Peptides for treating medical conditions

Abstract

The present invention provides compounds that are selective kappa-opioid receptor agonists, methods of making these compounds, compositions comprising these compounds, and methods for treating medical conditions associated with kappa-opioid receptor agonists.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an international application that claims the benefit of U.S. patent application serial number 17 / 862,086, filed on July 11, 2022, entitled “PEPTIDES FOR TREATMENT OF MEDICAL DISORDERS,” which is hereby incorporated by reference into this document. Technical Field

[0003] The present disclosure generally relates to compounds that are selective kappa-opioid receptor agonists, methods for preparing these compounds, compositions containing these compounds, and methods for treating medical conditions associated with kappa-opioid receptor agonists. Background Art

[0004] Opioid kappa receptors (KORs) are expressed in many parts of the body, such as the brain, spinal cord, and central and peripheral endings. KORs play an important role in signal transduction to maintain many physiological functions of the body. Like opioid μ receptors (MORs) and δ receptors (DORs), activation of KORs by agonist ligands leads to inhibition of adenylate cyclase and calcium channel activity and stimulation of potassium channel activity (Law PY, Wong YH, Loh HH. Molecular mechanisms and regulation of opioid receptor signaling. Annu Rev Pharmacol Toxicol, 2000; 40: 389-430).

[0005] Many physiological processes are associated with the activation of KOR, including analgesia, antipruritic activity (Inan S, Cowan A. Kappa opioid agonists suppress chloroquine-induced scratching in mice. Eur J. Pharmacol 2004; 502, 233-7), diuresis (Barber A, Gottschlich R. Novel developments with selective non-peptidic kappa-opioid receptor agonists. Exp Opinion Investigational drugs. 1997; 6: 1351-68; DeHaven-Hudkins DL, Dolls RE. Peripherally restricted opioid agonists are new analgesics (Current Drug Design Des (2004; 10: 743-57), inflammatory agents, immune system modulators, etc. Agonists offer great potential for KOR selective ligands for the treatment of various medical conditions such as pain, depression, autoimmune conditions, and neurological diseases. (Tyler C. Beck, Matthew A. Hapstack, Kyle R. Beck, and Thomas A. Dix. "Therapeutic Potential of Kappa Opioid Agonists", Pharmaceuticals (Basel). 2019 Jun; 12(2): 95).

[0006] Many KOR selective agonists have been synthesized and evaluated as potential analgesics that avoid the side effects associated with traditional opioid analgesics, such as respiratory depression, dependence, addiction, and constipation; some of these have been tested in clinical trials but have failed due to side effects such as diuresis, sedation, and dysphoria or lack of efficacy; examples include spiradoline mesylate (U62,066E) (Wadenberg ML, A review of the properties of spiradoline: a potent and selective kappa-opioid receptor agonist. CNS Drug Review (CNS Drug Rev.) 2003, Summer, 9(2):187-98), enadoline for potential analgesia (Walsh SL., Strain EC, Abreu M.E. Bigelow GE Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans) kappa opioid agonist: comparison with butorphanoland hydromorphone in humans). Psychopharmacology, 2001, 157, 151-162) and ADL-10-0101, etc.

[0007] TRK-820 (Nalfurafine) was originally developed as a potential analgesic, but has found success as an antipruritic agent and received regulatory approval in Japan under the trade name Remitch.

[0008] Highly opioid kappa receptor selective and potent D-amino acid tetrapeptide agonists were reported by Ferring BV (US005965701A) and are being further developed by Cara Therapeutics, Inc. Currently, Cara Therapeutics is developing the lead tetrapeptide compound CR-845 as an analgesic and antipruritic in clinical trials (Hesselink, JMKCR845 (Difelikefalin), A Kappa Receptors Agonist in Phase III by CARA Therapeutics: A Case of 'Spin' in Scientific Writing? J. Pharm. & Clinical Res. 2017 2(3), 001). Encouraged by the progress of CR-845 in clinical trials, several pharmaceutical companies have also been actively working on discovering peptide-based KOR selective agonist ligands by modifying the molecular structure of CR-845, hoping to discover new analgesics and potential antipruritic agents without the conventional side effects of morphine analgesics (CN107098871, WO2017211272A1, WO2018103624A1, WO2017210668A1, WO2018059331A1).

[0009] In addition, KOR agonists are also being developed for other indications; for example, both fedotozine and asimadoline are being tested as potential therapeutic agents for irritable bowel syndrome and dyspepsia.

[0010] What is needed are new kappa-opioid agonists for the treatment of a variety of medical conditions. Summary of the invention

[0011] In one aspect, disclosed herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0012]

[0013] in:

[0014] R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0015] R4 is selected from the group consisting of C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0016] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle;

[0017] R7 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0018] R8 and R9 are independently selected from the group consisting of H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, O-substituted C1-C8 alkyl, and O-unsubstituted C1-C8 alkyl;

[0019] R 10 for

[0020]

[0021] R 11 Choose Free OR 12 and NR 13 R 14 The group composed of;

[0022] R 12 Selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0023] R 13 and R 14 Independently selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2)n CH2CH2- and HO(CH2CH2) n CH2CH2-; and

[0024] n is an integer from 0 to 100.

[0025] In another aspect, disclosed herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0026]

[0027] in:

[0028] R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0029] R4 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0030] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl, C3-C 10 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle;

[0031] R7 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0032] R8 and R9 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, CH3O(CH2CH2O) n CH2CH2- and HO(CH2CH2O) n CH2CH2-;

[0033] R 10 for

[0034]

[0035] R 11 Choose Free OR12 and NR 13 R 14 The group composed of;

[0036] R 12 Selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2O) n CH2CH2- and HO(CH2CH2O) n CH2CH2-;

[0037] R 13 and R 14 Independently selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2O) n CH2CH2- and HO(CH2CH2O) n CH2CH2-; and

[0038] n is an integer from 0 to 100.

[0039] In another aspect, disclosed herein is a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0040]

[0041] The method comprises:

[0042] a) reacting a compound comprising formula (VI) in the presence of an acyl coupling reagent:

[0043]

[0044] and a compound comprising formula (VII):

[0045]

[0046] to form a compound comprising formula (VIII):

[0047]

[0048] b) contacting a compound comprising formula (VIII) with a proton acceptor to form a compound comprising formula (IX):

[0049]

[0050] c) reacting a compound comprising formula (IX) with a compound comprising formula (X) in the presence of an acyl coupling reagent:

[0051]

[0052] to form a compound comprising formula (XI):

[0053]

[0054] d) contacting a compound comprising formula (XI) with a proton acceptor to form a compound comprising formula (XII):

[0055]

[0056] e) reacting a compound comprising formula (XII) with a compound comprising formula (XIII) in the presence of an acyl coupling reagent:

[0057]

[0058] to form a compound comprising formula (XIV):

[0059]

[0060] f) contacting a compound comprising formula (XIV) with a proton acceptor to form a compound comprising formula (XV):

[0061]

[0062] g) contacting a compound comprising formula (XV) with a compound comprising formula (XVI) in the presence of an acyl coupling reagent to form a compound comprising formula (XVII):

[0063]

[0064] h) contacting a compound comprising formula (XVII) with a deprotecting reagent comprising an acid to form a compound comprising formula (I);

[0065] in:

[0066] R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0067] R4 and R7 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0068] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle;

[0069] R8 and R9 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0070] R 10 Selected from the group consisting of:

[0071]

[0072] R 11 Choose Free OR 12 and NR 13 R 14 The group composed of;

[0073] R 12 Selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, (CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0074] R 13 and R 14 Independently selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, (CH3O(CH2CH2) nCH2CH2- and HO(CH2CH2) n CH2CH2-; and

[0075] n is an integer from 0 to 100.

[0076] In yet another aspect, disclosed herein is a pharmaceutical composition comprising a compound comprising Formula (I).

[0077] In still another aspect, disclosed herein is a method for treating an opioid receptor agonist-associated medical condition, the method comprising administering to a subject in need thereof the pharmaceutical composition comprising a compound comprising formula (I).

[0078] Other features and iterations of the invention are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 is a chemical reaction scheme that can be used to prepare compounds comprising formula (I) according to embodiments of the present disclosure.

[0080] Figure 2 It is a bar graph showing the efficacy of the compound of formula (I) in inhibiting formalin-induced nociceptive responses in the formalin model. Data are presented in the form of mean ± SEM, n = 10 / group. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the vehicle group, by one-way ANOVA, followed by Dunnett's Multiple Comparison Test. MPE% is defined as 100% - ((total movement counts in each group - total movement counts in the original group) / (total movement counts in the vehicle group - total movement counts in the original group)).

[0081] Figure 3 is a bar graph showing the efficacy of compound 14 in the chloroquine-induced scratching model in mice. Data are presented as mean ± SEM, n = 10 / group. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle group by one-way ANOVA followed by Dunnett's multiple comparison test. DETAILED DESCRIPTION

[0082] The present disclosure provides compounds comprising formula (I) or pharmaceutically acceptable salts thereof, methods for preparing compounds comprising formula (I) or pharmaceutically acceptable salts thereof, compounds comprising compounds comprising formula (I) and methods for treating medical conditions associated with opioid receptor agonists.

[0083] (I) comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0084] In one aspect, the present disclosure encompasses a compound comprising formula (I) or a pharmaceutically acceptable salt thereof:

[0085]

[0086] in:

[0087] R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0088] R4 is selected from the group consisting of C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0089] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl, C3-C 10 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle;

[0090] R7 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0091] R8 and R9 are independently selected from the group consisting of H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, O-substituted C1-C8 alkyl, and O-unsubstituted C1-C8 alkyl;

[0092] R 10 for

[0093]

[0094] R 11 Choose Free OR 12 and NR 13 R 14 The group composed of;

[0095] R 12 Selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C24 alkyl CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0096] R 13 and R 14 Independently selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-; and

[0097] n is an integer from 0 to 100.

[0098] Typically, according to the embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 In some embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl and C3-C8 substituted cycloalkyl. In certain embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl and isopropyl. In specific embodiments, R1, R2 and R3 are H.

[0099] Typically, according to embodiments, R4 is selected from the group consisting of: C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, and C3-C8 substituted cycloalkyl. In some embodiments, R4 is selected from the group consisting of: methyl, ethyl, n-propyl, and isopropyl. In specific embodiments, R4 is methyl.

[0100] Typically, according to embodiments, R5 and R6 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, C3-C8 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle. In some embodiments, R5 and R6 are independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and phenyl. In specific embodiments, R5 is H, and R6 is phenyl.

[0101] Typically, according to embodiments, R7 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 In some embodiments, R7 is selected from the group consisting of: H, methyl, ethyl, n-propyl and isopropyl. In a specific embodiment, R7 is isopropyl.

[0102] Typically, according to embodiments, R8 and R9 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, O substituted C1-C4 alkyl, and O unsubstituted C1-C4 alkyl. In some embodiments, R8 and R9 are independently selected from the group consisting of: H, methyl, ethyl, propyl, and isopropyl. In specific embodiments, R8 and R9 are hydrogen.

[0103] Typically, according to an embodiment, R 10 for

[0104]

[0105] Typically, according to an embodiment, R 11 Choose Free OR 12 and NR 13 R 14 In some embodiments, R 11 Choose Free OR 12 and NR 13 R 14 In a specific embodiment, R 11 OR 12 .

[0106] Typically, according to an embodiment, R 12 Selected from the group consisting of: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl, O-substituted C1-C 12 Alkyl, O unsubstituted C1-C 12 Alkyl, CH3O(CH2CH2)n CH2CH2- and HO(CH2CH2) n CH2CH2-. In some embodiments, R 12 is selected from the group consisting of: H, methyl, ethyl, n-propyl and isopropyl. 12 It is H or Me.

[0107] Typically, according to an embodiment, R 13 and R 14 Independently selected from the group consisting of: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl, O-substituted C1-C 12 Alkyl, O unsubstituted C1-C 12 Alkyl, CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-. In some embodiments, R 13 and R 14 R is independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 13 and R 14 Does not exist

[0108] Typically, depending on the embodiment, n is an integer from 0 to 50. In some embodiments, n is an integer from 0 to 50. In certain embodiments, n is absent.

[0109] In an exemplary embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0110] R 10 for

[0111]

[0112] R 11 OR 12 ; R 12 is H; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (IV):

[0113]

[0114] In yet another exemplary embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0115] R 10for

[0116]

[0117] R 11 OR 12 ; R 12 for Me; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (V):

[0118]

[0119] Another aspect of the present disclosure encompasses compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0120]

[0121] R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0122] R4 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0123] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl, C3-C 10 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle;

[0124] R7 is selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0125] R8 and R9 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, CH3O(CH2CH2O) n CH2CH2- and HO(CH2CH2O) n CH2CH2-;

[0126] R 10 for

[0127]

[0128] R 11 Choose Free OR 12 and NR 13 R 14 The group composed of;

[0129] R 12 Selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2O) n CH2CH2- and HO(CH2CH2O) n CH2CH2-;

[0130] R 13 and R 14 Independently selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, CH3O(CH2CH2O) n CH2CH2- and HO(CH2CH2O) n CH2CH2-; and

[0131] n is an integer from 0 to 100.

[0132] Typically, according to the embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 In some embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl and C3-C8 substituted cycloalkyl. In certain embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl and isopropyl. In specific embodiments, R1, R2 and R3 are H.

[0133] Typically, according to an embodiment, R4 and R7 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl and C3-C8 substituted cycloalkyl. In some embodiments, R4 and R7 are independently selected from the group consisting of: H, methyl, ethyl, propyl and isopropyl. In a specific embodiment, R4 is methyl and R7 is isopropyl.

[0134] Typically, according to embodiments, R5 and R6 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, C3-C8 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle. In some embodiments, R5 and R6 are independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and phenyl. In specific embodiments, R5 is H, and R6 is phenyl.

[0135] Typically, according to embodiments, R8 and R9 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, O substituted C1-C4 alkyl, and O unsubstituted C1-C4 alkyl. In some embodiments, R8 and R9 are independently selected from the group consisting of: H, methyl, ethyl, propyl, and isopropyl. In specific embodiments, R8 and R9 are hydrogen.

[0136] Typically, according to an embodiment, R 10 Selected from

[0137]

[0138] Typically, according to an embodiment, R 11 Choose Free OR 12 and NR 13 R 14 In some embodiments, R 11 Choose Free OR 12 and NR 13 R 14 In a specific embodiment, R 11 OR 12 .

[0139] Typically, according to an embodiment, R 12 Selected from the group consisting of: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl, O-substituted C1-C 12 Alkyl, O unsubstituted C1-C 12 Alkyl, CH3O(CH2CH2)n CH2CH2- and HO(CH2CH2) n CH2CH2-. In some embodiments, R 12 is selected from the group consisting of: H, methyl, ethyl, n-propyl or isopropyl. 12 It is H or Me.

[0140] Typically, according to an embodiment, R 13 and R 14 Independently selected from the group consisting of: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl, O-substituted C1-C 12 Alkyl, O unsubstituted C1-C 12 Alkyl, CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-. In some embodiments, R 13 and R 14 R is independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 13 and R 14 Does not exist

[0141] Typically, depending on the embodiment, n is an integer from 0 to 50. In some embodiments, n is an integer from 0 to 50. In certain embodiments, n is absent.

[0142] In an exemplary embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0143] R 10 for

[0144]

[0145] R 11 OR 12 ; and R 12 is H; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (II):

[0146]

[0147] In another exemplary embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0148] R 10for

[0149]

[0150] R 11 OR 12 ; R 12 for Me; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (III):

[0151]

[0152] The compound comprising formula (I) can be a free alkali or a salt. When the compound is in salt form, the salt is preferably a pharmaceutically acceptable salt. Pharmaceutically acceptable salts may include but are not limited to hydrochloride, hydrobromide, phosphate, sulfate, mesylate, acetate, formates, tartaric acid, bitartrate, stearate, phthalate, hydroiodide, lactate, monohydrate, mucate, nitrate, phosphate, salicylate, phenylpropionate, isobutyrate, hypophosphite, maleic anhydride, malate, citrate, isocitrate, succinate, lactate, gluconate, glucuronate, pyruvate, oxalate, fumarate, propionate, aspartate, glutamate, benzoate, terephthalate, etc. In other embodiments, pharmaceutically acceptable salts include alkaline or alkaline earth metal ion salts. Specifically, sodium, potassium or other pharmaceutically acceptable inorganic salts are used. Salt form can be amorphous or various polymeric forms, including hydrates or solvates with alcohol or other solvents.

[0153] (II) A method for preparing a compound comprising formula (I) or a pharmaceutically acceptable salt thereof.

[0154] In another aspect, the present disclosure encompasses a method for preparing a compound comprising formula (I) or a pharmaceutically acceptable salt thereof:

[0155]

[0156] The method comprises, according to Figure 1 The reaction scheme depicted in:

[0157] a) reacting a compound comprising formula (VI) in the presence of an acyl coupling reagent:

[0158]

[0159] and a compound comprising formula (VII):

[0160]

[0161] to form a compound comprising formula (VIII):

[0162]

[0163] b) contacting a compound comprising formula (VIII) with a proton acceptor to form a compound comprising formula (IX):

[0164]

[0165] c) reacting a compound comprising formula (IX) with a compound comprising formula (X) in the presence of an acyl coupling reagent:

[0166]

[0167] to form a compound comprising formula (XI):

[0168]

[0169] d) contacting a compound comprising formula (XI) with a proton acceptor to form a compound comprising formula (XII):

[0170]

[0171] e) reacting a compound comprising formula (XII) with a compound comprising formula (XIII) in the presence of an acyl coupling reagent:

[0172]

[0173] to form a compound comprising formula (XIV):

[0174]

[0175] f) contacting a compound comprising formula (XIV) with a proton acceptor to form a compound comprising formula (XV):

[0176]

[0177] g) contacting a compound comprising formula (XV) with a compound comprising formula (XVI) in the presence of an acyl coupling reagent to form a compound comprising formula (XVII):

[0178]

[0179] h) contacting a compound comprising formula (XVII) with a deprotecting agent to form a compound comprising formula (I);

[0180] in:

[0181] R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0182] R4 and R7 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 substituted cycloalkyl;

[0183] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle;

[0184] R8 and R9 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl; CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0185] R 10 Selected from the group consisting of:

[0186]

[0187] R 11 Choose Free OR 12 and NR 13 R 14 The group composed of;

[0188] R 12 Selected from the group consisting of: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 Alkyl, (CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0189] R 13 and R 14 Independently selected from the group consisting of: H, C1-C24 Unsubstituted alkyl, C1-C 24 Substituted alkyl, O-substituted C1-C 24 Alkyl, O unsubstituted C1-C 24 alkyl; CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-;

[0190] n is an integer from 0 to 100;

[0191] P1 is a nitrogen protecting group, and P2 is a carboxylic acid protecting group.

[0192] Usually, according to Figure 1 In the reaction scheme depicted in the embodiment, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C3-C 10 Unsubstituted cycloalkyl and C3-C 10 In some embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl and C3-C8 substituted cycloalkyl. In certain embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl and isopropyl. In specific embodiments, R1, R2 and R3 are H.

[0193] Usually, according to Figure 1 In the reaction scheme depicted in , R4 and R7 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, and C3-C8 substituted cycloalkyl. In some embodiments, R4 and R7 are independently selected from the group consisting of: H, methyl, ethyl, propyl, and isopropyl. In specific embodiments, R4 is methyl; and R7 is isopropyl.

[0194] Usually, according to Figure 1 In the reaction scheme depicted in , R5 and R6 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, and C3-C8 substituted cycloalkyl; unsubstituted aryl, substituted aryl, unsubstituted heterocycle; and substituted heterocycle. In some embodiments, R5 and R6 are independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and phenyl. In specific embodiments, R5 is H; and R6 is phenyl.

[0195] Usually, according to Figure 1 The reaction scheme depicted in , R8 and R9 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, O-substituted C1-C4 alkyl, O-unsubstituted C1-C4 alkyl and (OCH2CH2O) n In some embodiments, R8 and R9 are independently selected from the group consisting of: H, methyl, ethyl, propyl and isopropyl. In specific embodiments, R8 and R9 are hydrogen.

[0196] Usually, according to Figure 1 The reaction scheme depicted in R 10 Selected from the group consisting of:

[0197]

[0198] Typically, according to an embodiment, R 11 Choose Free OR 12 and NR 13 R 14 In some embodiments, R 11 Choose Free OR 12 and NR 13 R 14 In a specific embodiment, R 11 OR 12 .

[0199] Usually, according to Figure 1 The reaction scheme depicted in R 12 Selected from the group consisting of: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl, O-substituted C1-C 12 Alkyl, O unsubstituted C1-C 12 Alkyl, CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-. In some embodiments, R 12 is selected from the group consisting of: H, methyl, ethyl, n-propyl and isopropyl. 12 It is H or Me.

[0200] Usually, according to Figure 1 The reaction scheme depicted in R 13 and R 14 Independently selected from the group consisting of: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl, O-substituted C1-C 12Alkyl, O unsubstituted C1-C 12 Alkyl; CH3O(CH2CH2) n CH2CH2- and HO(CH2CH2) n CH2CH2-. In some embodiments, R 13 and R 14 R is independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 13 and R 14 Does not exist.

[0201] Usually, according to Figure 1 , n is an integer from 0 to 50. In some embodiments, n is an integer from 0 to 50. In certain embodiments, n is absent.

[0202] Usually, according to Figure 1 In the embodiment of the reaction scheme described in , P1 is a suitable nitrogen protecting group. The nitrogen protecting group comprises carbamate. The non-limiting examples of these protecting groups can be tert-butyloxycarbonyl carbamate (BOC), 9-fluorenylmethyl carbamate (FMOC), benzyl carbamate (CBZ) etc. Suitable nitrogen protecting groups, methods for connecting these protecting groups and methods for removing these protecting groups are described in, for example, " Protective Groups in Organic Synthesis " of TW Greene, John Wiley & Sons, 2006. In a specific embodiment, P1 is BOC.

[0203] Usually, according to Figure 1 In the embodiment of the reaction scheme depicted in , P2 is a suitable carboxylic acid protecting group. The carboxylic acid (carboxyl) protecting group comprises an ester, an amide or a hydrazide. Non-limiting examples of carboxylic acid protecting groups can be methyl esters, ethyl esters, benzyl esters, N,N-dimethylamide, N-phenylhydrazide, etc. Suitable carboxylic acid protecting groups, methods for connecting these protecting groups, and methods for removing these protecting groups are described, for example, in TW Greene's "Protective Groups in Organic Synthesis", John Wiley & Sons, 2006. In a specific embodiment, P2 is a methyl group.

[0204] In a preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0205] R 10 for

[0206]

[0207] R 11 OR 12 ; and R 12 is H; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (II):

[0208]

[0209] In another preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0210] R 10 for

[0211]

[0212] R 11 OR 12 ; R 12 for Me; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (III):

[0213]

[0214] In still another preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0215] R 10 for

[0216]

[0217] R 11 OR 12 ; R 12 is H; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (IV):

[0218]

[0219] In yet another preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0220] R 10 for

[0221]

[0222] R11 OR 12 ; R 12 for Me; and R 13 , R 14 and n is absent, as shown in compounds comprising formula (V):

[0223]

[0224] Step (a)

[0225] As discussed above, step (a) of the eight-step method involves contacting a compound comprising formula (VI) with a compound comprising formula (VII) in the presence of an acyl coupling reagent to form a reaction mixture. After treatment and separation, the compound comprising formula (VIII) is isolated. This method step is referred to as "peptide coupling" or "acyl coupling".

[0226] The above details Figure 1 In some embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl and C3-C8 substituted cycloalkyl. In certain embodiments, R1, R2 and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl and isopropyl. In specific embodiments, R1, R2 and R3 are H.

[0227] In some embodiments, R4 is independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, and C3-C8 substituted cycloalkyl. In certain embodiments, R4 is selected from the group consisting of: H, methyl, ethyl, n-propyl, and isopropyl. In specific embodiments, R4 is methyl.

[0228] In some embodiments, P1 is a nitrogen protecting group. In specific embodiments, P1 is a BOC group. In a preferred embodiment, the compound comprising formula (VI) is (2R, 3R)-BOC-β-methyl-phenylalanine.

[0229] The above details Figure 1In some embodiments, R5 and R6 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, C3-C8 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle, and substituted heterocycle. In certain embodiments, R5 and R6 are independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and phenyl. In specific embodiments, R5 is H and R6 is phenyl.

[0230] In some embodiments, P2 is a carboxylic acid protecting group. In a specific embodiment, P2 is a methyl group. In a preferred embodiment, the compound comprising formula (VII) is D-phenylalanine methyl ester hydrochloride.

[0231] Typically, the equivalent ratio of the compound comprising formula (VI) to the compound comprising formula (VII) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (VI) to the compound comprising formula (VII) can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (VI) to the compound comprising formula (VII) can be about 1:0:1.1.

[0232] Step (a) of the method utilizes an acyl coupling reagent. The acyl coupling reagent converts the carboxylic acid moiety of the compound comprising formula (VI) into an activated acyl compound. Non-limiting examples of acyl coupling agents include trifluoromethanesulfonic anhydride, p-toluenesulfonic anhydride, methanesulfonic anhydride, thionyl chloride, oxalyl chloride, sulfonyl chloride, phosphorus oxychloride, phosphorus pentachloride, carbodiimide (e.g., N,N'-dicyclohexylcarbodiimide, 1,1'-carbonyldipiperidine, N,N'-diisopropylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), 1,1'-carbonyldiimidazole, 1,1'-carbonylbistriazole, cyanuric chloride, 2,4-dichloro-6-methoxy-1,3,5-triazine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, ethyl chloroformate, isobutyl chloroformate, acetic anhydride, trichloroacetic anhydride, or trifluoroacetic anhydride. In various embodiments, additional activators may be added. Non-limiting examples of additional activators may include 1-hydroxybenzotriazole, N-hydroxysuccinimide or N-hydroxyphthalamide. In a preferred embodiment, the acyl coupling reagent may be 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof.

[0233] Typically, the equivalent ratio of the compound comprising formula (VI) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (VI) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (VI) to the acyl coupling agent can be about 1:0:1.1.

[0234] Step (a) further comprises a proton acceptor. The proton acceptor will vary according to the starting substrate, the acyl coupling reagent and the reaction conditions. The proton acceptor can be inorganic or organic in nature. Non-limiting examples of suitable inorganic proton acceptors include sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, cesium carbonate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium borate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium methoxide, sodium tert-butoxide, potassium tert-butoxide, sodium acetate and potassium acetate. The proton acceptor can be an amine. The organic proton acceptor can be a secondary amine, a tertiary amine or a combination thereof. The amine can be chiral or achiral. Non-limiting examples of suitable secondary amines include methylethylamine, dimethylamine, diethylamine, dicyclohexylamine, methylcyclohexylamine, phenylethylamine, dibenzylamine, methylbenzylamine, ethylbenzylamine, cyclohexylphenylamine, dibutylamine, di-tert-butylamine, dipropylamine, dipentylamine, dicyclohexylamine, piperidine, 2-methylpiperidine, 2,5-dimethylpiperidine, 2,6-dimethylpiperidine, piperazine, 2-methylpiperazine, 2,6-dimethylpiperazine and morpholine. Non-limiting examples of suitable tertiary amines include trimethylamine, triethylamine, diisopropylethylamine, tripropylamine, tributylamine, 4-methylmorpholine, 4-ethylmorpholine, N-methylpyrrolidine, N-methylpiperidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyrazine, 4-dimethylaminopyridine, pyridine and 2,6-lutidine. Non-limiting examples of chiral secondary amines are (R)-α-methylbenzylamine, (S)-α-methylbenzylamine, (R)-α,α-diphenyl-2-pyrrolidinemethanol (DPP), (S)-α,α-diphenyl-2-pyrrolidinemethanol (DPP), (R)-α,α-diphenyl-2-pyrrolidinemethanol trimethylsilyl ether (DPPT) and (S)-α,α-diphenyl-2-pyrrolidinemethanol trimethylsilyl ether (DPPT). In a preferred embodiment, the proton acceptor is 4-methylmorpholine (N-methylmorpholine).

[0235] Typically, the equivalent ratio of the compound of formula (VI) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalent ratio of the compound of formula (VI) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5, about 1.0:1.0 to about 1.0:2.25, or about 1.0:2.2. In a preferred embodiment, the equivalent ratio of the compound of formula (VI) to the proton acceptor can be about 1:0:2.1.

[0236] Step (a) as described in detail herein comprises a solvent. As recognized by those skilled in the art, the solvent can and will vary depending on the starting substrate in the method. The solvent can be a polar protic solvent, a polar aprotic solvent, a non-polar solvent or a combination thereof. Suitable examples of polar protic solvents include, but are not limited to: water; alcohols such as methanol, ethanol, isopropanol, n-propanol, isobutanol, n-butanol, sec-butanol, tert-butanol, etc.; glycols such as propylene glycol; organic acids such as formic acid, acetic acid, etc.; amines such as trimethylamine or triethylamine, etc.; amides such as formamide, acetamide, etc.; and combinations of any of the above. Non-limiting examples of suitable polar aprotic solvents include acetonitrile, dichloromethane (DCM), diethoxymethane, N,N-dimethylacetamide (DMAC), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N-dimethylpropionamide, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxyethane (DME), dimethoxymethane, bis(2-methoxyethyl) ether, 1,4-dioxane, N-methyl-2-pyrrolidone (NMP), ethyl formate, formamide, hexamethylphosphoramide, N-methylacetamide, N-methylformamide, methylene chloride, nitrobenzene, nitromethane, propionitrile, sulfolane, tetramethylurea, tetrahydrofuran (THF), 2-methyltetrahydrofuran, chloroform, and combinations thereof. Suitable examples of non-polar solvents include, but are not limited to, alkanes and substituted alkanes (including cycloalkanes), aromatic hydrocarbons, esters, ethers, combinations thereof, and the like. Specific non-polar solvents that can be employed include, for example, benzene, butyl acetate, tert-butyl methyl ether, chlorobenzene, chloroform, methyl chloride, cyclohexane, methylene chloride, ethylene dichloride, diethyl ether, ethyl acetate, diethylene glycol, fluorobenzene, heptane, hexane, isopropyl acetate, methyltetrahydrofuran, amyl acetate, n-propyl acetate, tetrahydrofuran, toluene, and combinations thereof. In a preferred embodiment, the solvent can be dimethylformamide.

[0237] Typically, the volume to weight ratio of solvent to compound comprising formula (VI) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of solvent to compound comprising formula (VI) may range from about 0.5: 1 to about 500: 1, from about 2: 1 to about 250: 1, from about 5: 1 to about 200: 1, or from about 10: 1 to about 50: 1. In an exemplary embodiment, the volume to weight ratio of solvent to compound comprising formula (VI) may range from about 12: 1 to about 20: 1.

[0238] Typically, the reaction of step (a) will be carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction may range from about -20°C to about 25°C, from about -10°C to about 20°C, or from about -5°C to about 5°C. In one embodiment, the reaction may be carried out at a temperature of about 0°C. The reaction is typically carried out at ambient pressure. The reaction may also be carried out under an inert atmosphere, such as under nitrogen, argon, or helium.

[0239] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1 H NMR) determined. The duration of the reaction can range from about 5 minutes to about 2 hours. In certain embodiments, the duration of the reaction can range from about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour or about 1 hour to about 2 hours. In an exemplary embodiment, the reaction can be allowed to proceed for about 1 hour. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (VI) compound. Typically, the amount of formula (VI) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0240] The yield of the compound comprising formula (VIII) can be at least about 60%. In various embodiments, the yield of the compound comprising formula (VIII) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the yield of the compound comprising formula (VIII) can be about 90%.

[0241] Step (b)

[0242] Step (b) of the eight-step process involves contacting a compound comprising formula (VIII) with a proton acceptor, thereby forming a reaction mixture. After workup and isolation, a compound comprising formula (IX) is obtained. This process step is referred to as a "deprotection" reaction.

[0243] Compounds comprising formula (VIII) are described in more detail above.

[0244] Suitable proton acceptors are described in detail above in section (II)(a). In a preferred embodiment, the proton acceptor is NaOH.

[0245] Typically, the equivalent ratio of the compound comprising formula (VIII) to the proton acceptor can range from about 1.0:1.0 to about 1.0:5.0. In various embodiments, the equivalent ratio of the compound comprising formula (VIII) to the proton acceptor can range from about 1.0:1.0 to about 1.0:5.0, about 1.0:1.0 to about 1.0:3.0, or about 1.0:1.5. In a preferred embodiment, the equivalent ratio of the compound comprising formula (VIII) to the proton acceptor can range from about 1:0:2.0.

[0246] Step (b) further comprises a solvent. Suitable solvents are described in detail above in section (II)(a). In a preferred embodiment, the solvent is a combination of methanol and water.

[0247] Typically, the volume to weight ratio of solvent to compound comprising formula (VI) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of solvent to compound comprising formula (VI) may range from about 0.5: 1 to about 500: 1, from about 5: 1 to about 200: 1, from about 10: 1 to about 100: 1, or from about 15: 1 to about 50: 1. In a preferred embodiment, the volume to weight ratio of solvent to compound comprising formula (VIII) may be about 20: 1.

[0248] Typically, the reaction of step (b) will be carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about 0°C to about 50°C, from about 10°C to about 40°C, or from about 20°C to about 30°C. In one embodiment, the reaction can be carried out at a temperature of about 23°C (room temperature). The reaction is typically carried out under ambient pressure. The reaction can also be carried out under an inert atmosphere, for example, under nitrogen, argon or helium.

[0249] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1H NMR) is determined. The duration of the reaction can range from about 30 minutes to about 4 hours. In certain embodiments, the duration of the reaction can range from about 30 minutes to about 1 hour, about 1 hour to about 2 hours or about 2 hours to about 4 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 2 hours. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (VIII) compound. Typically, the amount of formula (VIII) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0250] After step (b) is completed, the pH of the reaction mixture is adjusted to a pH of less than about 6.0. In various embodiments, the pH is adjusted to less than about pH 6.0, less than about pH 5.0, less than about pH 4.0, less than about pH 3.0, less than about pH 2.0, or less than about pH 1.0. In a preferred embodiment, the pH range is adjusted to about pH 2.0 to about pH 2.5.

[0251] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids can be HCl, H2SO4, acetic acid, methanesulfonic acid or similar organic or inorganic acids. In a preferred embodiment, the acid that can be used is HCl.

[0252] The yield of the compound comprising formula (IX) can be at least about 60%. In various embodiments, the yield of the compound comprising formula (IX) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the yield of the compound comprising formula (IX) can be about 95%.

[0253] Step (c)

[0254] Step (c) of the eight-step process involves contacting a compound comprising formula (IX) with a compound comprising formula (X) in the presence of an acyl coupling reagent to form a reaction mixture. After workup and separation, a compound comprising formula (XI) is obtained.

[0255] Compounds comprising formula (IX) are described in detail above.

[0256] Compounds comprising formula (X) are described in detail above. In some embodiments, R7 is selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, and C3-C8 substituted cycloalkyl. In certain embodiments, R7 is selected from the group consisting of: H, methyl, ethyl, n-propyl, and isopropyl. In a preferred embodiment, R7 is isopropyl.

[0257] In some embodiments, P2 is a carboxylic acid protecting group. In specific embodiments, P2 is a methyl group. In a preferred embodiment, the compound comprising formula (X) is D-leucine methyl ester hydrochloride.

[0258] Typically, the equivalent ratio of the compound comprising (IX) to the compound comprising formula (X) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (IX) to the compound comprising formula (X) can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (IX) to the compound comprising formula (X) can be about 1:0:1.1.

[0259] The method of step (c) utilizes an acyl coupling reagent and a proton acceptor. Suitable acyl coupling reagents and proton acceptors are described in detail above in part (II) (a). In a preferred embodiment, the acyl coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or a salt thereof, and the proton acceptor is 4-methylmorpholine.

[0260] Typically, the equivalent ratio of the compound comprising formula (IX) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (IX) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (IX) to the acyl coupling agent can be about 1:0:1.1.

[0261] Typically, the equivalent ratio of the compound of formula (IX) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalent ratio of the compound of formula (IX) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5, about 1.0:1.0 to about 1.0:2.25, or about 1.0:2.2. In a preferred embodiment, the equivalent ratio of the compound of formula (IX) to the proton acceptor can be about 1:0:2.1.

[0262] Step (c) comprises a solvent. Suitable solvents are described in detail above in section (II)(a). In a preferred embodiment, the solvent that may be used in step (c) is dimethylformamide.

[0263] Typically, the volume to weight ratio of solvent to compound comprising formula (IX) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of solvent to compound comprising formula (IX) may range from about 0.5: 1 to about 500: 1, from about 2: 1 to about 250: 1, from about 5: 1 to about 200: 1, or from about 10: 1 to about 50: 1. In an exemplary embodiment, the volume to weight ratio of solvent to compound comprising formula (IX) may range from about 12: 1 to about 20: 1.

[0264] Typically, the reaction of step (c) will be carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about -20°C to about 25°C, from about -10°C to about 20°C, or from about -5°C to about 5°C. In one embodiment, the reaction can be carried out at a temperature of about 0°C. The reaction is typically carried out at ambient pressure. The reaction can also be carried out under an inert atmosphere, such as under nitrogen, argon, or helium.

[0265] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1 H NMR) is determined. The duration of the reaction can range from about 5 minutes to about 2 hours. In certain embodiments, the duration of the reaction can range from about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour or about 1 hour to about 2 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 1 hour. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (IX) compound. Typically, the amount of formula (IX) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0266] The yield of the compound comprising formula (XI) can be at least about 60%. In various embodiments, the yield of the compound comprising formula (XI) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the yield of the compound comprising formula (XI) can be about 96%.

[0267] Step (d)

[0268] Step (d) of the eight-step process involves contacting a compound comprising formula (XI) with a proton acceptor, thereby forming a reaction mixture. After workup and isolation, a compound comprising formula (XII) is obtained. This process step is referred to as a "deprotection" reaction.

[0269] Compounds comprising formula (XI) are described in more detail above.

[0270] Suitable proton acceptors are described in detail above in section (II)(b). In a preferred embodiment, the proton acceptor is NaOH or LiOH.

[0271] Typically, the equivalent ratio of the compound comprising formula (XI) to the proton acceptor can range from about 1.0:1.0 to about 1.0:5.0. In various embodiments, the equivalent ratio of the compound comprising formula (XI) to the proton acceptor can range from about 1.0:1.0 to about 1.0:5.0, about 1.0:1.0 to about 1.0:3.0, or about 1.0:1.5. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XI) to the proton acceptor can range from about 1:0:2.0.

[0272] Step (d) further comprises a solvent. Suitable solvents are described in detail above in section (II)(b). In a preferred embodiment, the solvent is a combination of methanol and water.

[0273] Typically, the volume to weight ratio of solvent to compound comprising formula (XI) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of solvent to compound comprising formula (XI) may range from about 0.5: 1 to about 500: 1, from about 5: 1 to about 200: 1, from about 10: 1 to about 100: 1, or from about 15: 1 to about 50: 1. In a preferred embodiment, the volume to weight ratio of solvent to compound comprising formula (XI) may be about 20: 1.

[0274] Typically, the reaction of step (d) will be carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about 0°C to about 50°C, from about 10°C to about 40°C, or from about 20°C to about 30°C. In one embodiment, the reaction can be carried out at a temperature of about 23°C (room temperature). The reaction is typically carried out under ambient pressure. The reaction can also be carried out under an inert atmosphere, for example, under nitrogen, argon or helium.

[0275] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1H NMR) is determined. The scope of the duration of reaction can be about 30 minutes to about 4 hours. In certain embodiments, the scope of the duration of reaction can be about 30 minutes to about 1 hour, about 1 hour to about 2 hours or about 2 hours to about 4 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 2 hours. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (XI) compound. Typically, the amount of formula (XI) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0276] After step (d) is completed, the pH of the reaction mixture is adjusted to a pH of less than about 6.0. In various embodiments, the pH is adjusted to less than about pH 6.0, less than about pH 5.0, less than about pH 4.0, less than about pH 3.0, less than about pH 2.0, or less than about pH 1.0. In a preferred embodiment, the pH range is adjusted to about pH 2.0 to about pH 2.5.

[0277] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids can be HCl, H2SO4, acetic acid, methanesulfonic acid or similar organic or inorganic acids. In a preferred embodiment, the acid that can be used is HCl.

[0278] The productive rate of the compound comprising formula (XII) can be at least about 60%. In various embodiments, the productive rate of the compound comprising formula (XII) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the productive rate of the compound comprising formula (XII) can be about 95%.

[0279] Step (e)

[0280] Step (e) of the eight-step method involves contacting a compound comprising formula (XII) with a compound comprising formula (XIII) in the presence of an acyl coupling reagent to form a reaction mixture. After workup and separation, a compound comprising formula (XIV) is obtained.

[0281] The compounds comprising formula (XII) are described in detail above.

[0282] Compounds comprising formula (XIII) are described in detail above. In some embodiments, R8 and R9 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, O substituted C1-C4 alkyl, O unsubstituted C1-C4 alkyl, (CH3O(CH2CH2) n CH2CH2-、HO(CH2CH2)n CH2CH2- and nitrogen protecting group (P1). In certain embodiments, R8 and R9 are independently selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl and nitrogen protecting group (P1). In specific embodiments, R8 and R9 are independently selected from the group consisting of: H and nitrogen protecting group (P1).

[0283] In some embodiments, n is an integer from 0 to 10. In specific embodiments, n is absent.

[0284] In some embodiments, P1 is a nitrogen protecting group. In specific embodiments, P1 is a BOC group.

[0285] In some embodiments, P2 is a carboxylic acid protecting group. In a specific embodiment, P2 is a methyl group. In a preferred embodiment, the compound comprising formula (XIII) is D-lysine methyl ester hydrochloride.

[0286] Typically, the equivalent ratio of the compound comprising (XII) to the compound comprising (XIII) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (XII) to the compound comprising formula (XIII) can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3 or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XII) to the compound comprising formula (XIII) can be about 1:0:1.1.

[0287] The method of step (e) utilizes an acyl coupling reagent and a proton acceptor. Suitable acyl coupling reagents and proton acceptors are described in detail above in part (II) (a). In a preferred embodiment, the acyl coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or a salt thereof, and the proton acceptor is 4-methylmorpholine.

[0288] Typically, the equivalent ratio of the compound comprising formula (XII) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (XII) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XII) to the acyl coupling agent can be about 1:0:1.1.

[0289] Typically, the equivalent ratio of the compound of formula (XII) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalent ratio of the compound of formula (XII) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5, about 1.0:1.0 to about 1.0:2.25, or about 1.0:2.2. In a preferred embodiment, the equivalent ratio of the compound of formula (XII) to the proton acceptor can be about 1:0:2.1.

[0290] Step (e) comprises a solvent. Suitable solvents are described in detail above in section (II)(a). In a preferred embodiment, the solvent that may be used in step (e) is dimethylformamide.

[0291] Typically, the volume to weight ratio of the solvent to the compound comprising formula (XII) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of the solvent to the compound comprising formula (XII) may range from about 0.5: 1 to about 500: 1, from about 2: 1 to about 250: 1, from about 5: 1 to about 200: 1, or from about 10: 1 to about 50: 1. In an exemplary embodiment, the volume to weight ratio of the solvent to the compound comprising formula (XII) may range from about 12: 1 to about 20: 1.

[0292] Typically, the reaction of step (e) will be carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about -20°C to about 25°C, from about -10°C to about 20°C, or from about -5°C to about 5°C. In one embodiment, the reaction can be carried out at a temperature of about 0°C. The reaction is typically carried out at ambient pressure. The reaction can also be carried out under an inert atmosphere, such as under nitrogen, argon, or helium.

[0293] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1 H NMR) is determined. The scope of the duration of reaction can be about 5 minutes to about 2 hours. In certain embodiments, the scope of the duration of reaction can be about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour or about 1 hour to about 2 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 1 hour. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (XII) compound. Typically, the amount of formula (XII) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0294] The yield of the compound comprising formula (XIV) can be at least about 60%. In various embodiments, the yield of the compound comprising formula (XIV) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the yield of the compound comprising formula (XIV) can be about 91%.

[0295] Step (f)

[0296] Step (f) of the eight-step process involves contacting a compound comprising formula (XIV) with a proton acceptor, thereby forming a reaction mixture. After workup and isolation, a compound comprising formula (XV) is obtained. This process step is referred to as a "deprotection" reaction.

[0297] Compounds comprising formula (XIV) are described in more detail above.

[0298] Suitable proton acceptors are described in detail above in section (II)(b). In a preferred embodiment, the proton acceptor is NaOH or LiOH.

[0299] Typically, the equivalent ratio of the compound comprising formula (XIV) to the proton acceptor can range from about 1.0:1.0 to about 1.0:5.0. In various embodiments, the equivalent ratio of the compound comprising formula (XIV) to the proton acceptor can range from about 1.0:1.0 to about 1.0:5.0, about 1.0:1.0 to about 1.0:3.0, or about 1.0:1.5. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XIV) to the proton acceptor can range from about 1:0:2.0.

[0300] Step (d) further comprises a solvent. Suitable solvents are described in detail above in section (II)(a). In a preferred embodiment, the solvent is a combination of methanol and water.

[0301] Typically, the volume to weight ratio of solvent to compound comprising formula (XIV) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of solvent to compound comprising formula (XIV) may range from about 0.5: 1 to about 500: 1, from about 5: 1 to about 200: 1, from about 10: 1 to about 100: 1, or from about 15: 1 to about 50: 1. In a preferred embodiment, the volume to weight ratio of solvent to compound comprising formula (XIV) may be about 20: 1.

[0302] Typically, the reaction of step (f) will be carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about 0°C to about 50°C, from about 10°C to about 40°C, or from about 20°C to about 30°C. In one embodiment, the reaction can be carried out at a temperature of about 23°C (room temperature). The reaction is typically carried out under ambient pressure. The reaction can also be carried out under an inert atmosphere, for example, under nitrogen, argon or helium.

[0303] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1 H NMR) determined. The duration of the reaction can range from about 30 minutes to about 4 hours. In certain embodiments, the duration of the reaction can range from about 30 minutes to about 1 hour, about 1 hour to about 2 hours or about 2 hours to about 4 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 2 hours. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (XIV) compound. Typically, the amount of formula (XIV) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0304] After step (f) is completed, the pH of the reaction mixture is adjusted to a pH of less than about 6.0. In various embodiments, the pH is adjusted to less than about pH 6.0, less than about pH 5.0, less than about pH 4.0, less than about pH 3.0, less than about pH 2.0, or less than about pH 1.0. In a preferred embodiment, the pH range is adjusted to about pH 2.0 to about pH 2.5.

[0305] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids can be HCl, H2SO4, acetic acid, methanesulfonic acid or similar organic or inorganic acids. In a preferred embodiment, the acid that can be used is HCl.

[0306] The yield of the compound comprising formula (XV) can be at least about 60%. In various embodiments, the yield of the compound comprising formula (XV) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the yield of the compound comprising formula (XV) can be about 92%.

[0307] Step (g)

[0308] Step (g) of the eight-step method involves contacting a compound of formula (XV) with a compound comprising formula (XVIa) or formula (XVIb) in the presence of an acyl coupling reagent to form a reaction mixture. After treatment and isolation, a compound comprising formula (XVII) is obtained.

[0309] Compounds comprising formula (XVII) are described in more detail above.

[0310] Compounds comprising formula (XVIa) and formula (XVIb) are described in more detail below:

[0311]

[0312] wherein P1 is a nitrogen protecting group and P2 is a carboxylic acid protecting group.

[0313] In a specific embodiment, P1 is a BOC group, and P2 is a methyl group. In a preferred embodiment, the compound comprising formula (XVIa) is 3-(BOC-amino)pyrrolidine-3-carboxylic acid methyl ester, and the compound comprising formula (XVIb) is 4-(BOC-amino)piperidine-4-carboxylic acid methyl ester.

[0314] Typically, the equivalent ratio of the compound comprising formula (XV) to the compound comprising formula (XVIa) or the compound comprising formula (XVIb) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (XV) to the compound comprising formula (XVIa) or the compound comprising formula (XVIb) can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XV) to the compound comprising formula (XVIa) or the compound comprising formula (XVIb) can be about 1:0:1.2.

[0315] The method of step (g) utilizes an acyl coupling reagent and a proton acceptor. Suitable acyl coupling reagents and proton acceptors are described in detail above in part (II) (a). In a preferred embodiment, the acyl coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or a salt thereof, and the proton acceptor is 4-methylmorpholine.

[0316] Typically, the equivalent ratio of the compound comprising formula (XV) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalent ratio of the compound comprising formula (XV) to the acyl coupling agent can range from about 1.0:1.0 to about 1.0:1.5, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XV) to the acyl coupling agent can be about 1:0:1.2.

[0317] Typically, the equivalent ratio of the compound comprising formula (XV) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalent ratio of the compound comprising formula (XV) to the proton acceptor can range from about 1.0:1.0 to about 1.0:2.5, about 1.0:1.0 to about 1.0:2.25, or about 1.0:2.2. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XV) to the proton acceptor can be about 1:0:2.1.

[0318] Step (g) comprises a solvent. Suitable solvents are described in detail above in section (II)(a). In a preferred embodiment, the solvent that may be used in step (g) is dimethylformamide.

[0319] Typically, the volume to weight ratio of solvent to compound comprising Formula (XV) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of solvent to compound comprising Formula (XV) may range from about 0.5: 1 to about 500: 1, from about 2: 1 to about 250: 1, from about 5: 1 to about 200: 1, or from about 10: 1 to about 50: 1. In an exemplary embodiment, the volume to weight ratio of solvent to compound comprising Formula (XV) may range from about 15: 1 to about 25: 1.

[0320] Typically, the reaction of step (g) will be carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about -20°C to about 25°C, from about -10°C to about 20°C, or from about -5°C to about 5°C. In one embodiment, the reaction can be carried out at a temperature of about 0°C. The reaction is typically carried out at ambient pressure. The reaction can also be carried out under an inert atmosphere, such as under nitrogen, argon, or helium.

[0321] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1H NMR) is determined. The scope of the duration of reaction can be about 5 minutes to about 2 hours. In certain embodiments, the scope of the duration of reaction can be about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour or about 1 hour to about 2 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 1 hour. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (XV) compound. Typically, the amount of formula (XV) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0322] The productive rate of the compound comprising formula (XVII) can be at least about 60%. In various embodiments, the productive rate of the compound comprising formula (XVII) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the scope of the productive rate of the compound comprising formula (XVII) can be about 88% to 93%.

[0323] Step (h)

[0324] Step (h) involves contacting the compound comprising formula (XVII) with a deprotection reagent comprising an acid, thereby forming a reaction mixture. After workup and isolation, a compound comprising formula (I) is obtained.

[0325] Step (h), after using a deprotecting agent comprising an acid and isolating the compound comprising formula (I), the method may further contact the compound comprising formula (I) with a proton acceptor to remove the carboxylic acid protecting group.

[0326] Compounds comprising formula (XVII) are described in more detail above.

[0327] Various acids can be used in this method step. In various embodiments, the deprotection reagent comprising the acid can be in pure form or in aqueous form. Non-limiting acid can be hydrochloric acid, hydrogen chloride, sulfuric acid, methanesulfonic acid or trifluoroacetic acid. In a preferred embodiment, the deprotection reagent comprising the acid is trifluoroacetic acid.

[0328] Typically, the deprotecting reagent comprising the acid is used in excess by volume to weight.

[0329] Step (h) comprises a solvent. Suitable solvents are described in detail above in section (II)(a). In a preferred embodiment, the solvent that may be used in step (h) is dichloromethane.

[0330] Typically, the volume to weight ratio of the solvent to the compound comprising Formula (XVII) will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of the solvent to the compound comprising Formula (XVII) may range from about 0.5: 1 to about 500: 1, from about 2: 1 to about 250: 1, from about 5: 1 to about 200: 1, or from about 10: 1 to about 100: 1. In an exemplary embodiment, the volume to weight ratio of the solvent to the compound comprising Formula (XVII) may range from about 40: 1 to about 80: 1.

[0331] Typically, the reaction of step (h) will be carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about -20°C to about 25°C, from about -10°C to about 20°C, or from about -5°C to about 5°C. In one embodiment, the reaction can be carried out at a temperature of about 0°C. The reaction is typically carried out at ambient pressure. The reaction can also be carried out under an inert atmosphere, such as under nitrogen, argon, or helium.

[0332] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1 H NMR) is determined. The scope of the duration of reaction can be about 5 minutes to about 2 hours. In certain embodiments, the scope of the duration of reaction can be about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour or about 1 hour to about 2 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 1 hour. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (XVII) compound. Typically, the amount of formula (XVII) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0333] The productive rate of the compound comprising formula (I) can be at least about 60%. In various embodiments, the productive rate of the compound comprising formula (I) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the productive rate of the compound comprising formula (I) can range from about 88% to 93%.

[0334] The second deprotection step utilizes a proton acceptor. Suitable proton acceptors are described in detail above in section (II)(b). In a preferred embodiment, the proton acceptor is NaOH or LiOH.

[0335] Typically, the equivalent ratio of the compound comprising formula (I) to the proton acceptor can range from about 1.0:1.0 to about 1.0:20.0. In various embodiments, the equivalent ratio of the compound comprising formula (I) to the proton acceptor can range from about 1.0:1.0 to about 1.0:20.0, about 1.0:5 to about 1.0:15.0, or about 1.0:8.0 to about 1.0:12.0. In a preferred embodiment, the equivalent ratio of the compound comprising formula (XIV) to the proton acceptor can range from about 1:0:10.0.

[0336] The second deprotection step in step (h) further comprises a solvent. Suitable solvents are described in detail above in section (II) (a). In a preferred embodiment, the solvent is a combination of methanol and water.

[0337] Typically, the volume to weight ratio of the solvent to the compound comprising Formula (I) in the second deprotection step will range from about 0.5: 1 to about 500: 1. In various embodiments, the volume to weight ratio of the solvent to the compound comprising Formula (I) may range from about 0.5: 1 to about 500: 1, from about 5: 1 to about 200: 1, from about 10: 1 to about 100: 1, or from about 15: 1 to about 50: 1. In a preferred embodiment, the volume to weight ratio of the solvent to the compound comprising Formula (I) in the second deprotection step may be about 20: 1.

[0338] Typically, the second deprotection step in step (h) will be carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent utilized. In various embodiments, the temperature of the reaction can range from about 0°C to about 50°C, from about 10°C to about 40°C, or from about 20°C to about 30°C. In one embodiment, the reaction can be carried out at a temperature of about 23°C (room temperature). The reaction is typically carried out at ambient pressure. The reaction can also be carried out under an inert atmosphere, such as under nitrogen, argon or helium.

[0339] Typically, the reaction is allowed to proceed for a sufficient period of time until the reaction is complete, such as by any method known to those skilled in the art, such as HPLC, TLC, or proton nuclear magnetic resonance (e.g., 1 H NMR) determined. The duration of the reaction can range from about 30 minutes to about 4 hours. In certain embodiments, the duration of the reaction can range from about 30 minutes to about 1 hour, about 1 hour to about 2 hours or about 2 hours to about 4 hours. In a preferred embodiment, the reaction can be allowed to proceed for about 2 hours. In this context, "completed reaction" generally means that the reaction mixture contains a significantly reduced amount of formula (I) compound. Typically, the amount of formula (I) compound remaining in the reaction mixture at the end of the reaction can be less than about 10%, less than about 5% or less than about 2%.

[0340] After completing step (h) using a proton acceptor, the pH of the reaction mixture is adjusted to a pH of less than about 6.0. In various embodiments, the pH is adjusted to less than about pH 6.0, less than about pH 5.0, less than about pH 4.0, less than about pH 3.0, less than about pH 2.0, or less than about pH 1.0. In a preferred embodiment, the pH range is adjusted to about pH 2.0 to about pH 2.5.

[0341] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids may be HCl, H2SO4, acetic acid, methanesulfonic acid or similar organic or inorganic acids.

[0342] The yield of the compound comprising formula (I) can be at least about 60%. In various embodiments, the yield of the compound comprising formula (I) can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%. In a preferred embodiment, the scope of the yield of the compound comprising formula (I) can be about 60%-70%.

[0343] (III) The pharmaceutical composition comprises the compound of formula (I) or a pharmaceutically acceptable salt of the compound of formula (I).

[0344] Another aspect of the present disclosure includes a pharmaceutical composition comprising a compound comprising Formula (I) or a pharmaceutically acceptable salt of a compound comprising Formula (I) and at least one pharmaceutically acceptable excipient.

[0345] (a) a compound of formula (I) or a pharmaceutically acceptable salt of a compound of formula (I)

[0346] The compounds of formula (I) or pharmaceutically acceptable salts of the compounds of formula (I) are described in detail in section (I) above.

[0347] Typically, the amount of the pharmaceutically acceptable salt of the compound of formula (I) or the compound of formula (I) used in the pharmaceutical composition can and will vary according to the age of the subject and the dosage used every day. Typically, the amount of the pharmaceutically acceptable salt of the compound of formula (I) or the compound of formula (I) used in the pharmaceutical composition can range from about 1.0 mg to about 100 mg. In various embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) or the compound of formula (I) used in the pharmaceutical composition can range from about 1.0 mg to about 100 mg, from about 5 mg to about 75 mg, or from about 10 mg to about 20 mg.

[0348] (b) at least one excipient

[0349] The compositions of the present disclosure may further comprise a pharmaceutically acceptable excipient. Non-limiting examples of suitable pharmaceutically acceptable excipients include diluents, adhesives, fillers, buffers, pH regulators, disintegrants, dispersants, preservatives, lubricants, taste masking agents, flavoring agents, coloring agents or combinations thereof. The amount and type of the excipients used to form the pharmaceutical composition can be selected according to known pharmaceutical principles.

[0350] In one embodiment, excipient can be diluent.Diluent can be compressible (that is, plastically deformable) or grinding brittle.The limiting examples of suitable compressible diluent include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose ester (that is, acetate and butyrate mixed ester), ethylcellulose, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphorus corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium glycolate starch, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, maltitol, sorbitol, xylitol, maltodextrin and trehalose.The limiting examples of suitable grinding brittle diluent include calcium hydrogen phosphate (anhydrous or dihydrate), tricalcium phosphate, calcium carbonate and magnesium carbonate.

[0351] In another embodiment, the excipient can be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinyl pyrrolidone, cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 -C 18 Fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, polypeptides, oligopeptides and combinations thereof.

[0352] In another embodiment, the excipient can be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. As non-limiting examples, the filler can be calcium sulfate (both dibasic and tribasic), starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, calcium hydrogen phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

[0353] In yet another embodiment, the excipient can be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

[0354] In various embodiments, the excipient can be a pH adjuster. As non-limiting examples, the pH adjuster can be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid or phosphoric acid.

[0355] In further embodiments, the excipient may be a disintegrant. Disintegrants may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof; sweeteners; clays such as bentonite; microcrystalline cellulose; alginates; sodium starch glycolate; gums such as agar, guar gum, locust bean gum, kana gum, pectin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include combinations of sodium bicarbonate and citric acid and combinations of sodium bicarbonate and tartaric acid.

[0356] In yet another embodiment, the excipient can be a dispersant or a dispersion enhancer. Suitable dispersants can include, but are not limited to, starch, alginic acid, polyvinyl pyrrolidone, guar gum, kaolin, bentonite, purified lignocellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

[0357] In another alternative embodiment, the excipient can be a preservative. Non-limiting examples of suitable preservatives include: antioxidants such as BHA, BHT, vitamin A, vitamin C, vitamin E or retinol palmitate, citric acid, sodium citrate; chelating agents such as EDTA or EGTA; and antimicrobial agents such as parabens, chlorobutanol or phenol.

[0358] In a further embodiment, the excipient can be a lubricant. Non-limiting examples of suitable lubricants include minerals, such as talc or silicon dioxide; and fats, such as vegetable stearin, magnesium stearate or stearic acid.

[0359] In yet another embodiment, the excipient can be a taste masking agent. Taste masking materials include cellulose ethers, polyethylene glycol, polyvinyl alcohol, copolymers of polyvinyl alcohol and polyethylene glycol, monoglycerides or triglycerides; acrylic acid polymers, mixtures of acrylic acid polymers and cellulose ethers, cellulose acetate phthalate, and combinations thereof.

[0360] In an alternative embodiment, the excipient may be a flavoring agent. The flavoring agent may be selected from synthetic flavoring oils and flavoring aromatics and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.

[0361] In yet another embodiment, the excipient can be a colorant. Suitable color additives include, but are not limited to, Food, Drug, and Cosmetic Colors (FD&C), Drug and Cosmetic Colors (D&C), or External Drug and Cosmetic Colors (Ext. D&C).

[0362] The weight fraction of an excipient or combination of excipients in the composition can be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less or about 1% or less of the total weight of the composition.

[0363] Compositions can be formulated into various dosage forms and applied by many different ways of delivering the active ingredient of a therapeutically effective amount. Such compositions can be administered orally, parenterally or topically in the form of dosage unit formulations containing the required conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Topical application can also involve the use of transdermal administrations such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques. For example, Gennaro, AR "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pa., Pennsylvania (18th edition, 1995) and Liberman, HA and Lachman, L. editors, "Pharmaceutical Dosage Forms", Marcel Dekker Inc., New York, New York (1980) discussed the formulation of the drug. In particular embodiments, the composition may be a food supplement or the composition may be a cosmetic.

[0364] Solid dosage forms for oral administration can include capsules, tablets, capsules, pills, powders, granules and particles. In such solid dosage forms, the active ingredient is usually combined with one or more pharmaceutically acceptable excipients, and the examples of the pharmaceutically acceptable excipients are described in detail above. Oral formulations can also be used as aqueous suspensions, elixirs or syrups. For this reason, the active ingredient can be combined with various sweeteners or flavorings, coloring agents, and if necessary, emulsifiers and / or suspending agents and diluents such as water, ethanol, glycerol and combinations thereof.

[0365] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal), the preparation can be an aqueous or oil-based solution. The aqueous solution can include: a sterile diluent such as water, a saline solution; a pharmaceutically acceptable polyol such as glycerol, propylene glycol or other synthetic solvents; an antibacterial and / or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, etc.; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and / or an agent for adjusting tension such as sodium chloride, dextran or a polyol such as mannitol or sorbitol. The pH of the aqueous solution can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The oil-based solution or suspension may further contain sesame, peanut, olive oil or mineral oil. The compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, microgranules and tablets.

[0366] For topical (e.g., transdermal or mucosal) administration, a penetrant suitable for the barrier to be penetrated is generally included in the formulation. Pharmaceutical compositions suitable for topical administration can be formulated into ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In certain embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated into an ointment, the active ingredient can be used together with paraffin or a water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream together with an oil-in-water cream base or an oil-in-water base. Pharmaceutical compositions suitable for topical administration to the eyes include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially in an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the oral cavity include lozenges, pastilles and mouthwashes. Mucosal administration can be accomplished by using nasal sprays, aerosol sprays, tablets or suppositories, and transdermal administration can be performed by ointments, salves, gels, patches or creams as generally known in the art.

[0367] In certain embodiments, a composition comprising formula (I) or a pharmaceutically acceptable salt of a compound comprising formula (I) is encapsulated in a suitable vehicle to help deliver the compound to a target cell, increase the stability of the composition, or minimize the potential toxicity of the composition. As will be appreciated by those skilled in the art, various vehicles are suitable for delivering the composition of the present invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating compositions into delivery vehicles are known in the art.

[0368] In an alternative embodiment, a liposome delivery vehicle can be utilized. In view of the structure and chemical properties of the liposome, depending on the embodiment, the liposome is suitable for delivering a composition comprising formula (I) or a pharmaceutically acceptable salt of a compound comprising formula (I). In general, a liposome is a spherical vesicle having a phospholipid bilayer membrane. The lipid bilayer of the liposome can be fused with other bilayers (e.g., cell membranes) to deliver the contents of the liposome to the cell. In this way, a composition comprising at least one antiviral therapeutic agent can be selectively delivered to the cell by encapsulating in a liposome fused with the membrane of the target cell.

[0369] Liposome can comprise various types of phospholipids with different hydrocarbon chain lengths.Phospholipids usually comprise two kinds of fatty acids connected with one of various polar groups by glycerophosphate.Suitable phospholipids comprise phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE).The scope of the length of the fatty acid chain comprising phospholipids can be about 6 to about 26 carbon atoms, and the lipid chain can be saturated or undersaturated. Suitable fatty acid chains include (common names are presented in brackets) n-dodecanoate (laurate), n-tetradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecanoate (palmitoleate), cis-9-octadecanoate (oleate), cis, cis-9,12-octadecadienoate (linoleate), all-cis-9,12,15-octadecadienoate (linolenate) and all-cis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acid chains of the phospholipid may be the same or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linalenyl PS and the like.

[0370] Phospholipids can be from any natural source, and thus, can comprise a mixture of phospholipids. For example, egg yolk is rich in PC, PG and PE, soybean contains PC, PE, PI and PA, and animal brain or spinal cord is rich in PS. Phospholipids can also be from synthetic sources. A mixture of phospholipids with a single phospholipid in different ratios can be used. A mixture of different phospholipids can produce a liposome composition with a favorable activity or stability of the active properties. The phospholipids mentioned above can be mixed with cationic lipids such as N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, 3,3'-deheptyloxacarbocyanine iodide, 1,1'-dedodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, perchloarate), 1,1'-dioleyl-3,3,3',3'-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1,1,-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate.

[0371] The liposomes may optionally comprise sphingolipids or cholesterol (a major component of animal cell membranes), in which sphingosine is the structural counterpart of one of the fatty acids of glycerol and phosphoglycerides. The liposomes may optionally contain PEGylated lipids, which are lipids covalently linked to a polymer of polyethylene glycol (PEG). The size of PEG may range from about 500 Daltons to about 10,000 Daltons.

[0372] The liposome may further comprise a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethyl sulfoxide (DMSO), methyl pyrrolidone, N-methyl pyrrolidone, acetonitrile, alcohol, dimethylformamide, tetrahydrofuran or a combination thereof.

[0373] Liposomes carrying compositions comprising compounds of formula (I) can be prepared by any known method for preparing liposomes for drug delivery, such as those described in detail in U.S. Patents Nos. 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837, 4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and 5,264,618, the disclosure of which is hereby incorporated by reference as a whole. For example, liposomes can be prepared by ultrasonic treatment of lipids, solvent injection, lipid hydration, reverse evaporation in aqueous solution or freeze drying by repeated freezing and thawing. In a preferred embodiment, liposomes are formed by ultrasonic treatment. Liposomes can be multilamellar or unilamellar, which have many layers like an onion. Liposomes can be large or small. Continuous high shear sonication tends to form smaller unilamellar liposomes.

[0374] As will be apparent to those of ordinary skill, all parameters controlling liposome formation may vary. These parameters include, but are not limited to, temperature, pH, concentration of the methionine compound, concentration and composition of lipids, concentration of multivalent cations, mixing rate, presence and concentration of solvents.

[0375] In another embodiment, the compositions of the present disclosure can be delivered to cells as microemulsions. Microemulsions are typically clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and an "oil". In this case, the "oil" is a supercritical fluid phase. The surfactant stays at the oil-water interface. Any of a variety of surfactants is suitable for use in microemulsion formulations, including microemulsion formulations described herein or otherwise known in the art. The characteristic structural size of aqueous microdomains suitable for use in the present invention is typically about 5 nm to about 100 nm. Aggregates of this size are weak scatterers of visible light, and therefore, these solutions are optically clear. As will be understood by those skilled in the art, microemulsions can and will have a variety of different microstructures including spherical, rod-shaped, or disc-shaped aggregates. In one embodiment, the structure can be a micelle, which is the simplest microemulsion structure that is typically a spherical or cylindrical object. A micelle is like a droplet of oil in water, and a reverse micelle is like a droplet in oil in water. In an alternative embodiment, the microemulsion structure is a thin layer. It comprises continuous water and oil layers separated by a surfactant layer. The "oil" of the microemulsion optimally comprises a phospholipid. Any of the phospholipids detailed above for liposomes are suitable for embodiments for microemulsions. The composition comprising at least one antiviral therapeutic derivative may be encapsulated in a microemulsion by any method generally known in the art.

[0376] In yet another embodiment, the composition of formula (I) or the pharmaceutically acceptable salt of the compound of formula (I) can be delivered in the form of a dendrimer or dendrimer. In general, a dendrimer is a branched dendritic molecule, wherein each branch is an interconnected chain of molecules that splits into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so dense that the canopy forms a sphere. Generally, the properties of the dendrimer are determined by the functional groups on its surface. For example, hydrophilic end groups such as carboxyl groups usually result in water-soluble dendrimers. Alternatively, phospholipids can be incorporated into the surface of the dendrimer to promote absorption across the skin. Any of the phospholipids used in the liposome embodiment described in detail is suitable for use in the dendrimer embodiment. Dendrimers can be prepared using any method generally known in the art and the composition of the present invention can be encapsulated in the dendrimer. For example, a dendrimer can be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Therefore, the dendrimer has a regular, highly branched 3D structure and has an almost uniform size and shape. Furthermore, the final size of the dendrimer is generally controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for the present invention. Typically, the size of the dendrimer can range from about 1 nm to about 100 nm.

[0377] (c) Dosage form

[0378] Compositions can be formulated into various dosage forms and applied by many different ways of delivering the active ingredient of a therapeutically effective amount. Such compositions can be administered orally, parenterally or topically in the form of dosage unit formulations containing the required conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Topical application can also relate to the use of transdermal administrations such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques. For example, Gennaro, AR "Remington's Pharmaceutical Sciences", Mike Publishing Company (18th edition, 1995) in Easton, Pennsylvania, and Liberman, HA and Lachman, L. editors, "Drug Formulations", Marcel Dekker Company (1980) in New York City, New York, discussed the formulation of the drug.

[0379] Solid dosage forms for oral administration include capsules, tablets, capsules, pills, powders, granules and particles. In such solid dosage forms, the active ingredient is usually combined with one or more pharmaceutically acceptable excipients, examples of which are described in detail above. Oral formulations can also be used as aqueous suspensions, elixirs or syrups. For this reason, the active ingredient can be combined with various sweeteners or flavorings, coloring agents, and, if necessary, emulsifiers and / or suspending agents and diluents such as water, ethanol, glycerol and combinations thereof.

[0380] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal), the preparation can be an aqueous or oil-based solution. The aqueous solution can include: a sterile diluent such as water, a saline solution; a pharmaceutically acceptable polyol such as glycerol, propylene glycol or other synthetic solvents; an antibacterial and / or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, etc.; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and / or an agent for adjusting tension such as sodium chloride, dextran or a polyol such as mannitol or sorbitol. The pH of the aqueous solution can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The oil-based solution or suspension may further contain sesame, peanut, olive oil or mineral oil.

[0381] (IV). Methods of treating medical conditions with kappa-opioid receptor agonists.

[0382] Yet another aspect of the present disclosure comprises a method of treating a κ-opioid receptor agonist medical condition. The method comprises administering to a subject in need thereof a pharmaceutical composition comprising formula (I) or a pharmaceutically acceptable salt of a compound of formula (I). κ-opioid receptor agonist-related diseases or conditions are pain, cardiovascular disease, pruritus, nausea, inflammatory disease, spinal anesthesia, cough suppression, stroke, hypoxic pulmonary hypertension, multiple sclerosis, addiction, and post-traumatic cartilage degeneration.

[0383] (a) Composition and Dosage Form

[0384] Compositions and dosage forms are described in more detail above in Section (III).

[0385] Such compositions can be administered orally, parenterally, by inhalation spray, rectally, intradermally, transdermally or topically in the form of dosage unit formulations containing the desired conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Topical administration can also involve the use of transdermal administrations such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques.

[0386] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal), the preparation can be an aqueous or oil-based solution. The aqueous solution can include: a sterile diluent such as water, a saline solution; a pharmaceutically acceptable polyol such as glycerol, propylene glycol or other synthetic solvents; an antibacterial and / or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, etc.; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and / or an agent for adjusting tension such as sodium chloride, dextran or a polyol such as mannitol or sorbitol. The pH of the aqueous solution can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The oil-based solution or suspension may further contain sesame, peanut, olive oil or mineral oil.

[0387] For topical (e.g., transdermal or transmucosal) administration, penetrants appropriate to the barrier to be permeated are generally included in the formulation. Transmucosal administration can be accomplished through the use of nasal sprays, aerosol sprays, tablets or suppositories, and transdermal administration can be performed by ointments, salves, gels, patches or creams as generally known in the art.

[0388] The amount of the medicament administered to the subject can and will vary depending on the type of medicament, the subject, and the particular mode of administration. It will be appreciated by those skilled in the art that dosages may also be determined under the guidance of Goodman and Goldman, The Pharmacological Basis of Therapeutics, 10th edition (2001), Appendix II, pages 475-493, and the Physicians' Desk Reference.

[0389] (b) Subjects

[0390] Suitable subjects include humans, livestock, companion animals, laboratory animals, or zoological animals. In one embodiment, the subject may be a rodent, for example, a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock. Non-limiting examples of suitable livestock animals may include pigs, cattle, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets, such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, "zoological animals" refer to animals that can be found in zoos. Such animals may include non-human primates, large cats, wolves, and bears. In a specific embodiment, the animal is a laboratory animal. Non-limiting examples of laboratory animals may include rodents, canines, felines, and non-human primates. In a specific embodiment, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In a preferred embodiment, the subject is a human.

[0391] (c) Dosage administration

[0392] Typically, the composition of formula (I) compound can be administered at a dosage rate of about 0.0001 mg / kg to about 100.0 mg / kg. In various embodiments, the compound of formula (I) compound can be administered at a dosage rate of about 0.0001 mg / kg to about 100.0 mg / kg, about 0.01 mg / kg to about 50.0 mg / kg, about 1.0 mg / kg to about 20.0 mg / kg, or about 5.0 mg / kg to about 10.0 mg / kg. In a preferred embodiment, the composition of formula (I) compound can be administered at a dosage rate of about 0.0001 mg / kg to about 10.0 mg / kg.

[0393] Depending on the severity of the subject's indication, the administration of the compound of formula (I) can be divided into a variety of treatment regimens. For example, in some embodiments, the pharmaceutical composition can be administered to a subject in need at least once a day, such as at least twice a day, at least three times a day, or multiple times a day.

[0394] definition

[0395] The compounds described herein have asymmetric centers. Compounds of the present disclosure containing asymmetrically substituted atoms can be isolated in optically active forms or racemic forms. Unless specifically indicated a specific stereochemistry or isomeric form, all chiral, diastereoisomer, racemic forms and all geometric isomeric forms of a structure are contemplated.

[0396] As used herein, the term "acyl" alone or as part of another group refers to a moiety formed by removing a hydroxyl group from the radical COOH of an organic carboxylic acid (e.g., RC(O)-) wherein R is R 1 , R 1 O-、R 1 R 2 N- or R 1 S-, R 1 is a hydrocarbon group, a heterosubstituted hydrocarbon group or a heterocycle, and R 2 is hydrogen, a hydrocarbon group or a substituted hydrocarbon group.

[0397] As used herein, the term "acyloxy" alone or as part of another group refers to an acyl group as described above bonded through an oxygen bond (O), for example RC(O)O-, wherein R is as defined in conjunction with the term "acyl".

[0398] As used herein, the term "allyl" refers not only to compounds containing a simple allyl group (CH2=CH-CH2-), but also to compounds containing a substituted allyl group or an allyl group forming a part of a ring system.

[0399] As used herein, the term "alkyl" describes a lower alkyl group preferably containing one to eight carbon atoms and up to 20 carbon atoms in the main chain. The group may be straight or branched or cyclic and includes methyl, ethyl, propyl, isopropyl, butyl, hexyl, etc.

[0400] The term "alkenyl" as used herein describes a lower alkenyl group preferably containing two to eight carbon atoms and up to 20 carbon atoms in the main chain. The group may be straight or branched or cyclic and includes ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, etc.

[0401] As used herein, the term "alkynyl" describes a lower alkynyl group preferably containing two to eight carbon atoms and up to 20 carbon atoms in the main chain. The group can be straight or branched and includes ethynyl, propynyl, butynyl, isobutynyl, hexynyl, etc.

[0402] As used herein, the term "aromatic" alone or as part of another group refers to an optionally substituted homocyclic or heterocyclic conjugated planar ring or ring system containing delocalized electrons. These aromatic groups are preferably monocyclic (e.g., furan or benzene) groups, bicyclic groups, or tricyclic groups containing 5 to 14 atoms in the ring portion. The term "aromatic" encompasses "aryl" groups defined below.

[0403] As used herein, the term "aryl" or "Ar" alone or as part of another group refers to an optionally substituted homocyclic aromatic group, preferably a homocyclic monocyclic or bicyclic group containing 6 to 10 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl.

[0404] As used herein, the term "carbocyclo" or "carbocyclic" alone or as part of another group refers to an optionally substituted, aromatic or non-aromatic homocyclic ring or ring system in which all atoms in the ring are carbon, preferably 5 or 6 carbon atoms in each ring. Exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amido, acetal, carbamoyl, carbocycle, cyano, ester, ether, halogen, heterocycle, hydroxy, ketone, ketal, phosphate, nitro, and thio.

[0405] As used herein, the term "halogen" or "halo" alone or as part of another group refers to chlorine, bromine, fluorine and iodine.

[0406] The term "heteroatom" refers to atoms other than carbon and hydrogen.

[0407] As used herein, the term "heteroaromatic" refers to an optionally substituted aromatic group having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring, either alone or as part of another group. The heteroaromatic group preferably has 1 or 2 oxygen atoms and / or 1 to 4 nitrogen atoms in the ring, and is bonded to the rest of the molecule by carbon. Exemplary groups include furanyl, benzofuranyl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzoxadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, isoindolene, indolizinyl, benzimidazolyl, indazolyl, benzotriazolyl, tetrazolyl pyridazinyl, carbazolyl, purinyl, quinolyl, isoquinolyl, imidazopyridinyl, etc. Exemplary substituents include one or more of the following: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amido, acetal, carbamoyl, carbocycle, cyano, ester, ether, halogen, heterocycle, hydroxy, ketone, ketal, phosphate, nitro, and thio.

[0408] As used herein, the term "heterocycle" or "heterocyclic" alone or as part of another group means an optionally substituted, fully saturated or unsaturated, monocyclic or bicyclic, aromatic or non-aromatic group having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring. The heterocyclic group preferably has 1 or 2 oxygen atoms and / or 1 to 4 nitrogen atoms in the ring and is bonded to the rest of the molecule through carbon or heteroatoms. Exemplary heterocyclic groups include aromatic heterocycles as described above. Exemplary substituents include one or more of the following groups: hydrocarbon, substituted hydrocarbon, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amide, acetal, carbamoyl, carbocycle, cyano, ester, ether, halogen, heterocycle, hydroxyl, ketone, ketal, phosphate, nitro and thio.

[0409] As used herein, the term "hydrocarbon" or "hydrocarbyl" describes an organic compound or group consisting only of the elements carbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl and aryl moieties. These moieties also include alkyl, alkenyl, alkynyl and aryl moieties substituted by other aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenyl and alkynyl. Unless otherwise indicated, these moieties preferably contain 1 to 20 carbon atoms.

[0410] As used herein, the term "protecting group" refers to a group capable of protecting a particular portion, wherein the protecting group can be removed after the reaction using protection without interfering with the remainder of the molecule. When the portion is an oxygen atom (and thus forming a protected hydroxyl), exemplary protecting groups include ethers (e.g., allyl, triphenylmethyl (triphenylmethyl / trityl or Tr), benzyl, p-methoxybenzyl (PMB), p-methoxyphenyl (PMP)), acetals (e.g., methoxymethyl (MOM), β-methoxyethoxymethyl (MEM), tetrahydropyranyl (THP), ethoxyethyl (EE), methylthiomethyl (MTM), 2-methoxy-2-propyl (MOP), 2-trimethylsilyloxymethyl (SEM)), esters (e.g., benzoate (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylethylsilyl carbonate), silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), trimethylisopropylsilyl (TIP) When the moiety is a nitrogen atom (and thus forms a protected amine), exemplary protecting groups include benzyl, p-methoxyphenyl (PMP), 3,4-dimethoxybenzyl (PMB), n-silyl, esters (e.g., benzoate (Bz), carbonyl (e.g., p-methoxybenzylcarbonyl (Moz), tert-butyloxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC)), acetyl, carbamate, n-silyl, etc. When the moiety is a carboxyl group, exemplary protecting groups include esters (methyl, substituted methyl esters, ethyl esters, substituted ethyl esters), etc. Various protecting groups and their synthesis can be found in "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts, John Wiley & Sons, 1999.

[0411] "Substituted hydrocarbyl" moieties as described herein are hydrocarbyl moieties substituted with at least one atom other than carbon, including moieties where carbon chain atoms are replaced with heteroatoms such as nitrogen, oxygen, silicon, phosphorus, boron or halogen atoms, and moieties where the carbon chain contains additional substituents. These substituents include alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amido, acetal, carbamoyl, carbocycle, cyano, ester, ether, halogen, heterocycle, hydroxy, ketone, ketal, phosphate, nitro and thio.

[0412] When introducing elements of the embodiments described herein, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0413] As various changes could be made in the above method without departing from the scope of the invention, it is intended that all matter contained in the above description and examples given below shall be interpreted as illustrative and not in a limiting sense.

[0414] Example 1: Synthesis of dipeptide intermediate (3)

[0415]

[0416] To a reaction flask under nitrogen was added (2R,3R)-Boc-β-methyl-phenylalanine (1) (15 g, 36.1 mmol, 1.0 eq) and DMF (212 mL). After the mixture became homogeneous under stirring, the resulting solution was cooled to 0°C in an ice bath. To the cooled reaction was added HOBt.HO (5.36 g, 39.7 mmol, 1.1 eq) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (7.60 g, 39.7 mmol, 1.1 eq). The resulting mixture was stirred for 30 minutes, then HD-Phe-OMe.HCl (2) (8.56 g, 39.7 mmol, 1.1 eq) and N-methylmorpholine (NMM) (7.58 mL, 75.7 mmol, 2.1 eq) were added. The reaction was stirred at 0°C for one hour and then at room temperature until HPLC analysis indicated the reaction was complete. Under stirring, the reaction solution was added dropwise to water (600 mL). After the water addition was completed, stirring was stopped and a precipitate began to form. The reaction mixture was allowed to stand for 1 hour, during which precipitation stopped. The precipitate (crystals) was collected by filtration and washed with water (400 mL x 3) and then dried in vacuo; the product (3) was obtained as a white solid, 14.36 g, yield = 90.8%. LCMS: m / z = 441.5 [M + H] + .

[0417] Example 2: Synthesis of dipeptide intermediate (4)

[0418]

[0419] Compound (3) (12 g, 27.2 mmol, 1.0 equiv) and methanol (240 mL) were added to the reaction flask. Stirring was started, and NaOH solution (60 mL, 1 M, 60 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for about 2 hours until HPLC analysis indicated that the reaction was complete. HCl solution (1.0 M) was added dropwise to the reaction mixture until the pH reached 2.0-2.5. Then, water (380 mL) was added dropwise to precipitate the product. The resulting mixture was allowed to stand for one hour, and the solid was then collected by filtration and washed with water (3 x 320 mL) and then dried under vacuum (30 ° C). The product was obtained as a white solid, 11 g, yield = 94.8%. LCMS m / z = 427.5 [M + H] + .

[0420] Example 3: Synthesis of tripeptide intermediate (6)

[0421]

[0422] Compound (4) (8.0 g, 18.8 mmol, 1.0 eq) and DMF (169 mL) were added to a reaction flask under nitrogen. The resulting solution was cooled to 0°C in an ice bath. HOBt.H2O (2.79 g, 20.6 mmol, 1.2 eq) and EDCI (3.96 g, 20.5 mmol, 1.1 eq) were added to the cooled reaction mixture. After the reaction mixture was stirred at 0°C for 30 minutes, HD-Leu-OMe.HCl (5) (20.6 mmol, 1.1 eq, 3.75 g) and N-methylmorpholine (NMM) (3.94 mL, 39.4 mmol, 2.1 eq) were added. The resulting reaction mixture was stirred at 0°C for 1 hour and then warmed to room temperature until HPLC analysis indicated that the reaction was complete. The reaction mixture was added dropwise to water (360 mL) with stirring. After the water addition was complete, stirring was stopped and a precipitate was formed. The reaction mixture was allowed to stand for one hour, during which precipitation stopped. The precipitate was then collected by filtration and washed with water (240 mL x 3), then dried in vacuo (30°C); the product (6) was obtained as a white solid, 9.96 g, yield = 96%. LCMS: m / z = 554.7 [M+H] + .

[0423] Example 4: Synthesis of tripeptide intermediate (7)

[0424]

[0425] Compound (6) (8 g, 14.4 mmol, 1.0 equiv) and methanol (160 mL) were added to the reaction flask under nitrogen. Stirring was started and then NaOH solution (40 mL, 1 M, 40 mmol) was added. The reaction mixture was stirred at room temperature for about 2 hours until HPLC analysis indicated that the reaction was complete. HCl solution (1.0 M) was added dropwise to the reaction mixture until the pH reached 2.0-2.5. Then, water (320 mL) was added dropwise to precipitate the product. The reaction mixture was allowed to stand for one hour, and then the solid was collected by filtration, washed with water (3 x 210 mL), and then dried under vacuum (30 ° C). The product (7) was obtained as a white solid, 7.41 g, yield = 95%. LCMS m / z = 540.7 [M + H] + .

[0426] Example 5: Synthesis of tetrapeptide intermediate (9)

[0427]

[0428] Compound (7) (6.0 g, 11.1 mmol, 1.0 eq) and DMF (127 mL) were added to a reaction flask under nitrogen. The resulting reaction mixture was cooled to 0°C in an ice bath. HOBt.H2O (1.81 g, 13.4 mmol, 1.2 eq) and EDCI (2.57 g, 13.4 mmol, 1.2 eq) were added to the cooled reaction mixture. After 30 minutes, HD-Lys(Boc)-OMe.HCl (8) (3.97, 13.7 mmol, 1.2 eq) and N-methylmorpholine (NMM) (2.37 g, 23.4 mmol, 2.1 eq) were added. The reaction mixture was stirred at 0°C for one hour and then warmed to room temperature until HPLC analysis indicated the reaction was complete. The reaction solution was added dropwise to water (360 mL) with stirring. After the addition was complete, stirring was stopped and a precipitate was formed. The reaction mixture was allowed to stand for one hour. The precipitate was collected by filtration, washed with water (240 mL x 3), and then dried under vacuum (30°C). The isolated product (9) was obtained as a white solid, 7.9 g, yield = 91%. LCMS: m / z = 783.0 [M+H] + .

[0429] Example 6: Synthesis of tetrapeptide intermediate (10)

[0430]

[0431] Compound (9) (5 g, 6.39 mmol, 1.0 equiv) and methanol (100 mL) were added to the reaction flask under nitrogen. Stirring was started and NaOH solution (25 mL, 1 M, 40 mmol) was added dropwise. The reaction mixture was stirred at room temperature for about 2 hours until HPLC analysis indicated that the reaction was complete. Then, HCl solution (1.0 M) was added dropwise until a pH of 2.0 to 2.5 was achieved. The reaction mixture was added dropwise to water (200 mL) to precipitate the product. The resulting mixture was allowed to stand for one hour. The solid was collected by filtration, washed with water (3 x 133 mL), and then dried under vacuum (30 ° C). The product (10) was obtained as a white solid, 4.51 g, yield = 91.9%. LCMS m / z = 769.0 [M + H] + .

[0432] Example 7: Synthesis of peptide analog (12)

[0433]

[0434] Compound (10) (1.0 g, 1.3 mmol, 1.0 eq) and DMF (21 mL) were added to a reaction flask under nitrogen. The reaction mixture was cooled to 0 ° C in an ice bath. HOBt.H2O (211 mg, 1.56 mmol, 1.2 eq) and EDCI (299 mg, 1.56 mmol, 1.2 eq) were added to the reaction mixture. After stirring the reaction mixture at 0 ° C for 30 minutes, 3-(Boc-amino)pyrrolidine-3-methyl carboxylate (11) (1.56 mmol, 1.2 eq, 381.1 mg) and N-methylmorpholine (NMM) (2.37 g, 23.4 mmol, 2.1 eq) were added. The reaction was stirred at 0 ° C for one hour and warmed to room temperature until HPLC analysis indicated that the reaction was complete. Under stirring, the reaction was added dropwise to water (60 mL). After the addition was complete, stirring was stopped. The reaction mixture was allowed to stand for one hour. The solid was then collected by filtration, washed with water (40 mL x 3), and then dried under vacuum (30°C). The product (12) was obtained as a white solid, 1.2 g, yield = 93.1%. LCMS: m / z = 995.2 [M+H] + .

[0435] Example 8 Synthesis of peptide analog (13)

[0436]

[0437] Compound (12) (200 mg, 0.201 mmol, 1 eq) and dichloromethane (4 mL) were added to the reaction flask under nitrogen. The resulting solution was cooled to -10 °C. A mixture of trifluoroacetic acid (TFA, 4 mL) and dichloromethane (8 mL) was added to the reaction mixture. The reaction was then stirred for one hour at -10 °C until HPLC analysis indicated that the reaction was complete. Volatile organic substances were removed on a rotary evaporator. The residue was dissolved in dichloromethane (4 mL), and the resulting solution was distilled to dryness on a rotary evaporator. This process was repeated three more times. The residue was dissolved in methanol (4 mL), and the resulting solution was evaporated to dryness on a rotary evaporator. This process was repeated three more times. The obtained residue was purified on reverse phase HPLC to give a white solid, 135.4 mg, yield = 65% product (13). LCMS: MS m / z = 694.4 [M + H] + .

[0438] Example 9 Synthesis of peptide analog (14)

[0439]

[0440] Under nitrogen, compound (13) (100 mg, 0.0965 mmol, 1 eq) and methanol (2 mL) were added to the reaction flask. NaOH solution (0.5 mL, 1.0 M, 1.0 mmol, 10 eq) was added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours until HPLC analysis indicated that the reaction was complete. HCl solution (1.0 M) was added dropwise to the reaction mixture until pH = 2.0 to 2.5 was reached. Volatile organic substances were removed on a rotary evaporator. The residue was purified on a reverse phase HPLC with 0.1% TFA acetonitrile / water as the mobile phase. The collected fractions were lyophilized to produce the product (14) as a white solid, 45.7 mg, yield = 60%. LC-MS m / z = 680.5 [M+H] + .

[0441] Example 9 Synthesis of peptide analog (16)

[0442]

[0443] Compound (10) (1.0 g, 1.3 mmol, 1.0 equiv) and DMF (21 mL) were added to a reaction flask under nitrogen. The reaction mixture was cooled to 0 ° C in an ice bath. HOBt.H2O (211 mg, 1.56 mmol, 1.2 equiv) and EDCI (299 mg, 1.56 mmol, 1.2 equiv) were added to the reaction mixture. After stirring at 0 ° C for 30 minutes, 4-(Boc-amino)piperidine-4-carboxylic acid methyl ester (15) (402.9 mg, 1.56 mmol, 1.2 equiv) and N-methylmorpholine (NMM) (0.273 mL, 2.73 mmol, 2.1 equiv) were added. The reaction mixture was stirred at 0 ° C for one hour and warmed to room temperature until HPLC analysis indicated that the reaction was complete. Under stirring, the reaction mixture was added dropwise to water (60 mL). After the addition was complete, stirring was stopped and a precipitate was formed. The reaction mixture was allowed to stand for one hour. The solid was then collected by filtration, washed with water (40 mL x 3), and then dried under vacuum (30°C). The product (16) was obtained as a white solid, 1.17 g, yield = 87.5%. LCMS: m / z = 1009.3 [M+H] + .

[0444] Example 10 Synthesis of peptide analog (17)

[0445]

[0446] Under nitrogen, compound (16) (500 mg, 0.496 mmol, 1 equivalent) and dichloromethane (10 mL) were added to the reaction flask. The reaction mixture was cooled to -10 ° C. A mixture of TFA (10 mL) and dichloromethane (20 mL) was added dropwise to the reaction mixture. The reaction mixture was then stirred at -10 ° C for one hour until HPLC analysis indicated that the reaction was complete. Volatile organic substances were removed on a rotary evaporator. The residue was dissolved in dichloromethane (10 mL) and distilled to dryness on a rotary evaporator. This process was repeated three more times. The residue was dissolved in methanol (10 mL) and evaporated to dryness on a rotary evaporator. This process was repeated three times. The obtained residue was purified on a reverse phase HPLC using 0.1% TFA acetonitrile / water as a mobile phase. The collected fractions were lyophilized to provide a white solid, 364.5 mg, product (17) with a yield of 70%. LCMS: MS m / z = 708.5 [M+H] + .

[0447] Example 11 Synthesis of peptide analog (18)

[0448]

[0449] Under nitrogen, compound (17) (100 mg, 0.0952 mmol, 1 eq) and methanol (2 mL) were added to the reaction flask. NaOH solution (0.5 mL, 1.0 M, 1.0 mmol, 10 eq) was added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours until HPLC analysis indicated that the reaction was complete. Then, HCl solution (1.0 M) was added dropwise until pH = 2.0 to 2.5 was achieved. Methanol was removed on a rotary evaporator to form a residue. The residue was purified on a reverse phase HPLC with 0.1% TFA acetonitrile / water as the mobile phase. The collected fractions were lyophilized to form a white solid, 45.8 mg, yield = 60% product (18). LC-MS m / z = 802.3 [M + H] + .

[0450] Example 12: Opioid Receptor Binding Assay

[0451] Opioid receptor binding affinity was measured using a radioligand binding assay on membranes prepared against HEK293 cells (human embryonic kidney cell line) heterologously expressing recombinant human μ, δ or κ opioid receptors.

[0452] The assay buffer used for opioid receptor binding studies was 50 mM Tris.HCl (pH 7.4) for KOR, 50 mM Tris.HCl (pH 7.4) with 5 mM MgCl2 for MOR, and 50 mM Tris.HCl (pH 7.4) with 10 mM MgCl2 plus 1 mM EDTA for DOR. The wash buffer contained 50 mM Tris.HCl at pH 7.4.

[0453] The opioid receptor binding affinities were compared to three known standards: naltrindole, U-50488 (trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]phenylacetamide, see M. Doi, T. Ishida and M, Inoue; Structure of K-agonist U-50488 Acta Cryst. (1990). C46, ​​676-678) and DAMGO (D-Ala2 MePhe4,Gly(ol)5]myelidine, see Allan D. Blake, George Bot, John C. Freeman and Terry Differential Opioid Agonist Regulation of the Mouse m Opioid Receptor*THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 272, No. 2, January 10, pp. 782-790, 1997).

[0454] Prepare radioligand at the following final concentrations: [ 3 H]DAMGO, 0.5 nM; [ 3 H] diprenorphine, 0.5 nM; and [ 3 H] DADLE, 0.5 nM, the radioligands were used as competitive radioligands for μ, κ, and δ receptors, respectively.

[0455] Cell membranes of HEK293 cells transfected with opioid receptors were prepared at 20 ug MOR, 6.7 ug KOR and 6.7 ug DOR per well, respectively. These membranes containing the receptors of interest were incubated with increasing concentrations of the test compound in the presence of a single concentration of radioligand. A fixed concentration of radioligand was used and serial dilutions of the test compound were prepared.

[0456] The assay was started with 10 uM of the test compound and continued through 4-fold serial dilutions for an 8-point assay. 1 μl of compound / high control / low control was transferred to a 96-well plate according to the plate map, and 100 μl of membrane stock solution was then dispensed into the plate, followed by the addition of 100 μl of radioligand solution. The plated wells were incubated for 1 hour at room temperature with gentle agitation at 300 rpm. The Unifilter-96GF / C filter plates were then soaked with 50 μl of 0.3% polyethyleneimine per well for at least 0.5 hour at room temperature and filtered using a FilterMate TM The reaction mixture was filtered through the plate by a harvester and each plate was then washed four times with cold wash buffer. The filter plates were then dried at 50°C for 1 hour. After drying, the filters were sealed in polyethylene and 50 μl of Perkin Elmer Microscint 20 mixture was added and the radioactivity was counted in a Perkin Elmer MicroBeta2 counter.

[0457] Specific binding was determined by subtracting the CPM values ​​of binding in the presence of 50-100x excess of cold ligand. The data were fit using the saturation analysis nonlinear curve fitting routine in . The inhibition was calculated using the following equation:

[0458] Inhibition % = (1-(measurement well-average value_LC) / (average value_HC-average value_LC))*100%

[0459] Binding data were analyzed using GraphPad Prism 5.0, and IC was generated by nonlinear regression from dose-response curves. 50 Data. The data were fit using the model "log(inhibitor) vs. response - variable slope". This data is shown in Table 1.

[0460] Table 1 Binding affinity of peptide ligands to recombinant human opioid receptors

[0461]

[0462] Example 13 FLIPR calcium assay in whole cells

[0463] The FLIPR Calcium Assay is used to measure the ability of opioid ligands to induce a functional response upon receptor binding. The opioid μ receptors (MOR), delta receptors (DOR), and kappa receptors (KOR) are G protein-coupled receptors (GPCRs) that play an important role in cellular signaling. The receptors are activated by ligands, which then trigger activation of G proteins within the cell. The activated G proteins induce various cascades of intracellular messengers including calcium flux. Functional cell-based assays are evaluated to detect changes in intracellular calcium levels, which are detected using a fluorescent calcium-sensitive reporter dye. The basic system for performing the calcium mobilization assay includes the FLIPR Calcium Assay Kit and the FLIPR A system was used to observe changes in intracellular calcium levels and determine dose responses in HEK293 cells transfected with recombinant human μ, δ, or κ opioid receptors.

[0464] The cells used in the assay are grown in the culture medium of 88% DMEM containing 10% FBS, 300ug / mL G418, 2ug / mL blasticidin, 1% GlutaMax and 1% penicillin / streptomycin (Hyclone-SV30010). 20000 cells in 20uL culture medium are inoculated into each well of assay plate (Greiner-781946), and cells are kept in an incubator with 5% CO at 37°C for 20 hours. Then, the compound is prepared with 5 times of serial dilutions, to obtain 10 doses, and 500nL of each concentration is transferred to the compound plate. Then 30uL assay buffer (20mM HEPES and 1XHBSS) is added to each well of the compound plate, and the plate is rotated 15 seconds at 1500rpm. Then 20uL of 2X Fluo-4DirectTM No-Wash Loading Buffer (Invitrogen - F10471) was gently dispensed into each well of the assay plate and spun at 1000rpm for 15 seconds followed by incubation at 37°C for 50 minutes. The assay plate was removed from the incubator and placed at room temperature for 10 minutes. The assay plate, compound plate, and tip box were placed directly in the FLIPR System. 10uL of each compound was transferred from the compound plate to the assay plate in the FLIPR Tetra fluorescent imaging plate reader. The plate was read 140 times; then the "Max-Min" starting from reading 1 to reading 140 was calculated to generate the final signal for the effect % calculation. Data were analyzed using Prism, the curve fitting equation "log (agonist) to response--variable slope". Table 2 shows the results of these assays.

[0465] Table 2. Opioid kappa receptor agonist FLIPR assay

[0466]

[0467] Example 14: Evaluation of the efficacy of Compound 14 in the formalin model in rats

[0468] 60 male Sprague-Dawley (SD) rats were randomly divided into six groups. These groups included a natural group, a vehicle group, a positive group (morphine 3 mg / kg) and three test vehicle groups (Compound 14: 0.1 mg / kg; 0.3 mg / kg; and 1.0 mg / kg). 15 minutes after the administration of the compound, 50 mL of 1% formalin was injected on the dorsum of the right hind foot of the rat. The rats in the natural group were not injected with 1% formalin. The movement counts of the rats were automatically recorded by an automatic pain analyzer for 60 minutes. The reactions of the animals, such as retracting the feet, licking the paws, and shaking the feet quickly, were observed. Therefore, the instrument counts and generates movement data. The results are described below and Figure 2 Presented in.

[0469] 15 minutes before injection of formalin, morphine (3mg / kg) was subcutaneously administered to the positive control group animals, and the movement counts were compared with the movement counts of the vehicle group animals. The total movement counts (p<0.001) within 60 minutes of the morphine (3mg / kg) positive control group were reduced, showing an inhibition rate of 82.9%. This result shows that the administration of 3mg / kg dose of morphine significantly inhibited the nociceptive response induced by formalin.

[0470] 15 minutes before injection of formalin, compound 14 (0.1 mg / kg) was subcutaneously administered to animals in the compound 14 group (0.1 mg / kg group), and the movement counts were compared with the movement counts of the vehicle group animals. The total movement counts in the compound 14 group within 60 minutes (p<0.001, ***) were reduced, showing an inhibition rate of 31.9%. This result shows that the administration of compound 14 at a dose of 0.1 mg / kg significantly inhibited the nociceptive response induced by formalin.

[0471] 15 minutes before injection of formalin, compound 14 (0.3 mg / kg) was subcutaneously administered to animals in the compound 14 group (0.3 mg / kg group), and the movement counts were compared with the movement counts of the vehicle group animals. The total movement counts in the compound 14 group within 60 minutes (p<0.001, ***) were reduced, showing an inhibition rate of 65.4%. This result shows that the administration of compound 14 at a dose of 0.3 mg / kg significantly inhibited the nociceptive response induced by formalin.

[0472] 15 minutes before injection of formalin, compound 14 (1.0 mg / kg) was subcutaneously administered to animals in the compound 14 group (1.0 mg / kg group), and the movement counts were compared with the movement counts of the vehicle group animals. The total movement counts within 60 minutes of the compound 14 group (p<0.001, ***) were reduced, showing an inhibition rate of 79.7%. This result shows that the administration of compound 14 at a dose of 1.0 mg / kg significantly inhibited the nociceptive response induced by formalin.

[0473] In this study, morphine (3 mg / kg) was administered subcutaneously to rats 15 minutes before formalin administration. This morphine administration inhibited the nociceptive response induced by formalin. Under the same conditions, 0.1 mg / kg, 0.3 mg / kg and 1 mg / kg of compound 14 inhibited the nociceptive response induced by formalin, and compound 14 showed dose-dependent analgesic activity in the dose range of 0.1-1 mg / kg.

[0474] Example 15: Evaluation of the efficacy of Compound 14 in the chloroquine-induced scratching model in mice

[0475] Sixty male ICR mice were randomly divided into 6 groups. These groups include a vehicle group, a positive control group (ICI204448 was administered at 3.0 mg / kg) and four dose groups (compound 14 was administered at the following doses: 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg and 3 mg / kg). After intravenous injection of the tested compound for 15 minutes, 50uL chloroquine (400 μg) was intradermally injected into the back of the neck of the mouse (except the positive control group). In the positive control group, 50uL chloroquine (400 μg) was intradermally injected into the back of the neck of the mouse after subcutaneous injection of ICI204448 for 30 minutes. After intradermal injection of chloroquine, the mouse was placed in a transparent Plexiglas box alone, and the number of scratches within 30 minutes was counted immediately. A scratch count refers to 3 or more separate rapid scratching movements of the hind paw around the injection site. Inhibition rate (MPE% = [(number of scratches in the vehicle group - number of scratches in each group) / number of scratches in the vehicle group] * 100%). The data of this study are shown in Figure 3 middle.

[0476] Compared with the vehicle group, the control positive compound ICI204448 administered at 3 mg / kg showed an inhibition rate of 53.05% (***p<0.001) on the number of scratches. For the compound 14 groups at doses of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg and 3 mg / kg, the inhibition rates of the number of scratches in mice were 28.26% (*p<0.05), 52.21% (***p<0.001), 94.96% (***p<0.001), and 99.96% (***p<0.001), respectively.

[0477] Compared with the vehicle group, the positive control group administered with compound ICI204448 showed inhibition of the number of scratches in the chloroquine-induced scratching model of ICR mice after 30 minutes of administration at a dose of 3 mg / kg. The compound 14 group showed inhibition of the number of scratches in the chloroquine-induced scratching model of ICR mice after 15 minutes of administration at doses of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Figure 3 As shown, in the chloroquine-induced ICR scratching model in mice, compound 14 had a dose-dependent antipruritic activity within the dose range of 0.1 to 3 mg / kg (intravenous injection).

Claims

1. A method for treating a κ-opioid receptor agonist-associated disease or condition, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a compound of formula (I).

2. The method of claim 1, wherein the pharmaceutical composition is administered subcutaneously, intradermally, intravenously, intramuscularly, orally or intraperitoneally.

3. The method of claim 1 or 2, wherein the pharmaceutical composition comprising the compound of formula (I) is administered at a dose rate of about 0.0001 mg / kg to about 100.0 mg / kg.

4. The method of claim 3, wherein the pharmaceutical composition comprising the compound of formula (I) is administered at a dosage rate of about 0.0001 mg / kg to about 10.0 mg / kg.

5. The method according to any one of claims 1 to 4, wherein the pharmaceutical composition comprising the compound of formula (I) is administered at least once a day, such as at least twice a day, at least three times a day or multiple times a day.

6. The method according to claim 5, wherein the pharmaceutical composition comprising the compound of formula (I) is administered at least once a day.

7. The method according to any one of claims 1 to 6, wherein the kappa opioid receptor agonist-associated disease or condition is selected from the group consisting of pain, cardiovascular disease, pruritus, nausea, inflammatory disease, spinal anesthesia, cough suppression, stroke, hypoxic pulmonary hypertension, multiple sclerosis, addiction, and post-traumatic cartilage degeneration.

8. The method according to any one of claims 1 to 7, wherein the subject is selected from the group consisting of humans, livestock, companion animals, laboratory animals, and zoological animals.

9. The method according to any one of claims 1 to 8, wherein the subject is a human.

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