl chloride (501 mL, 6864 mmol) was added via syringe. To the suspension, mesitylene (100 g, 832 mmol) was added. The flask was fitted with a condenser and connected to an oil bubbler, and the reaction mixture was heated in a 60 °C oil bath for 5 h. During this time, the solution turned red-orange in color, and HCl evolved from the solution. The reaction was monitored by LCMS. The flask was cooled in an ice bath, and excess thionyl chloride was removed under reduced pressure to give an orange liquid. To remove the catalyst, 2000 mL of pentane was added, stirred, and filtered through Celite, washing the bed with pentane (2 × 500 mL). The organic phase was collected and evaporated under reduced pressure to give 2,4,6-trimethylbenzenesulfinic acid chloride (151 g, 745 mmol, 90% yield) as a pale yellow solid, which was used in the next step without further purification. 1 H NMR (400MHz, CDCl3) δ 7.07-6.76 (m, 2H), 2.66 (s, 6H), 2.38-2.24 (m, 3H) ppm. [ka]

[0348] Step 2. The compound was synthesized using a procedure similar to that described in the reference: To a stirred solution of 2,4,6-trimethylbenzenesulfinic acid chloride (155 g, 765 mmol) in diethyl ether (1500 mL). After cooling to -40 °C, ammonia gas taken up in diethyl ether (900 mL) (2 L multi-neck RBF) was bubbled through in a separate setup at -40 °C for 30 minutes, and this purged solution was added to the above reaction mass at -40 °C. After warming to room temperature, the reaction mixture was stirred for 2 hours and monitored by open access LCMS for the absence of starting material. The reaction was stirred overnight at room temperature according to the given procedure. The reaction was monitored by TLC and open access LCMS (A1CE6-344-01), and the starting material was absent by TLC. Workup: The reaction mixture was diluted with ethyl acetate (3000 mL), washed with water (2000 mL), the organic layer was separated, and the aqueous phase was re-extracted with ethyl acetate (1 x 500 mL). The combined organic layers were washed with brine (1 x 800 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated under reduced pressure to give a light brown solid (235 g). The product (235 g) was recrystallized from 10% ethyl acetate / petroleum ether (500 mL), stirred, filtered, and dried to give mesitylenesulfinamide (125 g) racemate as a white solid. The compound was subjected to SFC method development. Two peaks were collected from SFC. The solvent was concentrated to give peak-1 (undesired): (R)-2,4,6-trimethylbenzenesulfinamide (51.6 g, 265 mmol, 34.6% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d) δ 7.01-6.68 (m, 2H), 6.23-5.77 (m, 2H), 2.52-2.50 (m, 6H), 2.32-1.93 (m, 3H) and peak-2 (desired): (S)-2,4,6-trimethylbenzenesulfinamide (51.6 g, 267 mmol, 35.0% yield) was obtained as a white solid. 1 H NMR (400MHz, DMSO-d6) δ 6.87 (s, 2H), 6.16-5.82 (m, 2H), 2.53-2.50 (m, 6H), 2.34-1.93 (m, 3H). [ka]

[0349] Step 3. The compound was synthesized using a procedure similar to that described in reference 1: To a well-stirred solution of (S)-2,4,6-trimethylbenzenesulfinamide (15.5 g, 85 mmol) in dichloromethane (235 mL) and 4A molecular sieves (84.5 g), 2-oxoethyl acetate in toluene (25.9 mL, 127 mmol) and pyrrolidine (0.699 mL, 8.46 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction was repeated, and two batches were combined for workup. The reaction was filtered through Celite, and the bed was washed with DCM. The solvent was removed under reduced pressure to give the crude product (55 g) as a brownish mass. The crude compound was purified by ISCO (column size: 300 g silica column, adsorbent: 60-120 silica mesh, mobile phase: 40% EtOAc / petroleum ether), and the product was recovered with 15-20% EtOAc. The fractions were concentrated to give ethyl (S,E)-2-((mesitylsulfinyl)imino)acetate (16.5 g, 57.4 mmol, 67.9% yield) as a colorless liquid, which slowly solidified as an off-white solid. 1 H NMR (400MHz, CDCl3) δ=8.27(s,1H),7.04-6.70(m,2H),4.59-4.21(m,2H),2.55-2.44(m,6H),2.36-2.23(m,3H),1.51-1.30(m,3H). 2.670 minutes 268.2(M+H).

[0350] Step 4. General Synthetic Procedure for TCNHPI Redox-Active Esters as in the Reference ACIE: TCNHPI esters were prepared according to the general procedure previously reported (the ACIE paper and its references): A round-bottom flask or culture tube was charged with carboxylic acid (1.0 equiv.), N-hydroxytetrachlorophthalimide (1.0–1.1 equiv.), and DMAP (0.1 equiv.). Dichloromethane was added (0.1–0.2 M), and the mixture was vigorously stirred. The carboxylic acid (1.0 equiv.) was then added. DIC (1.1 equiv.) was then added dropwise via syringe, and the mixture was stirred until the acid was consumed (as determined by TLC). Typical reaction times were 0.5–12 h. The mixture was filtered (through a thin pad of Celite®, SiO2, or a fritted funnel), and additional CHCl / EtO was added. The solvent was removed under reduced pressure, and the crude mixture was purified by column chromatography to yield the desired TCNHPI redox-active esters. If necessary, the TCNHPI redox active ester can be further recrystallized from CH2Cl2 / MeOH.

[0351] Step 5. Following the general procedure for the synthesis of TCNHPI redox-active esters on a 5.00 mmol scale, 4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl-4-((tert-butoxycarbonyl)amino)-2,2-dimethylbutanoate was obtained as a white solid. Purification by column chromatography (silica gel, gradient from CHCl to 10:1 CHCl:EtO) afforded 2.15 g (84%) of the title compound. 1 H NMR (400MHz, CDCl3): δ 4.89 (br s, 1H), 3.30 (q, J = 7.0Hz 2H), 1.98 (t, J = 7.6Hz, 2H), 1.42 (s, 15H) ppm. 13 C NMR (151MHz, CDCl3): δ 173.1,157.7,156.0,141.1,130.5,124.8,79.3,40.8,40.2,36.8,28.5,25.2 ppm. HRMS(ESI-TOF):C 19 H 20 Cl4N2NaO6[M+Na]+ Calculated value: 534.9968, measured value: 534.9973.

[0352] Step 6. Ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate was prepared using the general decarboxylation amino acid synthesis procedure from the reference ACIE. A culture tube was charged with TCNHPI redox-active ester A (1.0 mmol), sulfinimine B (2.0 mmol), Ni(OAc)2·4HO (0.25 mmol, 25 mol%), and zinc (3 mmol, 3 equiv). The tube was then evacuated and backfilled with argon (three times). Anhydrous NMP (5.0 mL, 0.2 M) was added using a syringe. The mixture was stirred overnight at room temperature. The reaction mixture was then diluted with EtOAc, washed with water, brine, and dried over MgSO4. After filtration, the organic layer was concentrated under reduced pressure (30 °C water bath) and purified by flash column chromatography (silica gel) to give the product. Purification by column chromatography (2:1 hexanes:EtOAc) afforded 327.6 mg (72%) of the title compound ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate as a colorless oil. 1 H NMR(600MHz,CDCl3):δ 6.86(s,2H),5.04(d,J=10.1Hz,1H),4.47(s,1H),4.28-4.16(m,2H),3.66(d,J=10.1Hz,1H),3.27-3.05( m,2H),2.56(s,6H),2.28(s,3H),1.54-1.46(m,2H),1.43(s,9H),1.30(t,J=7.2Hz,3H),0.96(s,6H)ppm. 13 C NMR (151MHz, CDCl3): δ 172.5,155.9,141.1,137.9,136.9,131.0,79.4,65.5,61.7,38.8,37.1,36.5,28.5,23.9,23.6,21.2,19.4,14.3ppm. HRMS(ESI-TOF):C 23 H39 N2O5S[M+H] + Calculated value: 455.2574, measured value: 455.2569. [ka]

[0353] Step 7. 2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((tert-butoxycarbonyl)amino)-3,3-dimethylpentanoic acid: A culture tube was charged with ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate (0.5 mmol, 1.0 equiv.), and HCl (4.0 equiv.) in MeOH (0.3 M) was added via syringe. The resulting mixture was stirred at room temperature for approximately 10 minutes (screened by TLC). After the reaction, EtN was added until pH = 7, and the solvent was removed under reduced pressure. LiOH (2 equiv.) in MeOH / HO (2:1, 0.04 M) was added to the crude mixture. The reaction was stirred at 60 °C overnight. Upon completion, HCl (0.3 M) in MeOH was added until pH = 7, and the solvent was removed under reduced pressure. The crude mixture was dissolved in 9% aqueous Na2CO3 (5 mL) and dioxane (2 mL). This was slowly added to a solution of Fmoc-OSu (1.2 equiv.) in dioxane (8 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h and then warmed to room temperature. After 10 h, the reaction mixture was quenched with HCl (0.5 M) to reach pH 3 and then diluted with EtOAc. The aqueous phase was extracted with EtOAc (3 × 15 mL), and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude mixture was then purified by flash column chromatography (silica gel, 2:1 hexanes:EtOAc) to give the product ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate in 68% overall yield and 95% ee as a colorless oil. 1H NMR(600MHz,CDCl3):δ 7.76(d,J=7.5Hz,2H),7.63-7.54(m,2H),7.39(td,J=7.3,2.6Hz,2H),7.33-7.28(m,2H),5.50(br s,1H),4.68(br s,1H),4.45-4.43(m,1H),4.38-4.35(m,1H),4.30(d,J=7.9Hz,1H),4.21(t,J=6.8Hz,1H),3.27(br s,1H),3.16(br s, 1H), 1.63-1.50 (m, 2H), 1.43 (s, 9H), 1.09-0.76 (m, 6H) ppm. 13 C NMR(151MHz,CDCl3):δ 185.8,174.3,156.5,144.0,143.9,141.5,127.9,127.2,125.24,125.21 ,120.2,120.1,79.8,67.2,60.9,47.4,39.2,36.8,29.9,28.6,23.9ppm. HRMS(ESI-TOF):C 27 H 35 N2O6[M+H] + Calculated value: 483.2490, measured value: 483.2489.

[0354] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4,4-difluorocyclohexyl)propanoic acid [ka] The final product was obtained following a similar procedure to ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate. The synthesis afforded the desired (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4,4-difluorocyclohexyl)propanoic acid (60 mg, 0.14 mmol, 27.9% yield) as a white solid after purification by reverse-phase HPLC. 1H NMR(500MHz,CDCl3)δ 7.79(br d,J=7.5Hz,2H),7.61(br s,2H),7.43(s,2H),7.36-7.31(m,2H),5.24-5.06(m,1H),4.57-4.36(m,3H),4.29-4.16(m,1H),2.19-1.99(m,2H),1.97-1.18(m,9H).

[0355] Preparation of (2S)-5-(tert-butoxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,3-dimethyl-5-oxopentanoic acid [ka]

[0356] Step 1. A solution of 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione (8.29 g, 58.3 mmol) in dry toluene (100 mL) was slowly added to a solution of (R)-2-amino-2-phenylethan-1-ol (10 g, 72.9 mmol) in dry toluene (100 mL) and CHCl (20 mL) at room temperature. The reaction mixture was then heated to 60 °C and reacted for 12 h. It was then cooled to room temperature until a white solid formed. The solid was filtered and washed with 1:1 EtOAc / CHCl to obtain the crude desired compound (R)-5-((2-hydroxy-1-phenylethyl)amino)-3,3-dimethyl-5-oxopentanoic acid (11.9 g, 41.0 mmol, 56.2% yield) without further purification. 1 H NMR(300MHz,DMSO-d6)δ 8.41(br d,J=7.9Hz,1H),7.44-7.32(m,2H),7.32-7.27(m,4H),7.26-7.18(m,1H),4.89-4.80(m,1H),4.14-3.98(m,1H),3.63 -3.43(m,3H),2.27-2.18(m,4H),2.08(s,1H),1.99(s,1H),1.17(t,J=7.2Hz,1H),1.00(d,J=4.5Hz,6H),0.92(s,1H).

[0357] Step 2. (R)-5-((2-hydroxy-1-phenylethyl)amino)-3,3-dimethyl-5-oxopentanoic acid (12 g, 43.0 mmol) was dissolved in a solution of benzyltrimethylammonium chloride (8.93 g, 48.1 mmol) in DMA (250 mL). KCO (154 g, 1117 mmol) was added to the above solution, followed by 2-bromo-2-methylpropane (235 mL, 2091 mmol). The reaction mixture was stirred at 55 °C for 24 h. The reaction mixture was then diluted with EtOAc (100 mL) and washed with HO (50 mL × 3) and brine (50 mL). The organic phase was dried over NaSO, concentrated in vacuo, and purified by flash column chromatography on silica gel (CHCl / MeOH, 15:1) to give (R)-5-((2-hydroxy-1-phenylethyl)amino)-3,3-dimethyl-5-oxopentanoate (6.0 g, 17.89 mmol, 41.6% yield). Analytical LC / MS conditions: M: 1.96 min, 336.3 [M+H] + . 1 H NMR(300MHz,DMSO-d6)d=8.14(br d,J=8.3Hz,1H),7.33-7.25(m,4H),7.25-7.17(m,1H),4.90-4.77(m,2H),3.52(br t,J=5.7Hz,2H),3.34(s,1H),2.94(s,1H),2.78(s,1H),2.20(d,J=14.0Hz,4H),1.97(d,J=9.8Hz,2H),1.41-1.31(m,9H),1.00(d,J=1.1Hz,6H). [ka]

[0358] Step 3. tert-Butyl (R)-5-((2-hydroxy-1-phenylethyl)amino)-3,3-dimethyl-5-oxopentanoate (6 g, 17.89 mmol) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (6.09 g, 26.8 mmol) were dissolved in dry dichloromethane (70 mL) under Ar. Triphenylphosphine (7.04 g, 26.8 mmol) was added to the above solution. The reaction mixture was stirred at room temperature for 2 h. The crude product was then concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc / hexane, 1:5) to give tert-butyl (R)-3,3-dimethyl-4-(4-phenyl-4,5-dihydrooxazol-2-yl)butanoate (5.6 g, 17.64 mmol, 99% yield). ESI-MS(+)m / z:318.3[M+H] + . 1 H NMR(300MHz,DMSO-d6)d=7.41-7.18(m,5H),5.18(t,J=9.1Hz,1H),4.59(dd,J=8.7,10.2Hz,1H),3.94-3.85(m,1H) ),3.94-3.85(m,1H),3.95-3.84(m,1H),4.10-3.84(m,1H),2.43-2.22(m,4H),1.40(s,9H),1.09(d,J=1.9Hz,6H).

[0359] Step 4. [ka] To a solution of tert-butyl (R)-3,3-dimethyl-4-(4-phenyl-4,5-dihydrooxazol-2-yl)butanoate (5.6 g, 17.64 mmol) in EtOAc (250 mL) was added selenium dioxide (4.89 g, 44.1 mmol) and refluxed for 2 hours. The reaction mixture was then cooled to room temperature and stirred for 12 hours. The crude product was then concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc / hexane, 1:7) to give tert-butyl (R)-3-methyl-3-(2-oxo-5-phenyl-5,6-dihydro-2H-1,4-oxazin-3-yl)butanoate (1.3 g, 3.92 mmol, 22.23% yield) as a colorless liquid. ESI-MS (+) m / z: 332.2 [M+H] + . 1H NMR(CDCl3)δ 1.37(s,3H),1.42(s,9H),1.44(s,3H),2.59(d,J=15.5Hz,1H),3.12(d,J=15.5Hz,1H),4.32(t,J= 11.1Hz,1H),4.47(dd,J=4.3Hz,J=6.7Hz,1H),4.80(dd,J=4.3Hz,J=6.7Hz,1H),7.35-7.39(m,5H). 13 C NMR(CD3Cl)δ 26.40,27.29,28.00,40.84,45.94,59.72,70.88,80.63,127.13,127.92,128.65,137.58,155.07,167.46,171.95.

[0360] Step 5. [ka] Platinum(IV) oxide monohydrate (130 mg, 0.530 mmol) was added to a solution of tert-butyl (R)-3-methyl-3-(2-oxo-5-phenyl-5,6-dihydro-2H-1,4-oxazin-3-yl)butanoate (1.3 g, 3.92 mmol) in methanol (50 mL). The reaction flask was purged with H (3×) and stirred under H for 24 h. After venting the vessel, the reaction mixture was filtered through Celite, and the filtrate was washed with EtOAc. The crude product was concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc / hexane, 1:8) to give tert-butyl 3-methyl-3-((3S,5R)-2-oxo-5-phenylmorpholin-3-yl)butanoate (1.2 g, 3.33 mmol, 85% yield). 1 H NMR(300MHz,DMSO-d6)δ 7.52-7.42(m,2H),7.41-7.26(m,3H),4.30-4.20(m,2H),4.13(d,J=10.6Hz,1H),3.80(d,J=7.6Hz,1H),3.07-2.98(m,1H),2.47(br s, 1H), 2.27 (d, J=13.6Hz, 1H), 1.43-1.35 (m, 9H), 1.17-1.07 (m, 5H).

[0361] Step 6. [ka] To a solution of tert-butyl 3-methyl-3-((3S,5R)-2-oxo-5-phenylmorpholin-3-yl)butanoate (1.2 g, 3.60 mmol) in methanol (50 mL) / water (3.13 mL) / TFA (0.625 mL) (40:2.5:0.5, v / v / v) was added Pearlman's carbon-supported catalyst Pd(OH) (1.264 g, 1.799 mmol, 20% w / w). The vessel was purged with H and stirred under H for 24 h. After venting the vessel, the reaction mixture was filtered through Celite, and the filtrate was washed with MeOH. The crude product ((S)-2-amino-5-(tert-butoxy)-3,3-dimethyl-5-oxopentanoic acid (0.83 g, 3.59 mmol, 100% yield) was concentrated under vacuum. This crude material was used in the next step without further purification. Analytical LC / MS conditions M: 1.13 min, 232.2 [M+H] + .

[0362] Step 7. [ka] The crude product (S)-2-amino-5-(tert-butoxy)-3,3-dimethyl-5-oxopentanoic acid (1 g, 4.32 mmol) was dissolved in water (30 mL). Na2CO3 (0.916 g, 8.65 mmol) was then added to the above solution. To this solution, fmoc n-hydroxysuccinimide ester (1.458 g, 4.32 mmol) in dioxane (30 mL) was added dropwise at 0 °C and stirred at room temperature for 16 hours. The reaction mixture was acidified to approximately pH 2 with 1N HCl, extracted with EtOAc (3 x 50 mL), dried over NaSO, concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc / petroleum ether, 35-39%) to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-3,3-dimethyl-5-oxopentanoic acid (0.73 g, 1.567 mmol, 36.2% yield) as a white solid. LCMS, analytical LC / MS conditions E, MS (ESI) R = 2.135 min, m / z 452.2 [M−H] - .1 H NMR(400MHz,DMSO-d6)δ 12.78-12.64(m,1H),7.90(d,J=7.5Hz,2H),7.77(dd,J=4.5,7.0Hz,2H),7.65(br d,J=9.5Hz,1H),7.46-7.39(m,2H),7.37-7.29(m,2H),4.32-4.15(m,4H ),2.39-2.31(m,1H),2.30-2.21(m,1H),1.39(s,9H),1.12-1.00(m,6H).

[0363] Preparation of (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(morpholin-4-yl)propanoic acid [ka]

[0364] Step 1. To a 2 L multi-necked round-bottom flask equipped with a thermal pocket was added (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (50 g, 245 mmol), dioxane (500 mL), followed by 1-bromo-2-(2-bromoethoxy)ethane (30.8 mL, 245 mmol) at room temperature. NaOH (367 mL, 734 mmol) solution was added, and the resulting clear yellow solution was heated to 110° C. (ambient, 85° C. internal) for 12 hours. An aliquot of the clear solution was subjected to LCMS (Polar method), which indicated completion, and then the dioxane was evaporated to give a pale red solution, which was acidified to pH 3. The resulting mixture was concentrated under high vacuum pump (approximately 4 mbar) at 60° C. to give (S)-2-((tert-butoxycarbonyl)amino)-3-morpholinopropanoic acid (67 g, 244 mmol, 100% yield) as a pale yellow solid. Analytical LC / MS conditions: M: 0.56 min, 275.2 [M+H] + .

[0365] Step 2. To a stirred suspension of (S)-2-((tert-butoxycarbonyl)amino)-3-morpholinopropanoic acid (100 g, 365 mmol) in dioxane (400 mL) at 0-5°C, HCl in dioxane (911 mL, 3645 mmol) was added slowly over 20 min. The resulting mixture was stirred at room temperature for 12 h. Evaporation of the volatiles gave a pale yellow, sticky crude (S)-2-amino-3-morpholinopropanoic acid (16 g, 92 mmol, 97% yield). This crude material was used in the next step without further purification. MS (ESI) m / z 175.2 [M+H] + .

[0366] Step 3. Then, the crude product (S)-2-amino-3-morpholinopropanoic acid (11 g, 63.1 mmol) was dissolved in water (250 mL), and Na2CO3 (13.39 g, 126 mmol) was added to the above solution. To this solution, Fmoc N-hydroxysuccinimide ester (21.30 g, 63.1 mmol) was added dropwise at 0 °C and stirred at room temperature for 16 h. The reaction mixture was acidified to approximately pH 2 with 1N HCl, extracted with EtOAc (3 x 500 mL), dried over NaSO, concentrated in vacuo, and purified by flash column chromatography on silica gel (petroleum ether / EtOAc, 0-100%, then MeOH / CHCl, 0-15%) to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-morpholinopropanoic acid (23 g, 55.9 mmol, 89% yield) as a brown solid. Analytical LC / MS conditions: E: 1.43 min, 397.2 [M+H] + . 1 H NMR(400MHz, methanol-d4)δ 7.78(br d,J=7.5Hz,2H),7.71-7.57(m,2H),7.42-7.34(m,2H),7.34-7.26(m,2H),4.71(br s,1H),4.54-4.32(m,2H),4.29-4.17(m,1H),3.90(br s,4H),3.76-3.62(m,1H),3.58-3.47(m,1H),3.41(br s,2H),3.36-3.32(m,2H),3.31-3.26(m,1H).

[0367] Preparation of (2S,3S)-3-{[(tert-butoxy)carbonyl]amino}-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoic acid [ka]

[0368] Step 1. [ka] To a solution of benzyl (tert-butoxycarbonyl)-L-threoninate (22 g, 71.1 mmol) in CHCl (600 mL) at −78° C., trifluoromethanesulfonic anhydride (24.08 g, 85 mmol) was added dropwise, followed by slow addition of 2,6-lutidine (10.77 mL, 92 mmol). After stirring at the same temperature for 1.5 hours and monitoring by TLC (hexane:EtOAc 8:2), tetrabutylammonium azide (50.6 g, 178 mmol) was added in small portions. After stirring at −78° C. for 1 hour, the cooling bath was removed and the reaction mixture was allowed to reach 23° C. over 1.5 hours. The reaction was repeated. Saturated aqueous NaHCO was added, and the aqueous phase was extracted with EtOAc. The crude product was purified by flash chromatography on silica gel (Hex: EtOAc 95:5 to 9:1) to give benzyl (2S,3S)-3-azido-2-((tert-butoxycarbonyl)amino)butanoate (20 g, 59.8 mmol, 84% yield) as a colorless liquid. Analytical LC / MS conditions: E: 3.13 min, 333.2 [MH] - .

[0369] Step 2. [ka] A solution of benzyl (2S,3S)-3-azido-2-((tert-butoxycarbonyl)amino)butanoate (20 g, 59.8 mmol), dichloromethane (300 mL), and TFA (50 mL, 649 mmol) was stirred at 23 °C for 2 hours and then evaporated to dryness to give the corresponding amine. The amine was redissolved in water (200 mL) and tetrahydrofuran (200 mL). At 0 °C, DIPEA (11.49 mL, 65.8 mmol) was added, followed by Fmoc chloride (17.02 g, 65.8 mmol). The mixture was warmed to room temperature and stirred for 3 hours. It was extracted with EtOAc and washed with 0.5 M HCl solution and then brine solution. It was concentrated to give a crude liquid. The crude product was purified by silica gel column chromatography. The product was eluted with 20% EtOAc in petroleum ether. The fractions were concentrated to give benzyl (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-azidobutanoate (23 g, 50.4 mmol, 84% yield) as a colorless liquid. Analytical LC / MS conditions E: 3.70 min, 479.3 [M+Na] + .

[0370] Step 3. A multi-necked round bottle flask was charged with benzyl (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-azidobutanoate (40 g, 88 mmol) in tetrahydrofuran (1200 mL). Pd / C (9.32 g, 8.76 mmol) was added under nitrogen, and the reaction was stirred under hydrogen for 12 hours. Sodium bicarbonate (11.04 g, 131 mmol) in 6 mL of water was added, followed by Boc anhydride (30.5 mL, 131 mmol). The mixture was stirred under nitrogen for 12 hours. The reaction mass was filtered through a celite bed, and the bed was washed with a THF / water mixture. The mother liquor was concentrated and washed with EtOAc. The pH of the aqueous layer was then adjusted to 7-6 using 1.5 N HCl solution. The resulting white solid was extracted with ethyl acetate. The above reaction was repeated three more times. The combined organic layers were washed with water and brine solution, dried over sodium sulfate, and concentrated to give (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)butanoic acid as a white solid (28 g). This was mixed with the previously obtained batch (8 g) in DCM (200 mL). n-Hexane (1 L) was added to the above solution and sonicated for 2 minutes. The solid was filtered, rinsed with hexane, and dried overnight to give (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)butanoic acid (36 g, 81 mmol, 92% yield) as a white powder. Analytical LC / MS conditions E: 1.90 min, 439.2 [MH] - . 1H NMR(400MHz,DMSO-d6)d 7.90(d,J=7.6Hz,2H),7.75(d,J=7.2Hz,2H),7.43(t,J=7.2Hz,2H),7.34(t,J=Hz,6.71(br.dJ=7.6Hz) ,1H),4.29-4.26(m,2H),4.25-4.21(m,1H),3.94-3.90(m,1H),1.37(s,9H),1.02(d,J=6.8Hz,3H) 13C. NMR(101Hz,DMSO-d6)δ 171.9, 156.3, 154.8, 143.7, 140.6, 127.6, 127.0, 125.3, 120.0, 77.7, 65.8, 57.8, 47.0, 46.6, 28.2, 16.2.

[0371] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)acetic acid [ka] The final product was obtained following a similar procedure for ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate. The synthesis afforded the desired product (0.65 g, 22% yield) as a white solid after purification by flash column chromatography (Red Sep, 40 g, SiO, 35-40% EtOAc:hexanes (compound ELSD active)). Analytical LC / MS conditions: E: 2.04 min, 465.2 [MH] - . 1 H NMR(300MHz,DMSO-d6)δ 7.90(d,J=7.6Hz,2H),7.71(m,3H),7.47-7.27(m,2H),6.98-6.71(m,2H),4.30 -4.17(m,3H),3.94-3.82(m,1H),3.20-2.90(m,2H),1.44-1.30(m,9H),0.48(br s,4H).

[0372] Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(1-(tert-butoxycarbonyl)azetidin-3-yl)acetic acid [ka] The final product was obtained following a similar procedure for ethyl (S)-5-((tert-butoxycarbonyl)amino)-2-(((S)-mesitylsulfinyl)amino)-3,3-dimethylpentanoate. The synthesis afforded the desired product (2.66 g, 20% yield) as a slightly tan solid after purification by reverse-phase HPLC. Analytical LC / MS conditions E: 1.87 min, 467.2 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ 7.89(d,J=7.6Hz,2H),7.69(m,2H),7.41(t,J=7.2Hz,2H),7.34-7.31(m,2H),6.71( br.dJ=7.6Hz,1H),4.29-4.23(m,3H),3.77-3.70(m,5H),2.80(m,1H),1.36(s,9H).

[0373] Linker / Tail Synthesis: Preparation of perfluorophenyl dodecanoate (C12) Scheme: [ka] To dodecanoic acid (1310 mg, 6.54 mmol) was added DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (1.454 mL, 8.50 mmol), and pyridine (1.322 mL, 16.35 mmol). The vial was capped and shaken at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was used directly in the next step.

[0374] Preparation of perfluorophenyl tetradecanoate (C14) Scheme: [ka] To tetradecanoic acid (345 mg, 1.511 mmol) was added DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (0.388 mL, 2.266 mmol), and pyridine (0.269 mL, 3.32 mmol). The vial was capped and shaken at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was used directly in the next step.

[0375] Preparation of perfluorophenyl pentadecanoate (C15) Scheme: [ka] To pentadecanoic acid (324 mg, 1.337 mmol) was added DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (0.343 mL, 2.005 mmol), and pyridine (0.238 mL, 2.94 mmol). The vial was capped, and the mixture was shaken at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was used directly in the next step.

[0376] Preparation of perfluorophenyl palmitate (C16) Scheme: [ka] To a pressure-sealed vial was added palmitic acid (1.026 g, 4 mmol, 1.0 equiv.), DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (1.369 mL, 8.00 mmol, 2.0 equiv.), and pyridine (0.712 mL, 8.80 mmol, 2.2 equiv.). The reaction mixture was maintained under a nitrogen atmosphere and stirred at room temperature for 16 h. The reaction was then poured into saturated citric acid solution and extracted three times with CHCl. ​​The organic layers were combined, washed with brine, dried over anhydrous NaSO, filtered, and evaporated in vacuo. The crude material was purified by silica gel chromatography (80 g) eluting with 100% hexane to 5% ethyl acetate / hexane. Appropriate fractions were combined to give perfluorophenyl palmitate (1.47 g, 0.348 mmol, 87% yield). 1H NMR (400 MHz, chloroform-d) δ 2.68 (t, J = 7.4 Hz, 2H), 1.80 (quin, J = 7.5 Hz, 2H), 1.44 (br.s., 3H), 1.29 (s, 21H), 0.93-0.87 (m, 3H).

[0377] Preparation of perfluorophenyl heptadecanoate (C17) Scheme: [ka] To heptadecanoic acid (304 mg, 1.124 mmol) was added DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (0.288 mL, 1.686 mmol), and pyridine (0.200 mL, 2.473 mmol). The vial was capped and shaken at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was used directly in the next step.

[0378] Preparation of perfluorophenyl stearate (C18) Scheme: [ka] To stearic acid (297 mg, 1.044 mmol) was added DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (0.268 mL, 1.566 mmol), and pyridine (0.186 mL, 2.297 mmol). The vial was capped and shaken at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was used directly in the next step.

[0379] Preparation of perfluorophenyl (R)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13 dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate Scheme: [ka] To (R)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13 dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoic acid (300 mg, 0.797 mmol) was added DMF (5 mL), perfluorophenyl 2,2,2-trifluoroacetate (0.204 mL, 1.195 mmol), and pyridine (0.142 mL, 1.753 mmol). The vial was capped and shaken at room temperature for 2 hours. The reaction mixture was concentrated, and the crude product was used directly in the next step.

[0380] Preparation of 1-tert-butyl 2,3,4,5,6-pentafluorophenyltetradecanedioate (C14) Scheme: [ka]

[0381] Step 1: 14-(tert-butoxy)-14-oxotetradecanoic acid N,N-Dimethylformamide di-tert-butyl acetal (24.78 mL, 103 mmol) was added to a suspension of tetradecanoic acid (10 g, 38.7 mmol) in toluene (62.5 mL) at 120° C. over 4 h. This was then heated at reflux (128° C.) for 16 h. After cooling to room temperature, all volatiles were removed in vacuo, and the residue was suspended in DCM / EtOAc (1:1, 130 mL) at room temperature for 30 min. The solid was collected by filtration and triturated with DCM (50 mL). After filtration, 14-(tert-butoxy)-14-oxotetradecanoic acid was obtained (4.02 g, 33% yield). 1 H NMR (400MHz, DMSO-d6) δ 11.89 (br s, 1H), 2.18 (br t, J=7.2Hz, 5H), 1.49 (br s, 5H), 1.39 (br s, 1H), 1.25 (br s, 19H).

[0382] Step 2: 1-tert-butyl 2,3,4,5,6-pentafluorophenyltetradecanedioate To a 250 mL round-bottom flask was added 14-(tert-butoxy)-14-oxotetradecanoic acid (5.27 g, 16.76 mmol), DMF (33.5 mL), pentafluorophenyl trifluoroacetate (5.76 mL, 33.5 mmol), and pyridine (3.39 mL, 41.9 mmol). The flask was sealed with a septum, kept under a nitrogen atmosphere, and stirred at room temperature overnight. The reaction mixture was poured into saturated citric acid solution and extracted three times with DCM. The organic layers were combined, washed with brine, dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified by silica gel chromatography eluting with 0% ethyl acetate / 100% hexane to 55% ethyl acetate / 45% hexane. The pure fractions were combined and evaporated in vacuo to give 1-tert-butyl 14-(perfluorophenyl)tetradecanedioate (8.05 g, 16.76 mmol, 100% yield). 1 H NMR (400 MHz, chloroform-d) δ 2.67 (t, J = 7.4 Hz, 2H), 2.22 (t, J = 7.5 Hz, 2H), 1.84-1.72 (m, 2H), 1.58 (t, J = 7.2 Hz, 2H), 1.45 (s, 9H), 1.44-1.37 (m, 2H), 1.28 (br.s., 14H).

[0383] 1-Tert-butyl 2,3,4,5,6-pentafluorophenyl dodecanedioate (C12) was made according to the procedure described above.

[0384] Preparation of 1-(tert-butyl) 15-(perfluorophenyl) pentadecanedioate (C15) Scheme: [ka]

[0385] Step 1: 15-(tert-butoxy)-15-oxopentadecanoic acid A three-neck flask was charged with pentadecanedioic acid (5.00 g, 18.36 mmol) and toluene (36.7 mL) to form a suspension. The mixture was heated to reflux until all solids were dissolved. With continued heating, 1,1-di-tert-butoxy-N,N-dimethylmethanamine (8.80 mL, 36.7 mmol) was added dropwise over 30 minutes. The mixture was heated under reflux overnight. The reaction mixture was cooled to room temperature and transferred to a 250 mL round-bottom flask. The volatiles were removed under reduced pressure to give an off-white solid. 150 mL of dichloromethane was added to the flask, and the suspension was stirred for 2 hours. The suspension was filtered by vacuum filtration. The recovered solid was again stirred with dichloromethane for 30 minutes and filtered. The filtrate was concentrated under reduced pressure to give a white solid. The product was dissolved in 200 mL of dichloromethane and concentrated. This procedure was performed twice. The resulting white solid was placed under vacuum for 72 hours to give 15-(tert-butoxy)-15-oxopentadecanoic acid (3.94 g, 65.3%). 1 H NMR (400 MHz, methanol-d4 δ 2.28 (t, J = 7.4 Hz, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.63-1.55 (m, 4H), 1.45 (s, 9H), 1.31 (s, 18H).

[0386] Step 2: 1-(tert-butyl) 15-(perfluorophenyl) pentadecanedioate To a 50 mL round-bottom flask was added 15-(tert-butoxy)-15-oxopentadecanoic acid (0.97 g, 2.95 mmol), DMF (11.81 mL), pyridine (0.836 mL, 10.34 mmol), and pentafluorophenyl trifluoroacetate (1.272 mL, 7.38 mmol). The flask was sealed with a septum, kept under a nitrogen blanket, and stirred at room temperature overnight. The next day, the reaction was poured into saturated citric acid solution and extracted three times with DCM. The organic layers were combined, washed with brine, dried over NaSO, and evaporated in vacuo. The crude product, 1-tert-butyl 15-(perfluorophenyl)pentadecanedioate, was used directly without purification.

[0387] Preparation of 1-(tert-butyl) 16-(perfluorophenyl) hexadecanedioate (C16) Scheme: [ka]

[0388] Step 1: Preparation of 16-(tert-butoxy)-16-oxohexadecanoic acid Hexadecanedioic acid (4.5 g, 15.71 mmol) was suspended in toluene (28.1 mL) and the mixture was heated to reflux. 1,1-Di-tert-butoxy-N,N-dimethylmethanamine (10.10 mL, 42.1 mmol) was added dropwise over 30 minutes. The mixture was left under reflux overnight. The solvent was removed in vacuo at 50°C, and the crude material was suspended in DCM / EtOAc (75 mL, 1:1) and stirred for 15 minutes. The solids were removed by filtration and washed with DCM (25 mL). The filtrate was evaporated in vacuo. The resulting material was suspended in DCM (6 mL), cooled on ice for 10 minutes, and filtered. The solvent was removed in vacuo to leave the crude product, which was purified by flash chromatography eluting with EtOAc / hexanes. After evaporation of the desired fractions, 16-(tert-butoxy)-16-oxohexadecanoic acid (2.56 g, 7.47 mmol, 47.6% yield) was obtained. 1 H NMR (500 MHz, methanol-d₄) δ 2.35-2.14 (m, 4H), 1.65-1.54 (m, 4H), 1.50-1.42 (m, 11H), 1.36-1.21 (m, 18H).

[0389] Step 2: Preparation of 1-(tert-butyl) 16-(perfluorophenyl) hexadecanedioate To a 1-dram vial was added 16-(tert-butoxy)-16-oxohexadecanoic acid (100 mg, 0.292 mmol), DMF (0.8 mL), perfluorophenyl 2,2,2-trifluoroacetate (164 mg, 0.584 mmol), and pyridine (0.052 mL, 0.642 mmol). The vial was sealed with a septum and stirred at room temperature overnight. The next day, the crude reaction mixture was loaded onto a silica gel column and purified by elution with 5% EtOAc / 95% hexane to 30% EtOAc / 70% hexane. Pure fractions were combined and evaporated in vacuo to afford 1-tert-butyl 16-(perfluorophenyl)hexadecanedioate (132 mg, 0.260 mmol, 89% yield) as a white solid. 1 H NMR (400 MHz, chloroform-d) δ 2.68 (t, J = 7.4 Hz, 2H), 2.22 (t, J = 7.5 Hz, 2H), 1.79 (quin, J = 7.4 Hz, 3H), 1.51-1.34 (m, 15H), 1.29 (br s, 16H).

[0390] Preparation of 1-(tert-butyl) 18-(perfluorophenyl) octadecandioate (C18) Scheme: [ka]

[0391] Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid Octadecanedioic acid (5.0 g, 15.90 mmol) was suspended in toluene (28.4 mL) and the mixture was heated to reflux. 1,1-Di-tert-butoxy-N,N-dimethylmethanamine (10.22 mL, 42.6 mmol) was added dropwise over 30 min. The mixture was refluxed overnight. The solvent was removed in vacuo at 50 °C, and the crude material was suspended in DCM / EtOAc (75 mL, 1:1) and stirred for 15 min. The solid was removed by filtration and washed with DCM (25 mL). The filtrate was evaporated in vacuo. The crude product was purified by flash chromatography (0–25% acetone / DCM) to give 18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol, 29.2% yield). 1 H NMR (500 MHz, methanol-d₄) shifts 2.32-2.17 (m, 4H), 1.66-1.54 (m, 4H), 1.50-1.30 (m, 32H).

[0392] Step 2: Preparation of 1-(tert-butyl) 18-(perfluorophenyl) octadecanedioate To a 50 mL round-bottom flask was added 18-(tert-butoxy)-18-oxooctadecanoic acid (807 mg, 2.178 mmol), N,N-dimethylformamide (8 mL), pyridine (379 mg, 4.79 mmol), and perfluorophenyl 2,2,2-trifluoroacetate (1220 mg, 4.36 mmol). The flask was sealed with a septum, kept under a nitrogen blanket, and stirred at room temperature overnight. The next day, the reaction was poured into saturated citric acid solution and extracted three times with DCM. The organic layers were combined, washed with brine, dried over NaSO, and evaporated in vacuo. The crude product, 1-tert-butyl 18-(perfluorophenyl)octadecandioate (1.1 g, 2.050 mmol, 94% yield), was used directly without purification. 1 H NMR(400MHz,chloroform-d)δ 2.70-2.63(m,2H),2.22(t,J=7.5Hz,2H),1.78(quin,J=7.5Hz,2H),1.63-1.52(m,2H),1.48-1.39(m,12H),1.37-1.20(m,21H).

[0393] Preparation of (S)-1-tert-butyl 5-(perfluorophenyl) 2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate Scheme: [ka]

[0394] Step 1: Preparation of resin-bound -(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid 2-Chloro-chlorotrityl resin mesh 50-150 (1.54 meq / gram, 1.94 grams, 3.0 mmol) was added to a glass reaction vessel equipped with a frit and allowed to swell in DCM (5 mL) for 5 minutes. A solution of Fmoc-Glu-OtBu (1.276 g, 3.00 mmol, 1.0 equiv.) in DCM (5 mL) was added to the resin, followed by DIPEA (2.61 mL, 15.00 mmol, 5.0 equiv.). The reaction was shaken at room temperature for 60 minutes. DIEA (0.5 mL) and methanol (3 mL) were added and shaken for an additional 15 minutes. The reaction solution was filtered through a frit, and the resin was rinsed with DCM (4 × 5 mL), DMF (4 × 5 mL), DCM (4 × 5 mL), diethyl ether (4 × 5 mL), and dried using a stream of nitrogen.

[0395] A resin sample (13.1 mg) was treated with 20% piperidine / DMF (v / v, 2.0 mL) by shaking for 10 minutes. 1 mL of this solution was transferred to a 25.0 mL volumetric flask and diluted with methanol to a total volume of 25.0 mL. A blank solution of 20% piperidine / DMF (v / v, 1.0 mL) was diluted to 25.0 mL with methanol in a volumetric flask. The UV was set to 301 nm and 0 on the blank solution, and the solution was subsequently read, resulting in an absorbance of 1.9411 (1.9411 / 20 mg) * 6.94 = 0.6736. The resin loading was determined to be 0.6736 mmol / g.

[0396] Step 2: Preparation of (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid To a glass reaction vessel equipped with a frit, the pre-loaded resin and 20% piperidine / DMF solution (v / v, 5 mL) were added, and the suspension was shaken for 5 min. The solution was filtered, and the resin was treated again with 20% piperidine / DMF (v / v, 5 mL) for an additional 5 min. The reaction solution was filtered through a frit, and the resin was washed with DMF (6 × 5 mL × 1 min shaking). A solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (593 mg, 1.6 mmol, 1.6 equiv) and 1-hydroxy-7-azabenzotriazole (218 mg, 1.600 mmol, 1.6 equiv) in DMF (4 mL) was added to the resin, followed by the addition of N,N'-diisopropylcarbodiimide (251 μL, 1.600 mmol, 1.6 equiv). The reaction was shaken at room temperature for 16 h. The resin was washed with DMF (4 x 5 mL x 1 min shaking).

[0397] The protected peptide was cleaved from the resin with 20% HexaFluoro IPA / DCM (v / v, 30 mL) at room temperature for 2 hours. The cleavage solution containing the crude product was obtained by filtration. The resin was rinsed with DCM (2 × 5 mL). The combined filtrate was evaporated and chased with DCM (2 × 5 mL) to give (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid as an oil (378 mg, 0.680 mmol, 68%). Analysis condition E: retention time = 1.32 min; ESI-MS (+) m / z [M+H] + :557.5.

[0398] Step 3: Preparation of 1-(tert-butyl) 5-(perfluorophenyl)(18-(tert-butoxy)-18-oxooctadecanoyl)-L-glutamate To a pressure-sealed vial was added (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid (378 mg, 0.680 mmol, 1.0 equiv), DMF (5 mL), perfluorophenyl-2,2,2-trifluoroacetate (233 μl, 1.360 mmol, 2.0 equiv), and pyridine (121 μl, 1.496 mmol, 2.2 equiv). The reaction mixture was kept under a nitrogen atmosphere and stirred at room temperature for 16 h. The reaction was poured into saturated citric acid solution and extracted with CHCl (3×). The organic layers were combined, washed with brine, dried over NaSO, and evaporated in vacuo. The crude material was purified by silica gel chromatography (40 g), eluting with 100 hexanes to 30% ethyl acetate / hexanes. The appropriate fractions were combined to give (S)-1-tert-butyl 5-(perfluorophenyl) 2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate (249 mg, 0.345 mmol, 50.7% yield). 1 H NMR(400MHz,chloroform-d)δ 6.08(d,J=7.8Hz,1H),4.63(td,J=7.8,5.0Hz,1H),2.89-2.65(m,2H),2.43-2.31(m ,1H),2.29-2.18(m,4H),2.14-2.02(m,1H),1.59-1.43(m,18H),1.37-1.22(m,24H).

[0399] Preparation of (R)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (N3(Dab)-FDA18) Scheme: [ka]

[0400] Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid Octadecanedioic acid (5.0 g, 15.90 mmol) was suspended in toluene (28.4 mL) and the mixture was heated to reflux. 1,1-Di-tert-butoxy-N,N-dimethylmethanamine (10.22 mL, 42.6 mmol) was added dropwise over 30 min. The mixture was refluxed overnight. The solvent was removed in vacuo at 50 °C, and the crude material was suspended in DCM / EtOAc (75 mL, 1:1) and stirred for 15 min. The solid was removed by filtration and washed with DCM (25 mL). The filtrate was evaporated in vacuo. The crude product was purified by flash chromatography (0–25% acetone / DCM) to give 18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol, 29.2% yield). 1 H NMR (500 MHz, methanol-d₄) δ 2.32-2.17 (m, 4H), 1.66-1.54 (m, 4H), 1.50-1.30 (m, 32H).

[0401] Step 2: Preparation of 1-(tert-butyl) 18-(perfluorophenyl) octadecanedioate To a 50 mL round-bottom flask was added 18-(tert-butoxy)-18-oxooctadecanoic acid (807 mg, 2.178 mmol), N,N-dimethylformamide (8 mL), pyridine (379 mg, 4.79 mmol), and perfluorophenyl 2,2,2-trifluoroacetate (1220 mg, 4.36 mmol). The flask was sealed with a septum, kept under a nitrogen blanket, and stirred at room temperature overnight. The reaction was poured into saturated citric acid solution and extracted with DCM (3x). The organic layers were combined, washed with brine, dried over NaSO, and evaporated in vacuo. The crude product, 1-tert-butyl 18-(perfluorophenyl)octadecandioate (1.1 g, 2.050 mmol, 94% yield), was used directly without purification. 1 H NMR(400MHz,chloroform-d)δ 2.70-2.63(m,2H),2.22(t,J=7.5Hz,2H),1.78(quin,J=7.5Hz,2H),1.63-1.52(m,2H),1.48-1.39(m,12H),1.37-1.20(m,21H).

[0402] Step 3: Preparation of (R)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid To a solution of 1-tert-butyl 18-(perfluorophenyl)octadecandioate (110 mg, 0.205 mmol) and (R)-2-amino-4-azidobutanoic acid (29.5 mg, 0.205 mmol) in DMF (7 mL) was added N-ethyl-N-isopropylpropan-2-amine (26.5 mg, 0.205 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo and purified via ISCO to give the desired (R)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (80 mg, 89%).

[0403] Preparation of (S)-1-Azido-16-carboxy-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oic acid (Azide-PEG3-γGlu-FDA18) Scheme [ka]

[0404] Step 1: Preparation of tert-butyl (S)-1-azido-16-(tert-butoxycarbonyl)-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oate To a solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (113 mg, 0.517 mmol, 1.1 equiv) in DMF (4498 μL) was added Hunig's base (314 μL, 1.799 mmol, 4.0 equiv), followed by (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid (250 mg, 0.450 mmol). HATU (342 mg, 0.900 mmol, 2.0 equiv) was then added, and the resulting solution was stirred at room temperature. The DMF was removed under high vacuum, and the residue was then applied to silica gel (40 g) and eluted with DCM (100 mL), followed by a gradient to 75% DCM / acetone over 540 mL, and finally held at 75% DCM / acetone over 150 mL. The desired fractions were combined, and the material was used directly in the next step.

[0405] Step 2: Preparation of (S)-1-azido-16-carboxy-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oic acid To a solution of (S)-tert-butyl 1-azido-16-(tert-butoxycarbonyl)-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oate (414.0 mg, 0.548 mmol) in DCM (5476 μL) was added TFA (1266 μL, 16.43 mmol, 30.0 equiv). LC / MS indicated a slow reaction, so an additional 14 equiv of TFA was added and the mixture was further stirred. After an additional 6 h, LC / MS indicated a nearly complete reaction. The solvent was removed in vacuo. The mixture was dissolved in Hunig's base / MeOH (approximately 1%). The reaction mixture was purified by PREP HPLC in five injections: (30 x 100 mm HPLC Luna Axia C18 50-100% A:B over 10 min, 100% B in 5 min (A is 90:10:0.1 water:MeOH:TFA; B is 90:10:0.1 MeOH:water:TFA)). The desired fractions were combined and concentrated to give (S)-1-azido-16-carboxy-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oic acid (112.4 mg, 0.124 mmol, 22.64% yield). LC / MS: (M+H) + =644.45.

[0406] Preparation of (S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15pentaoxa-18,23-diazapentacontan-41-oic acid (ELN) 17-{[(1S)-3-[(17-azido-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamoyl]-1-carboxypropyl]carbamoyl}heptadecanoic acid (IUPAC) (Azide-Peg5-γGlu-FDA18) Scheme [ka]

[0407] Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid Octadecanedioic acid (7.5 g, 23.85 mmol) was suspended in toluene (42.6 mL) and the mixture was heated to reflux. 1,1-Di-tert-butoxy-N,N-dimethylmethanamine (15.33 mL, 63.9 mmol, 2.7 equiv.) was added dropwise over 30 min. The mixture was refluxed overnight. The solvent was removed in vacuo at 50 °C, and the crude material was suspended in CHCl / EtOAc (110 mL, 1:1) and stirred for 15 min. The solids were removed by filtration and washed with CHCl (40 mL). The filtrate was evaporated in vacuo. The crude product was purified by flash chromatography (0–25% acetone / CHCl) to give 18-(tert-butoxy)-18-oxooctadecanoic acid (3.95 g, 10.66 mmol, 44.7% yield). Analysis conditions D: retention time = 5.04 minutes; ESI-MS(+)m / z 297.3 [M-OC(CH3)3]. 1 H NMR (500 MHz, methanol-d₄) δ 2.29 (t, J = 7.5 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.67-1.53 ​​(m, 4H), 1.50-1.42 (m, 9H), 1.40-1.25 (m, 24H).

[0408] Step 2: Preparation of 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate DCC (5.11 mL, 5.11 mmol, 1.1 equiv) was added to a solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol) and 1-hydroxypyrrolidine-2,5-dione (0.588 g, 5.11 mmol, 1.1 equiv) in DMF (48 mL). The mixture was stirred at room temperature overnight. The mixture was filtered and concentrated to give 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate, which was used directly in the next step.

[0409] Step 3: Preparation of (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid Water (5.80 mL) was added to a mixture of (S)-4-amino-5-(tert-butoxy)-5-oxopentanoic acid (1.038 g, 5.11 mmol, 1.1 equiv), 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate (2.171 g, 4.64 mmol), and sodium bicarbonate (0.468 g, 5.57 mmol, 1.2 equiv) in THF (17.41 mL). The resulting clear solution was stirred at room temperature for 4 h. The solvent was removed. HCl (6.04 mL, 6.04 mmol, 1.3 equiv) was added and the pH was adjusted to 2-3 at 0 °C. The resulting suspension was extracted with CHCl (3x). The organic layer was concentrated. The resulting crude product was purified by flash chromatography (acetone / CHCl 0-25%) to give (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid (2.29 g, 4.12 mmol, 89% yield) as a white solid. Analytical condition D: retention time = 2.74 min; ESI-MS (+) m / z 555.6 (M+H). + . 1 H NMR(500MHz, methanol-d4)δ 4.32(dd,J=9.0,5.3Hz,1H),2.45-2.33(m,2H),2.30-2.06(m,5H),1.99- 1.82 (m, 3H), 1.78-1.53 ​​(m, 2H), 1.53-1.44 (m, 18H), 1.44-1.26 (m, 24H).

[0410] Step 4: Preparation of (S)-tert-butyl 1-azido-22-(tert-butoxycarbonyl)-19,24-dioxo-3,6,9,12,15pentaoxa-18,23diazapentacontan-41-oate To a solution of (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecaneamido)-5-oxopentanoic acid (225 mg, 0.405 mmol) in DMF (4048 μL) was added Hunig's base (212 μL, 1.214 mmol, 3.0 equiv.) and HATU (308 mg, 0.810 mmol, 2.0 equiv.). 17-Azido-3,6,9,12,15-pentaoxaheptadecan-1-amine, HCl (139 mg, 0.405 mmol, 1.0 equiv.) was then added, and the solution was stirred at room temperature. The crude product was purified by flash chromatography (220 g, silica gel, 10-60% acetone / CHCl) to give (S)-tert-butyl 1-azido-22-(tert-butoxycarbonyl)-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazapentacontan-41-oate (330 mg, 0.391 mmol, 97% yield). Analytical condition D: retention time = 2.88 min; ESI-MS (+) m / z 844.7 (M+H). + . 1 H NMR(500MHz, methanol-d4)δ 4.30-4.23(m,1H),3.74-3.60(m,18H),3.60-3.52(m,2H),3.43-3.35(m,4H),2.34-2.28(m,2H),2.28-2.1 9(m,4H),2.15-2.08(m,1H),1.98-1.87(m,1H),1.69-1.53(m,4H),1.52-1.44(m,18H),1.41-1.27(m,24H).

[0411] Step 5: Preparation of (S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15pentaoxa-18,23-diazapentacontan-41-oic acid A mixture of (S)-tert-butyl 1-azido-22-(tert-butoxycarbonyl)-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23diazapentacontan-41-oate (330 mg, 0.391 mmol) and TFA (0.422 mL, 5.47 mmol, 14 equiv) in DCM (3.0 mL) was stirred at room temperature for 2 h. The resulting crude product was purified by preparative HPLC (Solvent A = 0% MeOH ~ 90% HO ~ 0.1% TFA, Solvent B = 90% MeOH ~ 10% HO ~ 0.1% TFA. Column: Phenomenex Luna 30 x 150 mm, S10, Flow rate: 40 mL / min, 50-100% B, Stop at 10 and 13 min) to give (S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazapentatetracontan-41-oic acid (101 mg, 0.138 mmol, 35.3% yield). Analysis condition D: Retention time = 2.42 min; ESI-MS (+) m / z 732.5 (M+H). + .

[0412] Preparation of (S)-1-Azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oic acid (Azide-PEG7-γGlu-FDA18) Scheme [ka]

[0413] Step 1: Preparation of tert-butyl (S)-1-azido-28-(tert-butoxycarbonyl)-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oate To a solution of 23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-amine (204 mg, 0.517 mmol, 1.15 equiv.) in DMF (4498 μL) was added Hunig's base (314 μL, 1.799 mmol, 4.0 equiv.), followed by (S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoic acid (250 mg, 0.450 mmol). HATU (342 mg, 0.900 mmol, 2.0 equiv.) was then added, and the resulting solution was stirred at room temperature. The DMF was removed under high vacuum, and the residue was applied to 40 g of silica gel and eluted with DCM (90 mL), followed by a gradient of 75% DCM / acetone over 540 mL and a final hold of 75% DCM / acetone over 150 mL. The desired fractions were combined to give (S)-tert-butyl 1-azido-28-(tert-butoxycarbonyl)-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oate (394.2 mg, 0.423 mmol, 94% yield).

[0414] Step 2: Preparation of (S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oic acid To a solution of (S)-tert-butyl 1-azido-28-(tert-butoxycarbonyl)-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oate (394.2 mg, 0.423 mmol) in DCM (4229 μL) was added TFA (456 μL, 5.92 mmol, 14 equiv). An additional 14 equiv of TFA was added, and the mixture was further stirred. After an additional 6 h, LC / MS indicated a nearly complete reaction. The solvent was removed in vacuo. The mixture was dissolved in MeOH. The reaction mixture was purified by PREP HPLC in seven injections: (30 x 100 mm HPLC Luna Axia C18 50-100% A:B over 10 min, 100% B in 5 min (A is 90:10:0.1 water:MeOH:TFA; B is 90:10:0.1 MeOH:water:TFA)) to give after concentration (S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oic a...

Claims

1. Compounds of formula (I) 【Chemistry 1】 [In the formula, R １ is C １ ~C ６ Alkyl, Amino C １ ~C ６ Alkyl, aryl C １ ~C ６ Alkyl, Heteroaryl C １ ~C ６ Alkyl, Hydroxy C １ ~C ６ Alkyl, -X-R ３１ , -(CH ２ ) ｚ -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ , and N.H. ２ C(X'')NHC １ ~C ６ alkyl, X″ is O or NH, and the aryl C １ ~C ６ The aryl portion of the alkyl and the heteroaryl C １ ~C ６ The heteroaryl portion of alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, -X-R ３１ , and —O—(CH ２ ) ｚ -Triazolyl-X-R ３５ and optionally substituted with one or two groups independently selected from R ２ is aryl C １ ~C ６ Alkyl and heteroaryl C １ ~C ６ alkyl, and said aryl C １ ~C ６ The aryl portion of the alkyl and the heteroaryl C １ ~C ６ The heteroaryl portion of alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, -X-R ３１ , and —O—(CH ２ ) ｚ -Triazolyl-X-R ３５ and optionally substituted with one or two groups independently selected from R ３ is carboxy C １ ~C ３ is alkyl, R ４ is aryl C １ ~C ６ Alkyl and Heteroaryl C １ ~C ６ alkyl, and said aryl C １ ~C ６ The aryl portion of the alkyl and the heteroaryl C １ ~C ６ The heteroaryl portion of the alkyl may have one or two C １ ~C ６ is optionally substituted with an alkyl group; R ５ is C １ ~C ６ Alkyl, aryl C １ ~C ６ Alkyl, -X-R ３１ , and -(CH ２ ) ｚ -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ wherein the aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, hydroxy, -X-R ３１ , and —O—(CH ２ ) ｚ -Triazolyl-X-R ３５ and optionally substituted with one or two groups independently selected from R ６ is aryl-aryl C １ ~C ３ is alkyl, R ７ is C １ ~C ６ Alkyl, aryl C １ ~C ６ Alkyl, -X-R ３１ , and -(CH ２ ) ｚ -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ wherein the aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, -X-R ３１ , and —O—(CH ２ ) ｚ -Triazolyl-X-R ３５ and optionally substituted with one or two groups independently selected from R ８ is C １ ~C ６ Alkyl, Amino C １ ~C ６ Alkyl, -X-R ３１ and -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ is selected from R ９ is C １ ~C ６ is alkyl, R １０ is amide C １ ~C ６ Alkyl, Amino C １ ~C ６ Alkyl, -X-R ３１ , and -(CH ２ ) ｚ -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ is selected from R １１ (C ３ ~C ８ Cycloalkyl)C １ ~C ６ is alkyl, R １２ is C １ ~C ６ alkyl, R １３ is aryl C １ ~C ６ Alkyl, Carboxy C １ ~C ６ Alkyl, Hydroxy C １ ~C ６ Alkyl, -X-R ３１ and -(CH ２ ) ｚ -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ wherein the aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, -X-R ３１ and -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ and optionally substituted with one or two groups independently selected from R １４ is -C(O)NH ２ Or -C(O)NHCHR １５ C(O)NHR ５０ and R １５ is hydrogen, C １ ~C ６ Alkyl and Amino C １ ~C ６ alkyl, R ５０ is hydrogen and NH ２ C(O)CH ２ (O.C.H. ２ CH ２ ) ２ - selected from R ３１ is -CO ２ H, —C(O)NR ｗ R ｘ , -CH ３ , alexa-5-SDP, and biotin; each z is independently 1, 2, 3, 4, 5, or 6; R ３５ is -CO ２ H, —C(O)NR ｗ R ｘ , C.H. ３ , biotin, 2-fluoropyridine, -C(O)-(CH ２ ) ２ -C(O)-vitamin E and -C(O)-vitamin E; R ｘ and R ｗ is hydrogen and C １ ~C ６ independently selected from alkyl, X is a chain of 1 to 172 atoms, said atoms being selected from carbon and oxygen, said chain optionally containing embedded therein 1, 2, 3, or 4 groups selected from -NHC(O)NH-, and -C(O)NH-, said chain being selected from -CO ２ H, —C(O)NH ２ , -CH ２ C(O)NH ２ , and -(CH ２ ) CO ２ H; However, R １ , R ２ , R ５ , R ７ , R ８ , R １０ , and R １３ At least one of is -X-R ３１ , -(CH ２ ) ｚ -O-(CH ２ ) ｚ -Triazolyl-X-R ３５ , and —O—(CH ２ ) ｚ -Triazolyl-X-R ３５ or is substituted with a group selected from the group or a pharma- ceutically acceptable salt thereof.

2. R １ But, C １ ~C ６ Alkyl, Amino C １ ~C ６ Alkyl, aryl C １ ~C ６ Alkyl, Heteroaryl C １ ~C ６ Alkyl, Hydroxy C １ ~C ６ Alkyl, HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ - and NH ２ C(X'')NHC １ ~C ６ alkyl, z is 1, 2, 3, or 4, X″ is O or NH, and said aryl C １ ~C ６ The aryl portion of the alkyl and the heteroaryl C １ ~C ６ The heteroaryl portion of alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ optionally substituted with 1 or 2 groups independently selected from O-; R ２ Aryl C １ ~C ６ Alkyl and Heteroaryl C １ ~C ６ alkyl, and said aryl C １ ~C ６ The aryl portion of the alkyl and the heteroaryl C １ ~C ６ The heteroaryl portion of alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ - and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ and optionally substituted with 1 or 2 groups independently selected from O-, and z is 1, 2, 3, or 4; R ５ But, C １ ~C ６ Alkyl, aryl C １ ~C ６ Alkyl, and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ OCH ２ -, wherein said aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, hydroxy, and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ optionally substituted with 1 or 2 groups independently selected from O-; R ７ But, C １ ~C ６ Alkyl, aryl C １ ~C ６ Alkyl, HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ --, H.O. ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ OCH ２ -, z is 1, 2, 3, or 4, and said aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ optionally substituted with 1 or 2 groups independently selected from O-; R ８ But, C １ ~C ６ Alkyl, Amino C １ ~C ６ Alkyl, HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ --, H.O. ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ OCH ２ -, z is 1, 2, 3, or 4; R １０ Amide C １ ~C ６ Alkyl, Amino C １ ~C ６ Alkyl, and HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ -, z is 1, 2, 3, or 4; R １３ Aryl C １ ~C ６ Alkyl, Carboxy C １ ~C ６ Alkyl, Hydroxy C １ ~C ６ Alkyl, HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ - (z is 1, 2, 3, or 4), and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ OCH ２ - selected from However, R １ , R ２ , R ５ , R ７ , R ８ , R １０ and R １３ At least one of the following is HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ O-, H-O ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ , and H.O. ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ OCH ２ - or substituted with a group selected from The compound of claim 1.

3. R １ , R ７ , R ８ , R １０ , and R １３ At least one of the ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ - and z is 1, 2, 3, or 4, or a pharma- ceutically acceptable salt thereof.

4. R １ , R ２ , R ５ , and R ７ At least one of the groups is aryl C １ ~C ６ Alkyl or heteroaryl C １ ~C ６ alkyl, and the aryl C １ ~C ６ The aryl portion of the alkyl and the heteroaryl C １ ~C ６ The heteroaryl portion of the alkyl is HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ O- or HO ２ C (CH ２ ) １０ C(O)NH(CH ２ ) ｚ 3. The compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, which is substituted with -.

5. R ５ , R ７ , R ８ , and R １３ At least one of the following is HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ OCH ２ 3. The compound according to claim 1 or 2, wherein: -, or a pharma- ceutically acceptable salt thereof.

6. R ２ Aryl C １ ~C ６ alkyl, and the aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, HO ２ C (CH ２ ) １０ C(O)NHCH ２ - and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ 6. The compound of any one of claims 1 to 5, or a pharma- ceutically acceptable salt thereof, optionally substituted with one or two groups independently selected from O-.

7. R ４ Aryl C １ ~C ６ alkyl, and the aryl C １ ~C ６ The aryl portion of the alkyl may have one or two C １ ~C ６ 10. The compound of claim 1 or 6, or a pharma- ceutically acceptable salt thereof, optionally substituted with an alkyl group.

8. R ５ Aryl C １ ~C ６ alkyl, and the aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, hydroxy, and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ 8. The compound of any one of claims 1 to 7, or a pharma- ceutically acceptable salt thereof, optionally substituted with one or two groups independently selected from O-.

9. R ７ Aryl C １ ~C ６ alkyl, and the aryl C １ ~C ６ The aryl portion of the alkyl is carboxy, carboxyC １ ~C ６ Alkoxy, and HO ２ C (CH ２ ) １６ C(O)NHCH(CO ２ H) (CH ２ ) ２ C(O)NH(CH ２ CH ２ O) １１ (CH ２ ) ２ Triazolyl CH ２ 9. The compound of any one of claims 1 to 8, or a pharma- ceutically acceptable salt thereof, optionally substituted with one or two groups independently selected from O-.

10. R ８ But Amino C １ ~C ６ 10. The compound according to any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, wherein R is an alkyl group.

11. R １０ But Amino C １ ~C ６ 11. The compound according to any one of claims 1 to 10, or a pharma- ceutically acceptable salt thereof, wherein R is an alkyl group.

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof.

13. 12. A method of enhancing, stimulating and / or increasing an immune response in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a compound according to any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof.

14. 12. A method of blocking the interaction of PD-1 and PD-L1 in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof.

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