Peptides for the treatment of 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 kappa-opioid receptor agonist-related medical conditions.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to 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. BACKGROUND

[0002] Opioid kappa receptors (KORs) are expressed in many parts of the body, such as the brain, spinal cord, and central and peripheral terminals. KORs play an important role in signal transduction to maintain many physiological functions of the body. Like opioid mu receptors (MORs) and delta receptors (DORs), activation of KORs by agonist ligands leads to inhibition of adenylyl 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).

[0003] Many physiological processes are associated with 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 novel analgesics (Curr Pharm Des 2004; 10: 743-57), anti-inflammatory agents, immune system modulators, etc. Agonists provide a great potential for KOR-selective ligands for the treatment of various medical conditions, such as pain, depression, autoimmune disorders, 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).

[0004] A number of KOR selective agonists have been synthesized and evaluated as potential analgesics that avoid side effects associated with traditional opioid analgesics, such as respiratory depression, dependence, addiction, and constipation; some of which have been tested in clinical trials but 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 Rev. 2003, Summer, 9(2): 187-98), enadoline for potential analgesics (Walsh SL., Strain EC, Abreu M.E. Bigelow G.E. Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans. Psychopharmacology 2001, 157, 151-162), and ADL-10-0101, among others.

[0005] TRK-820 (Nalfurafine) was originally developed as a potential analgesic, but was successful as an antipruritic agent and is approved in Japan under the trade name Remitch.

[0006] Ferring BV (US005965701A) reported highly opioid K-receptor selective and potent D-amino acid tetrapeptide agonists and were further developed by Cara therapeutics. Currently, Cara therapeutics is developing a lead tetrapeptide compound CR-845 as an analgesic and antipruritic agent in clinical trials (Hesselink, J. M. K. CR845 (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 are also actively committed to discovering peptide-based KOR selective agonist ligands by modifying the molecular structure of CR-845, hoping to find new analgesics and potential antipruritic agents without the conventional side effects of morphine analgesics (CN107098871, WO2017211272A1, WO2018103624A1, WO2017210668A1, WO2018059331A1).

[0007] In addition, KOR agonists have also been developed for other indications; for example, fedotozine and asimadoline were both tested as potential treatments for irritable bowel syndrome and indigestion.

[0008] What is needed are new kappa-opioid agonists to treat a variety of medical conditions. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 is a chemical reaction scheme that can be used to prepare compounds comprising Formula (I) according to embodiments of the present disclosure. SUMMARY

[0010] In one aspect, disclosed herein are compounds comprising Formula (I), or a pharmaceutically acceptable salt thereof:

[0011]

[0012] wherein:

[0013] R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0014] R4 and R7 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0015] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle or substituted heterocycle;

[0016] R8 and R9 are independently selected from the group consisting of: H, unsubstituted C1-C8 alkyl, substituted C1-C8 alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, or (OCH2CH2O). n ;

[0017] R 10 Choose from the following groups:

[0018]

[0019] R 11 Choose freely OR 12 or NR 13 R 14 The group formed;

[0020] R 12 Choose from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl or (OCH2CH2O) n ;

[0021] R 13 and R 14 Independently select from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C24 Alkyl or (OCH2CH2O) n ;and

[0022] n is an integer from 1 to 100.

[0023] On the other hand, this article discloses methods for preparing compounds of formula (I) or their pharmaceutically acceptable salts:

[0024]

[0025] The method includes:

[0026] a) In the presence of an acyl coupling agent, to make a compound including formula (VI):

[0027]

[0028] With compounds including formula (VII):

[0029]

[0030] Contact, to form a compound including formula (VIII):

[0031]

[0032] b) Contacting a compound including formula (VIII) with a proton acceptor to form a compound including formula (IX):

[0033]

[0034] c) In the presence of an acyl coupling agent, a compound comprising formula (IX) is reacted with a compound comprising formula (X):

[0035]

[0036] Contact, to form a compound including formula (XI):

[0037]

[0038] d) Contacting a compound comprising formula (XI) with a proton acceptor to form a compound comprising formula (XII):

[0039]

[0040] e) In the presence of an acyl coupling agent, a compound comprising formula (XII) is reacted with a compound comprising formula (XIII):

[0041]

[0042] Contact, to form compounds including formula (XIV):

[0043]

[0044] f) Contacting a compound including formula (XIV) with a proton acceptor to form a compound including formula (XV):

[0045]

[0046] g) In the presence of an acyl coupling agent, contact a compound comprising formula (XV) with a compound comprising formula (XVI) to form a compound comprising formula (XVII):

[0047] as well as

[0048] h) Contacting a compound including formula (XVII) with a deprotecting agent including an acid to form a compound including formula (I);

[0049] in:

[0050] R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0051] R4 and R7 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0052] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle or substituted heterocycle;

[0053] R8 and R9 are independently selected from the group consisting of: H, unsubstituted C1-C8 alkyl, substituted C1-C8 alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, or (OCH2CH2O). n ;

[0054] R 10 Choose from the following groups:

[0055]

[0056] R 11 Choose freely OR 12 or NR 13 R 14 The group formed;

[0057] R 12 Choose from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl or (OCH2CH2O) n ;

[0058] R 13 and R 14 Independently select from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl or (OCH2CH2O) n ;and

[0059] n is an integer from 1 to 100.

[0060] On the other hand, this document discloses pharmaceutical compositions comprising compounds of formula (I).

[0061] On the other hand, this document discloses a method for treating opioid receptor agonist-related medical conditions, the method comprising administering, to a subject in need, the pharmaceutical composition comprising a compound of formula (I).

[0062] Other features and iterations of the invention are described in more detail below. Detailed Implementation

[0063] This disclosure provides compounds comprising formula (I) or pharmaceutically acceptable salts thereof, methods for preparing compounds comprising formula (II) or pharmaceutically acceptable salts thereof, compounds comprising compounds comprising formula (I), and methods for treating opioid receptor agonist-related medical conditions.

[0064] (I) includes compounds of formula (I) or acceptable pharmaceutically acceptable salts thereof.

[0065] On the one hand, this disclosure covers compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0066]

[0067] in:

[0068] R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0069] R4 and R7 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0070] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl, C3-C 10 Substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle or substituted heterocycle;

[0071] R8 and R9 are independently selected from the group consisting of: H, unsubstituted C1-C8 alkyl, substituted C1-C8 alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, or (OCH2CH2O). n ;

[0072] R 10 Choose from the following groups:

[0073]

[0074] R 11 Choose freely OR 12 or NR 13 R 14 The group formed;

[0075] R 12 Choose from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl or (OCH2CH2O) n ;

[0076] R 13 and R 14 Independently select from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl or (OCH2CH2O) n ;and

[0077] n is an integer from 1 to 100.

[0078] Typically, according to embodiments, R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups. In some embodiments, R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C4 unsubstituted alkyl groups, C1-C4 substituted alkyl groups, C3-C8 unsubstituted cycloalkyl groups, or C3-C8 substituted cycloalkyl groups. In some embodiments, R1, R2, and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R1, R2, and R3 are H.

[0079] Typically, according to embodiments, R4 and R7 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl, or C3-C8 substituted cycloalkyl. In some embodiments, R4 and R7 are independently selected from the group consisting of: H, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R4 is methyl, and R7 is isopropyl.

[0080] 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, or 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, or phenyl. In a specific embodiment, R5 is H, and R6 is phenyl.

[0081] Typically, according to the 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, or (OCH2CH2O). n In some embodiments, R8 and R9 are independently selected from the group consisting of H, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R8 and R9 are hydrogen.

[0082] Typically, according to the embodiments, R 10 Choose from the following groups:

[0083]

[0084] Typically, according to the embodiments, R 11 Choose freely OR 12 or NR 13 R 14 A group consisting of [various components]. In some embodiments, R 11 Choose freely OR 12 or NR 13 R 14 A group consisting of [groups]. In a specific embodiment, R 11 OR 12 .

[0085] Typically, according to the embodiments, R 12 Choose from the following groups: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl groups, O-substituted C1-C groups 12 Alkyl, O-unsubstituted C1-C 12 Alkyl or (OCH2CH2O) n In some embodiments, R 12 The group consisting of H, methyl, ethyl, n-propyl, or isopropyl is selected. In a specific embodiment, R... 12 It can be H or Me.

[0086] Typically, according to the embodiments, R 13 and R 14 Independently select from the following groups: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl groups, O-substituted C1-C groups 12 Alkyl, O-unsubstituted C1-C 12 Alkyl or (OCH2CH2O) n In some embodiments, R 13 and R 14 Independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R 13 and R 14 It does not exist.

[0087] Typically, according to embodiments, n is an integer from 1 to 50. In some embodiments, n is an integer from 1 to 50. In some embodiments, n does not exist.

[0088] In one exemplary embodiment, R1, R2, R3, R5, R8, and R9 are H; R4 is methyl; R6 is phenyl; and R7 is isopropyl.

[0089] R 10 for

[0090]

[0091] R 11 OR 12 And R 12 For H; and R 13 R 14 And n does not exist, as shown in compounds including formula (II):

[0092]

[0093] In another exemplary embodiment, R1, R2, R3, R5, R8, and R9 are H; R4 is methyl; R6 is phenyl; and R7 is isopropyl.

[0094] R 10 for

[0095]

[0096] R 11 OR 12 ;R 12 For Me; and R 13 R 14 And n does not exist, as shown by compounds including formula (III):

[0097]

[0098] In yet another exemplary embodiment, R1, R2, R3, R5, R8, and R9 are H; R4 is methyl; R6 is phenyl; and R7 is isopropyl.

[0099] R 10 for

[0100]

[0101] R 11 OR 12 ;R 12 For H; and R 13 R 14 And n does not exist, as shown by compounds including formula (IV):

[0102]

[0103]

[0104] In yet another exemplary embodiment, R1, R2, R3, R5, R8, and R9 are H; R4 is methyl; R6 is phenyl; and R7 is isopropyl.

[0105] R 10 for

[0106]

[0107] R 11 OR 12 ;R 12 For Me; and R 13 R 14 And n does not exist, as shown in compounds including formula (V):

[0108]

[0109] Compounds comprising formula (I) can be free bases or salts. 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, hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, acetates, formates, tartaric acid, hydrogen tartrate, stearates, phthalates, hydroiodides, lactates, monohydrates, mucilages, nitrates, phosphates, salicylates, phenylpropionates, isobutyrates, hypophosphites, maleic anhydride, malates, citrates, isocitrates, succinates, lactates, gluconates, glucurons, pyruvates, oxalates, fumarates, propionates, aspartates, glutamates, benzoates, terephthalates, etc. In other embodiments, pharmaceutically acceptable salts comprise basic or alkaline earth metal ion salts. Specifically, sodium, potassium, or other pharmaceutically acceptable inorganic salts are used. Salts can be amorphous or in various polymeric forms, including hydrates or solvates with alcohols or other solvents.

[0110] (II) A method for preparing a compound comprising formula (I) or a pharmaceutically acceptable salt thereof.

[0111] On the other hand, this disclosure covers methods for preparing compounds comprising formula (I) or pharmaceutically acceptable salts thereof:

[0112]

[0113] The method includes, according to Figure 1 The reaction scheme described in the text:

[0114] a) In the presence of an acyl coupling agent, to make a compound including formula (VI):

[0115]

[0116] With compounds including formula (VII):

[0117]

[0118] Contact, to form a compound including formula (VIII):

[0119]

[0120] b) Contacting a compound including formula (VIII) with a proton acceptor to form a compound including formula (IX):

[0121]

[0122] c) In the presence of an acyl coupling agent, a compound comprising formula (IX) is reacted with a compound comprising formula (X):

[0123]

[0124] Contact, to form a compound including formula (XI):

[0125]

[0126] d) Contacting a compound comprising formula (XI) with a proton acceptor to form a compound comprising formula (XII):

[0127]

[0128] e) In the presence of an acyl coupling agent, a compound comprising formula (XII) is reacted with a compound comprising formula (XIII):

[0129]

[0130] Contact, to form compounds including formula (XIV):

[0131]

[0132] f) Contacting a compound including formula (XIV) with a proton acceptor to form a compound including formula (XV):

[0133]

[0134] g) In the presence of an acyl coupling agent, contact a compound comprising formula (XV) with a compound comprising formula (XVI) to form a compound comprising formula (XVII):

[0135] as well as

[0136] h) Contact a compound including formula (XVII) with a deprotecting agent to form a compound including formula (I);

[0137] in:

[0138] R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0139] R4 and R7 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups;

[0140] R5 and R6 are independently selected from the group consisting of: H, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle or substituted heterocycle;

[0141] R8 and R9 are independently selected from the group consisting of: H, unsubstituted C1-C8 alkyl, substituted C1-C8 alkyl, O-substituted C1-C8 alkyl, O-unsubstituted C1-C8 alkyl, (OCH2CH2O). n ;

[0142] R 10 Choose from the following groups:

[0143]

[0144] R 11 Choose freely OR 12 or NR 13 R 14 The group formed;

[0145] R 12 Choose from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl group, (OCH2CH2O) n ;

[0146] R 13 and R 14Independently select from the following groups: H, C1-C 24 Unsubstituted alkyl, C1-C 24 Substituted alkyl groups, O-substituted C1-C groups 24 Alkyl, O-unsubstituted C1-C 24 Alkyl group, (OCH2CH2O) n ;

[0147] n is an integer from 1 to 100;

[0148] P1 is a nitrogen protecting group, and P2 is a carboxylic acid protecting group.

[0149] Generally, according to Figure 1 In the reaction scheme described, R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C8 unsubstituted alkyl groups, C1-C8 substituted alkyl groups, C3-C 10 Unsubstituted cycloalkyl or C3-C 10 Substituted cycloalkyl groups. In some embodiments, R1, R2, and R3 are independently selected from the group consisting of: H, CN, Cl, F, C1-C4 unsubstituted alkyl groups, C1-C4 substituted alkyl groups, C3-C8 unsubstituted cycloalkyl groups, or C3-C8 substituted cycloalkyl groups. In some embodiments, R1, R2, and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R1, R2, and R3 are H.

[0150] Generally, according to Figure 1 In the reaction scheme described herein, R4 and R7 are independently selected from the group consisting of: H, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, unsubstituted C3-C8 cycloalkyl, or substituted C3-C8 cycloalkyl. In some embodiments, R4 and R7 are independently selected from the group consisting of: H, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R4 is methyl; and R7 is isopropyl.

[0151] Generally, according to Figure 1 In the reaction scheme described herein, R5 and R6 are independently selected from the group consisting of: H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C3-C8 unsubstituted cycloalkyl or C3-C8 substituted cycloalkyl, unsubstituted aryl, substituted aryl, unsubstituted heterocycle or 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 or phenyl. In a specific embodiment, R5 is H; and R6 is phenyl.

[0152] Generally, according to Figure 1In the reaction scheme described, R8 and R9 are independently selected from the group consisting of: H, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, O-substituted C1-C4 alkyl, O-unsubstituted C1-C4 alkyl, or (OCH2CH2O). n In some embodiments, R8 and R9 are independently selected from the group consisting of H, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R8 and R9 are hydrogen.

[0153] Generally, according to Figure 1 The reaction scheme described in R 10 Choose from the following groups:

[0154]

[0155] Typically, according to the embodiments, R 11 Choose freely OR 12 or NR 13 R 14 A group consisting of [various components]. In some embodiments, R 11 Choose freely OR 12 or NR 13 R 14 A group consisting of [groups]. In a specific embodiment, R 11 OR 12 .

[0156] Generally, according to Figure 1 The reaction scheme described in R 12 Choose from the following groups: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl groups, O-substituted C1-C groups 12 Alkyl, O-unsubstituted C1-C 12 Alkyl or (OCH2CH2O) n In some embodiments, R 12 The group consisting of: H, methyl, ethyl, n-propyl, isopropyl. In a specific embodiment, R... 12 It can be H or Me.

[0157] Generally, according to Figure 1 The reaction scheme described in R 13 and R 14 Independently select from the following groups: H, C1-C 12 Unsubstituted alkyl, C1-C 12 Substituted alkyl groups, O-substituted C1-C groups 12 Alkyl, O-unsubstituted C1-C 12 Alkyl group, (OCH2CH2O) n In some embodiments, R13 and R 14 Independently selected from the group consisting of: H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R 13 and R 14 It does not exist.

[0158] Generally, according to Figure 1 The reaction scheme described herein, where n is an integer from 1 to 50. In some embodiments, n is an integer from 1 to 50. In some embodiments, n does not exist.

[0159] Generally, according to Figure 1 In the embodiments of the reaction scheme described herein, P1 is a suitable nitrogen protecting group. The nitrogen protecting group includes a carbamate. Non-limiting examples of such protecting groups may be tert-butoxycarbonyl carbamate (BOC), 9-fluorenylmethyl carbamate (FMOC), benzyl carbamate (CBZ), etc. Suitable nitrogen protecting groups, methods for attaching such protecting groups, and methods for removing such protecting groups are described, for example, in TW Greene's "Protective Groups in Organic Synthesis," John Wiley & Sons, 2006. In a specific embodiment, P1 is BOC.

[0160] Generally, according to Figure 1 In the embodiments of the reaction scheme described herein, P2 is a suitable carboxylic acid protecting group. The carboxylic acid (carboxyl) protecting group includes esters, amides, or hydrazides. Non-limiting examples of carboxylic acid protecting groups may be methyl esters, ethyl esters, benzyl esters, N,N-dimethylamides, N-phenylhydrazides, etc. Suitable carboxylic acid protecting groups, methods for attaching these protecting groups, and methods for removing these protecting groups are described, for example, in TW Greene's "Protective Groups in Organic Synthesis," John Willie Parent & Son, 2006. In a specific embodiment, P2 is a methyl group.

[0161] In a preferred embodiment, R1, R2, R3, R5, R8, and R9 are H; R4 is methyl; R6 is phenyl; and R7 is isopropyl.

[0162] R 10 for

[0163]

[0164] R 11 OR 12 And R 12 For H; and R 13 R14 And n does not exist, as shown in compounds including formula (II):

[0165]

[0166] In another preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl.

[0167] R 10 for

[0168]

[0169] R 11 OR 12 ;R 12 For Me; and R 13 R 14 And n does not exist, as shown by compounds including formula (III):

[0170]

[0171] In another preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl.

[0172] R 10 for

[0173]

[0174] R 11 OR 12 ;R 12 For H; and R 13 R 14 And n does not exist, as shown by compounds including formula (IV):

[0175]

[0176]

[0177] In yet another preferred embodiment, R1, R2, R3, R5, R8 and R9 are H; R4 is methyl; R6 is phenyl; R7 is isopropyl;

[0178] R 10 for

[0179]

[0180] R 11 OR 12 ;R 12 For Me; and R 13R 14 And n does not exist, as shown in compounds including formula (V):

[0181]

[0182] Step (a)

[0183] 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 agent to form a reaction mixture. Following treatment and separation, the compound comprising formula (VIII) is separated. This method step is referred to as “peptide coupling” or “acyl coupling”.

[0184] The above details how Figure 1 The compounds of formula (VI) are depicted. 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, or C3-C8 substituted cycloalkyl. In some embodiments, R1, R2, and R3 are independently selected from the group consisting of: H, Cl, F, methyl, ethyl, propyl, or isopropyl. In a specific embodiment, R1, R2, and R3 are H.

[0185] 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, or C3-C8 substituted cycloalkyl. In some embodiments, R4 is selected from the group consisting of: H, methyl, ethyl, n-propyl, or isopropyl. In a specific embodiment, R4 is methyl.

[0186] In some embodiments, P1 is a nitrogen-protecting group. In a specific embodiment, P1 is a BOC group. In a preferred embodiment, the compound comprising formula (VI) is (2R,3R)-BOC-β-methyl-phenylalanine.

[0187] The above details how Figure 1 The compounds depicted include those of formula (VII). In 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, or 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, or phenyl. In a specific embodiment, R5 is H, and R6 is phenyl.

[0188] 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.

[0189] Typically, the equivalence ratio of compounds comprising (VI) to those comprising (VII) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of compounds comprising (VI) to those comprising (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 equivalence ratio of compounds comprising (VI) to those comprising (VII) can be about 1:0:1.1.

[0190] Step (a) of the method utilizes an acyl coupling reagent. The acyl coupling reagent converts the carboxylic acid moiety of a 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'-carbonylditriazole, 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. Other non-limiting examples of activators may include 1-hydroxybenzotriazole, N-hydroxysuccinimide, or N-hydroxyphthalamide. In a preferred embodiment, the acyl coupling agent may be 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof.

[0191] Typically, the equivalence ratio of formula (VI) to the acyl coupling agent ranges from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of formula (VI) to the acyl coupling agent ranges from about 1.0:1.0 to about 1.0:1.5, from about 1.0:1.0 to about 1.0:1.3, or from about 1.0:1.2. In a preferred embodiment, the equivalence ratio of formula (VI) to the acyl coupling agent is about 1:0:1.1.

[0192] Step (a) further includes a proton acceptor. The proton acceptor will vary depending on the starting substrate, the acyl coupling agent, 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-dimethylpyridine. Non-limiting examples of chiral secondary amines include (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).

[0193] Typically, the equivalence ratio of formula (VI) to proton acceptor can range from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalence ratio of formula (VI) to 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 equivalence ratio of formula (VI) to proton acceptor can be about 1:0:2.1.

[0194] Step (a) as detailed herein includes a solvent. As is 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 nonpolar 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 foregoing. 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-imidazolium ketone (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 nonpolar solvents include, but are not limited to, alkanes and substituted alkane solvents (including cycloalkanes), aromatic hydrocarbons, esters, ethers, and combinations thereof. Specific nonpolar solvents that may be used include, for example, benzene, butyl acetate, tert-butyl methyl ether, chlorobenzene, chloroform, chloromethane, cyclohexane, dichloromethane, dichloroethane, diethyl ether, ethyl acetate, diethylene glycol, fluorobenzene, heptane, hexane, isopropyl acetate, methyltetrahydrofuran, pentyl acetate, n-propyl acetate, tetrahydrofuran, toluene, and combinations thereof. In a preferred embodiment, the solvent may be dimethylformamide.

[0195] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (VI) ranges 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 (VI) can range from about 0.5:1 to about 500:1, about 2:1 to about 250:1, about 5:1 to about 200:1, or about 10:1 to about 50:1. In an exemplary embodiment, the volume-to-weight ratio of the solvent to the compound comprising formula (VI) can range from about 12:1 to about 20:1.

[0196] Typically, the reaction in step (a) is carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent used. In various embodiments, the reaction temperature 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 nitrogen, argon, or helium.

[0197] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 5 minutes to about 2 hours. In some 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 one exemplary embodiment, the reaction may 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 compound (VI). Typically, the amount of compound (VI) remaining in the reaction mixture at the end of the reaction may be less than about 10%, less than about 5%, or less than about 2%.

[0198] 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%.

[0199] Step (b)

[0200] Step (b) of the eight-step method involves contacting a compound comprising formula (VIII) with a proton acceptor to form a reaction mixture. Following treatment and separation, a compound comprising formula (IX) is obtained. This step is referred to as the "deprotection" reaction.

[0201] The compounds including formula (VIII) are described in more detail above.

[0202] Suitable proton acceptors have been described in detail in section (II)(a) above. In a preferred embodiment, the proton acceptor is NaOH.

[0203] Typically, the equivalence ratio of formula (VIII) to proton acceptor ranges from about 1.0:1.0 to about 1.0:5.0. In various embodiments, the equivalence ratio of formula (VIII) to proton acceptor ranges 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 equivalence ratio of formula (VIII) to proton acceptor is about 1:0:2.0.

[0204] Step (b) further includes a solvent. Suitable solvents have been described in detail above in section (II)(a). In a preferred embodiment, the solvent is a combination of methanol and water.

[0205] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (VI) 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 (VI) may range from about 0.5:1 to about 500:1, about 5:1 to about 200:1, about 10:1 to about 100:1, or about 15:1 to about 50:1. In a preferred embodiment, the volume-to-weight ratio of the solvent to the compound comprising formula (VIII) may be about 20:1.

[0206] Typically, the reaction in step (b) is carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent used. In various embodiments, the reaction temperature 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, such as nitrogen, argon, or helium.

[0207] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 30 minutes to about 4 hours. In some 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 compound (VIII). Typically, the amount of compound (VIII) 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%.

[0208] After step (b) is completed, the pH of the reaction mixture is adjusted to a pH 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 is adjusted to a range from about pH 2.0 to about pH 2.5.

[0209] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids include HCl, H₂SO₄, acetic acid, methanesulfonic acid, or similar organic or inorganic acids. In a preferred embodiment, HCl may be used.

[0210] The yield of compounds comprising formula (IX) can be at least about 60%. In various embodiments, the yield of compounds 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 compounds comprising formula (IX) can be about 95%.

[0211] Step (c)

[0212] Step (c) of the eight-step method involves contacting a compound comprising formula (IX) with a compound comprising formula (X) in the presence of an acyl coupling agent to form a reaction mixture. Following treatment and separation, a compound comprising formula (XI) is obtained.

[0213] The compounds including formula (IX) have been described in detail above.

[0214] Compounds comprising formula (X) have been described in detail above. In some embodiments, R7 is selected from the group consisting of: H, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, unsubstituted C3-C8 cycloalkyl, or substituted C3-C8 cycloalkyl. In some embodiments, R7 is selected from the group consisting of: H, methyl, ethyl, n-propyl, or isopropyl. In a preferred embodiment, R7 is isopropyl.

[0215] 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 (X) is D-leucine methyl ester hydrochloride.

[0216] Typically, the equivalence ratio of the compound comprising (IX) to the compound comprising (X) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of the compound comprising (IX) to the compound comprising (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 equivalence ratio of the compound comprising (IX) to the compound comprising (X) can be about 1:0:1.1.

[0217] The method in step (c) utilizes an acyl coupling agent and a proton acceptor. Suitable acyl coupling agents and proton acceptors are described in detail above in section (II)(a). In a preferred embodiment, the acyl coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof, and the proton acceptor is 4-methylmorpholine.

[0218] Typically, the equivalence ratio of formula (IX) to the acyl coupling agent ranges from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of formula (IX) to the acyl coupling agent ranges 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 equivalence ratio of formula (IX) to the acyl coupling agent is about 1:0:1.1.

[0219] Typically, the equivalence ratio of formula (IX) to proton acceptor ranges from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalence ratio of formula (IX) to proton acceptor ranges 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 equivalence ratio of formula (IX) to proton acceptor is about 1:0:2.1.

[0220] Step (c) involves a solvent. Suitable solvents have been described in detail above in section (II)(a). In a preferred embodiment, dimethylformamide may be used as a solvent in step (c).

[0221] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (IX) ranges 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 (IX) can range from about 0.5:1 to about 500:1, about 2:1 to about 250:1, about 5:1 to about 200:1, or about 10:1 to about 50:1. In an exemplary embodiment, the volume-to-weight ratio of the solvent to the compound comprising formula (IX) can range from about 12:1 to about 20:1.

[0222] Typically, the reaction in step (c) is carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent used. In various embodiments, the reaction temperature 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 nitrogen, argon, or helium.

[0223] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 5 minutes to about 2 hours. In some 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 compound (IX). Typically, the amount of compound (IX) 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%.

[0224] The yield of compounds comprising formula (XI) can be at least about 60%. In various embodiments, the yield of compounds 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 compounds comprising formula (XI) can be about 96%.

[0225] Step (d)

[0226] Step (d) in the eight-step method involves contacting a compound comprising formula (XI) with a proton acceptor to form a reaction mixture. Following treatment and separation, a compound comprising formula (XII) is obtained. This step is referred to as the "deprotection" reaction.

[0227] The compounds including formula (XI) are described in more detail above.

[0228] Suitable proton acceptors have been described in detail in section (II)(b). In a preferred embodiment, the proton acceptor is NaOH or LiOH.

[0229] Typically, the equivalence ratio of formula (XI) to proton acceptor can range from about 1.0:1.0 to about 1.0:5.0. In various embodiments, the equivalence ratio of formula (XI) to 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 equivalence ratio of formula (XI) to proton acceptor can be about 1:0:2.0.

[0230] Step (d) further includes a solvent. Suitable solvents have been described in detail above in section (II)(b). In a preferred embodiment, the solvent is a combination of methanol and water.

[0231] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (XI) ranges 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 (XI) can range from about 0.5:1 to about 500:1, about 5:1 to about 200:1, about 10:1 to about 100:1, or about 15:1 to about 50:1. In a preferred embodiment, the volume-to-weight ratio of the solvent to the compound comprising formula (XI) can be about 20:1.

[0232] Typically, the reaction in step (d) is carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent used. In various embodiments, the reaction temperature 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, such as nitrogen, argon, or helium.

[0233] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 30 minutes to about 4 hours. In some 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 compound (XI). Typically, the amount of compound (XI) 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%.

[0234] After step (d) is completed, the pH of the reaction mixture is adjusted to a pH 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 is adjusted to a range from about pH 2.0 to about pH 2.5.

[0235] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids include HCl, H₂SO₄, acetic acid, methanesulfonic acid, or similar organic or inorganic acids. In a preferred embodiment, HCl may be used.

[0236] The yield of compounds including formula (XII) can be at least about 60%. In various embodiments, the yield of compounds including 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 yield of compounds including formula (XII) can be about 95%.

[0237] Step (e)

[0238] 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 agent to form a reaction mixture. Following treatment and separation, a compound comprising formula (XIV) is obtained.

[0239] The above describes in detail compounds including formula (XII).

[0240] The compounds comprising formula (XIII) have been described in detail above. In some embodiments, R8 and R9 are independently selected from the group consisting of: H, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, O-substituted C1-C4 alkyl, O-unsubstituted C1-C4 alkyl, (OCH2CH2O). n Or a nitrogen-protecting group (P1). In some embodiments, R8 and R9 are independently selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, or a nitrogen-protecting group (P1). In specific embodiments, R8 and R9 are independently selected from the group consisting of: H and a nitrogen-protecting group (P1).

[0241] In some embodiments, n is an integer from 1 to 10. In a particular embodiment, n does not exist.

[0242] In some embodiments, P1 is a nitrogen-protecting group. In a specific embodiment, P1 is a BOC group.

[0243] 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.

[0244] Typically, the equivalence 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 equivalence 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, about 1.0:1.0 to about 1.0:1.3, or about 1.0:1.2. In a preferred embodiment, the equivalence ratio of the compound comprising (XII) to the compound comprising (XIII) can be about 1:0:1.1.

[0245] The method in step (e) utilizes an acyl coupling agent and a proton acceptor. Suitable acyl coupling agents and proton acceptors are described in detail above in section (II)(a). In a preferred embodiment, the acyl coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof, and the proton acceptor is 4-methylmorpholine.

[0246] Typically, the equivalence ratio of formula (XII) to the acyl coupling agent ranges from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of formula (XII) to the acyl coupling agent ranges from about 1.0:1.0 to about 1.0:1.5, from about 1.0:1.0 to about 1.0:1.3, or from about 1.0:1.2. In a preferred embodiment, the equivalence ratio of formula (XII) to the acyl coupling agent is about 1:0:1.1.

[0247] Typically, the equivalence ratio of formula (XII) to proton acceptor ranges from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalence ratio of formula (XII) to proton acceptor ranges 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 equivalence ratio of formula (XII) to proton acceptor is about 1:0:2.1.

[0248] Step (e) includes a solvent. Suitable solvents have been described in detail above in section (II)(a). In a preferred embodiment, dimethylformamide may be used as the solvent in step (e).

[0249] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (XII) ranges 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) can range from about 0.5:1 to about 500:1, about 2:1 to about 250:1, about 5:1 to about 200:1, or 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) can range from about 12:1 to about 20:1.

[0250] Typically, the reaction in step (e) is carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent used. In various embodiments, the reaction temperature 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 nitrogen, argon, or helium.

[0251] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 5 minutes to about 2 hours. In some 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 compound (XII). Typically, the amount of compound (XII) 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%.

[0252] The yield of compounds including formula (XIV) can be at least about 60%. In various embodiments, the yield of compounds including 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 compounds including formula (XIV) can be about 91%.

[0253] Step (f)

[0254] Step (f) in the eight-step method involves contacting a compound comprising formula (XIV) with a proton acceptor to form a reaction mixture. Following processing and separation, a compound comprising formula (XV) is obtained. This step is referred to as the "deprotection" reaction.

[0255] The compounds including formula (XIV) are described in more detail above.

[0256] Suitable proton acceptors have been described in detail in section (II)(b). In a preferred embodiment, the proton acceptor is NaOH or LiOH.

[0257] Typically, the equivalence ratio of formula (XIV) to proton acceptor ranges from about 1.0:1.0 to about 1.0:5.0. In various embodiments, the equivalence ratio of formula (XIV) to proton acceptor ranges 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 equivalence ratio of formula (XIV) to proton acceptor is about 1:0:2.0.

[0258] Step (d) further includes a solvent. Suitable solvents have been described in detail above in section (II)(a). In a preferred embodiment, the solvent is a combination of methanol and water.

[0259] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (XIV) ranges 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 (XIV) can range from about 0.5:1 to about 500:1, about 5:1 to about 200:1, about 10:1 to about 100:1, or about 15:1 to about 50:1. In a preferred embodiment, the volume-to-weight ratio of the solvent to the compound comprising formula (XIV) can be about 20:1.

[0260] Typically, the reaction in step (f) is carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent used. In various embodiments, the reaction temperature 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, such as nitrogen, argon, or helium.

[0261] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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., 1The reaction is determined by ¹H NMR. The duration of the reaction can range from about 30 minutes to about 4 hours. In some 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 compound (XIV). Typically, the amount of compound (XIV) 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%.

[0262] After step (f) is completed, the pH of the reaction mixture is adjusted to a pH 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 is adjusted to a range from about pH 2.0 to about pH 2.5.

[0263] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids include HCl, H₂SO₄, acetic acid, methanesulfonic acid, or similar organic or inorganic acids. In a preferred embodiment, HCl may be used.

[0264] The yield of compounds including formula (XV) can be at least about 60%. In various embodiments, the yield of compounds including 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 compounds including formula (XV) can be about 92%.

[0265] Step (g)

[0266] Step (g) of the eight-step method involves contacting a compound of formula (XV) with a compound comprising formula (XVIa) or (XVIb) in the presence of an acyl coupling agent to form a reaction mixture. Following treatment and separation, a compound comprising formula (XVII) is obtained.

[0267] The compounds including formula (XVII) are described in more detail above.

[0268] The compounds including formulas (XVIa) and (XVIb) are described in more detail below:

[0269] as well as

[0270]

[0271] P1 is a nitrogen protecting group, and P2 is a carboxylic acid protecting group.

[0272] 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 methyl 3-(BOC-amino)pyrrolidine-3-carboxylate, and the compound comprising formula (XVIb) is methyl 4-(BOC-amino)piperidine-4-carboxylate.

[0273] Typically, the equivalence ratio of the compound comprising (XV) to the compound comprising (XVIa) or the compound comprising (XVIb) can range from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of the compound comprising (XV) to the compound comprising (XVIa) or the compound comprising (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 equivalence ratio of the compound comprising (XV) to the compound comprising (XVIa) or the compound comprising (XVIb) can be about 1:0:1.2.

[0274] The method of step (g) utilizes an acyl coupling agent and a proton acceptor. Suitable acyl coupling agents and proton acceptors are described in detail above in section (II)(a). In a preferred embodiment, the acyl coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or a salt thereof, and the proton acceptor is 4-methylmorpholine.

[0275] Typically, the equivalence ratio of formula (XV) to the acyl coupling agent ranges from about 1.0:1.0 to about 1.0:1.5. In various embodiments, the equivalence ratio of formula (XV) to the acyl coupling agent ranges 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 equivalence ratio of formula (XV) to the acyl coupling agent is about 1:0:1.2.

[0276] Typically, the equivalence ratio of formula (XV) to proton acceptor ranges from about 1.0:1.0 to about 1.0:2.5. In various embodiments, the equivalence ratio of formula (XV) to proton acceptor ranges 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 equivalence ratio of formula (XV) to proton acceptor is about 1:0:2.1.

[0277] Step (g) includes a solvent. Suitable solvents have been described in detail above in section (II)(a). In a preferred embodiment, dimethylformamide may be used as the solvent in step (g).

[0278] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (XV) ranges 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 (XV) can range from about 0.5:1 to about 500:1, about 2:1 to about 250:1, about 5:1 to about 200:1, or about 10:1 to about 50:1. In an exemplary embodiment, the volume-to-weight ratio of the solvent to the compound comprising formula (XV) can range from about 15:1 to about 25:1.

[0279] Typically, the reaction in step (g) is carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent used. In various embodiments, the reaction temperature 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 nitrogen, argon, or helium.

[0280] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 5 minutes to about 2 hours. In some 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 compound (XV). Typically, the amount of compound (XV) 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%.

[0281] The yield of compounds including formula (XVII) can be at least about 60%. In various embodiments, the yield of compounds including 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 yield of compounds including formula (XVII) can range from about 88% to 93%.

[0282] Step (h)

[0283] Step (h) involves contacting a compound comprising formula (XVII) with a deprotecting agent comprising an acid to form a reaction mixture. Following treatment and separation, a compound comprising formula (I) is obtained.

[0284] In step (h), after using a deprotecting agent including an acid and separating the compound including formula (I), the method may further contact the compound including formula (I) with a proton acceptor to remove the carboxylic acid protecting group.

[0285] The compounds including formula (XVII) are described in more detail above.

[0286] Various acids can be used in this method step. In various embodiments, the deprotecting agent comprising the acid can be in pure or aqueous form. Non-limiting acids can be hydrochloric acid, hydrogen chloride, sulfuric acid, methanesulfonic acid, or trifluoroacetic acid. In a preferred embodiment, the deprotecting agent comprising the acid is trifluoroacetic acid.

[0287] Typically, excessive amounts of deprotecting agents, including acids, are used in the volume-to-weight ratio.

[0288] Step (h) includes a solvent. Suitable solvents have been described in detail above in section (II)(a). In a preferred embodiment, dichloromethane may be used as the solvent in step (h).

[0289] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (XVII) ranges 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) can range from about 0.5:1 to about 500:1, about 2:1 to about 250:1, about 5:1 to about 200:1, or 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) can range from about 40:1 to about 80:1.

[0290] Typically, the reaction in step (h) is carried out at a temperature ranging from about -20°C to about 25°C, depending on the solvent used. In various embodiments, the reaction temperature 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 nitrogen, argon, or helium.

[0291] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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., 1The reaction is determined by ¹H NMR. The duration of the reaction can range from about 5 minutes to about 2 hours. In some 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 compound (XVII). Typically, the amount of compound (XVII) 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%.

[0292] The yield of compounds comprising formula (I) can be at least about 60%. In various embodiments, the yield of compounds 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 yield of compounds comprising formula (I) can range from about 88% to 93%.

[0293] The second deprotection step utilizes a proton acceptor. Suitable proton acceptors have been described in detail in section (II)(b). In a preferred embodiment, the proton acceptor is NaOH or LiOH.

[0294] Typically, the equivalence ratio of formula (I) to proton acceptor can range from about 1.0:1.0 to about 1.0:20.0. In various embodiments, the equivalence ratio of formula (I) to 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 equivalence ratio of formula (XIV) to proton acceptor can be about 1:0:10.0.

[0295] The second deprotection step in step (h) further includes 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.

[0296] Typically, the volume-to-weight ratio of the solvent to the compound comprising formula (I) in the second deprotection step ranges 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) can range from about 0.5:1 to about 500:1, about 5:1 to about 200:1, about 10:1 to about 100:1, or 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 can be about 20:1.

[0297] Typically, the second deprotection step in step (h) is carried out at a temperature ranging from about 0°C to about 50°C, depending on the solvent used. In various embodiments, the reaction temperature 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, such as nitrogen, argon, or helium.

[0298] Typically, the reaction is allowed to proceed for a sufficient period of time until it 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 The reaction is determined by ¹H NMR. The duration of the reaction can range from about 30 minutes to about 4 hours. In some 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 compound (I). Typically, the amount of compound (I) 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%.

[0299] After step (h) using a proton acceptor, the pH of the reaction mixture is adjusted to 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 is adjusted to a range from about pH 2.0 to about pH 2.5.

[0300] This pH adjustment uses an aqueous acid. Non-limiting examples of suitable acids include HCl, H₂SO₄, acetic acid, methanesulfonic acid, or similar organic or inorganic acids.

[0301] The yield of compounds comprising formula (I) can be at least about 60%. In various embodiments, the yield of compounds 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 yield of compounds comprising formula (I) can range from about 60% to 70%.

[0302] (III) The pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt comprising a compound of formula (I).

[0303] Another aspect of this disclosure includes pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt of a compound of formula (I) and at least one pharmaceutically acceptable excipient.

[0304] (a) A compound comprising formula (I) or a pharmaceutically acceptable salt comprising formula (I).

[0305] The above section (I) details compounds of formula (I) or pharmaceutically acceptable salts of compounds of formula (I).

[0306] Typically, the amount of a compound comprising formula (I) or a pharmaceutically acceptable salt comprising formula (I) used in a pharmaceutical composition may and will vary depending on the age of the subject and the daily dose used. Typically, the amount of a compound comprising formula (I) or a pharmaceutically acceptable salt comprising formula (I) used in a pharmaceutical composition can range from about 1.0 mg to about 100 mg. In various embodiments, the range of a compound comprising formula (I) or a pharmaceutically acceptable salt comprising formula (I) used in a pharmaceutical composition can be 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.

[0307] (b) at least one excipient

[0308] The compositions disclosed herein may further include pharmaceutically acceptable excipients. Non-limiting examples of suitable pharmaceutically acceptable excipients include diluents, binders, fillers, buffers, pH adjusters, disintegrants, dispersants, preservatives, lubricants, flavor masking agents, flavoring agents, coloring agents, or combinations thereof. The amount and type of excipients used to form the pharmaceutical composition may be selected based on known pharmaceutical principles.

[0309] In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasive and brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., a mixture of acetate and butyrate esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, corn starch, phosphorylated 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. Non-limiting examples of suitable abrasive and brittle diluents include dicalcium phosphate (anhydrous or dihydrate), tricalcium phosphate, calcium carbonate, and magnesium carbonate.

[0310] In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethyl cellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, and C. 12 -C 18 Fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

[0311] In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. As a non-limiting example, the filler may be calcium sulfate (both di- and tri-alkali), starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, calcium hydrogen phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

[0312] In yet another embodiment, the excipient may 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).

[0313] In various embodiments, the excipient may be a pH adjuster. As a non-limiting example, the pH adjuster may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0314] In another embodiment, the excipient may be a disintegrant. The disintegrant 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, and their pregelatinized and modified forms; sweeteners; clays such as bentonite; microcrystalline cellulose; alginate; sodium glycolate; and gums such as agar, guar gum, locust bean gum, cannabinoid gum, pectin, and tragacanth gum. Non-limiting examples of suitable effervescent disintegrants include combinations of sodium bicarbonate and citric acid, and combinations of sodium bicarbonate and tartaric acid.

[0315] In yet another embodiment, the excipient may be a dispersant or a dispersion enhancer. Suitable dispersants may include, but are not limited to, starch, alginate, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified lignocellulose, sodium glycolate starch, heteroamorphous silicate, and microcrystalline cellulose.

[0316] In another alternative embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include: antioxidants such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelating agents such as EDTA or EGTA; and antibacterial agents such as parabens, chlorobutanol, or phenol.

[0317] In another embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate, or stearic acid.

[0318] In yet another embodiment, the excipient may be a flavor masking agent. The flavor masking material comprises cellulose ethers, polyethylene glycol, polyvinyl alcohol, copolymers of polyvinyl alcohol and polyethylene glycol, monoglycerides or triglycerides; acrylic polymers, mixtures of acrylic polymers and cellulose ethers, cellulose acetate phthalate, and combinations thereof.

[0319] In an alternative embodiment, the excipient may be a flavoring agent. The flavoring agent may be selected from synthetic flavoring oils and flavoring agents and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.

[0320] In yet another embodiment, the excipient may be a colorant. Suitable color additives include, but are not limited to, food, pharmaceutical and cosmetic colors (FD&C), pharmaceutical and cosmetic colors (D&C), or external pharmaceutical and cosmetic colors (Ext.D&C).

[0321] The weight fraction of excipients or combinations of excipients in the composition may 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.

[0322] Compositions can be formulated into various dosage forms and administered in many different ways by delivering a therapeutically effective amount of the active ingredient. Such compositions can be administered orally, parenterally, or topically in the form of formulations containing desired, conventionally non-toxic, pharmaceutically acceptable carriers, adjuvants, and mediators in dose units. Topical administration can also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. As used herein, parenterally administration includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques. Formulations of pharmaceuticals are discussed, for example, in *Remington's Pharmaceutical Sciences*, Gennaro, AR, Mack Publishing Co., Easton, PA (18th edition, 1995) and *Pharmaceutical Dosage Forms*, edited by Liberman, HA and Lachman, L., Marcel Dekker Inc., New York, NY (1980). In specific embodiments, the composition may be a food supplement or a cosmetic.

[0323] Solid dosage forms for oral administration can include capsules, tablets, pills, powders, granules, and pellets. In such solid dosage forms, the active ingredient is typically combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral formulations can also be administered as aqueous suspensions, elixirs, or syrups. For this purpose, the active ingredient can be combined with various sweeteners or flavorings, colorings, and, if desired, emulsifiers and / or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

[0324] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal), the formulation may be an aqueous or oil-based solution. Aqueous solutions may contain: sterile diluents such as water or saline solutions; pharmaceutically acceptable polyols such as glycerol, propylene glycol, or other synthetic solvents; antimicrobial and / or antifungal agents such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and / or agents for tension adjustment such as sodium chloride, dextran, or polyols such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further include sesame, peanut, olive oil, or mineral oil. The composition can be present in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under freeze-dried (lyophilized) conditions where a sterile liquid carrier, such as water for injection, is only required to be added immediately before use. Temporary injectable solutions and suspensions can be prepared from sterile powders, microparticles, and tablets.

[0325] For topical (e.g., transdermal or mucosal) application, the formulation typically contains a penetrant suitable for the barrier to be penetrated. Pharmaceutical compositions suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated as an ointment, the active ingredient can be used with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water or water-in-oil base. Pharmaceutical compositions suitable for topical application to the eyes comprise eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent. Pharmaceutical compositions suitable for topical application in the oral cavity comprise lozenges, soft lozenges, and mouthwashes. Mucosal application can be accomplished by using nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal application can be performed by ointments, creams, gels, patches, or creams as commonly known in the art.

[0326] In some embodiments, compositions comprising a pharmaceutically acceptable salt of a compound of formula (I) are encapsulated in a suitable medium to aid in the delivery of the compound to target cells, increase the stability of the composition, or minimize the potential toxicity of the composition. As those skilled in the art will understand, various mediums are suitable for delivering the compositions of the present invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendritic polymers, and other phospholipid-containing systems. Methods for incorporating compositions into delivery mediums are known in the art.

[0327] In an alternative embodiment, liposomes can be used to deliver the agent. Given the structure and chemical properties of liposomes, depending on the embodiment, liposomes are suitable for delivering compositions comprising a pharmaceutically acceptable salt of a compound of formula (I). Generally, liposomes are spherical vesicles 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 into cells. In this manner, compositions comprising at least one antiviral therapeutic agent can be selectively delivered to cells by encapsulation in liposomes fused to the membrane of target cells.

[0328] Liposomes can contain various types of phospholipids with different hydrocarbon chain lengths. Phospholipids typically consist of two fatty acids linked by a glycerophosphate to one of various polar groups. Suitable phospholipids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The length of the fatty acid chain comprising the phospholipid can range from about 6 to about 26 carbon atoms, and the lipid chain can be saturated or unsaturated. Suitable fatty acid chains include (common names are shown in parentheses) n-Lauryl ester, n-Tetradecanoate, n-Hexadecanoate, n-Stearate, n-Eicosanoate, n-Behenate, n-Tetradecanoate, cis-9-Hexadecanoate, cis-9-Octadecanoate, cis-9,12-Octadecanadienoate, cis-9,12,15-Octadecantrienoate, and cis-5,8,11,14-Eicosanotetraenoate. The two fatty acid chains of a phospholipid can be the same or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearyl PS, distearyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenic acid PS, etc.

[0329] Phospholipids can be derived from any natural source, and if so, mixtures of phospholipids can be included. For example, egg yolks are rich in PC, PG, and PE; soybeans contain PC, PE, PI, and PA; and animal brains or spinal cords are rich in PS. Phospholipids can also be derived from synthetic sources. Mixtures of phospholipids with different ratios of individual phospholipids can be used. Mixtures of different phospholipids can produce liposome compositions with favorable activity or stable active properties. The phospholipids mentioned above can be mixed with cationic lipids in an optimal ratio, such as N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, 1,1'-octadecyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate, 3,3'-deheptyloxacarbocyanine iodide, and 1,1'-docodecyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate. The following are listed as perchloarate, 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate.

[0330] Liposomes may optionally include sphingolipids or cholesterol (major components of animal cell membranes), in which sphingosine is a structural counterpart of one of the fatty acids, glycerol and glycerophosphate. Liposomes may optionally contain polyethylene glycol-modified lipids, which are lipids covalently linked to a polymer of polyethylene glycol (PEG). The size of the PEG can range from about 500 Daltons to about 10,000 Daltons.

[0331] Liposomes may further include a suitable solvent. The solvent may be an organic or inorganic solvent. Suitable solvents include, but are not limited to, dimethyl sulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetonitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.

[0332] Liposomes carrying compositions comprising compounds of formula (I) can be prepared by any known method for preparing liposomes for drug delivery, such as those detailed in U.S. Patent 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 disclosures of which are hereby incorporated by reference in their entirety. For example, liposomes can be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze-drying by repeated freezing and thawing. In a preferred embodiment, liposomes are formed by sonication. Liposomes can be multilayered or monolayered, with multilayered liposomes having many layers like an onion. Liposomes can be large or small. Sustained high-shear sonication tends to form smaller monolayer liposomes.

[0333] As is obvious to a person of ordinary skill in the art, all parameters controlling liposome formation can be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compounds, concentration and composition of lipids, concentration of polyvalent cations, mixing rate, and the presence and concentration of solvents.

[0334] In another embodiment, the compositions of this 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 remains at the oil-water interface. Any surfactant of a variety of surfactants is suitable for use in microemulsion formulations, including those described herein or otherwise known in the art. The characteristic structural size of the aqueous microregions suitable for this invention is typically from 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 those skilled in the art will understand, microemulsions can and will have a variety of different microstructures comprising spherical, rod-shaped, or disc-shaped aggregates. In one embodiment, the structure can be micelles, which are the simplest microemulsion structures, typically in the form of spherical or cylindrical objects. Micelles are like water-in-oil droplets, and antimicelles are like oil-in-water droplets. In alternative embodiments, the microemulsion structure is a thin layer. It comprises a continuous aqueous layer and an oil layer separated by a surfactant layer. The "oil" of the microemulsion preferably comprises phospholipids. Any phospholipids detailed above for liposomes are suitable for the embodiments for microemulsions. Compositions comprising at least one antiviral therapeutic derivative may be encapsulated in the microemulsion by any method generally known in the art.

[0335] In yet another embodiment, compositions comprising a pharmaceutically acceptable salt of formula (I) or a compound comprising formula (I) can be delivered in the form of dendritic macromolecules or dendritic structures. Generally, a dendritic structure is a branched dendritic molecule, where 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 spheres. Typically, the properties of a dendritic structure are determined by the functional groups on its surface. For example, hydrophilic end groups such as carboxyl groups typically yield water-soluble dendritic structures. Alternatively, phospholipids can be incorporated into the surface of the dendritic structure to facilitate transdermal absorption. Any phospholipids detailed in the liposome examples are applicable to the dendritic examples. Dendritic structures can be prepared using any method generally known in the art and the compositions of the present invention can be encapsulated within dendritic structures. For example, dendritic structures can be generated by an iterative sequence of reaction steps, in which each additional iteration results in a more advanced dendritic structure. Therefore, the dendrites have a regular, highly branched 3D structure and a nearly uniform size and shape. Furthermore, the final size of the dendrites is typically controlled by the number of iterative steps used during synthesis. Various dendrite sizes are suitable for this invention. Typically, the size of the dendrites can range from about 1 nm to about 100 nm.

[0336] (c) Dosage Form

[0337] Compositions can be formulated into various dosage forms and administered in many different ways by delivering a therapeutically effective amount of the active ingredient. Such compositions can be administered orally, parenterally, or topically in the form of formulations containing desired, conventionally non-toxic, pharmaceutically acceptable carriers, adjuvants, and mediators in dose units. Topical administration can also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. As used herein, the term parenterally includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques. Formulations of pharmaceuticals are discussed, for example, in Gennaro, AR., Remington’s Pharmaceutical Sciences, Mack Publishing, Easton, PA (18th edition, 1995) and Liberman, HA., and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY (1980).

[0338] Solid dosage forms for oral administration include capsules, tablets, pellets, powders, granules, and granules. In such solid dosage forms, the active ingredient is typically combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral formulations can also be administered as aqueous suspensions, elixirs, or syrups. For this purpose, the active ingredient can be combined with various sweeteners or flavorings, colorings, and, if desired, emulsifiers and / or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

[0339] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal), the formulation may be an aqueous or oil-based solution. Aqueous solutions may contain: sterile diluents such as water or saline solutions; pharmaceutically acceptable polyols such as glycerol, propylene glycol, or other synthetic solvents; antimicrobial and / or antifungal agents such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and / or agents for tension adjustment such as sodium chloride, dextran, or polyols such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further include sesame, peanut, olive oil, or mineral oil.

[0340] (IV). Methods for treating κ-opioid receptor agonist-related medical conditions.

[0341] Another aspect of this disclosure includes a method for treating κ-opioid receptor agonist-related medical conditions. The method includes administering to a subject in need a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound comprising formula (I) or a compound comprising formula (I). κ-opioid receptor agonist-related diseases or conditions include pain, cardiovascular disease, pruritus, nausea, inflammatory diseases, spinal anesthesia, cough suppression, stroke, hypoxic pulmonary hypertension, multiple sclerosis, addiction, and post-traumatic cartilage degeneration.

[0342] (a) Composition and dosage form

[0343] The compositions and dosage forms are described in more detail in Section (III) above.

[0344] Such compositions can be administered orally, parenterally, by inhalation spray, rectal, intradermal, transdermal, or topically in dosage units containing the desired conventional, non-toxic, pharmaceutically acceptable carrier, adjuvant, and mediator. Topical administration may also involve transdermal application, such as transdermal patches or iontophoresis devices. As used herein, parenterally includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques.

[0345] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal), the formulation may be an aqueous or oil-based solution. Aqueous solutions may contain: sterile diluents such as water or saline solutions; pharmaceutically acceptable polyols such as glycerol, propylene glycol, or other synthetic solvents; antimicrobial and / or antifungal agents such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and / or agents for tension adjustment such as sodium chloride, dextran, or polyols such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further include sesame, peanut, olive oil, or mineral oil.

[0346] For topical (e.g., transdermal or mucosal) application, the formulation typically contains a penetrant suitable for the barrier to be penetrated. Mucosal application can be accomplished by using nasal sprays, aerosol sprays, tablets, or suppositories, while transdermal application can be performed by ointments, creams, gels, patches, or creams as commonly known in the art.

[0347] The amount of medication administered to a subject can and will vary depending on the type of medication, the subject, and the specific method of administration. Those skilled in the art will understand that the dosage can also be determined with guidance from Goodman and Goldman's *The Pharmacological Basis of Therapeutics*, 10th edition (2001), Appendix II, pp. 475-493, and *The Physicians' Desk Reference*.

[0348] (b) Subjects

[0349] Suitable subjects include humans, livestock, companion animals, laboratory animals, or zoological animals. In one embodiment, the subject may be a rodent, such as a mouse, rat, guinea pig, etc. In another embodiment, the subject may be livestock. Non-limiting examples of suitable livestock 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 animal" refers to an animal that can be found in a zoo. Such animals may include non-human primates, large felines, 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.

[0350] Definitions

[0351] The compounds described herein have an asymmetric center. Compounds of this disclosure containing asymmetrically substituted atoms can be isolated in optically active or racemic forms. Unless a specific stereochemical or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, and geometrical isomers of a structure are expected.

[0352] As used herein, the term "acyl" alone or as part of another group refers to a portion formed by removing a hydroxyl group from the group COOH of an organic carboxylic acid (e.g., RC(O)–), where R is R 1 R 1 O-, R 1 R 2 N- or R 1 S-, R 1 It is a hydrocarbon group, a heterosubstituted hydrocarbon group, or a heterocyclic ring, and R2 It can be hydrogen, a hydrocarbon group, or a substituted hydrocarbon group.

[0353] As used herein, the term “acyloxy” alone or as part of another group refers to an acyl group bonded by an oxygen bond (O) as described above, such as RC(O)O–, where R is as defined in conjunction with the term “acyl”.

[0354] 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 that forms part of a ring system.

[0355] As used herein, the term "alkyl" describes a group preferably containing one to eight carbon atoms and up to 20 carbon atoms in the main chain of a lower alkyl group. The group may be straight-chain, branched, or cyclic and includes methyl, ethyl, propyl, isopropyl, butyl, hexyl, etc.

[0356] As used herein, the term "alkenyl" describes a group preferably containing two to eight carbon atoms and up to 20 carbon atoms in the main chain of a lower alkenyl group. The group may be linear, branched, or cyclic and includes vinyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, etc.

[0357] As used herein, the term "alkynyl" describes a group preferably containing two to eight carbon atoms and up to 20 carbon atoms in the main chain of a lower alkynyl group. The group may be straight-chain or branched and includes ethynyl, propynyl, butynyl, isobutynyl, hexynyl, etc.

[0358] As used herein, the term "aromatic" alone or as part of another group refers to a ring system of allotropic or heterocyclic conjoined planar rings or rings that include optionally substituted allotropic or heterocyclic rings with 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 moiety. The term "aromatic" encompasses "aryl" groups as defined below.

[0359] As used herein, the term "aryl" or "Ar" alone or as part of another group indicates an optionally substituted allocyclic aryl group, preferably an allocyclic monocyclic or bicyclic group containing 6 to 10 carbons in the ring moiety, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl.

[0360] As used herein, the term "carbocyclo / carbocyclic" alone or as part of another group refers to an optionally substituted, aromatic or non-aromatic allotropic ring or ring system in which all atoms in the ring are carbon, preferably 5 or 6 carbon atoms per ring. Exemplary substituents comprise one or more of the following groups: hydrocarbon, substituted hydrocarbon, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amide, acetal, carbamoyl, carbocyclic, cyano, ester, ether, halogen, heterocyclic, hydroxyl, ketone, ketal, phosphoric acid, nitro, and thio.

[0361] As used herein, the terms “halogen” or “halogen group” alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.

[0362] The term "heteroatoms" refers to atoms other than carbon and hydrogen.

[0363] As used herein, the term "heteroaromatic" alone or as part of another group refers to an optionally substituted aromatic group having at least one heteroatom in at least one ring, and preferably having 5 or 6 atoms in each ring. 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 remainder of the molecule via carbon atoms. Exemplary groups include furanyl, benzofuranyl, oxazolyl, isoxazolyl, oxadiazolyl, benzooxazolyl, benzooxadiazolyl, pyrroleyl, pyrazolyl, imidazoleyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, indoleyl, isoindoleenyl, indoleyl, benzimidazolyl, indazoleyl, benzotriazolyl, tetrazolopyridazinyl, carbazoleyl, purinyl, quinolinyl, isoquinolinyl, imidazolepyridyl, etc. Exemplary substituents include one or more of the following groups: hydrocarbon group, substituted hydrocarbon group, alkyl group, alkoxy group, acyl group, acyloxy group, alkenyl group, alkenyloxy group, aryl group, aryloxy group, amino group, amide group, acetal group, carbamoyl group, carbocyclic group, cyano group, ester group, ether group, halogen group, heterocyclic group, hydroxyl group, ketone group, ketal group, phosphoric acid group, nitro group, and thio group.

[0364] As used herein, the term "heterocyclic" or "heterocyclic" alone or as part of another group indicates 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 having 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 remainder of the molecule by carbon or heteroatoms. Exemplary heterocyclic groups comprise aromatic heterocycles as described above. Exemplary substituents comprise one or more of the following groups: hydrocarbon, substituted hydrocarbon, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amide, acetal, carbamoyl, carbocyclic, cyano, ester, ether, halogen, heterocyclic, hydroxyl, ketone, ketal, phosphoric acid, nitro, and thio.

[0365] As used herein, the term "hydrocarbon" or "hydrocarbon group" 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 with other aliphatic or cyclic hydrocarbon groups, such as alkylaryl, alkenyl, and alkynyl. Unless otherwise stated, these moieties preferably comprise 1 to 20 carbon atoms.

[0366] As used herein, the term "protecting group" refers to a group capable of protecting a particular moiety, wherein the protecting group can be removed after a reaction using protection without interfering with the remaining portion of the molecule. When the moiety is an oxygen atom (and thus forms a protected hydroxyl group), exemplary protecting groups include ethers (e.g., allyl, triphenylmethyl (triphenylmethyl / trirityl 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-trimethylsilylethyl carbonate), and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), trimethylisopropylsilyl (TIP)). S), triphenylsilyl (TPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBMPS), etc. 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-butoxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC)), acetyl, carbamate, n-silyl, etc. When the moiety is a carboxyl group, exemplary protecting groups include esters (methyl, substituted methyl ester, ethyl ester, substituted ethyl ester), etc. Various protecting groups and their synthesis can be found in "Protective Groups in Organic Synthesis" by TW Greene and PGMUTS, John Wiley & Sons, 1999.

[0367] The “substituted hydrocarbon group” described herein refers to a hydrocarbon group that is substituted with at least one atom other than carbon, including portions in which carbon chain atoms are substituted with heteroatoms such as nitrogen, oxygen, silicon, phosphorus, boron, or halogen atoms, and portions in which the carbon chain includes additional substituents. These substituents include alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenyloxy, aryl, aryloxy, amino, amide, acetal, carbamoyl, carbocyclic, cyano, ester, ether, halogen, heterocyclic, hydroxyl, ketone, ketal, phosphoric acid, nitro, and thio.

[0368] When describing the elements of the embodiments described herein, the articles “a,” “an,” “the,” and “said” are intended to mean one or more of the elements present. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements may be present besides the listed elements.

[0369] Since various changes can be made to the methods described above without departing from the scope of the invention, all content contained in the above description and the examples given below should be interpreted as illustrative rather than restrictive.

[0370] Example 1: Synthesis of dipeptide intermediate (3)

[0371]

[0372] (2R,3R)-Boc-β-methyl-phenylalanine (1) (15 g, 36.1 mmol, 1.0 equivalent) and DMF (212 mL) were added to a reaction flask under nitrogen atmosphere. After the mixture became homogeneous with stirring, the resulting solution was cooled to 0 °C in an ice bath. HOBt·H₂O (5.36 g, 39.7 mmol, 1.1 equivalent) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (7.60 g, 39.7 mmol, 1.1 equivalent) were added to the cooled reaction mixture. The resulting mixture was stirred for 30 min, and then HD-Phe-OMe·HCl (2) (8.56 g, 39.7 mmol, 1.1 equivalent) and N-methylmorpholine (NMM) (7.58 mL, 75.7 mmol, 2.1 equivalent) were added. The reaction was stirred at 0 °C for one hour and then at room temperature until HPLC analysis indicated that the reaction was complete. The reaction solution was added dropwise to water (600 mL) with stirring. After the water addition was complete, stirring was stopped and a precipitate began to form. The reaction mixture was allowed to stand for 1 hour, during which precipitation ceased. The precipitate (crystals) was collected by filtration, washed with water (400 mL x 3), and then dried under vacuum to give a product (3) as a white solid, 14.36 g, yield = 90.8%. LCMS: m / z = 441.5 [M+H] + .

[0373] Example 2: Synthesis of dipeptide intermediate (4)

[0374]

[0375] Compound (3) (12 g, 27.2 mmol, 1.0 equivalent) and methanol (240 mL) were added to a reaction flask. Stirring was initiated, and NaOH solution (60 mL, 1 M, 60 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for approximately 2 hours until HPLC analysis indicated completion. 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 collected by filtration, washed with water (3 x 320 mL), and then dried under vacuum (30 °C). 11 g of the product as a white solid was given, yield = 94.8%. LCMS m / z = 427.5 [M+H] + .

[0376] Example 3: Synthesis of tripeptide intermediate (6)

[0377]

[0378] Compound (4) (8.0 g, 18.8 mmol, 1.0 equivalent) and DMF (169 mL) were added to a reaction flask under nitrogen atmosphere. The resulting solution was cooled to 0 °C in an ice bath. HOBt·H₂O (2.79 g, 20.6 mmol, 1.2 equivalent) and EDCI (3.96 g, 20.5 mmol, 1.1 equivalent) were added to the cooled reaction mixture. After stirring the reaction mixture at 0 °C for 30 min, HD-Leu-OMe·HCl (5) (20.6 mmol, 1.1 equivalent, 3.75 g) and N-methylmorpholine (NMM) (3.94 mL, 39.4 mmol, 2.1 equivalent) were added. The resulting reaction mixture was stirred at 0 °C for 1 h, and then heated to room temperature until HPLC analysis indicated 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 formed. The reaction mixture was allowed to stand for one hour, during which precipitation ceased. The precipitate was then collected by filtration and washed with water (240 mL x 3), followed by drying under vacuum (30 °C); 9.96 g of a white solid was obtained, yield = 96% (6). LCMS: m / z = 554.7 [M+H] + .

[0379] Example 4: Synthesis of tripeptide intermediate (7)

[0380]

[0381] Compound (6) (8 g, 14.4 mmol, 1.0 equivalent) and methanol (160 mL) were added to a reaction flask under nitrogen atmosphere. 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 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). Product (7) was given as a white solid, 7.41 g, yield = 95%. LCMS m / z = 540.7 [M+H] + .

[0382] Example 5: Synthesis of tetrapeptide intermediate (9)

[0383]

[0384] Compound (7) (6.0 g, 11.1 mmol, 1.0 equivalent) and DMF (127 mL) were added to a nitrogen-filled reaction flask. The resulting reaction mixture was cooled to 0 °C in an ice bath. HOBt·H₂O (1.81 g, 13.4 mmol, 1.2 equivalent) and EDCI (2.57 g, 13.4 mmol, 1.2 equivalent) were added to the cooled reaction mixture. After 30 minutes, HD-Lys(Boc)-OMe·HCl (8) (3.97 g, 13.7 mmol, 1.2 equivalent) and N-methylmorpholine (NMM) (2.37 g, 23.4 mmol, 2.1 equivalent) were added. The reaction mixture was stirred at 0 °C for one hour and then heated 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 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). A 7.9 g, white solid was obtained, with a yield of 91% (9). LCMS: m / z = 783.0 [M+H] + .

[0385] Example 6: Synthesis of tetrapeptide intermediate (10)

[0386]

[0387] Compound (9) (5 g, 6.39 mmol, 1.0 equivalent) and methanol (100 mL) were added to a reaction flask under nitrogen atmosphere. 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 completion. Then, HCl solution (1.0 M) was added dropwise until a pH of 2.0–2.5 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). Product (10) was given as a white solid, 4.51 g, yield = 91.9%. LCMS m / z = 769.0 [M+H] + .

[0388] Example 7: Synthesis of peptide analogs (12)

[0389]

[0390] Compound (10) (1.0 g, 1.3 mmol, 1.0 equivalent) and DMF (21 mL) were added to a reaction flask under nitrogen atmosphere. The reaction mixture was cooled to 0 °C in an ice bath. HOBt·H₂O (211 mg, 1.56 mmol, 1.2 equivalent) and EDCI (299 mg, 1.56 mmol, 1.2 equivalent) were added to the reaction mixture. After stirring the reaction mixture at 0 °C for 30 min, methyl 3-(Boc-amino)pyrrolidine-3-carboxylate (11) (1.56 mmol, 1.2 equivalent, 381.1 mg) and N-methylmorpholine (NMM) (2.37 g, 23.4 mmol, 2.1 equivalent) were added. The reaction mixture was stirred at 0 °C for one hour and then heated to room temperature until HPLC analysis indicated that the reaction was complete. The reaction mixture was added dropwise to water (60 mL) with stirring. 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 dried under vacuum (30 °C). A product (12) was obtained as a white solid, 1.2 g in yield = 93.1%. LCMS: m / z = 995.2 [M+H] + .

[0391] Example 8 Synthesis of peptide analogs (13)

[0392]

[0393] Compound (12) (200 mg, 0.201 mmol, 1 equivalent) and dichloromethane (4 mL) were added to a reaction flask under nitrogen atmosphere. 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 at -10 °C for one hour until HPLC analysis indicated completion. Volatile organic matter was removed by rotary evaporation. The residue was dissolved in dichloromethane (4 mL), and the resulting solution was distilled to dryness by rotary evaporation. This process was repeated three more times. The residue was dissolved in methanol (4 mL), and the resulting solution was evaporated to dryness by rotary evaporation. This process was repeated three more times. The obtained residue was purified by reversed-phase HPLC to give product (13) as a white solid, 135.4 mg, yield = 65%. LCMS: MS m / z = 694.4 [M+H] + .

[0394] Example 9 Synthesis of peptide analogs (14)

[0395]

[0396] Under nitrogen atmosphere, compound (13) (100 mg, 0.0965 mmol, 1 equivalent) and methanol (2 mL) were added to a reaction flask. NaOH solution (0.5 mL, 1.0 M, 1.0 mmol, 10 equivalent) was added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours until HPLC analysis indicated completion. HCl solution (1.0 M) was added dropwise to the reaction mixture until pH 2.0–2.5 was reached. Volatile organic compounds were removed using a rotary evaporator. The residue was purified by reversed-phase HPLC using 0.1% TFA acetonitrile / water as the mobile phase. The collected fraction was lyophilized to yield product (14) as a white solid, 45.7 mg, in 60% yield. LC-MS m / z = 680.5 [M+H] + .

[0397] Example 9 Synthesis of peptide analogs (16)

[0398]

[0399] Compound (10) (1.0 g, 1.3 mmol, 1.0 equivalent) and DMF (21 mL) were added to a reaction flask under nitrogen atmosphere. The reaction mixture was cooled to 0 °C in an ice bath. HOBt·H₂O (211 mg, 1.56 mmol, 1.2 equivalent) and EDCI (299 mg, 1.56 mmol, 1.2 equivalent) were added to the reaction mixture. After stirring at 0 °C for 30 min, methyl 4-(Boc-amino)piperidine-4-carboxylate (15) (402.9 mg, 1.56 mmol, 1.2 equivalent) and N-methylmorpholine (NMM) (0.273 mL, 2.73 mmol, 2.1 equivalent) were added. The reaction mixture was stirred at 0 °C for one hour and then heated to room temperature until HPLC analysis indicated the reaction was complete. The reaction mixture was added dropwise to water (60 mL) with stirring. After the addition was complete, stirring was stopped and a precipitate 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 dried under vacuum (30 °C). A product (16) was obtained as a white solid, 1.17 g in weight, with a yield of 87.5%. LCMS: m / z = 1009.3 [M+H] + .

[0400] Example 10 Synthesis of peptide analogs (17)

[0401]

[0402] Under nitrogen atmosphere, compound (16) (500 mg, 0.496 mmol, 1 equivalent) and dichloromethane (10 mL) were added to a 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 matter was 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 by reversed-phase HPLC using 0.1% TFA acetonitrile / water as the mobile phase. The collected fraction was lyophilized to provide product (17) as a white solid, 364.5 mg, in 70% yield. LCMS: MS m / z = 708.5 [M+H] + .

[0403] Example 11 Synthesis of peptide analog (18)

[0404]

[0405] Under nitrogen atmosphere, compound (17) (100 mg, 0.0952 mmol, 1 equivalent) and methanol (2 mL) were added to a reaction flask. NaOH solution (0.5 mL, 1.0 M, 1.0 mmol, 10 equivalent) was added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours until HPLC analysis indicated completion. Then, HCl solution (1.0 M) was added dropwise until pH 2.0–2.5 was achieved. Methanol was removed on a rotary evaporator to form a residue. The residue was purified by reversed-phase HPLC using 0.1% TFA acetonitrile / water as the mobile phase. The collected fraction was lyophilized to form product (18) as a white solid, 45.8 mg, in 60% yield. LC-MS m / z = 802.3 [M+H] + .

[0406] Example 12: Opioid receptor binding assay

[0407] The affinity of membranes prepared from HEK293 cells (human embryonic kidney cell line) that heterologously express recombinant human μ, δ, or κ opioid receptors was measured using a radioligand binding assay.

[0408] The assay buffers used for opioid receptor binding studies are: 50 mM Tris.HCl (pH 7.4) for KOR; 50 mM Tris.HCl (pH 7.4) and 5 mM MgCl2 for MOR; and 50 mM Tris.HCl (pH 7.4) and 10 mM MgCl2 plus 1 mM EDTA for DOR. The wash buffer contains 50 mM Tris.HCl at pH 7.4.

[0409] The binding affinity of opioid receptors was compared with 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-Ala2MePhe4,Gly(ol)5]cerebroside, 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, 1997, pp. 782-790).

[0410] Radioligands were prepared at the following final concentrations: [ 3 H]DAMGO, 0.5nM; [ 3 [H] dipronofol, 0.5 nM; and [ 3 H]DADLE, 0.5 nM, uses the radioligands as competitive radioligands for μ, κ and δ receptors, respectively.

[0411] Cell membranes of HEK293 cells transfected with opioid receptors were prepared at concentrations of 20 μg MOR, 6.7 μg KOR, and 6.7 μg DOR per well, respectively. These membranes containing the receptors of interest were incubated with progressively increased concentrations of the test compound in the presence of a single concentration of the radioligand. Serial dilutions of the test compound were prepared using a fixed concentration of the radioligand.

[0412] The test was started with 10 μM of the test compound and continued until a 4-fold serial dilution was achieved for 8-point assays. 1 μL of compound / high control / low control was transferred to a 96-well plate according to the plate plot, followed by a 100 μL dispensing of the membrane stock solution, and then 100 μL of the radioligand solution was added. The plated wells were incubated for 1 hour at room temperature with gentle stirring at 300 rpm. The Unifilter-96GF / C filter plate was then soaked in 50 μL of 0.3% polyethyleneimine per well for at least 0.5 hours at room temperature, and the assay was performed using FilterMate. TM The collector filtered the reaction mixture through a plate filter, and then washed each plate four times with cold wash buffer. The filter plates were then dried at 50°C for 1 hour. After drying, the filter was sealed in polyethylene, and 50 μl of Perkin Elmer Microscint 20 mixture was added, and radioactivity was counted in a Perkin Elmer MicroBeta2 counter.

[0413] In the presence of 50-100x excess cold ligands, specific binding is determined by subtracting the CPM value of the binding. The saturation analysis nonlinear curve fitting routine is used to fit the data. Suppression is calculated using the following equation:

[0414] Inhibition % = (1 - (Measurement well - Average value LC) / (Average value HC - Average value LC)) * 100%

[0415] The combined data were analyzed using GraphPad Prism 5.0, and the IC was generated through nonlinear regression from the dose-response curves. 50 Data. The data were fitted using the model “log(inhibitor) vs. response – variable slope”. This data is shown in Table 1.

[0416] Table 1. Binding affinity of peptide ligands to recombinant human opioid receptors

[0417]

[0418] Example 13: FLIPR calcium assay in whole cells

[0419] The FLIPR calcium assay measures the ability of opioid ligands to induce functional responses upon receptor binding. Opioid μ receptors (MOR), δ receptors (DOR), and κ receptors (KOR) are G protein-coupled receptors (GPCRs) that play important roles in cell signaling. Receptor activation by ligands triggers intracellular G protein activation. Activated G proteins induce various cascades of intracellular messengers, including calcium flux. The assay is evaluated based on functional cells to detect changes in intracellular calcium levels using a fluorescent calcium-sensitive reporter dye. The basic system for performing calcium mobilization assays includes the FLIPR calcium assay kit and FLIPR... A system for observing changes in intracellular calcium levels and determining dose-response in HEK293 cells transfected with recombinant human μ, δ, or κ opioid receptors.

[0420] Cells used in the assay were grown in 88% DMEM medium containing 10% FBS, 300 μg / mL G418, 2 μg / mL blastomycin, 1% GlutaMax, and 1% penicillin / streptomycin (Hyclone-SV30010). 20,000 cells per well of the assay plate (Greiner-781946) were seeded in 20 μL of medium and incubated at 37°C in a 5% CO2 incubator for 20 h. The compound was then prepared by five-fold serial dilutions to obtain 10 doses, and 500 nmL of each concentration was transferred to the compound plate. 30 μL of assay buffer (20 mM HEPES and 1X HBSS) was then added to each well of the compound plate, and the plate was rotated at 1500 rpm for 15 seconds. Then, gently dispense 20 μL of 2X Fluo-4Direct™ wash-free loading buffer (Invitrogen-F10471) into each well of the assay plate and rotate at 1000 rpm for 15 seconds, followed by incubation at 37°C for 50 minutes. Remove the assay plate from the incubator and allow it to stand at room temperature for 10 minutes. Place the assay plate, compound plate, and tip box directly into the FLIPR. In the system, 10 μL of each compound was transferred from the compound plate to the assay plate in the FLIPR Tetra fluorescence imaging plate reader. The plate was rotated 140 times; then the “Max-Min” from reading 1 to reading 140 was calculated to produce the final signal for the effect % calculation. The data were analyzed using Prism, a curve fitting equation “log(agonist) vs. response – variable slope”. Table 2 shows the results of these assays.

[0421] Table 2. FLIPR assay for opioid κ receptor agonists

[0422]

Claims

1. A compound having any one of formulas (II)-(V) or a pharmaceutically acceptable salt thereof:

2. A pharmaceutical composition comprising the compound according to claim 1 and at least one pharmaceutically acceptable excipient.

3. Use of the compound of claim 1 or the pharmaceutical composition of claim 2 in the manufacture of a medicament for treating a κ-opioid receptor agonist-related disease or condition, the treatment comprising administering to a subject in need a pharmaceutical composition comprising a compound of formula (I); wherein the κ-opioid receptor agonist-related disease or condition is selected from the group consisting of: pain, cardiovascular disease, pruritus, nausea, inflammatory disease, spinal anesthesia, cough, stroke, multiple sclerosis, addiction, and post-traumatic cartilage degeneration.

4. The use according to claim 3, wherein the κ opioid receptor agonist-related disease or condition is hypoxic pulmonary hypertension.

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